prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/1941/1/957S.pdf · ii DECLARATION I Miss...
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i A THESIS TITLED Studies on the submerged fermentation of invertase by Saccharomyces cerevisiae Submitted to GC university Lahore in fulfillment of the requirements for the award of degree of Doctor of Philosophy in BIOTECHNOLOGY by AAFIA ASLAM 58 - G C U - P h D - Bio - 2 0 0 6 INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY GC UNIVERISITY LAHORE
Transcript of prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/1941/1/957S.pdf · ii DECLARATION I Miss...
i
A THESIS TITLED
Studies on the submerged fermentation of invertase by Saccharomyces cerevisiae
Submitted to GC university Lahore in fulfillment of the
requirements for the award of degree of
Doctor of Philosophy
in
BIOTECHNOLOGY
by
AAFIA ASLAM
58
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G
C
U
-
P
h
D
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Bio
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0
0
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INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY
GC UNIVERISITY LAHORE
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DECLARATION
I Miss Aafia Aslam Roll No. 58-Bio-2006 student of Ph.D in the subject of
Biotechnology, here by declare that the matter printed in the thesis titled “Studies on
the submerged fermentation of invertase by Saccharomyces cerevisiae” is my own
work and has not been printed, published and submitted as research work, thesis or
publication in any form in any university, research institution etc in Pakistan or abroad.
Date:_______________ _____________________ Aafia Aslam Signature of Deponent
iii
CERTIFICATE Certified that the research work contained in this thesis titled “Studies on the
submerged fermentation of invertase by Saccharomyces cerevisiae” has been carried
out and completed by Miss Aafia Aslam Roll No 58-Bio-PhD-06 under our supervision
during her Ph.D studies in the subject of Biotechnology.
_________________________
Prof. Dr. Ikram-ul-Haq (S.I)
Supervisor
________________ Date ________________________
Dr. Sikander Ali
Co- supervisor
Submitted through ___________________ _____________________
Prof. Dr. Ikram-ul-Haq (S.I) Controller of Examinations
Dean, Faculty and Science Technology.
Director,
Institute of Industrial Biotechnology
GC University, Lahore
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ACKNOWLEDGEMENTS
All praise for the, “ALMIGHTY ALLAH” who is the only supreme Authority and
whose presence has been figured on the two words i.e. “KUN FAYAKUN”. Every
tiny or massive entity moves with His permission. Countless thanks to Him for
accrediting me to accomplish this important task within this specified time. All my
respect and regards to the Holy Prophet Hazrat Muhammad (peace be upon him)
who is forever a torch of guidance and knowledge for humanity. In view of his
saying: “He who does not thank to people is not thankful to Allah”
I am highly obliged in paying deepest gratitude to my respected teacher and
research supervisor Prof. Dr. Ikram-ul-Haq, SI (Dean, Faculty of Science and
Technology; Director, Institute of Industrial Biotechnology, GCU, Lahore) for his
valuable guidance, encouragement, cooperation and discussion. His enthusiastic
inspiration and fatherly affection enabled me to attain the objectives without any
difficulty. I feel pleasure to express my sincere gratitude to my Co-supervisor Dr.
Sikander Ali, for his valuable suggestions and help during this tenure.
I most gratefully acknowledge my indebtedness to Dr. M. A. Qadeer, Dr.
Muhammad Yaqub (late), Dr. Mohsin Javed, Dr. Zahid, Dr. Mahmood, Dr.
Hamid Mukhtar, Dr. Numan Aftab, Dr. Bushra Munir (Assistant Professor,
GCU) and Ms. Uzma Hammed (Lecturer, GCU) for their scientific discussions and
generous advices as and when needed, during my research work.
I am grateful to Dr. Khalid Aftab, Vice Chancellor, GC University, Lahore for
providing me this opportunity to work in this great Institute.
I wish to express my thanks to Prof. Dr. Javed Iqbal (Director, School of
Biological Sciences), Prof. Dr. Naeem Rashid (SBS) and Dr. Nauman Rasool
(SBS) for the guiding and facilitating me for the completion of the purification part of
my work.
I am thankful to my friends Dr. Saba Butt, Dr. Aisha Saleem Khan (Assistant
Professor, Forman Christian College University) and Dr. Amber Shehzadi (Assistant
Professor, Kinnaird College for Women) for their sincere cooperation and moral
support during compilation of my work.
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The words are inadequate to express my heartfelt thanks to my fellows and friends
Dr. Roheena Abdullah, Dr. Zahid Butt, Shazia Malik and Dr. Mujahid Hussain,
for their cooperation and moral support in the research work.
I wish to acknowledge Dr. Shakeel Ahmad (Assistant Professor, Department of
Mycology and Plant Pathology, Punjab University) for helping in the identification of
strain.
I am also thankful to laboratory staff especially Mr. Fasial, Mr. Usman, Mr.
Rameez, Mr. Khawar and all others for their support during the whole period of my
research.
Although feelings are deep but unfortunately words are too shallow, that cannot
follow the depths of my deep gratitude to my loving mothers and late father. My
fortune is due to their best wishes and prayers.
Aafia Aslam
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CONTENTS
Titles Page No.
Abstract xvi-xvii
Chapter # 1
INTRODUCTION 1
Objectives 9
Chapter # 2
LITERATURE REVIEW 10
Chapter # 3
MATERIALS AND METHODS 37
3.1: Materials 37
3.2: Methods 37
3.2.1: Isolation of microorganism 37
3.3: Fermentation technique 37
3.3.1: Extracellular invertase production 37
3.3.1.1: Preparation of inoculum 37
3.3.1.2: Yeast viable count 38
3.3.1.3: Fermentation media 38
3.4: Shake flask studies 38
3.5: Fermentor studies 39
3.6: Significant parameters 39
3.6.1: Different fermentation media 39
3.6.2: Incubation period 39
3.6.3: Effect of initial pH 39
3.6.4: Effect of temperature 40
3.6.5: Effect of volume 40
3.6.6: Effect of inoculum size 40
3.6.7: Effect of agitation and aeration 40
3.6.8: Effect of dissolved oxygen 40
3.6.9: Effect of carbon sources 40
3.6.10: Effect of additional nitrogen sources 40
3.7: Strain improvement 41
3.7.1: UV irradiation 41
3.7.2: Nitrous acid treatment 41
3.7.3: EMS treatment 41
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3.7.4: 2-deoxy-D-glucose resistance 42
3.3.2: Intracellular invertase production 42
3.3.2.1: Extraction of intracellular invertase 42
3.8: Analytical techniques 42
3.8.1: Dry cell mass 42
3.8.2: Invertase activity 43
3.8.3: Protein estimation 43
3.9: Statistical analysis 43
3.10: Fermentation kinetic study 43
3.11: Immobilization Studies 44
3.11.1: Sucrose hydrolysis 44
3.11.1.1: Effect of sucrose concentrations 45
3.11.1.2: Effect of temperature 45
3.11.1.3: Effect of pH 45
3.11.1.4: Re-use of immobilized cells 45
3.12: Purification of invertase 45
3.12.1: Ammonium sulfate precipitation 45
3.12.2: Anion- exchange chromatography 45
3.12.3: Gel Filtration 46
3.12.4: Dialysis 46
3.12.5: Electrophoresis 46
3.12.6: Protein marker 46
3.12.7: Carbohydrate content 46
3.13: Gel preparation 46
3.13.1: Separating gel 46
3.13.2: Stacking gel 47
3.14: Characterization of purified invertase 47
3.14.1: Effect of pH and temperature on stability of invertase 47
3.14.2: Effect of additives on enzyme activity 47
3.14.3: Determination of kinetic constant (Km) 48
3.14.4: Determination of maximum velocity (Vmax) 48
3.15: Preparation of standard curves 48
3.15.1: Glucose curve 48
3.15.2: BSA curve 48
3.15.3: Mannose curve 49
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3.16: Preparation of solutions /reagents 49
3.17: Preparation of buffers 50
Chapter # 4
RESULTS
4.1: Isolation, identification and screening of yeast cultures 55
4.1.1: Optimization of cultural conditions for selected yeast isolate 60
4.1.1.1: Rate of invertase production 60
4.1.1.2: Effect of sucrose concentrations 60
4.1.1.3: Effect of pH 60
4.2: Strain improvement 64
4.2.1: Physical mutation 64
4.2.1.1: UV-irradiation 64
4.2.2: Chemical mutation 64
4.2.2.1: Nitrous acid treatment 64
4.2.2.2: EMS treatment 65
4.2.3: Mutant resistance to 2-deoxy-D-glucose 65
4.3: Enzyme production 78
4.3.1: Extracellular invertase 78
4.3.1.1: Shake flask 78
4.3.1.1.1: Rate of invertase production 78
4.3.1.1.2: Selection of culture media 78
4.3.1.1.3: Effect of different sugars 79
4.3.1.1.4: Effect of sucrose concentrations 87
4.3.1.1.5: Effect of incubation temperature 87
4.3.1.1.6: Effect of inoculum size 87
4.3.1.1.7: Effect of volume of the media 87
4.3.1.1.8: Effect of initial pH 88
4.3.1.1.9: Effect of additional organic nitrogen sources 94
4.3.1.1.10: Effect of additional inorganic nitrogen sources 94
4.3.1.1.11: Effect of additional agricultural byproducts 94 nitrogen sources
4.3.1.2: Fermentor studies 102
4.3.1.2.1: Rate of invertase production 102
4.3.1.2.2: Effect of sucrose concentrations 102
4.3.1.2.3: Effect of inoculum size 103
4.3.1.2.4: Effect of pH 111
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4.3.1.2.5: Effect of temperature 111
4.3.1.2.6: Effect of agitation intensity 111
4.3.1.2.7: Effect of aeration 115
4.3.1.2.8: Effect of dissolved oxygen 115
4.3.2: Intracellular invertase 118
4.3.2.1: Rate of invertase production 118
4.3.2.2: Effect of amplitudes 118
4.3.2.3: Effect of pH 119
4.4: Immobilization studies 124
4.4.1: Rate of sucrose hydrolysis 124
4.4.2: Effect of sucrose concentrations 124
4.4.3: Effect of temperature 124
4.4.4: Effect of pH 124
4.4.5: Re-use of immobilized cells in batch process 125
4.4.6: Storage stability 125
4.5: Purification 131
4.5.1: Extracellular invertase 131
4.5.2: Intracellular invertase 136
Chapter # 5
DISCUSSION 146
Conclusion 157
Chapter # 6
REFERENCES 158
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LIST OF TABLES
Table Titles Page No.
4.1 Screening of isolates of S. cerevisiae for extracellular invertase production by shake flask technique.
56
4.1.1 Sub-grouping of extracellular invertase producing isolates of S. cerevisiae
59
4.2 Production of extracellular invertase by S. cerevisiae IS-66 treated with UV in shake flask.
66
4.2.1 Sub-grouping of extracellular invertase producing UV-treated strains of S. cerevisiae.
68
4.3 Production of extracellular invertase by S. cerevisiae IS-66 treated with nitrous acid in shake flask.
70
4.3.1 Sub-grouping of extracellular invertase producing NA-treated strains of S. cerevisiae
72
4.4 Production of extracellular invertase by S. cerevisiae NA-45 treated with EMS in shake flask.
74
4.4.1 Sub-grouping of extracellular invertase producing EMS-treated strains of S. cerevisiae.
76
4.5 Kinetic parameters of rate of fermentation for the extracellular invertase production by wild and mutant strains of S. cerevisiae in shake flask.
82
4.6 Rate of biomass formation of wild and mutant strains of S. cerevisiae for extracellular invertase production in shake flask.
84
4.7 Effect of different additional organic nitrogen sources on the extracellular invertase production by S. cerevisiae EMS-42 in shake flask.
96
4.8 Effect of different additional inorganic nitrogen sources on extracellular invertase production by S. cerevisiae EMS-42 in shake flask.
98
4.9 Effect of different agricultural byproducts and its concentration on the biosynthesis of invertase by mutant strain of S. cerevisiae EMS-42 in shake flask.
100
4.10 Kinetic parameters of rate of fermentation for the extracellular invertase production by mutant strains of S. cerevisiae EMS-42 in shake flask and fermentor.
106
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4.11 Rate of biomass formation of mutant strains of S. cerevisiae EMS-42 for extracellular invertase production in shake flask and fermentor.
108
4.12 Purification steps of extracelluar invertase 135
4.13 Purification steps of intracelluar invertase (S and L forms) 140
4.14 Effect of additives on stability of purified glycosylated and non-glycosylated invertase.
143
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LIST OF FIGURES
Figure Title Page No.
3.1 Standard curve of glucose 52
3.2 Standard curve of bovine serum albumin (BSA) 53
3.3 Standard curve of mannose 54
4.1 Time course study for extracellular invertase production by S. cerevisiae IS-66 in shake flask.
61
4.2 Effect of sucrose concentration on the extracellular invertase production by S. cerevisiae IS-66 in shake flask.
62
4.3 Effect of pH on the extracellular invertase production by S. cerevisiae IS-66 in shake flask.
63
4.4 Survival curve of mutant strain of S. cerevisiae after UV irradiation.
69
4.5 Survival curve of mutant strain of S. cerevisiae NA-45 developed after nitrous acid treatment.
73
4.6 Survival curve of mutant strain of S. cerevisiae EMS-42 developed after EMS treatment.
77
4.7 Comparison of rate on the extracellular invertase production by wild and mutant strains of S. cerevisiae in shake flask.
80
4.8 Comparison of dry cell mass by wild and mutant strains of S. cerevisiae in shake flask.
81
4.9 Comparison of specific growth rate (μ h-1) of wild and mutant strain of S. cerevisiae for extracellular invertase production.
83
4.10 Selection of culture media for the extracellular invertase production and dry cell mass by S. cerevisiae EMS-42 in shake flask.
85
4.11 Effect of different sugars on the extracellular invertase production and dry cell mass by S. cerevisiae EMS-42 in shake flask.
86
4.12 Effect of different sucrose concentrations on the extracellular invertase production and dry cell mass by S. cerevisiae EMS-42 in shake flask.
89
4.13 Effect of different incubation temperatures on the extracellular invertase production and dry cell mass by S.
90
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cerevisiae EMS-42 in shake flask.
4.14 Effect of different inoculum size on the extracellular invertase production and dry cell mass by S. cerevisiae EMS-42 in shake flask.
91
4.15 Effect of different volumes of the media on the extracellular invertase production and dry cell mass by S. cerevisiae EMS-42 in shake flask.
92
4.16 Effect of different initial pH on the extracellular invertase production and dry cell mass by S. cerevisiae EMS-42 in shake flask.
93
4.17 Effect of different additional organic nitrogen sources on extracellular invertase production and dry cell mass by S. cerevisiae EMS-42 in shake flask.
97
4.18 Effect of different inorganic nitrogen sources on invertase production and dry cell mass by S. cerevisiae EMS-42 in shake flask.
99
4.19 Effect of different agricultural byproducts nitrogen sources on invertase production and dry cell mass by S. cerevisiae EMS-42 in shake flask.
101
4.20 Comparison of rate on the extracellular invertase production by S. cerevisiae EMS-42 in shake flask and stirred fermentor.
104
4.21 Comparison of rate on the production of dry cell mass of S. cerevisiae EMS-42 in shake flasks and stirred fermentor.
105
4.22 Comparison of specific growth rate (μ h-1) of mutant strain of S. cerevisiae EMS-42 in shake flask and fermentor for extracellular invertase production.
107
4.23 Effect of different sucrose concentrations on the extracellular invertase production by S. cerevisiae EMS-42 in the stirred fermentor.
109
4.24 Effect of different inoculum size on the extracellular invertase production by S. cerevisiae EMS-42 in the stirred fermentor.
110
4.25 Effect of pH on the extracellular invertase production by S. cerevisiae EMS-42 in the stirred fermentor.
112
4.26 Effect of different temperatures on the extracellular invertase production by S. cerevisiae EMS-42 in the stirred fermentor.
113
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4.27 Effect of different agitation intensity on the extracellular invertase production by S. cerevisiae EMS-42 in the stirred fermentor.
114
4.28 Effect of different aeration levels on the extracellular invertase production by S. cerevisiae EMS-42 in the stirred fermentor.
116
4.29 Effect of different concentrations of dissolved oxygen on the extracellular invertase production by S. cerevisiae EMS-42 in the stirred fermentor.
117
4.30 Comparison of rate on the intracellular invertase production by wild and mutant strains of S. cerevisiae in shake flask.
120
4.31 Comparison of rate on the intracellular invertase production by S. cerevisiae EMS-42 in shake flask and stirred fermentor.
121
4.32 Effect of different amplitudes to release intracellular invertase during sonication in S. cerevisiae EMS-42.
122
4.33 Effect of differenr pH on intracellular invertase release during sonication in S. cerevisiae EMS-42.
123
4.34 Time course study of sucrose hydrolysis by Calcium alginate immobilized yeast cells of S. cerevisiae EMS-42 in shake flask.
126
4.35 Effect of different sucrose concentrations on sucrose hydrolysis by Ca-alginate immobilized cells of S. cerevisiae EMS-42 in shake flask.
127
4.36 Effect of different temperatures on sucrose hydrolysis by Ca-alginate immobilized cells of S. cerevisiae EMS-42 in shake flask.
128
4.37 Effect of pH on sucrose hydrolysis by Ca-alginate immobilized cells of S. cerevisiae EMS-42 in shake flask.
129
4.38 Time course study of re-use of batch process for sucrose hydrolysis by Ca-alginate immobilized cells of S. cerevisiae EMS-42 in shake flask.
130
4.39 SDS-PAGE after ammonium sulfate treatment for extracellular invertase.
132
4.40 Elution pattern on DEAE-Sephadex for extracellular glycosylated invertase.
133
4.41 SDS-PAGE of purified extracellular invertase 134
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4.42 SDS-PAGE after sonication, ammonium sulfate treatment and chromatographies for intracellular invertase.
137
4.43 Elution Pattern on DEAE-Sephadex for intracellular non- glycosylated invertase.
138
4.44 Elution Pattern on Sephadex G-50 for intracellular non-glycosylated invertase.
139
4.45 Effect of pH on stability of purified glycosylated and non-glycosylated invertase.
141
4.46 Effect of temperature on stability of purified glycosylated and non-glycosylated invertase.
142
4.47 Lineweaver-Burk plot for intracellular non-glycosylated invertase.
144
4.48 Lineweaver-Burk plot for extracellular glycosylated invertase. 145
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ABSTRACT
In the present study, eighty six strains of Saccharomyces cerevisiae were isolated
from different samples of fruits and soil by serial dilution method. The strain IS-66 gave
maximum extracellular invertase production (1.10 U/ml). The enzyme activity reached to
5.6 U/ml when incubation time (48 h), sucrose concentration (5 g/l) and pH 5.5 were
optimized. The wild strain IS-66 was exposed to ultraviolet (UV) radiations to obtain a
mutant with improved enzyme activity. UV induced mutagenesis did not produce any
stable mutant and almost all of the mutants produced relatively lesser invertase than the
parental strain. Strain IS-66 was further subjected to chemical mutagenesis using nitrous
acid and ethyl methane sulphonate (EMS). After extensive screening, two mutants were
developed with increased enzyme activity NA-45 (20.74 U/ml) and EMS-42 (34.2 U/ml)
from the wild-culture (IS-66). The mutant strain EMS-42 was cultured on the medium
containing 2-deoxy-D-glucose (2dg) and its stability in invertase production was
determined at different concentrations of 2dg. The concentration of 0.04 mg/ml was
found optimal, as at this concentration EMS-42 showed consistent enzyme activity.
Six media were evaluated for the production of invertase in shake flasks. M1
medium (g/l) containing yeast extract 3, peptone 5 and sucrose 30 g/l gave better
production of invertase (25.28 U/ml) after 48 h of inoculation. Different sugars such as
sucrose, glucose, fructose, lactose, galactose, maltose, raffinose and molasses were
investigated on the enzyme production. Of these, sucrose was found to be best (44.03
U/ml) after optimizing the concentration at 10 g/l. Incubation temperature (30ºC),
inoculum size (2.0 %, v/v) and volume of the medium (50 ml/250 ml Erlenmeyer flask)
were optimized. The effect of different additional nitrogen sources such as organic,
inorganic and agriculture byproducts were also tested. Peptone at the concentration of 6
g/l gave maximum production of invertase (50 U/ml). The addition of inorganic nitrogen
sources and agricultural byproducts nitrogen were not found to have any impact on the
enhancement in enzyme production rather it was decreased from the control especially
in case of agricultural byproducts. In stirred fermentor (7.5 L), the scale up studies for
invertase production was carried out. The enzyme production (65.12 U/ml) was obtained
after 24 h of incubation. The overall increase in enzyme activity (15 U/ml) and
fermentation time was shortened by 24 h while scaling up enzyme production from shake
flask to fermentor. The maximum enzyme activity (80.06 U/ml) was achieved after
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optimization of cultural conditions such as sucrose (15 %, w/v), pH (4.5), inoculum size
(7.5 %, v/v), agitation intensity (240 rpm) and aeration rate (1 vvm, 10 % DO).
The intracellular enzyme activity was also determined by sonication. The
maximium enzyme activity (57 U/ml) was found in mutant strain of EMS-42 after 24 h
fermentation in the fermentor. During sonication, the maximum specific activity of 106
U/mg of protein was obtained with 0.5 duty cycle of impulses at amplitude of 40 % and
pH 5 for 60 min. The calcium alginate entrapment technique was used for
immobilization of whole cells of S. cerevisiae EMS-42 to form inverted syrup. It was
noticed that maximum sucrose hydrolysis (63.40 %) was achieved after 18 h of
incubation time. By optimization of cultural conditions for sucrose hydrolysis, the
maximum hydrolysis percentage (76.3 %) was obtained at 50ºC, pH 5.5 using sucrose
(60 %) as substrate.
An extracellular invertase was purified to homogeneity by two step purification
i.e., ammonium sulfate precipitation and DEAE-Sephadex A-50. The enzyme was
present in the supernatant of 85 % saturation being glycoprotein in nature. DEAE
column chromatography eluted the enzyme as single active fraction at 0.2 M NaCI. The
enzyme was purified by 15 fold with recovery of 38 %. The molecular mass of 110 kDa
was determined after SDS-PAGE. The carbohydrate content was found to be 48 %. The
intracellular invertase contains both forms of glycosylated (large) and non-glycosylated
(small). The same procedure was applied for glycosylated intracellular invertase (L-
form) while three purification steps were performed for non-glycosylated invertase (S-
form). The L-form was purified by 19 fold with recovery of 32 %. Like extracellular
invertase, the molecular weight (110 kDa) for L-form was found. Ammonium sulfate
precipitation separated the enzyme (S-form) as insoluble fraction. This form of enzyme
was eluted at 0.3 M NaCl using DEAE-Sephadex. A single band of molecular weight (55
kDa) was estimated after Sephadex G-50 with purification (16 fold) and recovery of 17
%. For both purified non-glycosylated and glycosylated invertase the optimum pH (5)
was same whereas optimal temperature, MnCl2 and the values of the Km and Vmax were
found to be as 50 and 60ºC, 109 and 111 %, 1.2 and 1.8 mM, 909 and 1429 U/ml/min,
respectively.
1
INTRODUCTION
Invertase (.D. fructofuranosidase, E.C. 3.2.1.26) cleaves -1,4 glycosidic linkage
between -D-glucose and -D-fructose molecules of sucrose by hydrolysis and releases
monosaccharides (Li et al., 1998; Mobini-Dehkordi et al., 2008). The enzyme attacks
beta-D-fructofuranoside (sucrose, raffinose, stachyose and inulin) from the fructose end
(Rubio et al., 2002; Rubio and Navarro, 2006; Gore et al., 2009). Invertase exists in two
forms, glycosylated periplasmic protein and cytosolic non- glycosylated protein (Vitolo
et al., 1995; Sezai and Turgut, 2002; Rashad et al., 2006). The secretion of enzyme
located intracellularly which corresponds to repressed forms of invertase and
extracellular one, containing nine or ten N-glycosidically linked oligosaccharides which
corresponds to the de-repressed form of the enzyme (Huffaker and Robins, 1983) are
regulated by catabolic repression. The culture medium containing glucose in higher
concentration completely repressed enzyme production, whereas the use of sucrose or
raffinose as carbon source allowed derepression of invertase synthesis (Olutiola and
Cole, 1980; Vainstein and Peberdy, 1991). In derepressed cells, the heavy (glycosylated)
invertase being major part (95 %) of the total enzyme is located chiefly outside the
cytoplasmic membrane (Lampen et al., 1967). It is known that both of these enzymes are
synthesized on the matrix of the same structural gene and their protein moieties have a
molecular mass of 60 kDa (Moreno et al., 1990). Mostly extracellular enzymes are
isolated as commercial products because extracellular enzymes are usually available in
purer and more stable form than intracellular enzymes (Chan et al., 1991).
There are many industrial applications of invertase in confectionary, beverage,
bakery and pharmaceutical formulations. It plays the role in the production of invert
syrup i.e. equimolar mixture of fructose and glucose and high fructose syrup (HFS) from
sucrose. Invert syrup and high fructose syrup produced by enzyme hydrolysis is
preferred over those syrups which is produced by acid hydrolysis of sucrose giving
undesirable by products, absence of sweet taste, low conversion efficiencies, high ash
contents and so are highly uneconomical. On the other hand, the enzymatic hydrolysis
gives high purity products that better in taste, stability, non-crystalizable and free from
any undesirable by-products (Rossi-Alva and Rocha-Leao, 2003; Tomotani and Vitolo,
2004; Kaur and Sharma, 2005; Aranda et al., 2006; Oztop et al., 2009; Celebi et al.,
2009). Inver sugar is used in many industries e.g., in the production of lactic acid and
ethanol (Acosta et al., 2000; Sanchez et al., 2001). The yeast invertase also plays an
2
important role in ethanol tolerance of yeast cells (Osho, 2005). The major use of this
enzyme in the preparation of creams, jams, candies, powder milk for infants, artificial
honey, digestive aid tablets and as plasticizing agents in cosmetics (Weber and Roitsch,
2000; Phadtare et al., 2004; Sungur and Al-Taweel, 2006; Marquez et al., 2008; Safarik
et al., 2009; Kotwal and Shankar, 2009).
A wide variety of microbial, animals or plant sources are used for the producion
of industrial enzymes. But most enzyme production processes rely on the microbial
source. Although invertase has been found in microbial, animals and higher plant sources
but microbes is the best choice being rapid growth and easy manipulation (Matrai et al.,
2000; Luxhoi et al., 2002; Gangadhara et al., 2008). The increasing demand for invertase
has stimulated its production from microbial sources like bacteria such as Arthrobacter
globiformis (Win et al., 2004), Lactobacillus reuteri (de Gines et al., 2000), Azotobacter
chroococum (de la Vega et al., 1991), fungi including yeasts such as Fusarium
oxysporium (Nishizawa et al., 1980), Aureobasidium pullulans (Yoshikawa et al., 2006),
Fusarium solani (Bhatti et al., 2006), Aspergillus niger (Zhang and Ge, 2006),
Aspergillus ochraceus (Ghosh et al., 2001), Aspergillus oryzae (Kurakake et al., 2010),
Thermomyces lanuginosus (Chaudhuri et al., 1999), Pichia anomala (Perez et al., 2001),
Rhodotorula glutinis (Rubio et al., 2002), Rhodotorula dairenensis (Gutierrez-Alonso et
al., 2009), Kluyveromyces fragilis (Workman and Day, 1983), Schizosaccharomyces
pombe (Moreno et al., 1990), Schwanniomyces occidentalis (Klein et al., 1989), Candida
utilis (Belcarz et al., 2002a) and Saccharomyces cerevisiae (Batista et al., 2004;
Andjelkovic et al., 2010). In spite of production by many bacteria and filamentous fungi,
yeasts are the most preferred source for this enzyme because of their high yields and
sucrose tolerance. One of yeasts, S. cerevisiae is used for invertase production due to
higher sucrose fermenting capability. Commercially invertase is produced by S.
cerevisiae using submerged fermentation as a rich source of both intracellular and
extracellular invertase utilization (Trumbly 1992; Silveira et al., 2000; Venkateshwar et
al., 2009).
Intracellular enzymes usually remain associated with the cell and therefore have
to be released, unless the microorganism itself is used as the catalyst. A number of
techniques i.e., sonication, hydrodynamic cavitation and high pressure homogenization
can be used for the release of intracellular invertase. Sonication is one of the most
commonly employed method has been used for cell disruption (James et al., 1972).
Ultrasound has been used to extract and release the intracellular enzymes such as
3
invertase being secretory protein to the periplasmic space from S. cerevisiae
(Balasundaram and Pandit, 2001), A. niger (Vargas et al., 2004), Phaffia rhodozyma
(Persike et al., 2002), ATPase and acid phosphatase from S. cerevisiae (Bucalon and
Palma, 1990) and ß- galactosidase from Lactobacillus (Wang and Sakakibara, 1997).
Although it is hardly useful for industrial purposes but it gives many interesting
advantages like it does not require sophisticated equipment and extensive technical
training (James et al., 1972).
The production of the enzyme mainly depends on the yeast strain employed,
nutritional requirements and cultural conditions. The conventional ways for random
mutagenesis are ultra violet (UV) radiation, use of alkylating agents like ethyl methane
sulphonate (EMS), N-methyl-N-nitro-N-nitroso guanidine (MNNG) or nitrous acid
(Nakajima et al., 1988; Azin and Noroozi, 2001; Szafraniec et al., 2003; Kig et al., 2005;
Haq et al., 2008; Mobini-Dehkordi et al., 2008). EMS is an alkylating agent that causes
point mutations by A-T transition to G-C (French et al., 2006). This mutagenic has been
used to enhance enzyme production and resistance to antifungal drugs (Khattab and
Bazaraa, 2005; Hapala et al., 2005). These mutagens proved successful after suitable
selection and screening of the resultant mutants. On the basis of catabolic repression,
mutants have been screened for resistance to 2-deoxy-D-glucose having improved
fermentative capacity on sugar medium (Rincon et al., 2001). Novak et al. (1990)
obtained a number of 2-deoxy-D-glucose (2-DOG) resistant mutants exhibiting resistance
to glucose repression in different strains of Saccharomyces spp. Ager and Haynes (1990)
indicated “the interaction between EMS and UV in S. cerevisiae might arise from the
inhibition of double-strand break repair by one, or both agents”.
Higher production of invertase depends to a great extent on the microorganism,
basal substrate and the microbial production process. A number of factors including
physiological stability, yield consistency, incubation temperature, agitation, incubation
time, aeration level, etc are required for the maximum enzyme production (Laluce et al.,
1991). The overall provision of appropriate fermentation conditions is vital for the proper
growth and subsequent higher yield of the desired product (Michael and Sarah, 1994).
The specific fermentation procedures adopted by manufacturers vary to a degree; there
remain only two main principle methods of cultivation, i.e. solid-state and submerged
fermentation. Most microbial enzymes are produced by aerobic submerged fermentation,
which allows greater control of growth factors than solid-state methods. Usually
submerged fermentation has been favored over solid-state for invertase production as it
4
gives higher yields, requires less manpower and environmentally friendly (Lambert et
al., 1983; Arguelles et al., 1995; Koo et al., 1998; Romero-Gomez et al., 2000).
Selection of suitable fermentation media has profound effect on enzyme
production. Yeasts are able to use a variety of compounds such as carbon and nitrogen
containing compounds (yeast-extract, peptone, carbohydrates, salt, or vitamin solution)
as basal medium for invertase production (Walker 1998; Arfi et al., 2003). The molasses
media with supplementation of ethanol and NaCl are also used for the production of
invertase (Zech and Goerisch, 1995). Many workers have optimised the cultural
conditions and nutritional requirements for the enhanced production of invertase by S.
cerevisiae in shake flask and stirred fermentor (Vitolo et al., 1995; Koo et al., 1998;
Abrahao- Neto et al., 1996; Herwig et al., 2002).
The presence of appropriate carbon source in a medium acts as critical nutrient
which stimulate growth and it has direct effects on the production of many enzymes
(Rodriguez et al., 1997; Herwig et al., 2001). Various carbon sources included glucose,
fructose, maltose, glycerol, ethanol, xylose, sucrose have been used for growth and
enzyme production in S. cerevisiae (Dworschack and Wickerham, 1961; Zhang and Ge,
2006; Martinezforce and Benitez, 1995). An extracellular invertase was secreted by S.
cerevisiae when it subjected to media containing β-fructofuranosides as substrates such
as sucrose or raffinose, sucrose or raffinose (Carlson, 1999; Mwesigye and Barford,
1996; Dynesen et al., 1998). In sucrose fermentation, the periplasmic extracellular
invertase hydrolyzed the sugar and produces glucose and fructose that transported into
the cells and metabolised (Batista et al., 2004). The selection of suitable carbon and
nitrogen sources out of the large diversity, the S. cerevisiae has sensing and regulation
mechanisms in the form of induction and repression of key systems (Cooper, 2002). The
addition of glucose and related sugars repress the transcription of genes encoding
enzymes required for the utilization of alternative carbon sources. Some of these genes
were also repressed by other sugars such as galactose, maltose and the process is known
as catabolite repression (Gancedo, 1998; Herwig et al., 2001). Catabolite repression by
glucose and fructose inhibit the formation of extracellular invertase, which are then
sequencially consumed by the organism (de Groot et al., 2003). Usually the sucrose
utilization was inhibited when the concentrations of glucose or fructose exceed from 5
g/l, and thus both sugars have equal capability for exerting catabolite repression
(Dynesen et al., 1998). Robledo-Olivo et al. (2009) suggested that an addition of lower
5
concentration of glucose was a viable option to increase the enzyme secretion by the
fungi.
Nitrogen sources as basic constituent of the media play a key role in synthesizing
enzymes because they supply amino acids. The S. cerevisiae encounters a wide variety of
nitrogen sources in its natural habitat. However, not all nitrogen sources support growth
equally well. The utilization of appropriate nitrogen sources promote higher growth rates
than poor nitrogen sources (Magasanik 1992). In most of the studies more than one
nitrogen sources are used for enhance the production of enzyme invertase in yeast. The
combined effect of organic nitrogen sources i.e., yeast extract and peptone with
appropriate carbon source gives the higher yield invertase than inorganic nitrogen
supplements (Rodriguez et al., 1995; Belcarz et al., 2000). The different organic nitrogen
sources have a marked influence on the ability of yeast to synthesize invertase. Yeast
extracts are concentrates of the soluble components of yeast cells and can be a good
source of supplement for protein deficient diet (Sommer, 1998). There exists a specific
physiological response of sucrose metabolism in the presence of nitrogen source
(Pitombo et al., 1994; Roitsch et al., 2003). The yeast periplasmic enzymes with
nutritional roles would be responsible for regulation of sensing and signalling pathways
that might respond to the quality of carbon and nitrogen sources (Oliveira et al., 2005).
The invertase synthesis is the best at pH 8.0, when sole nitrogen source was peptone
(Olusanya and Olutiola, 1994). On nitrogen starvation in the presence of sucrose, the
invertase activity in wild-type cells from midlog phase decreased three times, whereas in
stationary phase the activity declined eight times (Silveira et al., 2000). Improved
invertase production has been reported in the medium containing corn-steep liquor (Chan
et al., 1991).
Time course study determines the efficacy of the batch process and subsequent
product formation. The pattern of accumulated reducing sugar after specific incubation
time is characteristic to the species (Matrai et al., 2000). Maintenance of cell viability
throughout the fermentation process is an important factor that depends on the medium
composition and incubation conditions (Laluce et al., 1991). The incubation time for the
production of invertase by yeast has been optimized and the results have shown that 48 h
incubation is the best for invertase production by S. cerevisiae (Mizunaga et al., 1981).
The maximum invertase production in S. cerevisiae was obtained after the incubation
time period of 48-96 h (Barlikova et al., 1991).
6
The production of invertase is also influenced by the initial pH and incubation
temperature and exhibits marked stability towards them. Temperature of the reaction
mixture determines the rate of sucrose inversion by the active enzyme (Yusa and
Enokida, 1953; Vrabel et al., 1997). Dan and Teodorescu (1993) observed a marked
invertase secretion by yeast at 28C for 6 days. Regulation of fresh medium in response
to the metabolic activities of yeast population is controlled by the pH changes as it
enables in the attaining of high cell density with both high productivity and high yields
(Porro et al., 1991). Enzymes are only active in a restricted range of pH, and for most
cases, show a definite optimum pH where activity is maximal (Dixon and Webb, 1979;
Segel, 1975). Dworschack and Wickerham (1961) obtained invertase by Saccharomyces
spp. at temperature (30C), pH (6.0) and incubation period (24-48 h) of culture medium.
Optimal pH for invertase formation seems to correspond to that of sucrose fermentation
(L’Hocine et al., 2000).
Number of yeast cells introduced into the culture medium determines the extent
and quality of enzymes produced. So there exists a correlation between amount of
inoculum and substrate concentration for invertase production by S. cerevisiae. Influence
of inoculum age and size on invertase production is needed in-depth investigation before
scaling up a high-yielding fermentation process (Bokosa et al., 1992). High cell densities
minimize the effects of substrate and product inhibition, making it possible to carry out
fermentations in shorter periods (Riesenberg and Guthke, 1999). In shaking cultures,
Gancedo (1998) optimized 10 % of inoculum size for invertase production. A 16 h old
vegetative inoculum at a level of 2 % was used for maximum invertase production (45.65
U/ml) in shake flasks (Haq et al., 2008). In contrast, Roitsch et al. (2003) found that 48-h
old cells were as good as those from a 72-96 h old slant culture for invertase production,
which suggested that the age of yeast cells may not have a bearing on the enzyme
production. The lag associated with inoculum from the stationary phase of a culture may
be attributed to the reorganization necessary in the cell to reverse the changes caused by
cessation of growth.
Stirred fermentors of different working volumes may be used for the large scale
production of invertase as an industrially important enzyme under controlled conditions.
By optimizing the cultural conditions such as nutritional requirements, inoculum size,
temperature, pH, agitation, aeration, dissolved oxygen etc, the enzyme production can be
enhanced many folds. The maximum production of invertase was achieved by using
medium having composition (g/l) i.e., 20 ml glycerol, 5 KH2PO4, 4 (NH4)2SO4, 0.5 KCl,
7
0.5 MgSO4, 0.1 CaCl2, 5 ml trace element solution and vitamins in both 5 and 50 L
fermentors, respectively (Narciandi et al., 1995). The inoculum size of 106cells/ml in C.
utilis (7.8 %, v/v) with agitation speed of 900 rpm was inoculated in pineapple waste
medium of 1.5 L in a 2 L capacity fermentor set at 30оC for 30 h. Aeration rate and
agitation speed have major effects on the dissolved oxygen levels, which in turn affect
the cell growth and the intracellular protein content (Rosma and Ooi, 2006). The
maximum intracellular invertase activity (440 U/g dry cells) in S. cerevisiae was
obtained in a 1.5 L working volume fermentor using molasses medium with aeration (1.6
vvm) and pH of 4.5 (Bokosa et al. 1993).
The yeast S. cerevisiae cells as source of intracellular invertase can be used as
whole cell biocatalyst for immobilization. “Immobilization means associating the
biocatalysts with an insoluble matrix, so that it can retain in proper reactor geometry for
its economic reuse under stabilized conditions” (Akgol et al., 2001). There are many
advantages of immobilization process i.e., it extend the stability of the enzyme by
protecting the biological active material from deactivation, repeatedly use, low cost and
easy separation and recovery of the enzyme. One of the applications of immobilizated
whole yeast cells in food technology is the production of inverted syrup from sucrose.
Invertase has been immobilized by adsorption, microencapsulation, entrapment and
covalent immobilization (Mansour and Dawoud, 2003; Danisman et al., 2004; Amaya-
Delgado, 2006). In order to lower the production costs the whole S. cerevisiae cells can
be used as a biocatalyst. The various biopolymers (gelatine or alginate) are usually used
as matrix for cell entrapment that is a simple and cheap technique (Parascandola and
Scardi, 1982; Hasal et al., 1992). The calcium-alginate being hydrogel is considered to
be the best matrix for invertase immobilization because it does not affect enzyme activity
or its structure (Nakane et al., 2001; Milovanovic et al., 2007). The major limitation of
this technique for immobilization of enzymes is the possible slow leakage during
continuous use in view of the small molecular size compared to the cells (Tampion and
Tampion, 1987). The optimal culture conditions for attaining S. cerevisiae cells suitable
for invertase production were temperature and pH. The immobilized invertase as
intracellular hydrolytic enzyme from S. cerevisiae was used for maximum sucrose at pH
4.8 and at temperature 50C (Khobragade and Chandel, 2002). The system in fixed bed
reactor demonstrated a very good productivity at a temperature of 70ºC and a sugar
concentration of 2.0 M (Krastanov, 1997). The applications of enzyme in food and
pharmaceutical sector require high purity of invertase. The enzyme purification requires
8
the downstream processing techniques to remove as completely as possible all the
proteins except which possess the specific enzyme activity desired. For the purification
of proteins different precipitants such as ammonium sulphate, acetone, ethanol etc were
used as initial purifying agents (Pimpa, 2004). In Zymomonas mobilis, the maximum
recovery of invertase activity of 85 % with a 6-fold increase in activity upto 64 U/mg of
protein by the addition of 1.26 parts of ethanol/part of crude extract was found (Yanase
et al., 1995). The column chromatography has been the popular technique for isolation
and quantifying the components from mixture of the compounds. For purification of
invertase mostly anion exchange chromatography, gel filtration and affinity
chromatography techniques are used (de Gines et al., 2000; Belcarz et al., 2000;
Guimaraes et al., 2007; Milintawisamai et al., 2007; Uma et al., 2010). A 75 % recovery
of invertase with 9-fold purification was achieved when desorption occurred using 0.05
M Tris HCl buffer containing 0.5 M NaCl at 7.0 pH (Chan et al., 1992). Oda and
Tonomura (1994) found the molecular weight of invertase on SDS-PAGE as 130,000
daltons. The characterization and kinetic parameters show the efficiency of the purified
enzyme. A Lineweaver-Burk plot of the invertase affinity for sucrose gave a straight line
plot from which the Km as 0.23 mg/ml and Vmax was 15.8 U/mg in Rhodortorula glutinis
(Rubio et al., 2002). The optimum temperature of purified invertase isolated from
Candida utilis was 65оC and pH 5.5. The invertase present in A. flavus had an optimum
pH of 6.0 and Km of 133 mM for sucrose (Chavez et al. 1997).
9
OBJECTIVES
Invertase is an industrially important enzyme and its demand is increasing in line with
the growing markets of processed food, especially the confectionary and pharmaceutical
industries. Inverted syrup production by microbial invertase is not widespread because of
ease in chemical hydrolysis and high price of the enzyme. Almost all of the inverted
syrup used in country is imported or produced by acid hydrolysis. However, production
of invertase by yeast strain Saccharomyces cerevisiae is safer as compared to that
produced by the acidic hydrolysis. The purpose of the present studies was to develop a
process for the production of enzyme invertase by S. cerevisiae and to optimize cultural
conditions by submerged fermentation in shake flask prior to scale up studies in a stirred
fermentor.
The specific objectives of the present work are as follows:
1. Isolation and screening of the strains of S. cerevisiae from different fruits and soil
sample of Lahore District.
2. Random mutagenesis by UV or chemicals to enhance the invertase potential of
the strain.
3. Optimization of the cultural conditions of S. cerevisiae in shake flask as well as
in stirred fermentor.
4- Immobilization of mutant yeast cells for the production of inverted syrup.
5- Purification and characterization of invertase.
10
LITERATURE REVIEW
Dworschack and Wickerham (1961) reported that few strains of Candida utilis
produce remarkably large amounts of extracellular and total invertase. One C. utilis
strain (Y-900) showed high production of enzyme whether the carbon source was
glucose, xylose, sucrose or maltose and still higher production with ethyl alcohol,
glycerol and lactic acid. The total invertase in C. utilis was extracellular (20 to 30 %).
Strains of S. cerevisiae and S. carlsbergensis were proved to be inferior to C. utilis in
extracellular and total invertase production, the difference being accentuated in shaken
cultures. The invertase production from industrial yeasts was higher than the other yeasts
included in the survey.
Hoshino et al. (1964) separated three types of invertase (invertase I, II and III)
from the soluble and insoluble fractions (4,500 × g, 10 min supernatant and pellets of the
homogenate, respectively) of baker's yeast by a DEAE- cellulose column
chromatography. The invertases I and II were eluted with 0.1 M sodium acetate buffer
(pH 3.9) and 0.1 M sodium acetate buffer (pH 6.2) containing 0.1 M NaCl from DEAE-
cellulose respectively, whereas the invertase-III remained adsorbed on the cellulose
under these conditions. They were present in proportions of 2.5: 1: 0.06 in the soluble
fraction and 1.4: 1: 0.12 in the insoluble fraction of the fresh baker's yeast cells. While
invertase-II remained at a constant level, invertases I and III in the soluble fraction
increase upon incubation of cells for the formation of invertase under the continuous
supply of sucrose. Invertases I and II differ from each other considerably in the optimum
pH and slightly in the response to (activation and inactivation by) crude papain and were
identical with respect to the heat stability and probably to the affinity for sucrose.
Weimberg and Orton (1966) reported that invertase and acid phosphatase were
repressible extracellular enzymes in S. fragilis and S. cerevisiae. The conditions for the
release of these enzymes from both yeast were compared. Either β-mercaptoethanol or
KCl released the enzymes in varied amounts from S. fragilis according to the
physiological age of the yeast. These reagents were not only responsible for the release
of enzymes from the cells but also also caused the retention of large amount of enzyme
within the cells of S. fragilis. Invertase and acid phosphatase were not eluted from cells
of S. cerevisiae by β-mercaptoethanol or KCl. These enzymes were separated from S.
cerevisiae cells after the digestion of cell-wall by snail gut fluid.
11
Moreno et al. (1975) studied the molecular forms of yeast invertase by gel
filtration technique using Sephadex G-200. In this study not only the determination of
light invertase and heavy invertase with carbohydrate content of 50 % (carbohydrate
free)it was carried out but also showed a continuous spectrum of molecular forms
represented the sequential addition of mannose carbohydrate moity to the light form
during the secretion process, which resulted in the secretion of heavy enzyme from
cytoplasmic membrane. The elution volume-void volume ratio in Sephadex G-200 varied
from 1.75 of the light to 1.05 of the heavy invertase. The separation of invertase had also
been achieved by ion-exchange chromatography and by isoelectric focusing. It also
facilitated by removal of the heavy form by ammonium sulphate precipitation. With the
removal of the cell wall to get protoplasts, most of the heavy form of invertase was also
lost. The intermediate forms were entirely found inside the protoplast with the light
invertase and smaller quantity of heavy invertase. The difference between these
intermediate forms was only the extent of carbohydrate contents they possessed. The
effect of cycloheximide and 2-deoxy-glucose on the distribution of molecular forms of
yeast invertase and its production had also been studied. In the presence of glucose (10
mM), Saccharomyes (303-67) cells under repressive conditions readily produced
invertase during incubation period of 2 h. After the addition of 2-deoxy-D-glucose (75
pg/ml), the inhibition in the cells was observed to be as 60 %. But when activity was
calculated after breaking the cells, only an inhibition of 31 % was found, showing
presence of invertase inside the protoplast. At the expense of the formation of the heavy
enzyme, the 2-Deoxy-D-glucose collected a stack of the light and intermediate forms,
showing that the glycosylation process i.e., necessary for secretion of invertase was
inhibited.
Zimmermann and Scheel (1977) isolated mutants with defective carbon catabolite
repression in the yeast S. cerevisiae by applying a selective methodology. This was
derived from the fact that invertase was a glucose repressible cell wall enzyme which
slowly hydrolysed raffinose to produce fructose. The inhibitory effects of 2-deoxy-D-
glucose can be counteracted by fructose. Repressed cells were plated on a raffinose-2-
deoxy-D-glucose medium and the resistant cells growing up into colonies were tested for
glucose non-repressible maltase and invertase. In this way, a high percentage of
regulatory mutants (equally derepressed for maltase and invertase) was obtained. Not
even single mutant was obtained showing non-repressible invertase production which
was the selected function. A total of sixty one mutants isolated in different strains were
12
allele tested and could be attributed to three genes (all recessive). One class of mutants in
one gene showed reduced hexokinase activities. The other class, located in a centromere
linked gene, had high level of hexokinase and was inhibited by maltose. On the other
hand, mutants in a third gene were isolated on a 2-deoxy-D-glucose galactose medium
and had normal hexokinase levels. In all mutants, malate dehydrogenase being partially
derepressed was found while isocitrate lyase, however, was still fully repressible.
Elorza et al. (1977) reported that Saccharomyces cerevisiae-136 produced
invertase in media supplemented with sucrose and maltose. When the glucose
concentration was lower than 1 % enzyme production took place. On the other hand,
higher concentrations of glucose repressed the enzyme production. The effect of glucose
before mRNA inhibition showed the hexose interference with the transcription of DNA
into invertase mRNA and also inhibited the translation of invertase mRNA already
formed. If invertase activity was not affected by glucose higher concentration suggested
that the hexose did not cause catabolite repression for invertase. Inhibition of invertase
translation by glucose driven out to be reversible but the total of enzyme produced was
based on the duration of treatment. It was concluded that the catabolite repression of
invertase biosynthesis only act at the transcription and translation levels and produced an
increase in the rate of mRNA degradation and had no effect on catalytic activity and
secretion of invertase.
Trimble and Maley (1977) investigated that Saccharomyces cerevisiae had
external invertase with carbohydrate content of 50 %. It had been very difficult to gain an
accurate molecular weight of invertase by electrophoretic or centrifugal techniques.
However, on removing almost all of the oligosaccharide chains of invertase with the
endo-beta-N-acetyl-glucosaminidase H from Streptomyces plicatus, the carbohydrate-
free invertase composed of two 60 kDa subunits was obtained. Terminal sequence
analysis with carboxypeptidases A, B, and Y provided strong indication that the subunits
were identical.
Rodriguez et al. (1978) reported that the intracellular invertase of Saccharomyces
cerevisiae was largely found in a soluble form (91-95 %), while only small amounts
were found bound to the internal (4-8 %) and plasma membranes (less than 1 %). In the
processes of repression or derepression, inhibition of mRNA or protein synthesis, or in
the presence of 2-deoxy-D-glucose, the levels of the membrane-bound and external
activities were modified in a way in which their relation was apparent, while the soluble
13
enzyme proved to be unaffected. These findings, together with the fact that the
membrane-bound and the external invertase were glycoproteins, suggested a precursor-
product relationship between the different enzymic forms.
Lehle et al. (1979) studied the invertase released from broken cells of S.
cerevisiae X-2180 mm2 mannan mutant, was separated by into a in ammonium sulfate
saturation (75 %) in the form of insoluble fraction containing carbohydrate content of 36
% and a soluble fraction (53 % carbohydrate). “The soluble fraction was reacted with
antibodies specific for α-1 leads to 6-linked mannose of the mannoprotein outer chain,
whereas the insoluble fraction failed to react with this antiserum although it did react
with serum against terminal α-1 leads to 3-linked mannose units that was characteristic
of the mannoprotein core. A bacterial endo- α-1 leads to 6-mannanase removed the outer
chains from the soluble fraction of invertase and converted it to a form that was similar
in electrophoretic and immunochemical properties to the insoluble fraction of invertase,
whereas the endomannanase had little effect on the soluble fraction. The results
suggested that the insoluble fraction of invertase was a form of the enzyme to which only
the core oligosaccharide units had been added and the soluble invertase fraction to which
the polysaccharide outer chains were also attached”.
Olutiola and Cole (1980) described that an extracellular invertase in Aspergillus
flavus induced by sucrose containing liquid medium. The biosynthesis of invertase was
repressed when repressive hexose sugars (glucose or fructose) was added to sucrose-
metabolizing cells. It was induced in a glucose or fructose-metabolizing culture by the
addition of sucrose. The optimum pH (6.0) and Km (133 mM) for sucrose was optimized.
The enzyme needed optimum level of 250 mM of potassium phosphate for maximum
activity. The partial purification by ammonium sulphate precipitation procedure followed
by dialysis and finally separation of invertase by size exclusion showed three
components with molecular weights (40-55 kDa).
Matulaitite et al. (1980) isolated intracellular invertase from S. cerevisiae, race
XI, and purified it by using DEAE-cellulose and Sephadex G-200. The effect of
temperature, pH, metal ions, EDTA and thiolic agents on the invertase activity and
stability was studied. The molecular weight (270 kDa) of enzyme with carbohydrate
content (20-30 %) was estimated. The purified invertase was found to be heterogenous
by disc-electrophoresis and isoelectric focusing. The molecular forms of enzyme had
isoelectric points at 3, 4, 4.5 and 4.9.
14
Park and Sato (1982) performed comparative studies of the fermentation of cane
molasses into ethanol by S. cerevisiae in the presence or absence of fungal invertase. The
presence of the enzyme had no effect on ethanol production when cane molasses was
fermented by the yeast at 30°C and pH 5. At pH 3.5, ethanol production was increased
by the addition of invertase. At 40°C, the addition of invertase increased ethanol
production by 5.5 % at pH 5.0 and by 20.9 % at pH 3.5.
Bailey et al. (1982) isolated the mutant strains of S. cerevisiae (industrial-type)
which quickly and fully fermented equimolar mixtures of galactose and glucose to
ethanol. These mutants classified into two general phenotypic classes based upon their
enzyme induction patterns and fermentation kinetic. One class of mutants apparently
specifically showed the utilization of galactose in an anaerobic fermentation in a
sequential way i.e., utilization of first glucose and then galactose. The second class of
mutants was found to be resistant to catabolite repression and synthesized invertase,
galactokinase and maltase in the presence of repressive concentration of glucose.
Chu et al. (1983) investigated that yeast external invertase was a dimer (each
subunit of 60 kDa). It showed its maximum catalytic action at pH of 5.0. The major form
of external invertase was found to be as an octamer with an average size of 8 × 105 Da.
During ultracentrifugation the dissociation of octamer into lower molecular weight forms
(dimer, tetramer and hexamer) took place. The all forms of the enzyme that showed
identical specific activities had similar carbohydrate to protein ratio while the monomer
subunits (1 × 105 Da) were heterogenous in carbohydrate content. Each subunit had nine
oligosaccharide chains. The oligomeric form of the enzyme found to be active when it
was stained for protein and invertase activity after SDS-PAGE. Consequently, on
partially inactivating invertase with guanidine hydrochloride (4 M) both octamer and
monomer were apparent on the gels but only the former was active. In the same way, it
was incubated at pH of 2.5 in the presence of SDS produced inactive monomer. The
monomer, unlike the active oligomeric aggregate, was unable to hydrolyze sucrose after
SDS-PAGE. Consistent with the in vitro studies, freshly prepared yeast lysate had
octameric form of external invertase as the key active form of this enzyme. It was
concluded that the carbohydrate part of external invertase was not only responsible for
stabilizing the enzyme activity, but also maintained its oligomeric structure.
Workman and Day (1983) purified the ß-fructofuranosidase from Kluyverornyces
fragilis to single band on gel electrophoresis by three different methods. Two of the
15
preparations were observed to be impure by isoelectric focusing. This verified the need
for more than one criteria of homogeneity during purification of enzyme. The enzyme
being glycoprotein in nature showed optimum pH (4.5) and stability at 5ºC. The cations
Hg2+, Ag+, Cu2+ and Cd2+ exhibited a noticeable inhibition of the enzyme. Competitive
inhibition was observed with the fructose analog 2,5-anhydro-D-mannitol suggested that
the enzyme inhibition took place by the furanose form of fructose.
Moreno et al. (1985) studied the subcellular localization of the invertase in the
cells of both repressed and derepressed for poduction of the enzyme in
Schizosaccharomyces pombe. A large amount of the invertase was observed to be located
outside the plasma membrane and only a small amount was found to be associated to
membranes. A considerable portion of the external enzyme remained firmly bound to
cell wall material. The entire enzyme isolated in soluble form from cellular extracts
reacted with concanavalin A and with the lectin from Bandeiraea simplicifolia seeds.
This indicated that enzyme had carbohydrate moiety which perhaps showed terminal
mannosyl and galactosyl residues. The chance of the presence of two different forms of
invertase in S. pombe was considered. The intracellular soluble form of invertase,
carbohydrate-free, similar to the small invertase of the S. cerevisiae, was not present in
Sc. pombe. However, the Michaelis constant (Km) for sucrose of the invertase found in
repressed cells was smaller than that of the enzyme produced under derepressed
conditions. Although this difference could also be the result of a different pattern of
glycosylation of the invertase produced under different growth conditions.
Tammi et al. (1987) investigated the yeast external invertase as glycoprotein that
exists as a dimer that could associate to form tetramers, hexamers, and octamers (Chu et
al., 1983), a process that was facilitated by the attached oligosaccharide chains. This
association by HPLC on a gel filtration matrix, by which procedure wild-type bakers'
yeast invertase gave two peaks, and invertase from a core mutant (mnn1 mnn9) of S.
cerevisiae X2180 gave three peaks were studied. Concentration of an invertase solution
by freezing drived the dimers into higher aggregates that, at 30°C, re-equilibrated to a
mixture of smaller forms, the composition of which depended on pH, concentration and
time. The invertase from a mutant, mnn1 mnn9 dpg1, which underglycosylated its
glycoproteins and produced invertase with 4-7 oligosaccharide chains, forms oligomers
of much lower stability than the mnn1 mnn9 invertase, which had 8-11 carbohydrate
chains. Both of these mutants released external invertase from the periplasm into the
medium during growth. It was concluded that defects in the cell wall structure may be
16
more important in this release than an altered tendency of the invertases to aggregate.
Investigation of aggregate formation by electron microscopy revealed that all invertases
including the internal non-glycosylated enzyme, form octamers under appropriate
conditions.
Sanchez et al. (1988) studied the ability of the glycosylation system of
Schizosaccharomyces pombe to process heterologous glycoproteins, the expression of S.
cerevisiae invertase in the former yeast. Sc. pombe cells were able to produce
enzymatically active invertase from the S. cerevisiae SUC2 gene introduced by
transformation and the enzyme was glycosylated and secreted into the cell wall.
However, Sc. pombe transformants did not glycosylate the heterologous enzyme as their
own invertase since it was not bound by the lectin from Bandeiraea simplicifolia seeds.
This indicated the absence of terminal galactose residues. In addition, the electrophoretic
mobility of the heterologous invertase was similar to that of the large enzyme from S.
cerevisiae, both in its native form and after being deglycosylated with Endo H. These
results suggested that the polypeptide chain of invertase in S. cerevisiae was the major
factor for the glycosylation in the cells of Sc. pombe.
Schulke and Schmid (1988) reported that yeast invertase present in two different
forms. The cytoplasmic enzyme was non-glycosylated, whereas the external invertase
with about 50 % carbohydrate of the mannose type. The protein contents of both
enzymes were identical. The two invertases had been used previously as a model system
to study the effects of covalently linked carbohydrate chains on the stability of
glycoproteins, and controversial results were obtained. The thermal and denaturant-
induced unfolding by various probes, such as the changes in absorbance and fluorescence
and loss of enzymatic activity was measured. The ranges of stability of the two
invertases were found to be basically identical, suggested that the presence of a high
amount of carbohydrate did not appreciably contribute to the stability of external
invertase. The previous results that invertase was stabilized by glycosylation could not be
confirmed. The stability of this glycoprotein was apparently determined by the specific
interactions of the folded polypeptide chain. In contrast the glycosylated form, the
invertase devoid of carbohydrate was prone to aggregation in the denatured state at high
temperature and in a partially unfolded form in the presence of intermediate
concentrations of guanidinium chloride.
17
Buttner et al. (1990) investigated the intracellular and extracellular invertase
activity of two Trichosporon adeninovorans strains. Both strains (SBUG 724 and CBS
2844) secreted one invertase into the medium. The purification of external enzymes was
achieved by chromatography on hydroxylapatite. The molecular weight of the external
invertase of SBUG 724 (650 kDa) and of CBS 2844 (450 kDa) was found. The internal
invertases were separated by DEAE-cellulose chromatography. The molecular size of the
enzyme from CBS 2844 was determined to be 125 kDa. Two internal invertases had
molecular weight of 230 and 70 kDa in the strain SBUG 724. The activity of all
invertases showed similar properties. The optimal pH of the reaction was between 5 and
5.2 and temperature (60-70°C). The Km value for sucrose (71 to 83 mM) and for
raffinose (27 to 36 mM) was determined.
Novak et al. (1990) isolated a number of 2-deoxy-D-glucose (2-DOG) resistant
mutants showing resistance to glucose repression from various Saccharomyces yeast
strains. A large number of the mutants isolated were found to be enhanced maltose
uptake ability in the presence of glucose. Fermentation results indicated that maltose was
consumed faster than glucose in the mutant strains as compared to the wild strains, when
these sugar moities were fermented together. On the other hand, when these sugars were
fermented separately, only the 2-DOG resistant mutant obtained from S. cerevisiae strain
1190 exhibited alterations in glucose and maltose uptake compared to the wild strain.
Kinetic analysis of sugar transport employing radiolabelled glucose and maltose
indicated that both glucose and maltose were transported with higher rates in the mutant
strain. These findings suggested that the high affinity glucose transport system was
regulated by glucose repression in the wild strain but was derepressed in the mutant.
Vainstein and Peberdy (1991) investigated that Aspergillus nidulans produced an
extracellular invertase when cultured on a medium containing sucrose or raffinose. The
invertase production was found maximal on medium containing sucrose after 15 h of
incubation 28°C. The amount of invertase in the culture medium was declined after this
incubation time. During the linear growth phase of the fungus a high proportion of the
enzyme was found. Different sugars were tested for invertase induction, but only the two
(sucrose and raffinose) gave high production, while with the remaining sugars produced
the enzyme at a low constitutive level. Mycelium of Aspergillus nidulans grown under
repressive conditions i.e., by using glucose at the concentration of 1 %, rapidly produced
invertase when transferred to derepressive conditions e.g., the use of sucrose into the
18
fermentation medium. The invertase production was found to be higher by 26 fold after
80 min than the enzyme production at constitutive level.
Novak et al. (1991) reported that toxic and non-metabolizable glucose analogue
2-deoxy-D-glucose (2-DOG) is widely employed to screen for regulatory mutants which
lack catabolite repression. Several yeast mutants resistant to 2-DOG were isolated. One
mutant derived from a S. cerevisiae haploid strain, was found to be derepressed for
sucrose, galactose and maltose utilization. Moreover, kinetic analysis of glucose
transport indicated that the high affinity glucose transport system was also derepressed in
the mutant strain. Besides, the mutant had an increased intracellular concentration of
trehalose relative to the wild strain. These findings indicated that mutants showing the 2-
DOG resistantance are defective in general glucose repression.
Chan et al. (1991) studied a strain of Saccharomyces uvarum synthesized
extracellular invertase in a chemostat reactor using medium supplemented with corn
steep liquor and sugars. The invertase production was enhanced by increase in corn steep
liquor concentration. The rate of enzyme production was found to be maximum at a
dilution rate of 0.75 h–1. The rate of enzyme production was oberved to be affected by
fermentation temperature and the type of sugar substrate. The invertase from crude broth
was purified by one-step DEAE- chromatography. The enzyme was recovered (84 %)
with 9-fold purification. The overall 30 fold purification could be achieved using this
simple isolation procedure.
O'Mullan et al. (1992) reported that Zymomonas mobilis as Gram-negative
ethanologen ferment sucrose, fructose and glucose. Three enzymes that hydrolyze
sucrose were found in a zymogram of electrophoretically separated protein of Z. mobilis
CP4. Two were invertase, Inv I and Inv II; the latter was studied. Inv II was extracellular
and showed the saccharolytic activity (60 %) in the culture broth of Z. mobilis CP4. The
invertase was purified by 51 fold in 17 % recovery from culture broth of Z. mobilis
grown on medium containg sucrose. It was a β-D -fructofuranosidase, monomeric with a
molecular mass (47 kDa) and pI (4.3). Its K m value (86 mM) for sucrose and it had high
catalytic activity (V max = 1800 μ mol product/min/mg of protein). Bokossa et al. (1993)
investigated that biosynthesis of invertase by S. cerevisiae 01K32 was inversely
proportional to the concentration of sugarcane blackstrap molasses used in the
fermentation medium. The intracellular invertase activity (440 U/g dry cells) in a
fermenter was obtained.
19
Nakajima et al. (1993) developed a bioreactor which used immobilized enzyme
within a ceramic membrane support (1 mm thickness). Sucrose was forced through the
membrane by cross-flow filtration during the process of crossing the membrane. The
bioreactor was termed forced-flow membrane enzyme reactor. The immobilized
invertase membrane converted the sucrose (100 %) in a feed stream made up of molasses
solution (50 %) In addition to sucrose, molasses contained many other substances.
Therefore this method was appropriate to those processes which utilized substrates
present in ''impure'' feeds.
Chang et al. (1994) purified a fructooligosaccharide-producing beta-
fructofuranosidase from the crude extract of Aspergillus oryzae ATCC 76080 through
successive steps of ultrafiltration, DEAE-Sepharose CL-6B ion-exchange
chromatography, preparative isoelectric focusing electrophoresis and Sephacryl S-200
gel filtration. The purified enzyme had an optimal pH (5-6), temperature (50ºC) and a Km
value of 0.53 M for catalyzing selftransfer reaction from sucrose. The molecular weight
was 87 kDa by gel filtration. Randez-Gil and Sanz (1994) mutagenized the spore
progeny of baker's yeast as one of the industrial strain with UV and tested resistance to
2-deoxy-D-glucose for mutant isolated. Of the all isolates, one mutant (10a) exhibited
high levels of external invertase and maltase (alpha-glucosidase) and assimilated maltose
when grown under repressive conditions.
The synthesis of invertases was studied by Rodriguez in Pichia anomala
(Rodriguez et al., 1995). He reported that carbon is major component in culture media to
direct invertase synthesis. The invertase with 86.5 kDa was purified in P. anomala
derepressed cells for invertase synthesis and being glycoprotein (carbohydrate 30 %) it
was multimeric protein having identical subunits. The moiety accounts for approximately
of the total mass of the molecule and consists of manno-oligosaccharides N-linked to the
polypeptide. According to Rubio and Maldonado (1995) invertase produced by a strain
of Aspergillus niger showed the following main characteristics: maximum activity at
60°C, pH 5.0, K m with substrate of sucrose as 0.0625 mM, Vmax 0.013 mol/min and free
energy 9132 cal/mol. The metal ions and p-chloromercuribenzoate (PCMB) acted as
inhibitors, respectively.
Randez-Gil et al. (1995) reported that 2-deoxy-D-glucose (2-DOG), a non-
metabolize analogue of glucose, was taken up by yeast using the same transporter as
glucose and was phosphorylated by hexokinases producing 2-deoxy-D-glucose-6-P. It
20
was found that in DOGR yeasts, 2-DOG was not able to trigger glucose repression, even
at concentrations of 0.5 %. This result suggested that the specific 2-DOG-6P
phosphatase, the enzyme responsible for the DOGR phenotype might be involved in
inhibiting the process of catabolite repression mediated by 2-DOG.
According to Vitolo et al. (1995) S. cerevisiae was grown in a medium
containing blackstrap molasses in batch and fed-batch fermentation. The variable
parameters were pH (4.0-6.5), dissolved oxygen (0-5.0 mg O-2l-1) and sucrose feeding
rate. When glucose concentration was higher than 0.5 g/l, a reduction in the specific
activity of invertase intact cells and an oscillatory behavior during fermentation was
found. Both values could be related to the inhibitory effect of glucose on invertase
production. Best fermentation conditions to make S. cerevisiae cells suitable for
invertase production were pH (5), temperature (30°C), dissolved oxygen (3.3 mg O-2 l-1)
and substrate (0.5 g l-1).
Somiari and Bielecki (1995) described the total amount of novel oligosaccharides
produced by invertase at pH (7.5) increased 3-fold using a medium containing glucose
(0.1 M), fructose (0.5 M) and sucrose (1.2 M) as compared to that with only sucrose (1.8
M) solution. These three sugars at the concentration of 0.6 M reduced yield and
decreased the rate of sucrose hydrolysis by 72.7 %. Leite et al. (1995) developed a Flow
Injection Analysis (FIA) for sucrose using invertase, mutarotase and glucose oxidase.
The enzymes were immobilised on glass beads using glutaraldehyde. The sucrose
concentration was related to oxygen saturation. The decrease in oxygen concentration, as
a result of sucrose oxidation, was detected by a low cost, home-made oxygen electrode.
The system was able to measure sucrose from 0.025 to 100 mM with a response time of
6 min using 200 ul of sample, with an apparent Km of 42 mM of sucrose. The system had
been operated satisfactorily for 50 days without loss initial activity.
Yanase et al. (1995) investigated that Zymomonas mobilis IFO 13756 produced
three types of sucrose hydrolyzing enzymes (E1, E2, and E3). E2 and E3 were found to
be extracellular enzymes bound to the cell surface were released from cells by
suspension in potassium phosphate buffer (20 mM, pH 7.0) and incubation at 30C for
10 min. The cell suspension was centrifuged and E3 was isolated from the supernatant in
52 fold purification. The enzyme was found as monomeric protein with molecular mass
(58 kDa) and isoelectric point (3.2). The pH (5.5) and temperature (50C) were also
optimized. Thiol reagents caused the reduction in enzyme activity noticeably. According
21
to Muramatsu and Nakakuki (1995) an intracellular beta-D-fructofuranosidase produced
by Aspergillus sydowi IAM 2544 was purified by Q-Sepharose and Alkyl-Sepharose
chromatographies. The molecular mass was 50 kDa by SDS-PAGE analysis. The
optimum pH and temperature of sucrose hydrolyzing activity of the enzyme were 5.5
and 75C, respectively.
Zech and Gorisch (1995) reported that commercially available invertase
preparations from S. cerevisiae were reversibly deactivated in industrial molasses media
containing high NaCl and ethanol concentrations. A significant difference in stability
was found between the invertase activity of baker's yeast and of a highly specialized
ethanol, temperature and osmo-tolerant strain of S. cerevisiae. The invertase deactivation
by dissociation of the glycosylated enzyme into their subunits responsible for the
maximum ethanol concentration (80 g/l) which was obtained from industrial yeast
fermentations based on molasses.
Mwesigye and Barford (1996) investigated the growth of S. cerevisiae on
different concentrations of sucrose and glucose mixtures after adapting on sucrose. The
yeast cells were found to have two different mechanisms by which sucrose was utilised:
hydrolysis outside the cell membrane and direct transport into the cells. The mechanism
by which sucrose was utilised depended on the initial concentration of glucose in the
mixture and the adaptation state of the cells. In both cases, glucose was utilised first and
invertase secretion was repressed when the glucose concentration was greater than 2 gl-1.
The major finding was that, for fully sucrose-adapted cells, even in the presence of a
repressive glucose concentration, the yeast cells were able to utilise sucrose.
Chen et al. (1996) purified two forms of secreted invertase from Aspergillus
nidulans with the help of column chromatography i.e., ion-exchange and gel-filtration.
Slow-invertase being glycoprotein in nature gave a single broad, on native and SDS-
PAGE in the size of 185 and 78 kDa, respectively, as compared to Fast-invertase with 94
and 110 kDa, respectively. The major carbohydrate group of Slow-invertase was 14 % of
mannose and 5 % of galactose. The other Fast-invertase obtained was composed of 29 %
of galactose and 12 % of mannose. The Slow-invertase showed 3 fold higher specific
activity than Fast-invertase before and after deglycosylation. Both forms of invertase had
similar Km values. Chavez et al. (1997) were found a periplasmic invertase from the
yeast Candida utilis was purified to homogeneity from cells fully derepressed for
invertase synthesis. The enzyme was purified by successive Sephacryl S-300 and affinity
22
chromatography and shown to be a dimeric glycoprotein composed of two identical
monomer subunits with an apparent molecular mass of 150 kDa. After EndoH treatment,
the deglycosylated protein showed an apparent molecular weight of 60 kDa. The
apparent Km values for sucrose and raffinose were 11 and 150 mM, respectively, similar
to those reported in S. cerevisiae. The range of optimum temperature was 60-75°C. The
optimum pH was 5.5 and the enzyme was stable over pH range 3-6.
Krastanov (1997) immobilized the invertase as biocatalyst by adhesion of yeast
cells to wool by glutaraldehyde. When glutaraldehyde was used onto wool by treating
either the yeast cells or wool or both, the yeast cells were firmly immobilized.
Immobilized yeast cells were not desorbed by washing with KCl (1 M) or buffers (0.1 M,
pH 3.5). The immobilized biocatalyst showed a maximum enzyme activity at pH of 4.2
and 7.5. It was tested in a tubular fixed-bed reactor to study its promising application for
continuous large scale sucrose hydrolysis. The effect of sugar concentration,
temperature, and low rate on the productivity of the reactor and on the specific
productivity of the immobilized biocatalyst was investigated. The system showed a high
productivity at sugar concentration (2.0 M) and temperature (70°C). When volume of the
biocatalyst was increasd there was exponenttially increase in the productivity. During 60
days of continuous hydrolysis using sucrose of 2.0 M and temperature of 70 °C, the
productivity of the immobilized biocatalyst decreased no more than 50 %. But during the
first 30 days it remained constant. The resulted biocatalyst productivity for 60 days was
4.8 × 103 kg inverted sucrose/kg biocatalyst. The immobilized biocatalyst was found to
be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.
Gancedo (1998) investigated that glucose and related sugars caused the
repression of gene transcription that encoded the enzymes required for the consumption
of different carbon sources. A few of genes also caused repression by other hexoses and
the process is called catabolite repression. The various sugars produced signals which
modified the conformation of certain proteins directly or through a regulatory cascade
affected the expression of the genes subjected to catabolite repression. Although all
genes were not controlled by a single set of regulatory proteins, but there were different
circuits of repression for different groups of genes.
Dynesen et al. (1998) repoted that when S. cerevisiae was grown on the medium
containing a mixture of glucose and another fermentable sugar such as galactose, maltose
or sucrose, the metabolism was diauxic, i.e. glucose was metabolized first whereas the
23
other sugars metabolized when glucose was exhausted. This phenomenon was as the
result of glucose repression (catabolite repression). In addition to glucose, the other
hexoses such as mannose and fructose were also considered to be triggered catabolite
repression. The batch fermentations of S. cerevisiae in mixtures of sucrose and either
fructose, mannose or glucose was performed. It was observed that the utilization of
sucrose was inhibited by either fructose or glucose concentrations higher than 5 g/l. It
was suggested that fructose and glucose had the equal capability exerting catabolite
repression. However, sucrose after hydrolysis produced glucose and fructose, even when
the mannose high concentration (17 g/l) was used, indicated that mannose was not found
to be a repressing sugar. It was concluded that the capability to trigger catabolite
repression was connected to hexokinase PII, which was involved in the in vivo
phosphorylation of glucose and fructose.
de Alteriis et al. (1999) studied the expression of the gene encoding invertase
(SUC2) using free and gelatin-immobilized yeast cells in order to elucidate the high
activity of invertase exhibited by immobilized cells when grown in a nutrient medium.
The results indicated that there might be two reasons responsible for the accumulation of
invertase in immobilized cells. First, the expression of the gene (SUC2) was maintained
throughout growth in immobilized yeast cells, whereas its expression was only brief in
free cells. Second, invertase of immobilized cells was found to be less susceptible to
endogenous proteolysis than free cells. These findings had been interpreted, respectively,
in terms of diffusional limitations and changes in the pattern of invertase glycosylation
due to growth of yeast in an immobilized state. Melo and D’Souza (2000) precipitated all
the invertase by mixing of crude cell-free extract of yeast cells with sufficient quantity of
Jack bean meal extract. The precipitated enzymes were cross-linked by glutaraldehyde (2
%) that retained the enzyme activity over 60 %. The immobilized invertase could be
repeatedly use reused for over 10 batches without loss in activity.
de Gine et al. (2000) reported that the invertase in Lactobacillus reuteri (CRL
1100) was a glycoprotein consisted of single subunit with a molecular weight (58 kDa).
The enzyme was found to be stable below 45°C over a broad pH range (4.5-7.0) with
maximum activity at pH (6.0) and temperature (37°C). The invertase activity was
significantly inhibited by bivalent metal ions (Ca+2, Cu+2, Cd+2, and Hg+2), dithiothreitol
and beta-mercaptoethanol, and partially enhanced by ethylenediaminetetraacetic acid
(EDTA). The enzyme was purified by 32 fold over the crude extract with recovery (17
24
%) by gel filtration and ion-exchange chromatographies. The Km (6.66 mM) and Vmax
(0.028 μmol/min) values for sucrose were obtained.
Tanaka et al. (2000) investigated that the invertase production of S. cerevisiae
IFO 0309 protoplasts in a static culture was 35 times higher for extracellular and 9 times
for both extracellular and intracellular than those of cells. When S. cerevisiae protoplasts
were immobilized in strontium alginate gel (1 %) as an artificial provision of cell wall,
the protoplasts could be cultivated in a shake flask without breakage and invertase was
secreted into the broth. However, cell wall regeneration in the immobilized protoplasts
was detected at 24 h of cultivation. This implied that prevention of cell wall regeneration
was a prerequisite for long term process with protoplasts. When aculeacin A (0.5 μg/ml)
as an inhibitor of β-1, 3 glucan synthesis was added to the broth, active protoplasts were
maintained without cell wall regeneration for more than 24 h and invertase was produced
extracellularly. Immobilized S. cerevisiae T7 protoplasts were used for invertase
production in a bubble column reactor and a high and stable amount of invertase i.e., 45
U/ml) was consistent for 72 h.
L’Hocine et al. (2000) purified fructosyltransferase (EC. 2.4.1.9) and invertase
from the crude extract of Aspergillus niger AS0023 by successive chromatographies i.e.,
DEAE-sephadex A-25, sepharose 6B, sephacryl S-200, and concanavalin A-Sepharose
4B columns. On gel electophoresis the two enzymes, in native and denatured forms, gave
diffused glycoprotein bands with different electrophoretic mobility. On native-PAGE
and SDS-PAGE, both enzymes yielded broad and diffused bands being heterogeneous
glycoproteins. The glycoprotein nature of two enzymes was proved by adsorption on
concanavalin A lectin. Fructosyltransferase on native PAGE migrated as two
enzymatically active bands with different electrophoretic mobility, one around 600 kDa
and the other from 193 to 425 kDa. On SDS-PAGE, these two fractions yielded one band
corresponding to a molecular weight range from 81 to 168 kDa. Fructosyltransferase
seemed to undergo association-dissociation of its glycoprotein subunits to form
oligomers with different degrees of polymerization. Invertase showed higher mobility
with molecular range from 82 to 251 kDa, on native PAGE, and from 71 to 111 kDa on
SDS-PAGE. The two enzymes showed distinctly different pH and temperature profiles.
The optimum pH and temperature for fructosyltransferase were found to be 5.8 and
50°C, respectively, while invertase exhibited optimum activity at pH 4.4 and 55°C.
Metal ions and other inhibitors had different effects on the two enzyme activities.
Fructosyltransferase was completely abolished with 1 mM Hg+2 and Ag+2, while
25
invertase maintained 72 and 66 % of its original activity, respectively. Furthermore, the
two enzymes exhibited distinctly different kinetic constants e. g., the Km and Vm values
for each enzyme were calculated to be 44.38 mM and 1030 mmol ml-1 min-1 for
fructosyltransferase and 35.67 mM and 398 mmol ml-1 min-1 for invertase, respectively.
Fructosyltransferase and invertase catalytic activity was dependent on sucrose
concentration. Fructosyltransferase activity increased with increasing sucrose
concentrations, while invertase activity decreased markedly with increasing sucrose
concentration. Furthermore, invetase showed only hydrolytic activity producing
exclusively fructose and glucose from sucrose, while fructosyltransferase catalyzed
fructosyltransfer reaction producing glucose, 1-kestose, nystose and fructofuranosyl
nystose. In addition, at 50 % sucrose concentration fructosyltransferase produced
fructooligosaccharides at the yield of 62 % against 54 % with the crude extract.
Belcarz et al. (2000) investigated that extracellular invertase by using liquid
media produced in yeast strain of Candida utilis. The affinity chromatography was used
for one-step enzyme purification by applying optimum conditions. Three carbon sources:
maltose, sucrose and inulin were comparatively used as ligands in the affinity technique.
The partially purified invertase was afterwards immobilized on the newly described solid
matrix with keratin as an activator.
Akgol et al. (2001) prepared magnetic polyvinylalcohol microspheres by
glutaraldehyde as crosslinking agent. 1, 10-Carbonyldiimidazole, a carbonylating agent
was used for the activation of hydroxyl groups of polyvinylalcohol and invertase
immobilized onto the magnetic polyvinylalcohol microspheres by covalent bonding
through the amino group. The retained activity of the immobilized invertase was 74 %.
Kinetic analysis was performed for immobilized invertase and free enzyme as well. The
Km values (55 mM sucrose) for immobilized invertase were higher than that of the free
enzyme (24 mM sucrose), whereas Vmax values were smaller for the immobilized
invertase. The optimum temperature (5ºC) was higher for immobilized invertase than
that of the free enzyme. The operational inactivation rate constant of the immobilized
invertase at 35ºC with sucrose (200 mM) was 5.83 ×105 min-1. Storage and thermal
stabilities were found to be increase with immobilization.
According to Herwig et al. (2001) in S. cerevisiae, the expression of invertase as
hydrolyzing enzyme of sucrose, was controlled by the presence of hexoses such as
glucose and fructose referred to as carbon catabolite repression. Most of the efforts had
26
been made to identify the mechanism by which cells sense extracellular monosaccharide
concentrations and triggered the genes involved in the repression pathway. The aim of
the present work was to study the cellular regulation of invertase expression in the wild-
type strain S. cerevisiae during batch culture growth containing mixed sugar substrates
under different initial conditions. As a result of the high frequency and accurate online
analysis of multiple components, a tight control of invertase expression could be found
and threshold concentrations of the monosaccharides for derepression could be
determined to glucose concentration of 0.5 g/l and fructose (2 gl).
Tanriseven and Dogan (2001) immobilized S. cerevisiae invertase in alginate
capsules. The immobilization resulted in relative activity of 87 % for 36 days without
appreciable loss in activity. Immobilized invertase was found more stable at high pH and
temperatures. The kinetic analysis for free and immobilized invertase was also
determined. Because the process was simple and invertase did not leak out of capsules,
therefore this method can be used for the industrial production of inverted sugar. Ganeva
et al. (2002) detected invertase liberation from S. cerevisiae after application of series of
rectangular millisecond electric pulses. Maximal yield (60 % from the activity in crude
extract) was achieved within 8 h after pulsation. As shown by staining SDS-PAGE for
invertase activity, the main part of liberated enzyme was a high molecular weight
periplasmic invertase. Belcarz et al. (2002a) worked on Candida utilis yeast, which was
cultivated in liquid media enriched with saccharose, synthesizes the well-known
invertase of 300 kDa. This enzyme was present both intracellularly in the periplasmic
space and extracellularly in the culture broth. However, it was determined that the same
C. utilis strain cultured in certain conditions was simultaneously capable of producing
another, still unknown form of invertase with a molecular mass of 60 kDa. The presence
of the latter enzymatic form was detected in cells as well as in the liquid culture medium.
Both invertase forms were purified using a three-step process (ion-exchange
chromatography, affinity chromatography, and preparative column electrophoresis) and
named, due to their different migration ratio in polyacrylamide gel electrophoresis, F-
form (Fast; 60 kDa) and S-form (Slow; 300 kDa). The F-form of invertase was found to
be non-glycosylated as opposed to the well-known S-form of invertase from the same
source. The physio-chemical properties of the F-form of invertase (isoelectric point,
substrate specificity, pH, and temperature optima) were determined and compared with
those of the S-form of the enzyme.
27
Khobragade and Chandel (2002) reported that intracellular invertase commonly
used in the inversion of sucrose into glucose and fructose. Both sugars produced were the
primary compounds used as sweeteners in the food processing industries. The enzyme
was immobilized in sodium alginate gel by entrapment technique and its catalytic
activity was compared with the activity of native enzyme at various pH and
temperatures. The optimum activity for native enzyme was achieved at pH of 4.2 and
temperature of 30C, whereas for immobilized enzyme it was obtained at pH of 4.8 and
temperature of 50C.
Belcarz et al. (2002b) investigated that when grown on a sucrose-containing
medium; Candida utilis synthesized and secreted two invertases: one of molecular size
of 280 kDa (S-form -Slow-migrating) and a new form of molecular weight of 62 kDa (F-
form - Fast-migrating). Prior to immobilization, purification of S- and F-forms of
invertase increased the immobilization yield to 89-100 %, in comparison with that of
crude invertase preparation (52 %). The immobilized purified S- and F- form of invertase
remained partially active after 15 min at 100C; the F-form retained almost 30 % of its
maximum activity. The immobilized S- form or F-form of invertase almost completely
inverted (95 % hydrolysis) sucrose 60 % (w/v) over 5 h continuous reaction at 80C.
Moreover, at 90C the immobilized F-form hydrolysed 70 % of 60 % (w/v) sucrose over
5 h, while the capability of the immobilized S- form of inverting sucrose over 5 h
reaction decreased from 80 % to 45 %.
Warchol et al. (2002) characterized the invertase of Bifidobacterium infantis
(ATCC 15697) and to compare it with other bacterial invertases. It was 46.8 times
purified over the crude extract by anion exchange, ultrafiltration and gel filtration. The
sequence of 15 amino acid residues of the NH2 terminal was determined. This enzyme
being monomeric protein with molecular weight of 70 kDa exhibied invertase activities.
The isoelectric point was 4.3, the optimum pH (6.0) and pK (4.5 and 7.2) of two active
groups were obtained. The activities were inhibited by Hg2+ and p-chloromercuribenzoic
acid (pCMB). The optimal temperature was 37ºC and activities were unstable at 55ºC.
Invertase activity was more efficient than that of invertase with Vm ⁄Km ratios of 0.65 and
0.025 min-1mg-1, respectively. The enzyme catalysed the hydrolysis of
fructooligosaccharides, sucrose and inulin with relative velocities of 100, 10 and 6,
respectively. It was concluded that enzyme of B. infantis (ATCC 15697) was an exo-
28
inulinase which had invertase activities. This protein was different from the invertase of
another strain of B. infantis.
Mansour and Dawoud (2003) immobilized invertase from S. cerevisiae on celite
and polyacrylamide by an absorption procedure. Both immobilized and soluble invertase
was compared. The immobilized invertase showed activity on celite (92 %) and
polyacrylamide (81 %). The optimum pH 4.6 and temperature (60C) for both soluble
and immobilized invertase activity were recorded. The immobilized invertase was found
thermostable at higher temperatures (40 to 60C) and showed high stability at room
temperature upon storage of 90 days. It can be used repeatedly 20 times with operational
stability. In comparison, the immobilized invertase was more stable on celite than
polyacrylamide. The immobilization of invertase by absorption method showed marked
stability for altered temperature, pH with high storage and operational stability.
da Cruz et al. (2003) described that ethanol production and biomass by
commertial S. cerevisiae strains were strongly affected by the structural complexity of
the nitrogen source during fermentation. The fermentation media were supplemented
with galactose and nitrogen source varying from a single ammonium salt in the form of
ammonium sulfate to free peptides (peptone) and amino acids (casamino acids). Diauxic
was found to be observed when galactose was used in low concentration independent of
nitrogen supplementation. At high concentrations of sugar, altered patterns of galactose
utilisation were found. Ethanol production and biomass accumulation depended on the
nature of the nitrogen source and were different for brewing and baking processes.
Baking yeast exhibited improved galactose fermentation performance in the medium
containing casamino acids. A large biomass production was obtained when peptone and
casamino acids were used for the brewing strain. However, high ethanol production was
found only with casamino acids. On the contrary, peptone as nitrogen supplement
induced higher ethanol production and biomass for brewing strain. Ammonium salts
always induced poor yeast performance. The results with galactose differed from those
obtained with maltose and glucose showed that supplementation with a nitrogen source
in the form of peptide (peptone) was more encouraging for yeast metabolism. It was
suggested that sugar catabolite repression had a vital role in yeast performance in a
medium containing nitrogen sources with differing levels of structural complexity.
Rossi-Alva and Rocha-Leao (2003) studied the entrapped cells grown inside of a
calcium alginate matrix. In addition, free cells of S. cerevisiae mutant strains with regard
29
to their pattern of growth and invertase activity was under investigation. The selection
process of the mutants was performed by the catabolic repression of invertase with
utilization of glucose and its consumption ability. When entrapped mutant strain Q6R2
cells were grown within calcium alginate gel beads using sucrose plus glucose, a
maximum sucrose hydrolysis because of high invertase activity was found. After
optimization of culture conditions, 1 mg of dry weight of entrapped cells was able to
produce 20 μmol of inverted sugar with maximum activity of 20 U/mg in 3 min. The
experiments were carried out for six months without appreciable loss of either invertase
activity or bead integrity. The beads as biocatalyst were also stored at 4ºC for six months
without loss in invertase activity. It was shown that entrapped yeast cells with a weak
ability to consume sugar might be used to produce inverted sugar.
Batista et al. (2004) optimized the sucrose as major carbon source used by S.
cerevisiae during production of baker's yeast, fuel ethanol and several distilled
beverages. The previous studies indicated that sucrose fermentation proceeds through
extracellular hydrolysis of the sugar, mediated by the periplasmic invertase. The resulted
glucose and fructose were transported into the cells and metabolized. The contribution to
sucrose fermentation of a poorly characterized pathway of sucrose utilization by S.
cerevisiae cells, the active transport of the sugar through the plasma membrane and its
intracellular hydrolysis was observed. A yeast strain that lacks the major hexose
transporters (hxt1-hxt7 and gal2) was found to be incapable of growing or fermenting
fructose or glucose. The results showed that this hxt-null strain was still able to ferment
sucrose due to direct uptake of the sugar into the cells. Deletion of the AGT1 gene,
which encoded a high-affinity sucrose-H (+) symporter, rendered cells incapable of
sucrose fermentation. Since sucrose was not an inducer of the permease, expression of
the AGT1 must be constitutive that allowed growth of the hxt-null strain on sucrose.
Danisman et al. (2004) prepared Poly (2-hydroxyethyl methacrylate-glycidyl
methacrylate) (pHEMA-GMA) membrane by UV-initiated photopolymerization.
Invertase was immobilized by the condensation reaction of the epoxy groups of glycidyl
methacrylate in the membrane structure with amino groups of the enzyme. The Km
values were for free (22 mM) and immobilized (58 mM) enzyme, respectively.
Immobilization increased the pH and temperature stability of the enzyme. Thermal
stability was found to increase with immobilization. The half times for the activity decay
at 70C were found to be 11 and 38 min for the free and immobilized enzyme,
respectively.
30
Tomotani and Vitolo (2004) immobilized the industrial yeast invertase by
adsorption on anion-exchange resins, collectively named Dowex. The maximum binding
was obtained at pH of 5.5 and 32ºC. Of the all polystyrene beads used, the complex
Dowex-1x4-200/invertase showed a yield coupling and an immobilization coefficient
equal to 100 %. The thermodynamic and kinetic parameters for sucrose hydrolysis for
both soluble and insoluble enzyme were also investigated. The complex Dowex/invertase
was stable without any desorption of enzyme from the support during the reaction, and it
had thermodynamic parameters equal to the soluble form. The pH stability shown by
soluble and insoluble invertase was found to be between (4.0 and 5.0) and (5.0 and 6.0),
respectively. The Km and Vmax values of 40.3 mM and 0.032 U/ml for the soluble
invertase and 38.2 mM and 0.0489 U/ml for immobilized invertase were observed,
respectively.
Kovalenko et al. (2005) obtained comparatively high invertase activity in yeast
membranes after autolysis of different strains. Heterogeneous biocatalysts for sucrose
inversion were made of the yeast membranes and granulated carbon-containing supports
made of common natural materials, expanded clay aggregate, sapropel, and lignin. The
properties of these biocatalysts were investigated. It was found that the biocatalyst
activity and stability of the immobilized yeast membranes increased with reference to the
initial expanded clay aggregate, independent of the structure of the carbon layer
synthesized on the support surface. Heterogeneous biocatalysts prepared by adsorption of
yeast membranes on sapropel had the greatest activity and stability, whereas lignin-based
biocatalysts were relatively unstable.
Aslam et al. (2006) entrapped biocatalyst as whole mutant yeast cells of S.
cerevisiae NA-47 into calcium alginate for the production of inverted syrup i.e., glucose
and fructose from sucrose as substrate. Out of eight media (M1-M8), the medium M2
containing glucose (2 %) as carbon source was selected for cell growth and intracellular
invertase after the time interval of 48 h. The optimum conditions for sucrose hydrolysis
were as sucrose (50 %), alginate beads were repeatedly used of 26 days after every 18 h
of incubation time. The beads were also stored at 4°C for 6 months without appreciable
loss of the invertase activity.
Yoshikawa et al. (2006) found five types of invertases (I, II, III, IV and V) in the
cell wall of Aureobasidium pullulans DSM2404 grown in a medium containing sucrose.
The fungus first catalyzed the transfructosylation of sucrose, and produced
31
fructooligosaccharide and glucose in the culture. In this process, the dominant type (I) of
invertase was found. The released fructioligosaccharides were consumed together with
glucose, and finally fructose was produced. In the fructioligosaccharide degrading
period, the levels of other invertase types II, III, IV and V were increased. These results
suggested that the expression of invertase type I was not repressed by glucose, but those
of invertases II–V were strongly inhibited in the presence of glucose. It was concluded
that invertase type I played a key role in fuctioligosaccharide production by this fungus,
whereas invertase type IV especially might function as a fructioligosaccharide degrading
enzyme with its strong hydrolyzing activity.
Sungur and Al-Taweel (2006) investigated a new biocatalyst possessed medium
feeding capability was prepared by immobilized S. cerevisiae cells into gelatin by
crosslinking with chromium salts. The optimum levels of chromium salt were found to
be as 0.016 mol/dm3 chromium III acetate and 0.008 mol/dm3 chromium III sulfate. The
resulted biocatalyst was characterized with regarding to its pH, temperature tolerances,
kinetic parameters and reusability. It was indicatd that immobilization shifted the pH for
maximum activity from 4.6 to 7.2. Thermal stabilities below 60оC were positively
affected by immobilization. Vmax values obtained for immobilized samples were 1.85 and
1.87 μmol sucrose/mg.min, whereas the value was 0.0262 μmol sucrose/mg.min for free
cells. Thus, a 70-fold increase of Vmax was obtained by immobilization and reuse
experiments showed no activity declined for 10 reuses in 28 days which were attributed
to continuous cell growth of immobilized whole cells using glucose (from sucrose
hydrolysis) and gelatin as nutrients. Karandikar et al. (2006) immobilized
Kluyveromyces marxianus, theromotolerant yeast was on glasswool reinforced silica
aerogel. Silica aerogel, being nanoporous, had extremely low density with large open
pores. High surface area and biocompatibility made it a promising material for
immobilization of biologically active molecules notably enzymes. However its brittle
nature limited the performance as support material. Reinforcement of silica aerogel with
glasswool increased its strength and flexibility maked it useful for such applications.
Electron microscopy and invertase activity measurements indicated that aerogel provided
a suitable plat form for cell immobilization and could be reused without degradation.
Bhatti et al. (2006) described the purification and thermal characterization of an
acid invertase produced by Fusarium solani in submerged culture. When culture medium
was supplemented with molasses (2 %) and peptone (1 %), the maximum enzyme
activity (9.90 U/ml) was achieved after incubation period of 96 h at pH 5.0 and 30ºC.
32
The enzyme was purified to homogeinity by ammonium sulfate precipitation and column
chromatography i.e., DEAE-cellulose and Sephadex G-200. The molecular mass (65
kDa) of the enzyme was noticed after SDS-PAGE. The optimum pH and temperature for
activity were 2.6 and 50оC, respectively. The Km value of 3.57 mM was determined for
sucrose.
Mahmoud (2007) adopted a new technique using wood waste as a carrier for
adsorption of invertase. The novel method enhanced activity, pH and thermal stability of
the immobilized invertase from S. cerevisiae and showed resistance against washing by
concentrated NaCl (6 M) solution. This finding encouraged the use of sawdust as matrix
for invertase purification or might be for other enzymes. The optimum cultural
conditions for enzyme activity were not found to be affected by immobilization.
However, the optimum (pH 5.6) and temperature (60оC) for either free or immobilized
enzymes were same. Immobilized invertase showed more stablity at high pH and
temperatures. There was no leakage of the invertase for storage of two months. The
immobilized invertase being less sensitive to inhibition by impurities present in molasses
were applicable for continuous sucrose hydrolysis in column bioreactor. It was suggested
that this technique can be used for the production of industrial inverted syrup.
Milintawisamai et al. (2007) worked on invertase from 2 yeast strains, Candida
humicolus 5M2 and S. cerevisiae 7M, isolated from Mitra Phuveing Sugar Industry.
They were cultured aerobically in a complete medium enriched with 10 % sucrose at
30°C and subjected to partial purification for extracellular and intracellular invertase.
The purification was done by (NH4)2SO4 precipitation and HPLC with IEC-DEAE ion
exchange column. The major fraction of extracellular invertase from C. humicolus could
be separated by 60-100 % (NH4)2SO4, followed by DEAE column chromatography as a
single active fraction at the concentration of 0.3-0.35 M NaCl with molecular mass of
110 kDa. The intracellular enzyme was partially separated by DEAE column
chromatography at the same conditions. Two forms of intracellular invertase, high (>200
kDa) and low molecular weight forms (58 and 64 kDa), were found in S. cerevisiae. The
high molecular weight form from the crude cell extract of S. cerevisiae was further
separated by DEAE column chromatography at the concentration of 0.21-0.24 M NaCl.
Guimaraes et al. (2007) reported that the filamentous fungus Aspergillus
ochraceus gave higher production of thermostable extracellular invertase when grown on
Khanna medium supplemented with sugar cane bagasse at 40°C for 96 h. The enzyme
33
was purified (7.1 fold) with a recovery of 24 % by using DEAE-cellulose and Sephacryl
S-200 chromatographies. The invertase enzyme after gel filtration was found to be as
homodimeric glycoprotein that showed carbohydrate content (41 %) with molecular
mass of 135 kDa. The optima of pH and temperature were 4.5 and 60°C, respectively.
The enzyme activity was stimulated by Ba2+ (20 %), Na+ (35 %) %), Mg2+ (50 %) and
Mn2+ (57 %) and inhibited by Hg2+ and Cu2+.
Haq et al. (2008) improved the yeast strain of S. cerevisiae through random
mutagenesis for better production of invertase using sucrose as substrate. Sixty isolates
of S. cerevisiae were screened for invertase production. The isolate showing highest
activity (1.10 U/ml) was subjected to ultraviolet (UV) radiation and chemical mutagen
i.e., MNNG (N-methyl N-nitro N-nitroso guanidine). One mutant produced maximum
enzyme activity (17.8 U/ml) that was further exposed to EMS (ethyl methane
sulphonate). The maximum enzyme activity (25.56 U/ml) was obtained. When cultural
and nutritional conditions such as incubation time (48 h), sucrose concentration (5 g/l),
initial pH (6) and inoculum size (2.0 %, v/v) were optimized, the invertase production
reached to 45.65 U/ml. It was about 40 fold increase in enzyme production than parent
culture.
Pawar and Thaker (2009) worked on Aspergillus niger for its acid phosphatase
(E.C 3.1.3.2) and invertase production in media containing different concentrations of
sucrose (1 %, 3 %, or 5 %). Both these enzymes played a key role in phosphate and
carbon metabolism in microorganisms, animals and plants and thus were interesting from
the perspective of biotechnological applications. Ontogenic changes in cytoplasmic,
wall-bound and extracellular enzyme activities of were investigated. The growth (fresh
weight) of Aspergillus niger exhibited inverse correlation with pH. When pH was
increased, both enzyme activities were found to be higher in the medium containing
sucrose in low concentration. It was indicated that decrease in the fresh weight of fungi
caused the increase in enzyme activity. It was concluded that both enzymes might
participate in autolysis of fungi.
Kotwal and Shankar (2009) studied the importance of invertase for the sucrose
hydrolysis that yielded an equimolar mixture of glucose and fructose called as inverted
syrup, also commonly used in beverage and food industries. Its major use in
manufacturing of artificial honey, pharmaceutical and paper industries, as plasticizing
agents in cosmetics, and enzyme electrodes for the detection of sucrose was studied.
34
Immobilization of invertase and its biotechnological applications were also investigated.
Gutierrez-Alonso et al. (2009) characterized biochemically an extracellular invertase
from the yeast Rhodotorula dairenensis. The molecular mass of the enzyme was found to
be as 680 kDa by analytical gel filtration and 172 kDa on SDS-PAGE. The N-linked
carbohydrate content (16 %) of the total mass was found. The optimum activity was
achieved at pH 5 and temperature of 55-60оC. The enzyme showed wide-ranging
substrate specificity and hydrolyzed sucrose, nystose, 1-kestose, leucrose and inulin.
Robledo-Olivo et al. (2009) studied invertase production by Aspergillus niger in
submerged culture using different concentrations of glucose and sucrose. When the
initial concentration of sucrose was increased from 6.25 to 50 g/l, a higher biomass
production (6.1 g/l) was achieved. The biomass production was increased four times
more when a glucose-sucrose combination was used as substrate (26.31 g/l). The strain
A. niger produced extracellular invertase activity at all tested concentrations of the
substrate. The highest enzymatic activity (3873 U/l) was found when sucrose was used at
12.5 g/l. However, with a glucose-sucrose concentration of 25 g/l the beta-
fructofuranosidase activity was of 23706 U/l. The maximum rate of invertase enzyme
production in presence of sucrose by A. niger in submerged culture was 3.67 U/l/h at
12.5 g/l concentration, while in the case of glucose-sucrose mixture, it was 13.95 U/l/h at
a concentration of 25 g/l. It was observed that the enzyme yield (YE/X) was 1.25 times
more in presence of sucrose than with combined action of glucose-sucrose. In addition,
the results suggested that an addition of lower concentration of glucose was a viable
option to increase the enzyme secretion by the fungi.
Resa et al. (2009) studied the use of a low-intensity ultrasonic technique (non-
invasive, non-destructive, on-line, and able to assess opaque samples) to monitor the
kinetics of invertase hydrolysis. Adiabatic compressibility had been shown to be
sensitive to sugar species, ultrasonic velocity increasing as saccharose was transformed
into glucose and fructose. The effect of initial sucrose concentration (2-60 %), pH (3.5-
6.5), temperature (25-55оC) and number of microorganisms (105-109 yeasts/ml) on the
reaction rate, catalyzed by the extracellular invertases of intact S. cerevisiae cells, had
measured. The findings were verified in strict agreement with the optimal kinetic
parameters of the invertase. The variations of ultrasonic velocity were elucidated in
terms of changes of the solute concentrations in the mixture water-saccharose-
glucose/fructose. The calculations were made from the velocity of ultrasound in the
35
corresponding pure sugar solutions. A linear relationship between the initial rate of
ultrasonic velocity and the number of yeasts (enzymes) was also shown.
Venkateshwar et al. (2009) investigated that invertase is an important enzyme of
the fructose syrup industry fructose syrup industry andf confectionery produced by a
number of microorganisms. Among these, yeasts showed the highest sucrose
fermentation potential. In the present study, invertase production was carried out in
submerged fermentation using a high enzyme-producing yeast strain. Plackett–Burman
statistical experimental design was applied to evaluate the fermentation medium
components. The effects of ten nitrogen sources were studied in a 16-run experimental
design. Yeast extract, N-Z-amine, tryptone, Beef extract, ammonium acetate and meat
extract were found to have significant effects on enzyme production. Among these, yeast
extract, N-Z-amine, and ammonium acetate were the most significant. A maximum
invertase activity (299.4 U/ml) was obtained after fermentation period of 24 h.
Safarik et al. (2009) reported that inverted sugar (an equimolar mixture of
glucose and fructose prepared by sucrose hydrolysis) was a very important food
component. Magnetically responsive alginate microbeads contained entrapped S.
cerevisiae cells was prepared and magnetite microparticles which can be easily
separatedin an appropriate magnetic separator. The microbeads of diameter between 50
and 100 mm were prepared using the water-in-oil emulsification process. The prepared
microbeads contained yeast cells with invertase activity enabled deficient sucrose
conversion. The biocatalyst was quite stable, the same catalytic activity was found after
one month storage at 4ºC and the microbeads could be used at least six times.
Andjelkovic et al. (2010) purified four external invertase isoforms from S.
cerevisiae by isoelectric precipitation, ethanol precipitation, QAE-Sephadex and using
Sephacryl S-200. In contrast to earlier published work for extracellular purification of
invertase, a specially designed elution step was applied on QAE-Sephadex which
enabled the separation of four isoforms. The isoforms had the same molecular weight
and catalytic properties: Km value (25.6 mM) for sucrose, optimum pH (3.5-5) and
optimum temperature (60оC). But they exhibited significant difference in isoelectric
values, thermal stability and chemical reactivity. Deglycosylation studies showed that the
observed differences between isoforms occurred from posttranslational modifications.
Results showed that external invertase was a mixture of at least four isoforms, but in
order to improve the efficiency of food industry processes, only the most stable isoform
36
(E1) was purified and utilised. Substantially different chemical reactivity of the isoforms
could be used to improve the yield of covalent immobilization procedures.
Uma et al. (2010) produced high levels of invertase in A. flavus under optimized
culture conditions on fourth day of incubation at an optimum pH (5), temperature (30оC),
and inoculum size (3 %) in Czapek Dox medium using fruit peel waste as a substrate by
submerged fermentation. Improved enzyme production was obtained when nutritional
factors such as sucrose and yeast extract were added into the medium. The enzyme
invertase was purified to 5.8 fold with recovery of 3.2 % by DEAE-column
chromatography. The molecular weight of the enzyme was estimated to be 67 kDa by
SDS-PAGE. It had a Vmax value of 15.8 U/mg and Km 0.23 mg/ml at pH of 6.0. The
invertase activity was found to be stable at 50оC for 30 min and it was stimulated by
metal ions like Na+ and Ca+2 and inhibited by Zinc.
37
MATERIALS AND METHODS
3.1: MATERIALS
The chemicals used in this study such as mannose, 3,5-dinitro salicylic acid, sodium
potassium tartarate, phenol, diammonium hydrogen phosphate, sodium metabisulphate,
yeast extract, peptone, acrylamide, bisacrylamide, Trizmabase, potassium dihydrogen
phosphate, Tris HCl, sodium dodecyle sulfate (SDS), Bovine serum albumen (BSA), β-
mercaptoethanol, ethyl methane sulphonate (EMS), 2-deoxy-D-glucose, sodium
alginate, bromophenol blue, DEAE-Sephadex A-50, Coomassie brilliant blue (G-250),
ammonium per sulphate (APS), N,N,N’,N’-tetramethylenediamine (TEMED) were
obtained from BDH (UK), Merck (Germany), Sigma (USA), Fluka (Switzerland) and
E-Acros (Belgium). All chemicals were of analytical grade.
3.2: METHODS
3.2.1: Isolation of microorganism
Eighty six strains of Saccharomyces cerevisiae were isolated from different soil samples
and fruits like apple, plum, peach, date, banana, mango and guava collected aseptically
from different areas of Lahore District in polythene bags. Isolation was carried out by
serial dilution method on YPS agar medium containing (g/l): yeast extract 3, peptone 5,
sucrose 20 and agar 20 (pH 6) after modified method of Dworschock and Wickerham,
1961. The petriplates were incubated in incubator (Model: MIR-153, Sanyo Japan) at
30ºC for 2-3 days. The colonies were transferred to the YPS agar slants. The isolates
were identified after Wickerham (1951), Lodder and Rij (1952) and Barnett et al. (1979)
by determining cultural and morphological characteristics. Sub-culturing of the isolates
was carried out every 2 weeks. These strains were screened for invertase production and
preserved in sterile 20 % (v/v) glycerol solution at -80C.
3.3: Fermentation technique
3.3.1: Extracellular invertase production
3.3.1.1: Preparation of vegetative inoculum
Fifty millilitre of the YPS broth medium was transferred to the individual 250 ml
Erlenmeyer flasks. The flasks were cotton plugged and sterilized in an autoclave (Model:
KT-40 L, ALP, Japan) at 15 lbs/in2 pressure (121оC) for 15 min and cooled at room
38
temperature. Cell suspension was prepared from a 2-3 day old slant culture by adding 10
ml of sterilized distilled water and shaking vigorously. One millilitre of the cell
suspension was aseptically transferred into the flask and incubated at 30оC in a rotary
shaking incubator (Model: 10X400.XX2.C, SANYO Gallenkamp, PLC, UK) at 200 rpm
for 24 h.
3.3.1.2: Yeast viable count
The yeast cell count was made by Haemacytometer using Trypan Blue (0.4 %) as
indicator to mark dead and viable cells. The reactivity of trypan blue is based on the fact
that the chromopore is negatively charged and does not interact with the cell unless the
membrane is damaged. Therefore, all the cells which exclude the dye are viable. Each
milliliter of cell suspension contained 2.1 ×10 3 CFU.
3.3.1.3: Fermentation media
Different media were tested for the production of extracellular invertase by S. cerevisiae.
These include (g/l):
M1: Sucrose 30, yeast extract 3 and peptone 5, pH 6.0 (Dworschock and Wickerham,
1961).
M2: Cane molasses 30 (as equivalents of total reducing sugar), (NH4)2SO4 5,
Na2HPO4.12H2O 2.4 and MgSO4.7H2O 0.07, pH 4.5 (Bokosa et al., 1993).
M3: Cane molasses 36, (NH4)2SO4 5, (NH4)2HPO4 3, MgSO4.7H2O 3 and yeast extract
3, pH 4.8 (Linko et al., 1980).
M4: Yeast extract 3, peptone 5, glucose 2, sucrose 15, (NH4)2SO4 51, MgSO4.7H2O
0.075 and Na2HPO4.12H2O 2.4, pH 4.5 (Abrahao-Neto et al., 1996).
M5: Yeast extract 4, peptone 4, sucrose 50, K2HPO4 1, CaCl2.2H2O 0.1, MgSO4.7H2O
0.1 and (NH4)2SO4 1.5, pH 4.5 (Marques et al., 2006).
M6: Sucrose 20, peptone 20, NH4H2PO4 12, NaCl 5, MgSO4. 7H2O 0.5 and FeSO4.
7H2O 0.01, pH 6.0 (Zhang and Ge, 2006).
3.4: Shake flask studies
Fifty millilitre of the YPS medium was transferred to the individual 250 ml Erlenmeyer
flasks. The pH value adjusted to 5.0 with 5 N HCl before sterilization was carried out for
15 min at 121оC. Flasks were cooled, inoculated with 1 ml of yeast culture and incubated
39
in a rotary shaking incubator at 30оC for 48 h. The agitation rate was kept at 200 rpm.
The experiments were run parallel in triplicate. The yeast cells were separated by
spinning at 6000×g for 15 min. The supernatant was used for further analysis of
extracellular invertase.
3.5: Fermentor studies
Production of invertase was carried out in a laboratory scale stirred fermentor of 7.5 L
capacity with working volume of 5 L. The working vessel containing YPS broth medium,
was sterilized in an autoclave at 121оC for 20 min. The inoculum was transferred at
different levels (5-12.5 %, v/v). The solutions of 0.1 N HCl or 0.1 N NaOH were used
for pH adjustment. The temperature was kept at 30оC. Agitation speed of the stirrer was
maintained at 200 rpm while aeration rate was set at 1.0 l/l/m (vvm). The dissolved
oxygen (DO) was maintained by the proportional integral derivitive (PID) cascade
controller, which changed the speed of agitation. Pure air was automatically supplied to
the fermentor to keep the DO level at the set point after the agitation speed reached the
maximum allowable set point.
3.6: Significant parameters
In the present study, among significant parameters, the following parameters of
nutritional and cultural requirements of S. cerevisiae in terms of optimum growth (dry
cell mass) and enzyme production were studied.
3.6.1: Different fermentation media
Six different media were evaluated for the extracellular enzyme production in shake
flasks.
3.6.2: Incubation period
The effect of different incubation time period was carried out (8-72 h) in shake flasks
and (8-48 h) in fermenter at 30оC. The sample was collected with regular time intervals.
3.6.3: Effect of initial pH
A range of pH (3.5-6.5) in shake flasks and fermenter was tested for invertase
production (Martinezforce and Benitez, 1995).
40
3.6.4: Effect of temperature
The effect of different temperature (20-50оC) on the biomass and enzyme production
was investigated (Inan et al, 1999).
3.6.5: Effect of volume
The volume of fermentation medium such as 25, 50, 75 and 100 ml was evaluated in
shake flask fermentation.
3.6.6: Effect of inoculum size
Vegetative inoculum at varying amount (1-4 %, v/v) in shake flasks and (2.5-10 %)
fermenter studies was investigated.
3.6.7: Effect of agitation and aeration
Different agitation (120-240 rpm) and aeration (0.5-2.0 vvm) levels were investigated for
optimum invertase production (Bernardo et al., 2005; Rosma and Ooi, 2006).
3.6.8: Effect of dissolved oxygen
Different concentrations of dissolved oxygen (5-20 %) were used in control with
agitation for enhancement of the enzyme invertase in stirred fermenter (Abrahao-Neto
et al., 1996).
3.6.9: Effect of carbon sources
The various carbon sources such as sucrose, glucose, lactose, maltose, fructose,
galactose and molasses were evaluated for the production of invertase (Granot and
Snyder, 1993).
3.6.10: Effect of additional nitrogen sources
Different additional nitrogen sources in addition to yeast extract (3 g/l) such as organic
sources (peptone, meat extract, urea, casein) and different inorganic nitrogen sources
(ammonium nitrate, ammonium sulfate, ammonium chloride, diammonium hydrogen
phosphate and potassium nitrate) were evaluated for the enzyme production (da Cruz et
al. 2003). Different agricultural wastes including soybean meal, sunflower meal and
corn steep liquor were also investigated.
41
3.7: Strain improvement
3.7.1: UV irradiation:
Five millilitre of 8 h old inoculum of S. cerevisiae IS-66 was added into a sterile
centrifuge tube and spun at 6,000×g for 15 min. The cells were suspended in 5.0 ml of
sterilized 0.5 % sucrose acetate buffer at pH 4.5 and washed twice; cells were
resuspended in 50 ml of the buffer. Five millilitre each of the suspension was transferred
to the individual sterile petriplates and exposed to UV light (Model: Mineral light, UVS-
12, California, USA) for different time intervals (10-60 min) at a fixed distance of 5 cm
(dose 1.2102 J/m2/S) to obtain cell viability loss greater than 90 %. Approximately 0.1
ml each of the UV-irradiated cell suspension was transferred to the petriplates containing
YPS agar medium. A sterile colony spreader was gently used to uniformly distribute the
irradiated cell suspension onto the medium. The initial colonies that appeared within 48
h of incubation at 30oC were transferred to the YPS agar slants and screened individually
for invertase activity.
3.7.2: Nitrous acid treatment
Different (0.05-0.20 M) solutions of NaNO2 prepared in acetate buffer (0.2 M, pH 4.5)
were added to the washed and centrifuged pellet of S. cerevisiae. The solution was
thoroughly shaken for 2-10 min. One millilitre of solution was withdrawn and diluted 5
fold in phosphate buffer (0.2 M, pH 7.0) to stop the reaction. Approximately, 0.1 ml of
the yeast suspension was inoculated to the YPS agar plates were kept for 48-72 h of
incubation at 30oC. The control was also run parallel. The colonies having > 90 % cell
viability loss were tested for enzyme activity.
3.7.3: EMS treatment:
Different concentrations (50 to 100 μl) of EMS were added to the individual centrifuge
tubes containing 5 ml of yeast cells and shaken to a homogenous suspension. In the
control, 0.05 ml phosphate buffer (pH 7) instead of EMS was added. After specific time
interval (5-20 min), 8 ml of sterilized 5 % (w/v) sodium thiosulphate solution was added
to inactivate the EMS. The yeast cells were centrifuged washed thrice in phosphate
buffer. The EMS treated yeast cells were resuspended in 5 ml of sterilized distilled
water. Approximately, 0.1 ml of the cell suspension was inoculated to the YPS agar
plates. The colonies showing > 90 % cell viability loss, appearing between 48-72 h of
incubation at 30oC were screened for enzyme activity (Haq et al., 2008).
42
3.7.4: 2-deoxy-D-glucose resistance:
Mutagenized cells were plated onto YPS agar medium containing 12 % (w/v) sucrose.
Colonies exhibiting the better growth were replica plated, and one set of the colonies was
exposed to a glucose measuring kit solution (Sigma, St. Louis, USA). Colonies that were
surrounded by the largest pinkish zones were selected for further study. The potential
mutant strains were cultured overnight on the YPS agar medium, harvested during the
exponential phase of growth (1×103 cells/ml), washed with sterilized distilled water and
plated on the 2dg-YPR agar medium containing (mg/ml): yeast extract 3, peptone 5,
raffinose 20, agar 20 and 2-deoxy-D-glucose (0.02-0.10). Raffinose was used replacing
sucrose because sucrose hydrolysis by yeast invertase results into glucose, which
competes the toxin (Rincon et al., 2001). Colonies appearing between 1-2 days were sub-
cultured on the same medium and colonies exhibiting the most vigorous growth were
tested for stability in invertase production by shake flask fermentation. Samples were
drawn periodically, washed and plated on YPR agar medium to select the strains
resistant to higher level of 2dg. The master culture was preserved in sterilized 20 % (v/v)
glycerol at - 80оC.
3.3.2: Intracellular invertase production:
3.3.2.1: Extraction of intracellular invertase
The yeast cells of strain EMS-42 were harvested from stirred fermentor after spinning at
6,000×g for 15 min. Cells were washed once with acetate buffer, pH 5.0, resuspend in
the same buffer with 40 µM phenylmethylsulfonylfluride (PMSF). All subsequent steps
were carried out at 4oC. The cell suspension (500 ml) having pH (3-6.5) was sonicated
with 0.5 duty cycle of impulses at different amplitude (20-80 %) for varying time
interval (15-90 min), using probe (Horn H22 D) immersed 2.5 cm in the suspension. The
crude extract after sonication was spun at 12,000×g for 15 min at 4oC. The supernatant
was used for intracellular invertase determination and purification.
3.8: Analytical techniques
3.8.1: Dry cell mass:
Yeast dry cell mass was determined by spinning of the fermented broth at 6000×g for 15
min using preweighed centrifuge tubes. After decanting off the supernatant, cell mass
43
was washed twice with distilled water. The tubes containing cell mass were oven dried at
105оC for 1 h. Final weight was then taken to obtain dry cell mass.
3.8.2: Invertase activity:
Invertase activity was determined after Akgol et al. (2001). “One invertase unit is
defined as the amount of enzyme, which releases 1.0 mg of inverted sugar per milliliter
in 5 min at 35оC and pH 5.5”.
For invertase activity, 2.5 ml acetate buffer (50 mM, pH 5.5) and 0.1 ml sucrose (300
mM) was added into the individual test tubes. The tubes were pre-incubated at 35оC for 5
min. After the addition of 0.1 ml of appropriately diluted enzyme solution, incubation
was continued for another 5 min. The reaction mixture was placed in a boiling water bath
for 5 min to stop the reaction and then allowed to cool at room temperature. A blank was
also run parallel replacing the enzyme solution with distilled water. To 1 ml of each
reaction mixture 1 ml of DNS was added and placed the tubes in boiling water for 5 min.
After cooling to an ambient temperature, volume was raised up to 10 ml. Transmittance
was measured at 546 nm using spectrophotometer (Model: CECIL CE-7200 Aquarius,
UK).
3.8.3: Protein estimation:
Protein concentration was estimated in the enzyme solution after Bradford (1976).
Bradford reagent (5 ml) was added to a test-tube containing 0.1 ml of the diluted enzyme.
A blank was run parallel. The tubes were vortexed. The absorbance was noted at 595 nm
on a spectrophotometer. The amount of protein in each sample was obtained using the
BSA standard curve.
3.9: Statistical analysis
Treatment effects were compared by the method (Costate, cs6204W.exe) after
Snedecor and Cochran (1980). Post-Hoc Multiple comparison tests were applied under
one-way ANOVA. Significance has been presented in the form of probability (p<0.05)
values.
3.10: Fermentation kinetic study
Kinetic parameters for batch fermentation were determined according to the method
describe by Pirt (1975) and Lawford and Rouseau (1993). The following parameters of
kinetics were studied.
44
Specific growth rate:
The value of specific growth rate i.e., µ (h-1) was calculated from plot of In (x) vs time
of fermentation.
Product yield coefficient:
Product yield coefficient namely Yp/x was determined by the equation:
Yp/x = dP/dx
Volumetric rates:
The volumetric rate of product formation Qp (U/l/h) was determined from the
maximum slope of enzyme produced vs time of fermentation. The volumetric rate for
biomass formation Qx (g cell mass /l/h) was determined from the maximum slope of
cell mass formation vs time of fermentation.
Specific rate constant:
Specific rate constant for product formation was determined by the equation
qp =µ × Y p/x
3.11: Immobilization Studies
The collected cells of S. cerevisiae EMS-42 were resuspended in 0.05 M acetate buffer,
pH 5 to give a final concentration of 16 mg of dry weight/ml and were mixed with an
equal volume of 4 % (v/v) sodium alginate. This mixture was dropped into 1 % CaCl2
solution with constant gentle shaking. The so-formed beads were left for 1 h in this
solution, then filtered and washed three times with sterile distilled water before use. The
size of beads was estimated as 3 mm in diameter.
3.11.1: Sucrose hydrolysis:
Batch reactors were run in 250 ml Erlenmeyer flask containing 800 beads in 100 ml of
40 % (w/v) sucrose solution. The inverted sugar (glucose and fructose) was estimated by
DNS method (Miller, 1959). The pH of sucrose solution was adjusted to 5. The flasks
were incubated at 30oC for maximum sucrose hydrolysis. After incubation, the beads
were recovered, washed with distilled water and re-used in a new batch with fresh
sucrose solution. This operation was repeated until beads retained their integrity.
45
3.11.1.1: Effect of sucrose concentrations
The effect of different sucrose concentrations (30-70 %, w/v) was carried out on sucrose
hydrolysis.
3.11.1.2: Effect of temperature
The effect of different temperature (30-70oC) on sucrose hydrolysis was investigated.
3.11.1.3: Effect of pH
The production of invertase for sucrose hydrolysis by immobilized cells at different pH
(3-6) in shake flasks was studied.
3.11.1.4: Re-use of immobilized cells
The effect of the re-use of the biocatalyst in the batch process on sucrose hydrolysis was
studied till 48 batches.
3.12: Purification of invertase
Both enzymes (extracellular and intracellular) from S. cerevisiae EMS-42 were purified
to homogeneity by following purification steps.
3.12.1: Ammonium sulfate precipitation:
The ammonium sulfate was added at varying amounts (20-85 %) to cell free broth for
extracellular invertase and sonicated crude extract (supernatant after spinning) for
intracellular invertase. They were spun at 18,000×g for 30 min to get the precipitates of
all fractions. The resultant precipitates and supernatant were dissolved in 0.5 M Tris-HCl
buffer, pH 7.5 and dialyzed against same buffer.
3.12.2: Anion- exchange chromatography:
For the purpose of anion exchange chromatography, 1 g DEAE-Sephadex A-50 (Sigma,
USA) was swollen in 100 ml of the 0.05 M Tris-HCl buffer, pH 7.5 in a boiling water
bath for 2 h. After cooling, it was poured into the column and made final bed volume
(1.5 × 15.0 cm). The dialyzed enzyme solution was applied to column that pre-
equilibrated with five column volumes of the 0.05 M Tris-HCl buffer, pH 7.5. A linear
NaCl gradient from 0 to 1 M in 150 ml of the same buffer was applied. Fractions of 3 ml
were collected at a flow rate of 0.5 ml/min. The collected fractions were assayed for
46
protein at 280 nm and invertase activity by performing enzyme assay. The fractions
containing enzyme activity were pooled, dialyzed and analyzed on SDS-PAGE.
3.12.3: Gel filtration:
Sephadex G-50 (Phamacia Fine Chemical), 2 g was swollen in 50 ml of 0.05 M Tris-HCl
buffer, pH 7.5 in a boiling water bath for 2 h. The gel slurry was poured along the side of
tilted column by taking care that no air bubble was entrapped. The column (1.5 × 20 cm)
was equilibrated with five column volumes of the 0.05 M Tris-HCl buffer, pH 7.5 in
order to stabilize the bed. The enzyme sample (3 ml) was eluted with the same buffer;
adjusting flow rate at 0.5 ml/min. The collected fractions were assayed for protein and
invertase activity. The active enzyme fractions were pooled, dialyzed and used for
enzyme characterization.
3.12.4: Dialysis
The salts were removed from precipitates and pooled samples by using 12,000 molecular
weight cut off dialyzing bag, which was placed in one liter of the 0.05 M Tris-HCl buffer
(pH 7.5) for 5-6 h at 4ºC. The process was repeated 4-5 times until all salts were
removed from the enzyme solution.
3.12.5: Electrophoresis
At each step of purification, sodium dodecyle sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) was performed by the method of Hames (1990).
3.12.6: Protein marker
The molecular weight of the invertase was estimated by SDS- polyacrylamide gel with
protein marker (BIORAD, Catalog #161-0363).
3.12.7: Carbohydrate content
The total carbohydrate content was detected by the phenol sulfuric acid method (Dubois
et al., 1956) with mannose as standard.
3.13: Gel preparation
3.13.1: Separating gel:
The separating gel (10 %) was prepared by adding all the ingredients i.e. 3.3 ml
acrylamide (30 %), 2.5 ml 1.5 M Tris HCl (pH 8.8), 0.1 ml SDS (10 %), 0.1 ml APS (10
47
%), 0.006 ml TEMED, 4 ml distilled water and poured in the gel assembly leaving one
inch vacant space at the top. Almost 100 µl of distilled water was layered at the top of
the gel to give a flat surface and to remove oxygen which inhibited polymerization. The
gel was allowed to polymerize for 30 min.
3.13.2: Stacking gel:
The staking gel was prepared by adding 0.5 ml acrylamide (30 %), 0.38 ml 1M Tris HCl
(pH 6.8), 0.03 ml SDS 10 %, 0.03 ml APS (10 %), 0.004 ml TEMED and 2.1 ml distilled
water. The water was removed from top of the separating gel and staking gel was poured
in the gel assembly. Comb was inserted and gel was allowed to polymerize at the room
temperature for 10 min. When complete polymerization took place, gel comb was taken
out and wells were washed with tank buffer four times by means of a syringe. After
removing the bottom spacer the gel assembly was settled in the gel chamber and made
contact top and bottom with tank buffer which was previously diluted in the ratio of 1:5
with distilled water.
The enzyme solution (6 µl) and loading buffer (4 µl) were denatured by heating in
boiling water bath for 3 min. The samples were loaded along the protein marker and
electrophoressed at a constant voltage of 150 v potential difference and 20 mA current
supply for about 4 h.
3.14: Characterization of purified invertase
3.14.1: Effect of pH and temperature on stability of invertase:
It was observed by taking hundreds microlitres of appropriately diluted enzyme solution
incubated in 0.05 M citrate/ 0.05 M acetate buffer at different pH values ranging for 2-8
at 40оC for 15 min. At optimal pH, the invertase activity was tested at varying
temperature values (20-80оC). For this, the reaction mixture was incubated for 15 min at
different temperatures and residual activity in both parameters was determined under
standard conditions (Akgol et al., 2001).
3.14.2: Effect of additives on enzyme activity:
Different chemicals and metal ions such as NaCl, KCl, MnCl2, EDTA, BaCl2, MgCl2,
CuSO4, HgCl2, CoCl2, FeSO4, CaCl2 and ZnSO4 preincubated with the purified enzyme
at 1 mM at 30оC for 30 min. before determination of the enzyme activity. Blank was
taken showing relative activity (100 %) before adding the metals.
48
3.14.3: Determination of kinetic constant (Km):
The Km value of the invertase is determined using sucrose as a substrate and using
Lineweaver-Burk plot (Lineweaver and Burk, 1934) by following conditions of sucrose
(10-100 mM) in 0.05 M acetate buffer (pH 4.5), incubation time (15 min) and
temperature (35оC). The amount of liberated reducing sugars was measured by Miller,
1959.
3.14.4: Determination of maximum velocity (Vmax):
The maximum velocity (Vmax) of sucrose hydrolysis of invertase under same optimal
conditions of kinetic constant was calculated.
3.15: Preparation of standard curves:
3.15.1: Glucose curve:
One gram of anhydrous glucose was dissolved in a small quantity of distilled water and
volume was made up to 100 ml. This solution contained 10 mg of glucose per milliliter.
Further dilute it 10 times by taking 1 ml of solution into 9 ml of distilled water
containing 1.0 mg of glucose per milliliter. This stock solution was used to make 10
appropriate dilutions from 0.1 to 0.6 mg/ml. One milliliter of each dilution was taken in
separate test tubes followed by the addition of 1 ml of DNS reagent. The blank was made
with 1 ml of distilled water and 1 ml of DNS solution. The tubes were boiled in a water
bath for 5 min prior to cooling at room temperature. Transmittance was measured at 546
nm spectrophotometrically following the method of Haq et al., 2006. Graph (Fig 3.1)
was plotted, taking transmittance at ordinate and sugar concentration at abscissa.
3.15.2: BSA curve:
Bovine serum albumen (BSA) in amount of 10 mg was dissolved in approximately 8.0
ml of distilled water and final volume was made up to 10 ml to get 1 μg/μl concentration.
This solution was further diluted up to 100 μl with distilled water to contain 20, 40, 60,
80 and 100 µg of BSA. Then 5.0 ml of Bradford reagent was added. The absorbance was
noted by a spectrophotometer (Model: CECIL CE-7200 Aquarius, UK) at 595 nm and
compared it with blank. A graph was plotted between the absorbance and BSA
concentration (Fig. 3.2) following the method of Bradford (1976). The slope of the curve
was used for protein estimation.
49
Protein (mg/ml) = Slope (µg) × 10 × Dilution factor 1000
3.15.3: Mannose curve:
The sugar mannose was dissolved in amount of 10 mg in approximately 8.0 ml of
distilled water and final volume was made up to 10 ml to get 1 μg/μl concentration. This
solution was further diluted up to 100 μl with distilled water to contain 20, 40, 60, 80 and
100 µg of mannose. To 1 ml of 5 % (w/v) phenol solution and 5 ml of concentrated
sulfuric acid was added. The absorbance was noted by a spectrophotometer at 490 nm
and compared it with blank. A graph (Fig. 3.3) was plotted between the absorbance and
mannose concentration. The slope of the curve was used for carbohydrate estimation.
3.16: Preparation of solutions /reagents:
3.16.1: Trypan blue solution (0.4 %):
It was prepared by dissolving 0.4 g trypan blue in 100 ml distilled water and filter.
3.16.2: Acrylamide bisacrylamide (30 %):
It was prepared by dissolving 29 g of acrylamide and 1 g bisacrylamide in 100 ml of
distilled water. The solution was filtered and stored at 4оC.
3.16.3: SDS solution (10 %):
It was prepared by dissolving 10 g of SDS in hot water. The solution was stirred and
final volume was made upto 100 ml.
3.16.4: APS solution (10 %):
It was prepared by dissolving 1 g of ammonium per sulfate in distilled water and raised
the final volume upto 10 ml. This solution freshly was used.
3.16.5: Phenol solution (5 %):
It was prepared by mixing 5 g phenol in 50 ml distilled water and final volume was
raised upto 100 ml.
3.16.6: Staining solution:
It was prepared by dissolving 0.5 g of Coomassie brilliant blue R-250 in 250 ml of
methanol and allowed to stand at room temperature for 20 min, filtered, added 50 ml of
acetic acid and raised the final volume upto 500 ml with distilled water.
50
3.16.7: Destaining solution:
It was prepared by mixing 150 ml methanol, 50 ml acetic acid and volume was raised
upto 500 ml with distilled water.
3.16.8: DNS reagent:
“It was prepared by dissolving 3, 5-dinitrosalicylic acid (10.6 g) and sodium hydroxide
(19.5 g) in approximately 600-800 ml of distilled water and gently heated in a water bath
at 80oC until a clear solution was obtained. Sodium potassium tartarate (306 g), phenol
melted at 60oC (7.5 ml) and sodium metabisulfate (8.3 g) were also added. After
dissolving the chemicals, final volume was raised upto 1416 ml with distilled water. The
solution was filtered through a large coarse sintered glass filter and stored at room
temperature in an amber colored bottle to avoid photo-oxidation. It was stable for about
six months”.
3.16.9: Bradford reagent:
Hundred milligram of Coomassie brilliant blue (G-250) was added in 50 ml of 95 %
ethanol. This solution was poured into 100 ml of 85 % (w/v) phosphoric acid and the
final volume was raised up to 1 L with distilled water. It was shaking well and filtered
through Whatman filter paper (No. 1) to obtain a clear solution. The reagent was stored
in an amber colored bottle to avoid photo-oxidation.
3.17: Preparation of buffers
3.17.1: Sodium acetate buffer (50 mM, pH 5.5):
Solution A: It was prepared by dissolving 27.22 g/l of sodium acetate
(CH3COONa.3H2O) to make 200 mM stock solution.
Solution B: It was prepared by diluting 12.05 ml of glacial acetic acid to 1 L to make
200 mM stock solution.
Solution A (45.95 ml) and solution B (4.05 ml) were mixed in 100 ml volumetric flask
and raised the final volume upto mark.
3.17.2: Phosphate buffer (pH 7):
It was prepared by dissolving 1.8 g of KH2PO4 and 3.5 g of Na2HPO2 in 100 ml of
distilled water and volume was raised upto 1 L. This was phosphate buffer of pH 7.
51
3.17.3: Preparation of 0.05 M Tris-HCl buffer (pH 7.5):
It was prepared by dissolving 6.25 g of Tris in 700-800 ml of distilled water and adjusted
pH 7.5 with 5 N HCl with constant stirring. Finally volume was raised to 1000 ml with
distilled water.
3.17.4: Separating buffer (1.5 M Tris HCl, pH 8.8):
It was prepared by dissolving 36.3 g Trizma in 150 ml of distilled water with constant
stirring to adjust the pH 8.8 by adding concentrated HCl (32 %) dropwise. After pH
adjustment, the final volume was raised upto 200 ml with distilled water.
3.17.5: Stacking buffer (1 M Tris HCl, pH 6.8):
It was prepared by dissolving 12.1 g Trizma in 70 ml of distilled water with constant
stirring to adjust the pH at 6.8 by adding concentrated HCl (32 %) dropwise. After pH
adjustment, the final volume was raised upto 100 ml with distilled water.
3.17.6: Tank buffer (10 X, pH 8.3):
It was prepared by dissolving 15 g of Trizma base, 72 g of glycine and 5 g of SDS in
distilled water and raising the final volume up to 1000 ml. The solution was stored at
4оC.
3.17.7: Gel loading buffer:
It was prepared by mixing 1 ml Tris HCl buffer (pH 6.8), 1 ml glycerol, 0.4 ml SDS (10
%), 15 µl β-mercaptoethanol and 0.02 g bromophenol blue dye. The final volume was
raised upto 10 ml with distilled water and stored at -20оC.
52
Fig 3.1: Standard curve of glucose
0
10
20
30
40
50
60
70
80
90
100
0 0.1 0.2 0.3 0.4 0.5 0.6
Glucose conc. (mg/ml)
Tra
nsm
itta
nce
(%)
53
Fig 3.2: Standard curve of bovine serum albumin
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 20 40 60 80 100 120
Bovine Serum Albumin (µg/100 µl)
Abs
orba
nce
(595
nm
)
54
Fig 3.3: Standard curve of Mannose
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 20 40 60 80 100 120 140 160 180 200
Mannose (µg/µl)
Abs
orba
nce
(490
nm
)
55
RESULTS
4.1: Isolation, identification and screening of yeast cultures
Eighty six strains of Saccharomyces cerevisiae were isolated from different samples of
fruits and soil by serial dilution method. They were identified according to conventional
yeast identification methods based on the morphology, sporulation and fermentation
characteristics, as well as the assimilation of a wide range of nitrogen and carbon
sources. The identification of S. cerevisiae was carried out (Wickerham 1951; Lodder
and Rij, 1952; Barnett et al., 1979). Colonies were flat, smooth, moist, glistening, and
creamy in color. They were appeared on YPS agar plates after 24 h, rapidly grew and
fully matured within 3 days. Microscopic studies showed that S. cerevisiae is one of the
budding yeast. Cells in the sediment of YPS broth were unicellular, globose, and
ellipsoid to elongate in shape measuring (4, 5-10 μ) × (7-11 μ) with a ratio between
length and width varing from 1-2. The bud arised on different parts of the cell surface on
a narrow base as said to be multilateral (multipolar) budding was typical. The spores
formed by S. cerevisiae were 1-4 spores per ascus. They were rounded and slightly oval
in shape. Temporary psedomycelium was also seen in old static cultures. In fermentation
tests with the S. cerevisiae gave positive results with glucose, sucrose, maltose,
galactose, fructose and raffinose but negative for lactose. It was able to use different sole
nitrogen sources but nitrate was not assimilated. Screening of S. cerevisiae isolates was
carried out in shake flask by submerged fermentation for invertase production. All the
isolates were screened out for their invertase synthesizing ability (Table 4.1). Of all the
strains examined, IS-66 isolated from dates gave maximum enzyme production and it
was selected as the wild strain for the subsequently studies i.e., optimization in shake
flask, mutagenesis, optimization of mutant in shake flask and fermentor, immobilization
studies and enzyme purification.
56
Table 4.1: Screening of isolates of S. cerevisiae for extracellular invertase production by shake flask technique*.
Isolates of S.
cerevisiae
Sources Enzyme activity
(U/ml)
DCM (g/l)
IS-1 Soil 0.2 ±0.03 10.2±1.2
IS-2 Soil 0.2±0.06 12.3±0.6
IS-3 Soil 0.15±0.01 10.63±1.2
IS-4 Soil 0.09±0.01 9.84±0.16
IS-5 Soil 0.1±0.04 11.65±0.56
IS-6 Soil 0.9±0.02 13.30±2.07
IS-7 Soil 0.08±0.01 9.05±0.73
IS-8 Soil 0.3±0.07 12.64±1.27
IS-9 Soil 0.23±0.1 12.37±0.91
IS-10 Soil 0.7±0.09 11.57±1.30
IS-11 Soil 0.1±0.02 10.48±1.29
IS-12 Soil 0.3±0.04 6.46±0.83
IS-13 Apple 0.5±0.1 8.23±0.90
IS-14 Apple 0.4±0.15 12.93±0.66
IS-15 Apple 0.8±0.15 5.62±1.23
IS-16 Apple 0.1±0.03 7.65±0.26
IS-17 Apple 0.5±0.1 10.44±0.18
IS-18 Apple 0.2±0.08 9.22±0.19
IS-19 Apple 0.6±0.1 10.59±1.20
IS-20 Apple 0.07±0.01 12.93±1.01
IS-21 Apple 0.1±0.1 13.54±0.47
IS-22 Apple 0.7±0.1 14.06±0.85
IS-23 Apple 0.3±0.1 12.25±0.55
IS-24 Plum 0.7±0.3 13.90±1.09
IS-25 Plum 0.09±0.01 12.42±0. 46
IS-26 Plum 0.06±0.02 7.16±0.37
IS-27 Plum 0.02±0.01 5.69±0.26
IS-28 Plum 0.4±0.1 10.25±0.22
IS-29 Plum 0.5±0.1 13.74±0.17
57
IS-30 Banana 0.3±0.06 11.69±0.08
IS-31 Banana 0.02±0.01 9.94±0.20
IS-32 Banana 0.2±0.04 10.9±0.1
IS-33 Banana 0.06±0.01 13.2±0.4
IS-34 Banana 0.02±0.01 14.3±0.2
IS-35 Banana 0.1±0.08 10.3±0.15
IS-36 Banana 0.01±0.03 5.1±0.3
IS-37 Banana 0.08±0.01 13.3±0.09
IS-38 Peach 0.03±0.01 11.5±1.03
IS-39 Peach 0.4±0.09 14.1±0.2
IS-40 Peach 0.7±0.02 7.85±0.12
IS-41 Peach 0.03±0.02 9.41±0.27
IS-42 Peach 0.03±0.01 12.21±0.10
IS-43 Peach 0.7±0.1 10.95±0.06
IS-44 Peach 0.06±0.02 8.40±0.12
IS-45 Peach 0.2±0.05 9.83±0.05
IS-46 Mango 0.4±0.04 4.87±0.08
IS-47 Mango 0.6±0.05 9.59±0.05
IS-48 Mango 0.8±0.1 11.87±0.06
IS-49 Mango 0.7±0.1 9.20±0.17
IS-50 Mango 0.1±0.03 10.20±0.18
IS-51 Mango 0.3±0.05 12.82±0.11
IS-52 Mango 0.05±0.01 4.26±0.07
IS-53 Mango 0.5±0.1 11.76±0.05
IS-54 Mango 0.3±0.07 13.28±1.2
IS-55 Mango 0.2±0.04 10.77±0.19
IS-56 Mango 0.01±0.01 13.87±0.35
IS-57 Mango 0.02±0.01 14.08±0.26
IS-58 Mango 0.04±0.02 14.32±0.13
IS-59 Mango 0.1±0.06 9.34±0.08
IS-60 Mango 0.02±0.01 9.30±0.70
IS-61 Mango 0.2±0.09 13.41±1.02
IS-62 Mango 0.5±0.11 12.98±0.05
58
IS-63 Dates 0.03±0. 5.62±0.03
IS-64 Dates 0.7±0.2 4.12±0.01
IS-65 Dates 0.1±0.02 13.33±0.05
IS-66 Dates 1.0±0.1 14.10±0.25
IS-67 Dates 0.4±0.15 13.92±0.13
IS-68 Dates 0.3±0.05 14.60±0.05
IS-69 Dates 0.6±0.1 13.9±0.09
IS-70 Dates 0.7±0.2 6.7±0.30
IS-71 Dates 0.06±0.02 5.69±0.22
IS-72 Dates 0.2±0.05 9.25±0.06
IS-73 Dates 0.5±0.15 10.74±0.17
IS-74 Dates 0.1±0.04 11.69±0.41
IS-75 Dates 0.06±0.01 9.94±0.20
IS-76 Dates 0.2±0.03 9.9±0.69
IS-77 Dates 0.4±0.1 4.2±0.09
IS-78 Guava 0.03±0.01 7.3±0.02
IS-79 Guava 0.09±0.03 10.3±0.13
IS-80 Guava 0.2±0.04 12.1±0.26
IS-81 Guava 0.03±0.01 13.10±0.1
IS-82 Guava 0.1±0.05 6.5±0.20
IS-83 Guava 0.05±0.01 12.3±0.5
IS-84 Guava 0.04±0.01 13.87±0.18
IS-85 Guava 0.2±0.05 14.08±0.04
IS-86 Guava 0.08±0.03 9.32±0.02
*Sucrose 30 g/l, incubation time 48 h, temperature 30C, initial pH 6, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05, indicates the standard deviation (sd) among the three parallel replicates (within each column).
59
Table 4.1.1: Sub-grouping of extracellular invertase producing isolates of S. cerevisiae
Number of isolates Range of enzyme activity (U/ml)
51 0-0.2
14 0.2-0.4
9 0.4-0.6
9 0.6-0.8
3 0.8-1
*IS-66 was selected as hyperproducer of invertase for physical mutation (UV-irradiations) studies.
60
4.1.1: Optimization of cultural conditions for selected yeast isolate
4.1.1.1: Rate of invertase production
The effect of incubation period on the extracellular invertase production by wild strain
of S. cerevisiae IS-66 in shake flask was optimized (Fig 4.1). The fermentation was
carried out for the time period of 72 h and enzyme production was calculated after
every 8 h. After 8 h of incubation, the minimum enzyme activity (0.04±0.01 U/ml) and
dry cell mass (1.6 g/l) was observed, respectively. The production of enzyme was
increased with the increase in the incubation period and reached maximum at 48 h after
inoculation with enzyme activity of 1.13±0.03 U/ml. At this incubation time (48 h) the
dry cell mass (13.1 g/l) was achieved. Above this time period, enzyme activity was
gradually declined upto 0.73±0.08 U/ml at 72 h of incubation possibly due to the
decrease in nutrient availability in the medium or carbon catabolite repression. The
enzyme production after 48 h was selected in subsequent studies.
4.1.1.2: Effect of sucrose concentrations
The effect of different sucrose concentrations (0-30 g/l) on enzyme production by wild
culture was evaluated (Fig 4.2). The maximum enzyme activity (5.6±0.6 U/ml) with dry
cell mass of 5.4±0.1 g/l was noticed at sucrose concentration of 5 g/l. This Increase in
invertase activity was noticed when sucrose concentration was decreased in the
fermentation medium from 30 to 5.0 g/l. Enzyme production increased about 5 fold at
5.0 g/l sucrose concentration. Above this concentration, a gradual decrease in enzyme
activity was observed. At the highest sucrose concentration (30 g/l), the minimum
enzyme activity (1.4±0.5 U/ml) was achieved. On the other hand, a gradual increase in
dry cell mass was observed. The maximum dry cell mass (13.5±0.5 g/l) was achieved at
the sucrose concentration of 30 g/l.
4.1.1.3: Effect of pH
The effect of pH (3.0-6.5) on the inverttase production by wild culture was optimized
(Fig 4.3). At low pH (3.0), the minimum enzyme activity (1.0±0.4 U/ml) and dry cell
mass (2.0±0.24 g/l) was found. Further gradual increase was observed for enzyme
activity and dry cell mass and reached maximum at pH (5.5) with 5.6±0.7 U/ml and 5.1
g/l, respectively. The higher pH (6.5) also caused decline both in enzyme activity
(4.5±0.13 U/ml) and dry cell mass (4.0±0.14 g/l).
61
Fig 4.1: Time course study for extracellular invertase production by S. cerevisiae IS-66 in shake flask*.
0
2
4
6
8
10
12
14
16
18
20
0 8 16 24 32 40 48 56 64 72 80
Incubation period (h)
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
*Sucrose 30 g/l, temperature 30C, initial pH 6, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
62
Fig 4.2: Effect of sucrose concentration on the extracellular invertase production by S. cerevisiae IS-66 in shake flask*.
0
2
4
6
8
10
12
14
16
18
20
0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25 27.5 30 32.5
Sucrose conc. (g/l)
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
*Incubation time 48 h, temperature 30C, initial pH 6, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
63
Fig 4.3: Effect of pH on the extracellular invertase production by S. cerevisiae IS-66 in shake flask*.
0
2
4
6
8
10
12
14
16
18
20
2.5 3 3.5 4 4.5 5 5.5 6 6.5 7pH
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
*Sucrose 5 g/l, incubation time 48 h, temperature 30C, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
64
4.2: Strain improvement
The selected yeast isolate was mutagenized by following mutagens
4.2.1: Physical mutation
4.2.2: Chemical mutation
4.2.1: Physical mutation
4.2.1.1: UV-irradiation
The wild strain S. cerevisiae IS-66 was exposed to UV irradiation for improvement in the
invertase production. UV irradiated forty seven isolates obtained were screened for
invertase production (Table 4.2), however, it was noted that UV treatment did not induce
any stable mutation that might lead to increased enzyme production, while most of the
UV-induced mutants produced less invertase as compared to the wild culture. Fig 4.4 is
depicted the data on survival curve of S. cerevisiae UV-44 developed after UV-
irradiation at different time intervals (10-70 min). After 60 min of UV exposure, the
death rate > 90 % was achieved. Because UV mutagenesis did not gave stable mutants
therefore wild strain (IS-66) was selected for chemical mutation using nitrous acid and
ethyl methane sulphonate (EMS).
4.2.2: Chemical mutation
4.2.2.1: Nitrous acid treatment
The wild strain S. cerevisiae IS-66 was further treated with different concentrations of
nitrous acid (0.05-0.2 M) for varying time intervals (2.0-10 min). Nitrous acid treated
forty seven isolates were screened for invertase production (Table 4.3). For nitrous acid
treated strain S. cerevisiae NA-45 in varying concentrations (0.05- 0.2 M) for different
time intervals (2-10 min). The survival curve of S. cerevisiae NA-45 showed death rate >
90 % after 10 min of exposure treatment with nitrous acid at 0.05 M (Fig 4.4). Out of
forty seven, mutant strain NA-45 gave maximum production of invertase (20.74±0.65
U/ml) with 4 fold increase as compared to wild strain. As the concentration of nitrous
acid increased, the number of the mutant isolates decreased as shown in the Table 4.3.
Nitrous acid at 0.05 M concentration gave 21 mutant isolates, the number of the mutant
isolates reduced to 16 at 0.1 M, still higher concentration i.e. 0.15 M again caused to
reduce the number of the mutant isolates to only 10 and at the finally used 0.2 M
65
concentration of nitrous acid a complete death of organism occurred. The varying
exposure time of interval for each nitrous acid concentration (0.05-0.15 M) also caused
the reduction for mutant isolates as it was increased from 2 to 10 min.
4.2.2.2: EMS treatment
The nitrous acid mutant S. cerevisiae NA-45 was subjected to ethyl methane sulphonate
(EMS) induced mutagenesis for varying time intervals (5-20 min) at various
concentrations (50-100 μl/ml). EMS treated forty four isolates were screened for enzyme
production as shown in Table 4.4. At the result of EMS treatment, survival curve of S.
cerevisiae EMS-42 was obtained showing the death rate > 90 % after 15 min at EMS
concentration of 100 μl/ml (Fig 4.6). Of all the screened strains, one mutant EMS-42
showed 6 folds higher enzyme production (34.2±0.29 U/ml) as compared to IS-66
(5.6±0.7 U/ml). One mutant EMS-42 was achieved after treating with EMS
concentration of 100 μl/ml at 15 min exposure time.
4.2.3: Mutant resistance to 2-deoxy-D-glucose
The mutant strain EMS-42 was cultured on the medium containing 2-deoxy-D-glucose
(2dg) and its stability in terms of invertase production was determined at various
concentrations. Initially, high yielding colonies were obtained at 2dg concentration of
0.02 mg/ml, however, these cultures lost stability after a couple of weeks. The
concentration of 0.04 mg/ml was found optimal, as at this level EMS-42 gave consistent
invertase production.
66
Table 4.2: Production of extracellular invertase by S. cerevisiae IS-66 treated with UV in shake flask*.
UV irradiated S.
cerevisiae strains
Exposure time
(min)
Enzyme activity
(U/ml)
DCM
(g/l)
IS-66 - 5.6±0.7 5.40±0.1
UV-1 10 0.08±0.02 4.58±0.08
UV-2 2.04±1.2 4.77±0.06
UV-3 3.4±0.02 4.63±0.22
UV-4 4.2±1.2 5.84±0.16
UV-5 1.05±0.02 4.65±0.56
UV-6 3.7±1.2 5.30±0.07
UV-7 4.3±1.2 6.05±0.03
UV-8 2.09±1.2 4.64±0.27
UV-9 0.5±0.02 5.37±0.01
UV-10 0.07±0.02 4.57±0.30
UV-11 0.6±0.02 6.48±0.29
UV-12 0.4±0.02 4.46±0.13
UV-13 0.8±0.02 4.23±0.30
UV-14 3.3±1.2 4.93±0.06
UV-15 0.5±0.02 5.62±0.23
UV-16 20 0.07±0.02 5.65±0.26
UV-17 5.1±1.0 5.44±0.18
UV-18 1.2±0.02 5.22±0.19
UV-19 0.3±0.02 4.59±0.20
UV-20 0.05±0.02 4.93±0.01
UV-21 0.08±0.02 4.54±0.47
UV-22 0.7±0.02 6.06±0.05
UV-23 1.25±0.02 6.25±0.10
UV-24 2.3±1.2 5.90±0.09
UV-25 2.4±1.0 5.42±0.09
UV-26 30 4.15±1.2 5.16±0.06
UV-27 2.8±1.2 5.69±0.26
67
UV-28 3.1±1.1 5.25±0.02
UV-29 2.3±1.5 5.74±0.07
UV-30 3.2±1.2 5.69±0.08
UV-31 4.15±1.2 5.94±0.20
UV -32 0.55±1.2 1.9±0.1
UV-33 3.9±1.2 4.2±0.1
UV-34 40 2.8±1.7 4.3±0.2
UV-35 3.85±1.2 4.3±0.1
UV-36 2.7±1.2 5.1±0.3
UV-37 1.9±2.6 1.3±0.1
UV-38 1.3±0.02 5.5±0.1
UV-39 3.25±1.2 2.1±0.2
UV-40 0.43±0.11 4.85±0.12
UV-41 50 1.9±0.3 4.41±0.27
UV-42 1.0±0.15 2.06±0.11
UV-43 0.05±0.01 4.95±0.06
UV-44 1.3±0.1 5.61±0.10
UV-45 5.04±0.13 4.83±0.05
UV-46 0.07±0.01 4.87±0.08
UV-47 60 0.8±0.02 3.59±0.15
UV-48 2.11±0.05 3.18±0.07
UV-49 3.07±0.24 2.20±0.01
UV-50 1.69±0.18 3.39±0.02
UV-51 4.56±0.87 4.0±0.05
UV-52 4.11±0.09 3.10±0.12
UV-53 1.0±0.2 2.1±0.01
UV-54 70 3.15±0.9 3.5±0.03
UV-55 1.16±0.3 1.0±0.22
*Sucrose 5 g/l, incubation period 48 h, temperature 30C, initial pH 5.5, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05, indicates the standard deviation (sd) among the three parallel replicates (within each column).
68
Table 4.2.1: Sub-grouping of extracellular invertase producing UV-treated strains of S.
cerevisiae
Number of UV-treated strains Range of enzyme activity (U/ml)
28 0-2
19 2-4
8 4-6
*Almost all the UV-treated strains showed relatively less invertase activity as compare to wild strain. Thus
IS-66 was treated for chemical mutation.
69
Fig 4.4: Survival curve of mutant strain of S. cerevisiae after UV irradiation.
0
10
20
30
40
50
60
70
80
90
100
110
0 10 20 30 40 50 60 70
UV exposure time (min)
Surv
ival
freq
uenc
y (%
)
*The mutants were picked up from the YPS agar plates having at least 90 % death rate. The duration of UV irradiation was varied from 15-105 min at 1.2102 J/m2/S.
70
Table 4.3: Production of extracellular invertase by S. cerevisiae IS-66 treated with nitrous acid in shake flask*.
Nitrous acid induced
mutant strains
Conc. (M) Exposure time
(min)
Enzyme activity
(U/ml)
DCM
(g/l)
IS-66 - - 5.6±0.7 5.40±0.1
NA-1 0.05 2 17.94±0.04 6.31±0.01
NA-2 11.37±0.31 6.17±0.01
NA-3 18.24±0.30 5.84±0.05
NA-4 13.20±0.37 6.14±0.05
NA-5 11.11±0.12 5.39±0.06
NA-6 12.0±0.09 6.25±0.06
NA-7 4 14.13±0.10 6.60±0.08
NA-8 1.87±0.08 6.57±0.05
NA-9 1.20±0.05 5.60±0.31
NA-10 6.09±0.17 5.56±0.30
NA-11 6 15.26±0.23 6.08±0.12
NA-12 18.74±0.01 6.11±0.10
NA-13 0.79±0.01 5.72±0.11
NA-14 5.42±0.15 5.55±0.32
NA-15 8 16.21±0.02 5.25±0.07
NA-16 13.73±0.04 5.66±0.29
NA-17 2.60±0.30 5.17±0.15
NA-18 9.32±0.14 5.14±0.02
NA-19 10 7.74±002 4.78±0.01
NA-20 9.83±0.01 4.97±0.17
NA-21 16.11±0.25 5.78±0.09
NA-22 0.1 2 0.27±0.04 6.20±0.02
NA-23 18.69±0.08 5.39±0.04
NA-24 14.56±0.28 5.88±0.09
NA-25 14.21±0.15 5.94±0.02
NA-26 1.23±0.12 5.32±0.01
NA-27 3.45±0.31 5.59±0.02
71
NA-28 4 1.76±0.29 5.30±0.02
NA-29 4.91±0.45 5.85±0.03
NA-30 12.26±0.23 6.48±0.29
NA-31 11.54±0.17 4.46±0.13
NA-32 6 16.63±0.34 4.23±0.30
NA-33 13.28±0.51 4.93±0.06
NA-34 15.30±0.07 5.62±0.23
NA-35 8 11.25±0.25 5.65±0.26
NA-36 15. 57±0.38 5.44±0.18
NA-37 10 16.43±0.17 5.22±0.19
NA-38 0.15 2 11.83±0.48 4.59±0.20
NA-39 18.25±0.49 4.93±0.01
NA-40 16.52±0.48 4.54±0.47
NA-41 4 11.34±0.29 6.06±0.05
NA-42 13.36±0.46 6.25±0.10
NA-43 6 15.64±0.34 5.90±0.09
NA-44 12.25±0.26 5.42±0.09
NA-45 8 20.74±0.65 6.06±0.04
NA-46 11.50±0.44 5.69±0.26
NA-47 10 1.76±0.29 5.30±0.02
* Fermentation time 48 h, sucrose 5 g/l, incubation temperature 30C, initial pH 5.5, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05, indicates the standard deviation (sd) among the three parallel replicates (within each column).
A complete death (≈100 %) of the organism was noticed at 0.20 M nitrous acid.
72
Table 4.3.1: Sub-grouping of extracellular invertase producing NA-treated strains of S.
cerevisiae
Number of NA-treated strains Range of enzyme activity (U/ml)
10 0-5
5 5-10
17 10-15
15 15-21
*The mutant strain NA-45 as hyperproducer of invertase was selected for chemical mutation by EMS.
73
Fig 4.5: Survival curve of mutant strain of S. cerevisiae NA-45 developed after nitrous acid treatment.
0
10
20
30
40
50
60
70
80
90
100
110
0 2 4 6 8 10
NA exposure time (min)
Surv
ival
freq
uenc
y (%
)
NA (0.05 M) NA (0.1 M) NA (0.15 M)
*The mutants were picked up from the YPS agar plates having at least 90 % death rate.
74
Table 4.4: Production of extracellular invertase by S. cerevisiae NA-45 treated with EMS in shake flask*.
EMS treated S.
cerevisiae strains
EMS conc.
(μl/ml)
Exposure time
(min)
Enzyme activity
(U/ml)
DCM (g/l)
NA-45 - - 20.74±0.65 6.06±0.04
EMS -1 50 5 4.0±0.3 4.58±0.08
EMS -2 5.2±0.2 4.77±0.06
EMS -3 0.31±0.1 4.63±0.22
EMS -4 0.8±0.15 5.84±0.16
EMS -5 3.25±0.10 4.65±0.56
EMS -6 5.55±0.02 5.30±0.07
EMS -7 17.23±0.25 6.05±0.03
EMS -8 10 18.15±0.16 4.64±0.27
EMS -9 12.60±0.27 5.37±0.01
EMS -10 17.60±0.32 4.57±0.30
EMS -11 20.26±0.23 6.48±0.29
EMS -12 11.54±0.17 4.46±0.13
EMS -13 24.63±0.34 4.23±0.30
EMS -14 15 13.28±0.51 4.93±0.06
EMS -15 15.30±0.07 5.62±0.23
EMS -16 11.25±0.25 5.65±0.26
EMS -17 20. 57±0.38 5.44±0.18
EMS -18 20 21.43±0.17 5.22±0.19
EMS -19 11.83±0.48 4.59±0.20
EMS -20 18.25±0.49 4.93±0.01
EMS -21 75 5 16.52±0.48 4.54±0.47
EMS -22 3.0±0.12 2.2±0.2
EMS -23 13.36±0.46 6.25±0.10
EMS -24 20.64±0.34 5.90±0.09
EMS -25 12.25±0.26 5.42±0.09
EMS -26 20.14±0.15 5.16±0.06
EMS -27 10 2.76±0.19 5.30±0.20
75
EMS -28 4.91±0.45 5.85±0.13
EMS -29 20.26±0.23 6.48±0.32
EMS -30 11.0±0.68 4.0±0.4
EMS -31 15 21.1±0.24 4.23±0.30
EMS -32 3.28±0.60 4.93±0.57
EMS -33 15.30±0.87 4.62±0.20
EMS -34 20 11.20±0.90 5.25±0.16
EMS -35 20. 57±0.38 5.0±0.28
EMS -36 100 5 11.43±0.75 5.20±0.19
EMS -37 11.80±0.50 5.59±0.30
EMS -38 8.25±0.49 5.63±0.1
EMS -39 6.52±0.38 5.04±0.27
EMS -40 10 11.24±0.29 6.26±0.08
EMS -41 7.2±0.9 5.77±0.02
EMS -42 15 34.2±0.29 6.27±0.15
EMS -43 14.0±0.85 4.58±0.08
EMS -44 20 9.0±0.6 3.90±0.10
* Fermentation time 48 h, sucrose 5 g/l, incubation temperature 30C, initial pH 5.5, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05, indicates the standard deviation (sd) among the three parallel replicates (within each column).
76
Table 4.4.1: Sub-grouping of extracellular invertase producing EMS-treated strains of S. cerevisiae
Number of EMS-treated strains Range of enzyme activity (U/ml)
14 0-10
20 10-20
9 20-30
1 30-40
*The mutant strain EMS-42 as hyperproducer of invertase was selected for subsequent studies.
77
Fig 4.6: Survival curve of mutant strain of S. cerevisiae EMS-42 developed after EMS treatment.
0
10
20
30
40
50
60
70
80
90
100
110
0 5 10 15 20
EMS exposure time (min)
Surv
ival
freq
uenc
y (%
)
EMS (50μl/ml) EMS (75μl/ml) EMS (100μl/ml)
*The mutants were picked up from the YPS agar plates having at least 90 % death rate.
78
4.3: Enzyme production
4.3.1: Extracellular invertase
4.3.2: Intracellular invertase
4.3.1: Extracellular invertase
4.3.1.1: Shake flask
4.3.1.1.1: Rate of invertase production
Fig 4.7 & 4.8 shows the effect of rate on the enzyme production by the wild (IS-66) and
mutants (NA-45 and EMS-42) strains of S. cerevisiae in shake flasks. The invertase
production was estimated at 8 h intervals (8-72 h). Maximum enzyme production was
noticed after 48 h of incubation in all the five tested strains. IS-66 showed maximum
enzyme activity (5.1±0.07 U/ml) and dry cell mass (4.08±0.23 g/l) when incubation time
reached at 48 h. Two developed mutants (NA-45 and EMS-42) also showed maximum
enzyme activity as 20±0.89 and 34.3±0.62 U/ml, respectively. In comparison with (IS-
66), the mutants NA-45 and EMS-42 caused increase in enzyme activity by 4 and 6 fold.
No further increase in the invertase activity and dry cell mass was obtained after 48 h of
incubation. Of all the strains EMS-42 was selected for subsequent studies. Table 4.5
depicts the above data for kinetic analysis by calculations of Qp (enzyme produced/l/h),
Qx (g cell mass formation/l/h), Yp/x (enzyme produced/g cell mass formation, qp (Enzyme
produced/g cell/h). The value of specific growth rate of wild (IS-66) and mutant EMS-42
was found to be 0.01and 0.025 h-1, repectively (Fig 4.9).
4.3.1.1.2: Selection of culture media
Six media (M1-M6) were evaluated for the production of extracellular invertase (Fig
4.10). M1 medium having 30 g/l (w/v) sucrose concentration gave maximum production
of invertase (25.28±1.72 U/ml) and dry cell mass (15.4±0.71 g/l). Of all the six media, in
two media M1 and M2 cane molasses was used as carbon source gave enzyme activity
and dry cell mass as 16.9±0.9 and 18.1±0.5 U/ml, 14±0.39 and 14.2±0.12 g/l,
respectively. The second best medium was M4 slightly better than M3. In this medium
two carbon sources i.e., sucrose and glucose were used. It showed the enzyme activity
(19.3±1.18 U/ml) and dry cell mass (7.7±0.29 g/l). The maximum dry cell mass
(21.6±0.22 g/l) was found to be observed in M5 having 50 g/l sucrose as carbon
source. Medium M6 showed minimum enzyme activity (12.8±0.67 U/ml) with dry cell
79
mass of 12.36±0.39 g/l. However, this medium was found to be as least effective to
support invertase activity. Therefore, M1 was selected for subsequent studies.
4.3.1.1.3: Effect of different sugars
Effect of different sugars such as sucrose, glucose, fructose, lactose, galactose, maltose,
raffinose and molasses on the extracellular enzyme production by S. cerevisiae EMS-42
in shake flasks was under taken (Fig 4.11). Sugar level was kept constant at 2.0 % (w/v).
Optimal invertase activity (37±1.17 U/ml) was obtained when sucrose was added into the
fermentation medium with dry cell mass of 7.1±0.93 g/l. Other than sucrose, remaining
sugars gave less significant results in terms of enzyme activity. In case of glucose and
fructose the enzyme activity and dry cell mass were 12.9±0.34 and 11.2±1.06 U/ml,
8.0±0.1 and 7.86±0.6 g/l, respectively. After sucrose, molasses showed enzyme
activity (19.73±1.5 U/ml) and dry cell mass (6.8±0.19). When raffinose was used as
sole carbon source it gave enzyme activity of 17.42±0.91 U/ml and dry cell mass of
5.3±0.3 g/l. However, the least invertase activity (3.19±0.46 U/ml) was obtained with
lactose when it was added as a sole carbon source. So sucrose was optimized for further
study.
80
Fig 4.7: Comparison of rate on the extracellular invertase production by wild and mutant strains of S. cerevisiae in shake flask*.
0
5
10
15
20
25
30
35
40
45
50
0 8 16 24 32 40 48 56 64 72 80
Incubation period (h)
Enz
yme
acti
vity
(U/m
l)
IS-66 NA-45 EMS-42
* Sucrose 5 g/l, incubation temperature 30C, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
81
Fig 4.8: Comparison of dry cell mass by wild and mutant strains of S. cerevisiae in shake flask*.
0
1
2
3
4
5
6
7
0 8 16 24 32 40 48 56 64 72 80
Incubation period (h)
DC
M (g
/l)
IS-66 NA-45 EMS-42
* Sucrose 5 g/l, incubation temperature 30C, agitation intensity 200 pm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
.
82
Table 4.5: Kinetic parameters* of rate of fermentation for the extracellular invertase production by wild and mutant strains of S. cerevisiae in shake flask.
Kinetic parameters
IS-66
EMS-42
Yp/x (U/g)
Qp (U/g/h)
Qx (g/l/h)
qp (U/g/h)
1.25
0.026
0.085
0.013
5.91
0.123
0.120
0.148
* Yp/x= Enzyme produced/g cell mass formation, Qp= Enzyme produced/l/h. Qx= g cell mass formation/l/h, qp= Enzyme produced/g/h.
83
Fig 4.9: Comparison of specific growth rate (μ h-1) of wild and mutant strain of S. cerevisiae for extracellular invertase production.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 8 16 24 32 40 48 56 64 72 80
Incubation time (h)
ln (X
)
IS-66 EMS-42
*X= Dry weight of cell mass
84
Table 4.6: Rate of biomass formation of wild and mutant strains of S. cerevisiae for extracellular invertase production in shake flask.
Time (h) DCM (X)
(IS-66)
ln (X) DCM (X)
(EMS-42)
ln (X)
8 1.2 0.18 2.4 0.87
16 2.3 0.83 3.4 1.22
24 3.15 1.15 4.06 1.4
32 3.66 1.3 4.3 1.46
40 3.82 1.34 4.8 1.57
48 4.08 1.41 5.8 1.76
56 4.0 1.39 5.76 1.75
64 3.97 1.38 5.71 1.74
72 3.92 1.36 5.32 1.67
85
Fig 4.10: Selection of culture media for the extracellular invertase production and dry cell mass (DCM) by S. cerevisiae EMS-42 in shake flask*.
0
5
10
15
20
25
30
35
40
45
50
M1 M2 M3 M4 M5 M6
Media
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
* Fermentation time 48 h, incubation temperature 30C, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
86
Fig 4.11: Effect of different sugars on the extracellular invertase production and DCM by S. cerevisiae EMS-42 in shake flask*.
0
5
10
15
20
25
30
35
40
45
50
Contro
l
Sucrose
Gluco
se
Fructo
se
Lacto
se
Galacto
se
Malt
ose
Raffino
se
Mola
sses
Sugars
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
* Fermentation time 48 h, sugar concentration 20 g/l, incubation temperature 30C, initial pH 5.5, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
87
4.3.1.1.4: Effect of sucrose concentrations
Fig 4.12 shows the effect of sucrose concentrations on the extracellular enzyme
production by S. cerevisiae EMS-42. Different concentrations of sucrose (2-20 g/l) were
used in the media. Maximum enzyme activity (44.03±0.30 U/ml) was obtained when
sucrose concentration in the fermentation medium was kept at 10 g/l. At this sucrose
concentration, dry cell mass (6.5±0.35 g/l) was observed. Further increase in sucrose
concentration caused a gradual decrease in enzyme activity. On the contrary, a gradual
increase in dry cell mass was observed by using the sucrose concentration ranged from 2
to 20 g/l. At maximum sucrose concentration (20 g/l) a less enzyme activity (36.0±0.48
U/ml) but maximum dry cell mass (7.9±0.42 g/l) was obtained.
4.3.1.1.5: Effect of incubation temperature
Figure 4.13 shows the effect of varying incubation temperature (20-60°C) on the
enzyme production by mutant strain. At low temperature (20°C), the enzyme activity
and dry cell mass was found to be as 10.5±1.2 U/ml and 4.1±0.2 g/l, respectively. The
optimal enzyme activity (44±1.5 U/ml) and dry cell mass (6.7±0.17 g/l) was obtained at
30°C. When incubation temperature was increased from 30°C, the enzyme activity was
decreased. At 60°C, the minimum enzyme activity (4±0.6 U/ml) and dry cell mass
(1.7±0.9 g/l) was achieved.
4.3.1.1.6: Effect of inoculum size
The effect of different sizes of vegetative inoculum (1-4 %) after 24 h of inoculation on
the production of extracellular enzyme by S. cerevisiae EMS-42 was studied (Fig 4.14).
Enzyme activity was ranged from 38.46±1.2 to 47.33±0.61 U/ml. Maximum enzyme
production (47.33±0.61 U/ml) was obtained when 2 % inoculum was used to inoculate
50 ml of the fermentation medium. At this inoculum size, the dry cell mass was
6.66±0.22 g/l. Beyond 2 % inoculum, the enzyme activity was found to be 44.27±0.8
U/ml.
4.3.1.1.7: Effect of volume of the media
Fig 4.15 is summarized the results on the effect of different volume (25-100 ml) of
fermentation media in shake flasks of 250 ml flask on the extracellular enzyme
production by mutant strain. In 25 ml fermentation medium, the minimum enzyme
activity (28.16±0.8 U/ml) and dry cell mass (4.76±0.1 g/l) was observed. The maximum
enzyme activity (46.87±0.71 U/ml) with dry cell mass (6.29±0.07 g/l) was obtained
88
when volume of fermentation medium was kept at 50 ml. With increase in volume of
fermentation media the enzyme activity (30.99±0.90 U/ml) was decreased. However,
with increase in fermentation volume, the dry cell mass was observed to be increased.
Thus maximum dry cell mass (7.80±0.08 g/l) was obtained when 100 ml fermentation
medium was used. However, 50 ml fermentation medium was selected for subsequently
studies.
4.3.1.1.8: Effect of initial pH
The effect of initial pH (3-7) on the enzyme production by mutant strain in shake flasks
was studied (Fig 4.16). Maximum enzyme production (48.86±0.80 U/ml) was obtained
when initial pH was 5. At this pH, the dry cell mass was 6.33±0.46 g/l. At low pH of 3,
the enzyme activity and dry cell mass were also observed to be as minimum 21.30±1.30
U/ml and 4.53±0.12 g/l, respectively. Beyond pH 5, a gradual reduction in enzyme
activity (42.09±1.04 to 23.09±1.02 U/ml) was obtained.
89
Fig 4.12: Effect of different sucrose concentrations on the extracellular invertase production and DCM by S. cerevisiae EMS-42 in shake flask*.
0
5
10
15
20
25
30
35
40
45
50
0 2 4 6 8 10 12 14 16 18 20 22
Sucrose conc.(g/l)
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
*Fermentation time 48 h, incubation temperature 30C, initial pH 5.5, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
90
Fig 4.13: Effect of different incubation temperatures on the extracellular invertase production and DCM by S. cerevisiae EMS-42 in shake flask*.
0
5
10
15
20
25
30
35
40
45
50
10 20 30 40 50 60 70
Temperature (ºC)
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
* Sucrose 5 g/l, incubation time 48 h, agitation intensity 200 pm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
91
Fig 4.14: Effect of different inoculum sizes on the extracellular invertase production and DCM by S. cerevisiae EMS-42 in shake flask*.
0
5
10
15
20
25
30
35
40
45
50
0 1 2 3 4 5
Inoculum size (%, v/v)
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
* Fermentation time 48 h, sucrose 10 g/l, incubation temperature 30C, initial pH 5.5, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
92
Fig 4.15: Effect of different volumes of the media on the extracellular invertase production and DCM by S. cerevisiae EMS-42 in shake flask*.
0
5
10
15
20
25
30
35
40
45
50
0 25 50 75 100 125
Volume of medium (ml) / 250 ml flask
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
* Fermentation time 48 h, sucrose 10 g/l, incubation temperature 30C, initial pH 5.5, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
93
Fig 4.16: Effect of different initial pH on the extracellular invertase production and DCM by S. cerevisiae EMS-42 in shake flask*.
0
5
10
15
20
25
30
35
40
45
50
2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5pH
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
* Fermentation time 48 h, sucrose 10 g/l, incubation temperature 30C, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
94
4.3.1.1.9: Effect of additional organic nitrogen sources
The effect of different additional organic nitrogen sources such as urea, peptone, meat
extract and casein in various amounts (2-8 g/l) on extracellular enzyme production by S.
cerevisiae EMS-42 were evaluated (Table 4.7). Of all the nitrogen sources tested,
peptone at the level of 6 g/l gave maximum production of invertase (50±1.11 U/ml) with
dry cell mass of 6.72±0.01 g/l. The urea at the concentration of 3 g/l proved as second
best producer of invertase (26.01±0.91 U/ml). The least enzyme activity (10.27±0.77
U/ml) was observed when 5 g/l casein was used as an organic nitrogen source. A
comparison of the all organic nitrogen sources used at the level of 6 g/l in terms of
enzyme activity and dry cell mass as shown in Fig 4.17. In addition to yeast extract
(3g/l), the peptone (6 g/l) as additional nitrogen source was optimized and used for
subsequently studies.
4.3.1.1.10: Effect of additional inorganic nitrogen sources
Table 4.8 shows the effect of different additional inorganic nitrogen sources such as
(NH4)2HPO4, NH4NO3, NH4CI, (NH4)2SO4 and KNO3 in varying concentrations (2-10
g/l) on the extracellular enzyme production by mutant strain in shake flasks. Of all the
nitrogen sources tested, (NH4)2HPO4 at the amount of 6 g/l gave maximum production of
enzyme (40.51±0.86 U/ml). The dry cell mass was achieved as 6.05±0.13 g/l,
respectively. The second best enzyme activity (36.42±1.6 U/ml) was obtained in the
fermentation medium when (NH4)2SO4 was used at a concentration of 6 g/l. In
comparison with the control, all the tested inorganic nitrogen sources showed greater
enzyme activity except KNO3. The comparison of all inorganic nitrogen sources used at
the concentration of 6 g/l is depicted in Fig 4.18. Addition of KNO3 was found to be
least effective for enzyme production among all the inorganic nitrogen sources.
4.3.1.1.11: Effect of additional agricultural byproducts nitrogen sources
In Table 4.9 is depicted the data on the effect of different agricultural byproducts such as
soybean meal, sunflower meal and corn steep liquor in varying concentration (1-10 g/l)
on the extracellular enzyme production by mutant strain in shake flasks. The best
producer of enzyme was found to be corn steep liquor (21.56±0.55 U/ml) at a
concentration of 6 g/l. At this concentration, the dry cell mass (6.37±0.21 g/l) was
observed. In case of soybean meal, the enzyme activity (18.30±0.91 U/ml) was obtained
at the concentration of 6 g/l. In comparison with the control, all the tested inorganic
95
nitrogen sources showed greater enzyme activity. Fig 4.19 depicts the comparison of
all agricultural by products as nitrogen sources used at the concentration of 6 g/l. Of
these, sunflower meal was found to be the least effective in enzyme production.
96
Table 4.7: Effect of different additional organic nitrogen sources on the extracellular invertase production by S. cerevisiae EMS-42 in shake flask*.
Organic nitrogen
source
Concentrations
(g/l)
Enzyme activity
(U/ml)
DCM (g/l)
Control - 12.06±0.56 4.10±0.09
Peptone
2 33.01±1.03 5.71±0.15
3 38.03±1.40 6.20±0.11
4 43.27±2.47 6.35±0.2
5 48.19±1.00 6.36±0.23
6 50.0±1.11 6.72±0.09
7 47.26±1.84 6.38±0.1
8 43.0±1.41 6.27±0.13
Urea
2 18.77±0.68 5.14±0.06
3 26.01±0.91 6.13±0.26
4 14.83±0.76 6.0±0.11
5 12.92±0.71 5.77±0.20
6 8.32±0.79 5.44±0.12
7 6.27±0.46 5.31±0.16
8 5.28±0.41 5.27±0.06
Meat extract
2 14.17±0.38 5.19±0.03
3 16.08±0.24 5.55±0.17
4 18.49±0.49 5.63±0.14
5 17.07±0.10 5.47±0.10
6 15.51±0.45 5.41±0.11
7 14.52±0.46 5.36±0.24
8 12.56±0.50 5.31±0.14
Casein
2 8.09±0.64 4.13±0.13
3 12.69±1.96 5.35±0.16
4 9.92±0.98 5.35±0.12
5 10.27±0.77 5.20±0.07
6 7.93±0.60 5.41±0.08
7 6.54±0.76 4.72±0.05
8 5.96±0.98 4.32±0.06
* Fermentation time 48 h, sucrose 10 g/l, incubation temperature 30C, initial pH 5, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05, indicates the standard deviation (sd) among the three parallel replicates.
97
Fig 4.17: Effect of different additional organic nitrogen sources on extracellular invertase production and DCM by S. cerevisiae EMS-42 in shake flask*.
0
5
10
15
20
25
30
35
40
45
50
Control Peptone Urea Meat extract Casein
Organic N-source (6 g/l)
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
* Fermentation time 48 h, sucrose 10 g/l, incubation temperature 30C, initial pH 5, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
98
Table 4.8: Effect of different additional inorganic nitrogen sources on extracellular
invertase production by S. cerevisiae EMS-42 in shake flask*.
Inorganic nitrogen source
Concentrations (g/l)
Enzyme activity
(U/ml)
DCM
(g/l)
Control - 12.06±0.56 4.10±0.09
(NH4)2HPO4
2 32.79±0.80 5.83±0.15
4 36.80±0.55 5.18±0.03
6 40.51±0.86 6.05±0.13
8 37.0±1.07 6.12±0.12
10 31.85±0.79 6.43 ±0.24
NH4NO3
2 16.30±0.88 4.21±0.10
4 18.30±1.57 4.43±0.19
6 20.53±1.10 4.53±0.05
8 16.23±1.20 4.76±0.1
10 15.20±1.11 4.66±0.04
NH4CI
2 10.78±0.70 4.62±0.13
4 16.73±0.64 4.88±0.04
6 15.88±1.40 5.20±0.14
8 18.21±1.30 5.31±0.04
10 14.10±1.29 5.33±0.17
(NH4)2SO4
2 3.95±0.13 5.19±0.08
4 22.10±2.0 5.23±0.20
6 36.42±1.6 5.73±0.15
8 34.43±1.03 5.70±0.12
10 32.10±1.23 5.23±0.06
KNO3
2 1.78±0.50 1.62±0.23
4 3.0±0.64 1.80±0.14
6 8.10±0.40 2.20±0.08
8 3.40±1.00 2.31±0.15
10 3.60±0.29 2.33±0.17
* Fermentation time 48 h, sucrose 10 g/l, incubation temperature 30C, initial pH 5, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05, indicates the standard deviation (sd) among the three parallel replicates.
99
Fig 4.18: Effect of different inorganic nitrogen sources on production of invertase and DCM by S. cerevisiae EMS-42 in shake flask*.
0
5
10
15
20
25
30
35
40
45
50
Control DAHP** Ammoniumnitrate
Ammoniumchloride
Ammoniumsulfate
Potassiumnitrate
Inorganic N-source (6 g/l)
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
* Fermentation time 48 h, sucrose 10 g/l, incubation temperature 30C, initial pH 5, agitation intensity 200 rpm.
** Diammoniumhydrogen phosphate
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
100
Table 4.9: Effect of different agricultural byproducts and its concentration on the biosynthesis of invertase by mutant strain of S. cerevisiae EMS-42 in shake flask*.
Agricultural byproducts
Concentrations (%)
Enzyme activity
(U/ml)
DCM
(g/l)
Control 12.06±0.56 4.10±0.09
Soybean meal
0.1 10.59±0.65 2.53±0.17
0.2 12.73±1.64 3.23±0.05
0.3 13.21±2.3 5.19±0.10
0.4 16.56±0.60 5.12±0.19
0.5 17.45±0.60 5.25±0.23
0.6 18.30±0.91 5.35±0.13
0.7 8.44±1.41 3.57±0.08
0.8 11.29±0.90 2.59±0.15
Sunflower meal
0.1 12.30±0.66 2.16±0.15
0.2 13.51±0.48 2.28±0.14
0.3 10.59±1.08 2.82±0.11
0.4 9.56±0.68 2.82±0.08
0.5 6.62±0.74 2.22±0.19
0.6 7.62±1.40 2.47±0.26
0.7 7.65±0.73 2.52±0.20
0.8 9.04±0.63 2.56±0.23
Corn steep liquor
0.1 7.52±0.50 3.11±0.15
0.2 12.36±1.14 3.24±0.02
0.3 15.07±0.74 3.96±0.05
0.4 17.25±0.34 4.95±0.12
0.5 18.72±0.64 5.78±0.14
0.6 21.56±0.55 6.37±0.21
0.7 18.76±0.60 6.35±0.02
0.8 15.48±0.63 6.24±1.0
* Fermentation time 48 h, sucrose 10 g/l, incubation temperature 30C, initial pH 5, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05, indicates the standard deviation (sd) among the three parallel replicates.
101
Fig 4.19: Effect of different agricultural byproducts nitrogen sources on invertase production and DCM by S. cerevisiae EMS-42 in shake flask*.
0
5
10
15
20
25
30
35
40
45
50
Control Soybean meal Sunflower meal cornsteep liquor
Agricultural N-source (6 g/l)
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
* Fermentation time 48 h, sucrose 10 g/l, incubation temperature 30C, initial pH 5, agitation intensity 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
102
4.3.1.2: Fermentor studies
4.3.1.2.1: Rate of invertase production
The rate was studied on the enzyme production by S. cerevisiae EMS-42 in a stirred
fermentor with comparison of shake flask as shown in Fig 4.20 & 4.21. The enzyme
activity and dry cell mass was estimated at 8 h intervals (8-56 h). In shake flask, the
enzyme activity in the fermented broth increased gradually with increase in incubation
period from 8 to 48 h. At 48 h incubation period, the maximum enzyme activity (47.6
0.9 U/ml) was observed in shake flask. A marked increase in enzyme activity (65.12
U/ml) after 24 h of incubation was recorded when it was scaled up in stirred fermentor as
shown in Fig 4.20. In addition to maximal enzyme activity (65.12 U/ml) achieved, the
reduction in fermentation incubation period by 24 h in stirred fermentor was also
recorded. The difference between shake flask and stirred fermentor regarding enzyme
activity was about 17 U. Fig 4.21 depicts the data of comparison of dry cell mass in both
shake flask and fermentor. However, the dry cell mass in shake flask and fermentor was
found to be observed as 6.19 and 6.4 g/l, respectively.
Data obtained from above experiment were subjected to kinetic analysis for
calculations of qp (unit product produced/g cell/h), Qp (enzyme produced/l/h), Qx (g cell
mass formation/l/h), Yp/x(enzyme produced/g cell mass formation) as shown in Table
4.10. From the kinetic findings, it was also shown that optimum fermentation period for
enzyme production was 48 h in shake flask and 24 h in stirred fermentor was highly
significant. The specific growth rate (µ h-1) was observed to be high significantly in
fermentor (0.04 h-1) as compared to shake flask (0.02 h-1) as shown in Fig 4.22.
4.3.1.2.2: Effect of sucrose concentrations
In Fig 4.23 is depicted the data on the effect of different sucrose concentrations (10-25
g/l) on invertase production by mutant strain in stirred fermentor. At 10 g/l of sucrose
concentration, the enzyme activity (65 U/ml) with least dry cell mass (6.85 g/l) was
found to be observed. Further increase in sucrose concentration (15 g/l) caused the
increase in enzyme activity (69 U/ml). Beyond 15 g/l, a gradual decline in enzyme
activity was found. The minimum enzyme activity (50.2 U/ml) was observed when
sucrose concentration was kept at 25 g/l. On the other hand, a gradual increase in dry cell
mass was recorded when sucrose concentration was maintained from 10 to 25 g/l.
However, the maximum dry cell mass (9.3 g/l) was obtained at highest sucrose
103
concentration (25 g/l) but with least enzyme activity i.e., 50.2 U/ml. The sucrose
concentration of 15 g/l was optimized for enzyme production and subsequent parameters.
4.3.1.2.3: Effect of inoculum size
The effect of different sizes of inoculum (5-12.5 %, v/v) on the enzyme production by
mutant strain in stirred fermentor was studied (Fig 4.24). At 5 % inoculum size, the
enzyme activity was found less (69 U/ml) with dry cell mass of 7.7 g/l. The maximum
enzyme activity (71.2 U/ml) was obtained when the size of inoculum was kept at 7.5 %.
The dry cell mass (7.9 g/l) at inoculum size of 7.5 % was observed. Further increase of
inoculum size such as 10 and 12.5 % caused a gradual decline in both enzyme activity
(66 and 59 U/ml) and dry cell mass (6.9 and 6 g/l), respectively. An inoculum size of 7.5
% was optimized for enzyme production and subsequent parameters.
104
Fig 4.20: Comparison of rate on the extracellular invertase production by S. cerevisiae EMS-42 in shake flask* and stirred fermentor**.
0
10
20
30
40
50
60
70
80
90
0 8 16 24 32 40 48 56 64
Incubation period (h)
Enz
yme
acti
vity
(U/m
l)
Shake flask Fermentor
* Sucrose 10 g/l, incubation temperature 30C, agitation intensity 200 rpm, pH 5.
** Sucrose 10 g/l, incubation temperature 30C, agitation intensity 200 rpm, aeration 1 vvm, pH 5.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
105
Fig 4.21: Comparison of rate on the production of dry cell mass of S. cerevisiae EMS-42 in shake flask* and stirred fermentor**.
0
1
2
3
4
5
6
7
8
0 8 16 24 32 40 48 56 64
Incubation period (h)
DC
M (g
/l)
Shake flask Fermentor
* Sucrose 10 g/l, incubation temperature 30C, agitation intensity 200 rpm, pH 5.
** Sucrose 10 g/l, incubation temperature 30C, agitation intensity 200 rpm, aeration 1 vvm, pH 5.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
106
Table 4.10: Kinetic parameters* of rate of fermentation for the extracellular invertase production by mutant strains of S. cerevisiae EMS-42 in shake flask* and fermentor*.
Kinetic parameters Shake flask
Fermentor
Yp/x (U/g)
Qp (U/g/h)
Qx (g/l/h)
qp (U/g/h)
8.06
0.17
0.13
0.08
11.6
0.48
0.29
0.44
* Yp/x= Enzyme produced/g cell mass formation, Qp= Enzyme produced/l/h. Qx= g cell mass formation/l/h, qp= Enzyme produced/g/h.
107
Fig 4.22: Comparison of specific growth rate (μ h-1) of mutant strain of S. cerevisiae EMS-42 in shake flask and fermentor for extracellular invertase production.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 8 16 24 32 40 48 56 64
Incubation time (h)
ln (X
)
Shake flask Fermentor
* X = Dry weight of cell mass
108
Table 4.11: Rate of biomass formation of mutant strains of S. cerevisiae EMS-42 for extracellular invertase production in shake flask and fermentor.
Time (h) DCM (X)
(Shake flask)
ln (X) DCM (X)
(Fermentor)
ln (X)
8 3 1.09 4.1 1.41
16 4.1 1.41 5.5 1.7
24 5.0 1.6 6.9 1.93
32 5.3 1.67 6.7 1.89
40 5.6 1.72 6.5 1.87
48 6.2 1.82 6.4 1.86
56 6.1 1.8 6.3 1.84
109
Fig 4.23: Effect of different sucrose concentrations on the extracellular invertase production by S. cerevisiae EMS-42 in the stirred fermentor*.
0
10
20
30
40
50
60
70
80
90
5 10 15 20 25 30
Sucrose conc. (g/l)
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
*Fermentation time 24 h, incubation temperature 30C, initial pH 5, agitation intensity 200 rpm, aeration 1 vvm.
110
Fig 4.24: Effect of different inoculum size on the extracellular invertase production by S. cerevisiae EMS-42 in the stirred fermentor*.
0
10
20
30
40
50
60
70
80
90
2.5 5 7.5 10 12.5 15
Inoculum size (%, v/v)
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
*Fermentation time 24 h, sucrose 15 g/l, incubation temperature 30C, initial pH 5, agitation intensity 200 rpm, aeration 1 vvm.
111
4.3.1.2.4: Effect of pH
The data summarized in the Fig 4.25 shows the effect of different initial pH (3.5-6.5) on
the enzyme production by S. cerevisiae EMS-42 in stirred fermentor. The minimum
enzyme activity (49.6 U/ml) and dry cell mass (4.22 g/l) were found at pH 3.5. At pH
4.5, the maximum enzyme activity (73.41 U/ml) with dry cell mass (7.81 g/l) was
achieved. Further increase in pH caused the reduction in enzyme activity and dry cell
mass. At higher pH 6.5, a less enzyme activity (54 U/ml) and dry cell mass (5.1 g/l) was
recorded. A pH 4.5 was optimized for enzyme production and subsequent parameters.
4.3.1.2.5: Effect of temperature
The effect of different incubation temperature (20-50C) on enzyme production by
mutant strain in stirred fermentor was investigated (Fig 4.26). From the data it shows that
extreme low and high temperature caused the marked decrease in enzyme activity. At the
temperature of 20C, the enzyme activity (42 U/ml) and dry cell mass (5.1 g/l) was
obtained. An optimal enzyme activity (73 U/ml) and dry cell mass (7.9 g/l) was noticed
when incubation of temperature was adjusted at 30C. Further increase in incubation
temperature caused decrease in enzyme activity. At 40 and 50C the enzyme activity and
dry cell mass was found to be as 47 and 23 U/ml, 5.3 and 2 g/l, respectively.
4.3.1.2.6: Effect of agitation intensity
Fig 4.27 shows the data on the effect of different agitation intensity (120-280 rpm) on the
enzyme production by S. cerevisiae EMS-42 in stirred fermentor. At low agitation
intensity (120 rpm), the minimum enzyme activity (50.2 U/ml) and dry cell mass (5.29
g/l) was recorded. Further increase in agitation intensity there was also increase in
enzyme activity and dry cell mass. When the agitation intensity was set at 240 rpm then
the maximum enzyme activity (76.36 U/ml) and dry cell mass (7.8 g/l) was obtained.
Beyond this level, a marked decline in enzyme activity was noticed at 280 rpm. A less
enzyme activity (65 U/ml) with dry cell mass of 7.69 g/l was achieved at this agitation
intensity. An agitation intensity of 240 rpm was optimized for enzyme production and
subsequent parameters.
112
Fig 4.25: Effect of pH on the extracellular invertase production by S. cerevisiae EMS-42 in the stirred fermentor*.
0
10
20
30
40
50
60
70
80
90
3 3.5 4 4.5 5 5.5 6 6.5 7
pH
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
*Fermentation time 24 h, sucrose 15 g/l, incubation temperature 30C, agitation intensity 200 rpm, aeration 1 vvm.
113
Fig 4.26: Effect of different temperatures on the extracellular invertase production by S. cerevisiae EMS-42 in the stirred fermentor*.
0
10
20
30
40
50
60
70
80
90
10 20 30 40 50 60
Temperature (ºC)
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
*Fermentation time 24 h, sucrose 15 g/l, pH 4.5, agitation intensity 200 rpm , aeration 1 vvm.
114
Fig 4.27: Effect of different agitation intensity on the extracellular invertase production by S. cerevisiae EMS-42 in the stirred fermentor*.
0
10
20
30
40
50
60
70
80
90
80 120 160 200 240 280 320Agitation intensity (rpm)
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
*Fermentation time 24 h, sucrose 15 g/l, incubation temperature 30C, initial pH 4.5, aeration 1 vvm.
115
4.3.1.2.7: Effect of aeration
The effect of different aeration levels (0.5-2.0 vvm) on the enzyme production by mutant
strain in stirred fermentor was recorded (Fig 4.28). At the 0.5 vvm, the minimum enzyme
activity (57.45 U/ml) and dry cell mass (4.69 g/l) were found. The maximum enzyme
activity (76.71 U/ml) was achieved when the aeration rate was maintained at 1 vvm. At
this aeration level, the dry cell mass (7.8 g/l) was observed. No further increase in
enzyme activity and dry cell mass beyond this optimum level (1 vvm). Increased levels
of aeration such as 1.5 and 2 vvm caused reduction in enzyme activity and dry cell mass
as 73 and 66.39 U/ml, 7.46 and 7.4 g/l, respectively. An aeration level of 1 vvm was
optimized for enzyme production and subsequent parameters.
4.3.1.2.8: Effect of dissolved oxygen
Fig 4.29 shows the data on the effect of different saturation levels (5-20 %) of dissolved
oxygen (DO) on enzyme production by mutant strain in stirred fermentor. At 0.5 % of
DO, the enzyme activity (77.24 U/ml) and dry cell mass (7.27 g/l) was achieved. The
maximum enzyme activity (80.06 U/ml) was obtained when DO level was adjusted at 10
% DO. At this optimal level, the dry cell mass (7.82 g/l) was recorded. Further increase
in DO caused decline in enzyme activity and dry cell mass. The minimum enzyme
activity (70.63 U/ml) and dry cell mass (7.25 g/l) were found to be observed at dissolved
oxygen of 20 %.
116
Fig 4.28: Effect of different aeration levels on the extracellular invertase production by S. cerevisiae EMS-42 in the stirred fermentor*.
0
10
20
30
40
50
60
70
80
90
0 0.5 1 1.5 2 2.5
Aeration levels (vvm)
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
*Fermentation time 24 h, sucrose 15 g/l, initial pH 4.5, incubation temperature 30C, agitation intensity 240 rpm.
117
Fig 4.29: Effect of different concentrations of dissolved oxygen on the extracellular invertase production by S. cerevisiae EMS-42 in the stirred fermentor*.
0
10
20
30
40
50
60
70
80
90
0 5 10 15 20 25
DO (%)
Enz
yme
acti
vity
(U/m
l), D
CM
(g/l
)
Enzyme activity (U/ml) DCM (g/l)
*Fermentation time 24 h, sucrose 15 g/l, initial pH 4.5 incubation temperature 30C, agitation intensity 240 rpm, aeration 1 vvm.
118
4.3.2: Intracellular invertase
4.3.2.1: Rate of invertase production
Fig 4.30 shows the effect of different time course on the intracellular enzyme extraction
by sonication from wild (IS-66) and mutants (NA-45 and EMS-42) strains of S.
cerevisiae in shake flask. The invertase production was estimated at 8 h intervals (8-72
h). Maximum enzyme production was noticed after 48 h of incubation in all the five
tested strains. IS-66 showed maximum enzyme activity (33±1.4 U/ml) when incubation
time reached at 48 h. Two developed mutants (NA-45 and EMS-42) also showed
maximum enzyme activity as 38.1±1.2 and 43±1.6 U/ml, respectively. No further
increase in the invertase activity was obtained after 48 h of incubation. Of all the strains
EMS-42 was selected for subsequent optimization of intracellular invertase.
Fig 4.31 shows the data on the rate (8-56 h) of intracellular enzyme production by
mutant strain of S. cerevisiae EMS-42 in shake flask and in stirred fermentor. The
production of enzyme was gradually increased with the increase in incubation period
from 8 to 48 h in shake flask and 8 to 24 h in stirred fermentor. The least enzyme activity
7±1.02 and 16 U/ml were recorded after 8 h incubation period in shake flask and stirred
fermentor, respectively. Maximum enzyme production (47±1.0 U/ml) was achieved after
48 h of inoculation in shake flask. On the other hand, the maximum enzyme production
(57 U/ml) with dry cell mass of 12 g/l was recorded after 24 h of inoculation in stirred
fermentor. When fermentation was scaled up from shake flask to laboratory scale stirred
fermentor, an increase of approximately 10 U/ml was observed. The fermentation time
period from shake flask to fermentor was also shortened (48 to 24 h) by 24 h.
4.3.2.2: Effect of different amplitudes
During sonication the effect of different amplitudes (Amp) i.e., 20, 40, 60 and 80 % in
varying intervals (15-90 min) with 0.5 duty cycle on the enzyme release by mutant strain
was investigated (Fig 4.32). The specific activity was obtained by dividing the total
enzyme activity by total protein of the cell extract. At Amp-20 %, the specific activity
(101±2.0 U/mg of protein) was achieved when cell extract was sonicated for 75 min. The
maximum specific activity (105±1.5 U/mg of protein) was recorded at Amp-40 % after
60 min of sonication. Further increase in amplitude resulted in decreased specific
activity. At Amp-60 %, the maximum specific activity was (98±1.5 U/mg of protein) but
less than optimal (105±1.5 U/mg of protein) which was achieved at Amp-40 %. Above
119
Amp-60 % a decline in specific activity was recorded. After 45 min of sonication at
Amp-80 %, a less specific activity (94±1.0 U/mg of protein) was observed. Thus Amp-
40 % was optimized for subsequent parameters.
4.3.2.3: Effect of different pH
The effect of pH (3.0-6.5) on the intracellular enzyme release through sonication by
mutant strain was studied (Fig 4.33). At low pH 3, the minimum specific activity (49±2.0
U/g of dry cell mass) was achieved. Further increase in pH caused increase in specific
activity. The maximum specific activity (106±2.1 U/mg of protein) was recorded at pH
5. Beyond optimal pH 5, there was a decrease in specific activity was found to be
observed. At pH 6.5, the specific activity was found to be as 82±0.8 U/ mg of protein.
120
Fig 4.30: Comparison of rate on the intracellular invertase production by wild and mutant strains of S. cerevisiae in shake flask*.
0
10
20
30
40
50
60
0 8 16 24 32 40 48 56 64 72 80
Incubation time (h)
Enz
yme
acti
vity
(U/m
l)
IS-66 NA-45 EMS-42
*Incubation temperature 30ºC, sucrose 5 g/l, initial pH 5, agitation 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
121
Fig 4.31: Comparison of rate on the intracellular invertase production by S. cerevisiae EMS-42 in shake flask* and stirred fermentor**.
0
10
20
30
40
50
60
0 8 16 24 32 40 48 56 64
Incubation period (h)
Enz
yme
acti
vity
(U/m
l)
Shake flask Fermentor
* Sucrose 10 g/l, incubation temperature 30C, agitation intensity 200 rpm, pH 5.
** Sucrose 15 g/l, pH 4.5, incubation temperature 30C, agitation intensity 240 rpm, aeration 1 vvm, DO 10 %.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
122
Fig 4.32: Effect of different amplitudes to release intracellular invertase during sonication* in S. cerevisiae EMS-42
0
20
40
60
80
100
120
0 15 30 45 60 75 90 105
Time (min)
Spec
ific
act
ivit
y (U
/mg
of p
rote
in)
Amp-20 % Amp-40 % Amp-60 % Amp-80 %
* Temperature 4ºC, pH 4.5, 0.5 duty cycle.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
123
Fig 4.33: Effect of different pH on intracellular invertase release during sonication* in S. cerevisiae EMS-42.
0
20
40
60
80
100
120
2.5 3 3.5 4 4.5 5 5.5 6 6.5 7pH
Spec
ific
act
ivit
y (U
/mg
of p
rote
in)
* Temperature 4ºC, Amp. 40 %, 0.5 duty cycle, time 60 min.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
124
4.4: Immobilization studies
4.4.1: Rate of sucrose hydrolysis
Time course of sucrose hydrolysis (2-22 h) for invertase production by Ca-alginate
immobilized yeast cells of mutant strain of S. cerevisiae EMS-42 was also studied in
shake flasks (Fig 4.34). A gradual increase in sucrose hydrolysis was observed with the
incubation period from 2 to 18 h. After 2 h of incubation period, the minimum sucrose
hydrolysis (4.4±0.3 %) was recorded. Maximum sucrose hydrolysis (63.40±1.5 %) was
achieved 18 h after suspending the beads in sucrose solution. A steady decline in sucrose
hydrolysis was observed after 18 h of incubation. So, in the present studies, an
incubation period of 18 h was optimized for maximum invertase activity.
4.4.2: Effect of different sucrose concentrations
Fig 4.35 is depicted the data on the effect of different sucrose concentrations (30-70 %,
w/v) on the production of invertase by mutant strain in the form of Calcium-alginate
entrapped yeast cells. A lower enzyme production for sucrose hydrolysis (49.50±0.78 %)
was noticed at an initial sucrose concentration of 30 % (w/v). Maximum hydrolysis
(68±2.0 %) was however, achieved at sucrose concentration of 60 % (w/v). With the
increase in sucrose concentration beyond 60 % (w/v), a reduction in sucrose hydrolysis
(53.0±0.9 %) was observed. Thus a sucrose concentration of 60 % (w/v) was optimized
for subsequent experiments.
4.4.3: Effect of different temperature
The production of invertase for sucrose hydrolysis by Ca-alginate immobilized yeast
cells of mutant strain was studied at different temperatures (30-70ºC) in shake flasks (Fig
4.36). A lower sucrose hydrolysis (51.3±1.15 %) was recorded at a temperature of 30ºC.
However, sucrose hydrolysis was increased and became maximum (74.6±1.4 %) at 50ºC.
Further increase in the temperature caused a marked decrease in sucrose hydrolysis. Thus
the hydrolysis became 1.2 fold lower (27.8 %) than optimal between temperature range
of 60-70ºC.
4.4.4: Effect of different pH
The production of invertase for sucrose hydrolysis by Ca-alginate immobilized mutant
cells was studied at different pH (3.0-6.0) in shake flasks (Fig 4.37). At lower pH (3.0) a
lower sucrose hydrolysis (42.0±1.0 %) was noticed. Further increase in pH, a gradual
increase in sucrose hydrolysis till pH of 5.5 was recorded. At this pH a maximum
sucrose hydrolysis (76.3±0.45 %) was achieved. Beyond optimal pH (5.5), a gradual
125
decrease in sucrose hydrolysis. At higher pH 6.5, the sucrose hydrolysis (61.0±1.06 %)
was found to be observed. Among all the pH (3-6), pH 5.5 was found to be optimal for
higher sucrose conversion.
4.4.5: Re-use of immobilized cells in batch process
The effect of the re-use of the biocatalyst in the batch process on sucrose hydrolysis is
shown in Fig 4.38. The value of maximum sucrose hydrolysis (76.3±0.45 %) activity
obtained at 50oC after 18 h of incubation time. After every 18 h of incubation till 28
batches, the biocatalyst retained about 95 % of its original activity. The maximum
activity became gradually low (85 %) after 36 batch.
4.4.6: Storage stability
Immobilized mutant yeast cells did not lost their activity till six months, and were stored
in 0.05 M acetate buffer (pH 5.5) retained 90 % of their initial activity.
126
Fig 4.34: Time course study of sucrose hydrolysis by Calcium alginate immobilized yeast cells of S. cerevisiae EMS-42 in shake flask*.
0
10
20
30
40
50
60
70
80
0 2 4 6 8 10 12 14 16 18 20 22 24
Incubation period (h)
Sucr
ose
hydr
olys
is (%
)
*Temperature 30ºC, sucrose 40 % (w/v), pH 5, agitation 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
127
Fig 4.35: Effect of different sucrose concentrations on sucrose hydrolysis by
Ca-alginate immobilized cells of S. cerevisiae EMS-42 in shake flask*.
0
10
20
30
40
50
60
70
80
20 30 40 50 60 70 80
Sucrose conc. (%, w/v)
Sucr
ose
hydr
olys
is (%
)
*Incubation time 18h, temperature 30ºC, pH 5, agitation 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
128
Fig 4.36: Effect of different temperatures on sucrose hydrolysis by Ca-alginate immobilized cells of S. cerevisiae EMS-42 in shake flask*.
0
10
20
30
40
50
60
70
80
20 30 40 50 60 70 80
Temperature (ºC)
Sucr
ose
hydr
olys
is (%
)
*Incubation time 18 h, sucrose 60 % (w/v), agitation 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
129
Fig 4.37: Effect of pH on sucrose hydrolysis by Ca-alginate immobilized cells of S. cerevisiae EMS-42 in shake flask*.
0
10
20
30
40
50
60
70
80
2.5 3 3.5 4 4.5 5 5.5 6 6.5 7pH
Sucr
ose
hydr
olys
is (%
)
*Incubation time 18 h, sucrose 60 % (w/v), temperature 50ºC, agitation 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
130
Fig 4.38: Time course study of re-use of batch process for sucrose hydrolysis by Ca-alginate immobilized cells of S. cerevisiae EMS-42 in shake flask*.
84
86
88
90
92
94
96
98
100
102
0 3 6 9 12 15 18 21 24 27 30 33 36 39
Batch Number
Ret
enti
on in
vert
ase
acti
vity
(%)
.
*Incubation time 18 h, sucrose 60 % (w/v), temperature 50ºC, agitation 200 rpm.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
131
4.5: Purification
4.5.1: Extracellular invertase:
An extracellular invertase from S. cerevisiae EMS-42 was purified through successive
steps of ammonium sulfate (40-85 %) precipitation and DEAE-Sephadex A-50 (Table
4.12). The key step involved a fractionation of insoluble and soluble forms of invertase
apparently due to differences in carbohydrate content of the enzymes. The ammonium
sulfate at the 85 % saturation level separates the external (glycosylated) invertase as
soluble fraction while giving insoluble in precipitated form. Both precipitated and
soluble fractions (85 % ammonium sulfate supernatant) were dialyzed and concentrated
after freeze drying. They were run on gel electrophoresis (SDS-PAGE). The protein
profile of the precipitated fraction as shown in Fig 4.39 from lane 2-7 while lane 8
showing the protein profile of soluble extracellular invertase. The one major peak as
shown in Fig 4.40 was eluted by using 0.2 M NaCI. When this peak was tested by
electrophoresis, only one broader band (Fig 4.41) was found with molecular mass of 110
kDa. The purified extracellular was glycoprotein in nature and 48 % carbohydrate found
to be observed by Dubois et al. (1956).
The results obtained from the purification steps are given in Table 4.12. The specific
activity of the purified extracellular invertase was estimated to be 1915 U/mg, which is
about 15 fold than that of the crude enzyme with final activity recovery of 38 %. The
optimum pH and temperature were found to be as 5 and 60оC, respectively (Fig 4.45 &
4.46). Table 4.14 depicts the data on the effect of additives such as NaCl, KCl, MnCl2,
EDTA, BaCl2, MgCl2, CuSO4, HgCl2, CoCl2, CuCl2, FeSO4, CaCl2 and ZnSO4 at the
concentration of 1 mM on glycosylated invertase. Of the all, MgCl2, MnCl2 and CoCl2
was found to be as slightly stimulatory with relative activity from 102-111 % while
remaining caused reduction in relative activity. The addition of HgCl2, CuSO4 and CuCl2
completely inhibited the enzyme. By using Lineweaver-burk plot, the Km and Vmax values
were observed to be as 1.8 mM and 1429 U/ml/min, respectively (Fig 4.48).
132
Fig 4.39: SDS-PAGE after ammonium sulfate treatment for extracellular invertase.
8 7 6 5 4 3 2 1 kDa
250
150
100
75
50
37
25
20
*Lane 1, Protein marker
Lanes 2-7, 60 -70 % ammonium sulfate precipitates
Lane 8, 85 % ammonium sulfate supernatant
133
Fig 4.40: Elution Pattern on DEAE-Sephadex for extracellular glycosylated invertase.
0
500
1000
1500
2000
2500
0 10 20 30 40 50 60
Fraction No.
Enz
yme
acti
vity
(U/m
l)
0
0.5
1
1.5
2
2.5
Abs
orba
nce
(280
nm
)
Enzyme activity (U/ml) Absorbance (280 nm)
134
Fig 4.41: SDS-PAGE of purified extracellular invertase
*Lane 1, Protein marker.
Lanes 2-3, purified extracellular invertase
250 150
100
75
50
37
25
20
kDa 3 2 1
135
Table 4.12: Purification steps of extracelluar invertase
Purification
steps
Volume
(ml)
Total
activity
(U)
Total Protein
(mg)
Specific
activity
(U/mg)
Fold
Purification
Activity
recovery (%)
Crude broth
Freeze dried
ammonium
sulfate
supernatant
(85%)
DEAE-
Sephadex
1000
200
8
53120
34016
20110
404
210
10.5
131
162
1915
-
1.2
15
100
64
38
136
4.5.2: Purification of intracellular invertase (S and L forms):
The intracellular invertase from mutant strain was purified successively through three
step i.e., ammonium sulfate (20-85 %) saturation, DEAE-Sephadex A-50 and Sephadex
G-50 (Table 4.13). The procedure of ammonium sulfate was also used for the separation
of two forms of intracellular invertase, one in small amount (S-form) being non-
glycosylated was recovered from (20-85 %) saturation in precipitated form. On the other
hand, the second form in large amount (L-form) was found from supernatant of 85 %
ammonium sulfate saturation being glycoprotein in nature. It means that extracellular
invertase isolated from the cell free broth was secretory periplasmic enzyme. It can be
extracted from the cells in the same glycosylated form as shown in Fig 4.39 (lane 8,
arrow indicated) & Fig 4.42 (lanes 2-3). In both cases the bands were broad having same
molecular weight of 110 kDa. The further purification of L-form invertase was obtained
in the same manner as extracellular invertase. For the purification of S-form invertase,
the collected precipitates were dialyzed and then loaded on DEAE-Sephadex column.
After anion-exchange, out of four protein peaks one peak was eluted at 0.3 M NaCl
showing enzyme activity as shown in Fig 4.43. The active fractions showing maximum
enzyme activity were pooled, dialyzed and concentrated by freeze drier. The
concentrated protein was further purified on Sephadex G-50 column. As that result only
one protein peak showing invertase activity was obtained (Fig 4.44). After SDS-PAGE
only one protein band was found having approximately molecular weight of 55 kDa as
shown in (Fig 4.42, lane 5). After Phenol-Sulfuric test the L-invertase was found to be
glycoprotein and S- invertase as carbohydrate-free protein.
The specific activity of the purified intracellular S-invertase and L-invertase were
estimated to be as 1670 U/mg, 1964 U/mg and fold purification of 16, 19 with recovery
of 17 % and 32 %, respectively as shown in Table 4.13. The optimum pH (5) and
temperature (50оC) of non-glycosylated invertase was found to be observed (Fig 4.45 &
4.6). The effect of chemicals and metal ions such as NaCl, KCl, MnCl2, EDTA, BaCl2,
MgCl2, CuSO4, HgCl2, CoCl2, CuCl2, FeSO4, CaCl2 and ZnSO4 on non-glycosylated
invertase was also investigated (Table 4.14). The findings after adding all additives was
almost similar to glycosylated invertase but with slight decrease of relative activity.
From Lineweaver-burk plot, the Km and Vmax values for intracellular non-glycosylated
were found to be as 1.2 mM and 909 U/ml/min, respectively (Fig 4.47).
137
Fig 4.42: SDS-PAGE after sonication, ammonium sulfate treatment and chromatographies for intracellular invertase.
20
25
37
50
75
100
150
12345
250
kDa
* Lane 1, protein marker
Lanes 2-3, intracellular sonicated crude extract
Lane 4, 60 % ammonium sulfate precipitates
Lane 5, purified non-glycosylated intracellular invertase
138
Fig 4.43: Elution Pattern on DEAE-Sephadex for intracellular non-glycosylated invertase.
0
500
1000
1500
2000
2500
0 10 20 30 40 50
Fraction No.
Enz
yme
acti
vity
(U/m
l)
0
0.5
1
1.5
2
2.5
Abs
orba
ce (2
80 n
m)
Enzyme activity (U/ml) Absorbance (280 nm)
139
Fig 4.44: Elution Pattern on Sephadex G-50 for intracellular non-glycosylated invertase.
0
500
1000
1500
2000
2500
0 5 10 15 20 25 30 35
Fraction No.
Enz
yme
acti
vity
(U/m
l)
0
0.5
1
1.5
2
2.5
Abs
orba
nce
(280
nm
)
Enzyme activity (U/ml) Absorbance (280 nm)
140
Table 4.13: Purification steps of intracelluar invertase (S and L forms)
Foms of
Intracellular
invertase
Purification steps
Volume
(ml)
Total
activity
(U)
Total
protein
(mg)
Specific
activity
(U/mg)
Fold
purification
Activity
recovery
(%)
Crude extract
500
29700
287
103
-
100
S-form
(55 kDa)
invertase
Ammonium
sulfate (20-80%)
DEAE-Sephadex
Sephadex G-50
100
28
1.5
10346
7300
5011
95
28
03
109
260
1670
1.1
2.5
16
35
25
17
L-form
(110 kDa)
invertase
Freeze dried
ammonium
sulfate
supernatant
(85%)
DEAE-Sephadex
100
2.5
14221
9820
72
05
98
1964
1.9
19
46
32
141
Fig 4.45: Effect of pH on stability of purified glycosylated and non-glycosylated invertase.
0
50
100
150
200
250
300
350
2 3 4 5 6 7 8 9
pH
Enz
yme
acti
vity
(U/m
l)
Glycosylated Non-glycosylated
*Hundreds microlitres of each enzyme solution was incubated in 0.05 M citrate/ 0.05 M acetate buffer at pH values ranging for 2-8 at 40оC for 15 min. The residual activity was determined under standard conditions.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
142
Fig 4.46: Effect of temperature on stability of purified glycosylated and
non-glycosylated invertase.
0
50
100
150
200
250
300
350
10 20 30 40 50 60 70 80 90
Temperature (ºC)
Enz
yme
acti
vity
(U/m
l)
Glycosylated Non-glycosylated
*The enzyme activity was measured in the temperature range of 20-80оC. The reaction mixture (pH 5.0) was incubated for 15 min and residual activity was determined under standard conditions.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
143
Table 4.14: Effect of additives on stability of purified glycosylated and non-glycosylated invertase.
Additives
(1 mM)
Relative activity (%)
Non- glycosylated Glycosylated
Control
NaCl
KCl
MnCl2
EDTA
BaCl2
MgCl2
CuSO4
HgCl2,
CoCl2
FeSO4
CaCl2
CuCl2
ZnSO4
100
93±0.2
96±1.0
109±0.04
102±1.5
97±0.3
105±1.4
17±1.5
2.9±2.0
102±1.0
86±2.2
78±1.8
19±1.2
88±2.4
100
96±0.5
96±1.1
111±0.09
103±2.5
98±1.3
107±0.9
17±1.0
3.0±1.9
104±0.8
89±1.3
80±1.6
21±0.8
90±2.5
144
Fig 4.47: Lineweaver-Burk plot for intracellular non-glycosylated invertase
y = 0.0013x + 0.0007
R2 = 0.7294
0
0.005
0.01
0.015
0.02
0.025
2 2.2 2.5 2.9 3.3 4 5 6.7 10 20
1/[S] mM
1/V
(U/m
l/m
in)
The intercept on the y-axis corresponding to 1/Vmax = 0.0007, Slope = 0.0013.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
145
Fig 4.48: Lineweaver-Burk plot for extracellular glycosylated invertase
y = 0.0013x + 0.0011
R2 = 0.7333
0
0.005
0.01
0.015
0.02
0.025
2 2.2 2.5 2.9 3.3 4 5 6.7 10 20
1/[S] mM
1/V
(U/m
l/m
in)
The intercept on the y-axis corresponding to 1/Vmax = 0.0011, Slope = 0.0013.
The mean difference is significant at p≤0.05. Y bars indicate the standard deviation (sd) among the three parallel replicates.
146
DISCUSSION
In the present study, eighty six isolates were isolated and screened for their
extracellular invertase synthesizing ability. One isolate (IS-66) gave better enzyme
activity (1.130.03 U/ml). A marked increase (5.60.6 U/ml) in enzyme activity was
found after optimizing cultural conditions including 48 h of incubation period, 5 g/l of
sucrose and pH (5.5). Enzyme production increased about 5 fold when sucrose
concentration was decreased in the fermentation medium from 30 to 5 g/l. The reason for
this increase of sucrose in the medium after hydrolysis produces glucose and fructose
which if not fully utilized induced carbon catabolite repression of invertase. When the
sucrose concentration was adjusted to 5 g/l the reducing sugar released by sucrose
hydrolysis was almost completely utilized and a little residual sugar was left behind,
invertase feedback repression was removed and its production increased (Gancedo,
1998).
It was quite apparent from the results that UV mutagenesis gave no stable mutant
with improved enzyme activity rather a lesser enzyme production was recorded when
compared to the wild strain. In contrary, when one mutant developed by UV mutation
which resistant to 2-deoxy-D-glucose was grown under the conditions of catabolic
repression, a high level of extracellular invertase produced in baker’s yeast is reported by
Randez-Gil and Sanz (1994). Therefore, chemical mutation using nitrous acid and EMS
were carried out. One nitrous acid treated mutant of S. cerevisiae, NA-45 out of forty
seven showed highest enzyme activity (20.740.65 U/ml) with dry cell mass of
6.060.04 g/l. This mutant was further chemical mutagenized by EMS. Forty four EMS
treated isolates were screened for invertase production. In the present work the cell
survival rate of IS-66 decreased with increase in concentration of nitrous acid or EMS
and exposure time. The mutant EMS-42 showed about 6 fold higher enzyme activity
(34.20.29 U/ml) as compared to IS-66 (5.60.7 U/ml). Ager and Haynes (1990)
indicated “the interaction between EMS and UV in S. cerevisiae might arise from the
inhibition of double-strand break repair by one, or both agents”. UV-irradiation, EMS
and nitrous acid were commonly used for strain improvement (Azin and Noroozi, 2001;
Szafraniec et al., 2003; Kig et al., 2005; Haq et al., 2008). The resistance for catabolic
repression to 2-deoxy-D-glucose for mutant strain EMS-42 was determined at the
concentration of 0.04 mg/ml. At this optimal concentration, EMS-42 gave consistent
enzyme activity. On the basis of catabolic repression, many workers had screened
147
mutants for resistance to 2-deoxy-D-glucose having improved fermentative capacity on
sugar medium (Heredia and Heredia 1988; Randez-Gil et al., 1995 and Rincon et al.,
2001).
When comparison on the rate of enzyme was made for IS-66, NA-45 and EMS-
42, the incubation time period of 48 h was found to be optimal for all tested strains.
Similarly, other workers have reported optimal invertase production by S. cerevisiae
incubated for 36-48 h (Atiyeh and Duvnjak, 2002). The mutant strain EMS-42 showed
maximum enzyme activity (34.20.29 U/ml). No further increase in the enzyme activity
was obtained beyond optimal incubation time. It was due to the decreased availability of
nutrients in the medium, the age of organism, the addition of inhibitors produced by
yeast itself, the protease production and less budding capacity of yeast (Cochran, 1961).
According to Herwig et al. (2001) the enzyme activity declined due to nutrient
deficiency in the medium or carbon catabolite repression. The kinetic parameters
regarding the yield of the enzyme by biomass formation in terms of rates on enzyme
production by mutant (EMS-42) as compared to wild (IS-66) was significant after 48 h
of fermentation incubation time. At this time period the value of specific growth rate (μ
h-1) was found to be significantly high than wild strain.
The selection of suitable fermentation medium is of great importance for enzyme
production. Among the six media tested for invertase production in shake flask, M1
medium gave maximum enzyme production (25.28±1.72 U/ml). Camacho-Ruiz (2003)
also studied and practiced the effects of media composition on yeast growth. M1 medium
was supplemented by different sugars as sole carbon sources such as sucrose, fructose,
lactose, galactose, maltose, raffinose and molasses at concentration of 20 g/l. The
optimal enzyme activity (37±1.17 U/ml) was obtained when sucrose was added into the
fermentation medium. Other sugars gave relatively less enzyme activity. However, the
medium containing lactose showed least enzyme activity (3.19±0.46 U/ml). The order of
utilization of four sugars as glucose, fructose, sucrose and maltose by S. cerevisiae is
reported by Meneses et al. (2002). Paine et al. (1925) described that sucrose had certain
advantages over some other sugars because of its high degree of sweetness, relatively
low degree of hygroscopicity, crystal form, solubility, and facility of crystallization and
readily hydrolyzed with formation of invert sugar.
The maximum enzyme activity (44.03±0.30 U/ml) was obtained when sucrose
concentration was kept at concentration of 10 g/l. Above optimal concentration of
148
sucrose, a gradual decline in enzyme activity but increase in dry cell mass was recorded.
The increase in enzyme production was due to that sucrose in higher concentration in the
medium hydrolyzes into glucose and fructose that may not be fully utilized due to the
induced carbon catabolic repression of invertase (Carlson, 1999). At low concentration
of fructose and glucose, the products of sucrose hydrolysis by invertase, induced the
expression of an invertase coding gene in S. cerevisiae as reported by Ozcar et al. (1997).
Increased concentration of glucose caused decreased transcription and thus a decrease in
their translation rate or an increase in the degradation rate of the protein. Another reason
of decreased invertase production at higher sucrose concentration was due to
transfructosylation that produce fructo-oligosaccharides (Gancedo, 1998). Win et al.
(2004) used the 5 % sucrose for transfructosylation. The transfructosylation of thiol
group by invertase is reported by Nakano et al. (2000). At low sucrose concentration,
transfructosylating activity competes with invertase activity in sucrose utilization as
reported by Kim et al. (2000).
The optimization of the volume of fermentation medium play a key role for air
and nutrient supply, proper agitation, designing inoculum size, growth of microorganism
and enzyme production. The maximum enzyme activity (47.33±0.61 U/ml) was obtained
when 2 % vegetative inoculum was used to inoculate 50 ml of the fermentation medium
after 24 h of inoculation. The number of yeast cells introduced into the medium
determines the quality and extent of enzymes produced. Cells have the capacity for
altering their metabolism with changes in environment as reported by Ordaz et al.
(2001). Above and below optimal level of inoculum size, a decrease in enzyme activity
was achieved. It was due to non-compatible ratio between sucrose concentration and the
number of cells, so in that way sucrose acted as a repressor. Inoculum size larger than
optimal caused overgrowth of yeast and thus, nutrient imbalance resulting in less enzyme
production (Bokosa et al., 1992; Haq et al., 2002). In contrary, Roitsch et al. (2003)
reported that 48 h old cells were as good as those from 72 to 96 h old slant culture for
invertase production.
The optimal production of enzyme was determined as a function of pH and
temperature. The enzyme production was found to be optimal at 30ºC. At temperature
other than optimal, a decline in enzyme activity was recorded. The higher temperature
than optimal caused decrease in rate of reaction due to thermal denaturation of enzyme as
reported by Resa et al. (2009). Uma et al. (2010) kept the 250 ml Erlenmeyer flasks each
containing 50 ml fermentation medium for invertase production by Aspergillus flavus at
149
30ºC for 7 days. At pH 5, maximum enzyme production (48.86±0.80 U/ml) was
obtained. Other than optimal caused decline in enzyme activity. The extreme pH (high or
low) caused detrimental effects to the growth of S. cerevisiae cells thus, reducing
enzyme activity (Kurakake et al., 1996; Kim et al., 2000; Persike et al., 2002). Silvia et
al. (2000) and Liu et al. (2001) observed optimal growth and enzyme activity by S.
cerevisiae at pH 5.0. A less enzyme production at higher pH was due to obstructed
enzyme secretion from the yeast cells as reported by Porro et al. (1991) and Costaglioli
et al. (1997).
The addition of proper nitrogen source into the fermentation medium has major
effect on enzyme production. In all the fermentation media used, the yeast extract at the
concentration of 3 g/l was added. Further nutrition requirement in terms of additional
nitrogen source was investigated. Among all organic nitrogen sources used the peptone
at the concentration of 6 g/l gave the maximum enzyme activity (50±1.1 U/ml) with dry
cell mass (6.72±0.01 g/l). The combined effect of yeast extract and peptone into
fermentation medium is reported by many workers (Poonawalla, 1965; Rodriguez et al.,
1995; Abrahao-Neto et al., 1996; Tanaka et al., 2000; Marques et al., 2006).
Rouwenhorst et al. (1991) studied the effect of different nitrogen sources on invertase
production and found peptone as the best source. In the present study, the effect of
different inorganic nitrogen sources such as NH4NO3, (NH4)2HPO4, NH4CI, (NH4)2SO4
and KNO3 was evaluated. At 6 g/l, (NH4)2HPO4 gave less enzyme activity (40.51±0.86
U/ml) as compare to peptone. It was due to ammonium salts induced poor yeast
performance (da Cruz et al., 2003). A least enzyme activity was achieved by adding
agricultural by products such as soybean meal, sunflower meal and cornsteep liquor. The
cornsteep liquor gave enzyme activity (21.56±0.55 U/ml) at the concentration of 6 g/l
while remaining showed less enzyme activity even than control (12.06 U/ml). The role
of ammonia metabolism was investigated in nitrogen catabolite repression by S.
cerevisiae (Schure et al., 2001). In order to choose the best out of a large variety of
available nitrogen sources, the yeast had developed molecular mechanisms consisted of a
sensing and a regulatory mechanism in which induction of required systems and
repression of systems that were not useful. The periplasmic enzymes of yeast which play
their roles in nutrition would be responsible to take action towards different sources of
carbon and nitrogen by sensing and signalling pathways (Oliveira et al., 2005).
It would seem easy enough to culture S. cerevisiae for invertase production in a
shake flask but can pose a number of problems, including pH and temperature control,
150
oxygen and nutrient limitation etc. By switching from a shake flask to a fermentor with
controlled conditions as mentioned above there was an enhanced invertase production in
fermentor than shake flask. The maximum enzyme production in shake flask (50±0.9
U/ml) and fermentor (65.12 U/ml) was found after 48 and 24 h, respectively. The
fermentation time was shortened by 24 h but with overall increase of 15 U/ml in enzyme
activity. The reduction in the incubation time period in fermentor as compared to shake
flasks probably due to the controlled factors such as temperature, pH, agitation, aeration
which caused the better growth of organism and production of enzyme as reported by
Gigras et al. (2002). The present work was found to be significant not only in terms of
higher production but also in reduction of fermentation time period that made the
enzyme production more economical by saving the energy requirements. The kinetic
parameters regarding the yield of the enzyme by biomass formation in terms of rates on
enzyme production by mutant (EMS-42) in fermentor was significant after 24 h of
fermentation incubation time as compared to shake flask. At this time period the value of
specific growth rate (μ h-1) in fermentor was significantly high than shake flask.
The maximum enzyme activity (69 U/ml) was found at 15 g/l of sucrose. Above
optimal no increase in enzyme activity but gradual increase in dry cell mass was
recorded. The addition of sucrose in higher concentration successively attributed to the
limiting water. The kinetic behaviour of invertase become deviated from the Michaelis-
Menten equation, when sucrose concentrations was kept beyond 0.2 M that caused
gradual decrease in biocatalyst hydrolysis as reported by Farine et al. (2001). At higher
sucrose concentration (up to 2.34 M), the action of invertase followed by High
Performance Liquid Chromatography (HPLC) resulted in quantitative determination of
mixtures of D-fructose, D-glucose, 6-kestose, sucrose, inulobiose (Straathof et al., 1986).
The size of inoculum plays an important role in the fermentation of enzymes. The
optimal enzyme activity (71.2 U/ml) was recorded at inoculum size of 7.5 % with dry
cell mass (7.9 g/l). As the inoculum size was further increased, the production of the
enzyme gradually decreased due to the fact that at high level of inoculum size yeast grow
fast by consuming the essential nutrients at the initial stages and rapid accumulation of
by product into the fermentation medium. The reason for the low production of enzyme
at the inoculum size below than optimal was due to the slow growth of the organism and
extended time period to utilize nutrients properly. Thus the production of invertase was
affected at higher and lower size of the inoculum as well. Toda (1976) obtained
maximum invertase production when 5 % (v/v) inoculum size was used in 1.2 L working
151
volume of fermentor. Abrahao-Neto et al. (1996) poured a volume of 0.45 L (15 %, w/v)
of inoculum (0.70 g dry cell/L) into 5L NBS-MF 200 fermentor containing 2.55 L of
culture medium.
The maximum enzyme activity (73.41 U/ml) was recorded at pH 4.5. At extreme
pH, a less invertase activity than optimal was found. Dixon and Webb (1979) and Segel
(1975) reported that enzymes normally contain various amino acids residues in their
active site, and the interaction among them, and with the substrate, influences the
catalytic process. Consequently, enzymes were only active in a restricted range of pH,
and for most cases, show a definite optimum pH where activity was maximal. As the pH
was varied from 4-5, the alteration in the intracellular pH would be unlikely due to
internal buffering capability of S. cerevisiae (Weitzel et al., 1987). The pH affected
invertase activity by altering the tertiary or quaternary structures of the protein, leading
to an inadequate insertion of the macromolecule in the cell wall during budding (Reddy
et al., 1990). Enzyme production is largely dependent on temperature of the fermentation
medium. When effect of different temperature was work out in stirred fermentor, the
optimal production of enzyme (73 U/ml) was found to be at 30ºC. Vitolo et al. (1995)
found the optimal temperature of 30C for invertase production by S. cerevisiae. On a
growth medium contained sucrose, extracellular invertase production was maximum at
15 h in cultures incubated at 28C is reported by Vainstein and Peberdy (1991).
When agitation intensity was kept at 240 rpm, the maximum enzyme activity and
dry cell mass was found to be as 76.36 U/ml and 7.8 g/l, respectively. The higher
agitation speed above than optimal resulted in oxidative and mechanical stress,
disruption, excessive foaming and physiological disturbance of cells. On the other hand
lower agitation speed reduced the oxygen supply along with the imbalance nutrient
distribution due to non-homogenized fermentation medium. Rosma and Ooi (2006)
reported the highest yield of protein content (1.2 g/l) and biomass (7.8 g/l) from Candida
utilis with agitation speed (900 rpm). Narciandi et al. (1995) obtained high production of
recombinant invertase in Hansenula polymorpha when cells growing in exponential
phase were inoculated into fermentors containing media (pH 5.5) and cultured at 37C
with 1 vvm aeration and 350 rpm for 90 h.
The maximum enzyme activity (76.71 U/ml) was found when aeration level was
kept at 1 vvm. The unavailability of proper air supply to microorganism greatly disturbed
the physiology and metabolism of organism and thus a lower enzyme production was
152
achieved. In addition, the formation of any toxic byproduct in the culture medium caused
reduction in enzyme activity. On the other hand, excessive air supply also caused some
harmful effects on the growth of microorganism and enzyme production during
fermentation (Ionita et al., 2001). The maximum invertase activity of 200 U/g dry cells
in the yeast by using 2.0 L fermentor containing 1.5 L molasses medium with aeration at
1.6 vvm and pH 4.5 is reported by Bokosa et al. (1993).
The supply of oxygen is very essential for the aerobic fermentation. The oxygen
in dissolved form into the medium becomes available to growth of microorganism.
Dissolved oxygen at the level of 10 % enhanced the production of invertase (80.06 U/ml)
with dry cell mass (7.8 g/l). A 5 fold increase in specific invertase activity was obtained
when cultural conditions were shifted from anaerobic to aerobic in recombinant S.
cerevisiae as reported by Pyun et al. (1999). Vitolo et al. (1995) optimized the dissolved
oxygen (3.3 mg/l) for the production of invertase in S. cerevisiae at temperature (30C)
and pH (5.0). Thus, the process became economical with decreased input and
comparatively higher production.
In the present study, the intracellular invertase was also determined in S.
cerevisiae as reported by many workers (Gascon et al., 1968, Moreno et al., 1975,
Iglesias et al., 1980 and Vitolo et al., 1991). The intracellular enzyme activity exhibited
by EMS-42 in shake flasks and fermentor was found to be as 43 and 57 U/ml after 48
and 24 h, respectively. Balasundaram and Pandit (2001) released intracellular invertase
enzymes from S. cerevisiae being secretory protein to the periplasmic space. By using
sonication technique, the maximum specific activity (105±1.5 U/mg of protein) was
recorded at amplitude of 40 % after 60 min. Above optimal amplitude (Amp-40 %) a
decline in specific activity was observed. Vargas et al. (2003) obtained greatest invertase
activity (1.08 U/ml) after 5 min of sonication in batch culture of S. cerevisiae. On the
other hand, Marques et al. (2006) irradiated the culture of S. cerevisiae at a frequency of
20 kHz and amplitude 20. Vargas et al. (2004) obtained the highest value of total
invertase from Aspergillus niger by ultrasound irradiation for periods of 2 to 10 min with
an amplitude of 20. The ultrasound application at frequency of 24 kHz and power input
of 2 W caused an increase in proteinase activity by 24 % on S. cerevisiae batch culture
(Lanchun et al., 2003). The maximum specific activity (106±2.1 U/mg of protein) was
obtained after optimizing the pH 5. At an alkaline pH enzyme was not stable, thus
sucrose inversion efficiency affected in direct way (Balasundaram and Pandit, 2001).
153
Calcium alginate gel has been used as an entrapment matrix for whole microbial
cells due to its excellent characteristics (Nakane et al., 2001; Milovanovic et al., 2007).
The incubation time period of 18 h was found to be optimal for maximum sucrose
hydrolysis (63.40 %). When sucrose concentration was increased from 40 to 50 % (w/v)
there was an increase in sucrose hydrolysis (68.20 %) by about 5 %. The lower
concentrations of sucrose as sole carbon source limit yeast growth and caused a lesser
production of invertase (Arfi et al., 2003). Dynesen et al. (1998) and Chi et al. (2004)
suggested that when the concentration was adjusted so that the reducing sugar released
by sucrose hydrolysis was almost completely utilized and very little residual sugar was
left behind, invertase feedback repression was removed and hence enzyme production
increased. The maximum sucrose hydrolysis (74.6 %) was recorded at 50ºC. When the
temperature was increased from 30-50oC, there was an increase in hydrolysis percentage
(25 %). Immobilized invertase was more active in higher temperature range because of
the prevention of thermal denaturation by rigid immobilization. However, when the
temperature was set beyond optimal, there was decline in the invertase activity. This
decrease in activity was due to a reduction in the enzyme stability caused by protein
denaturation. At high temperature the enzyme activity was not significant because of
denaturation of enzyme active site as reported by Russo et al. (1996). Similarly, heat
inactivation enthalpy from 200 to 300 KJ/mol caused the denaturation of enzyme
through the unfolding of its tertiary or quaternary structure as reported by Owusu and
Makhzoum (1992). Secondly, the reason for stability at somewhat high temperature than
free cells was due to less susceptibility to endogenous proteolysis, diffusional limitations
and changed pattern of glycosylation of invertase in immobilized cells (de Alteriis et al.,
1999). At pH 5.5, the maximum sucrose hydrolysis (76.3 %) was found. When pH
increased from 3.0-5.5, there was an average gain of 35 % in the hydrolysis percentage.
This result suggested that microbiological enzymes at pH (4.0-5.5) are more active.
Similarly, Manston and Rodgers (1987) obtained optimum enzyme hydrolysis at pH 5.5
and 50°C using sucrose (50 %, w/v). Tomotani and Vitolo (2004) reported that
DOWEX/invertase complex at pH (4.5-6.0) was functionally stable.
The beads after every 18 h of incubation time period were repeatedly used for 26
batches and thus retained 95 % invertase activity but further use of beads caused the
reduction in invertase activity with loss of integrity of the beads. Tanriseven and Dogan
(2001) obtained relative activity (87 %) for 36 days without decline in enzyme activity
by immobilizing S. cerevisiae in alginate capsules. The beads were stored as biocatalyst
154
at 4oC in acetate buffer (0.05 M, pH 5.0) for six months without appreciable loss of the
enzyme activity. This finding was similar to Rossi-Alva and Rocha-Leao (2003) who
also reported the storage duration of 6 month for retaining maximum activity.
An extracellular invertase was purified to homogeneity by two step purification.
The ammonium sulfate saturation (85 %) separated the external (glycosylated) invertase
as soluble fraction in the supernatant. Gascon and Lampen (1968) separated the external
invertase from the internal by ammonium sulfate precipitation method. They obtained
the internal invertase (non-glycosylated) in precipitated form at ammonium sulfate
saturation (70 %) whereas most of the external form (glycosylated) remained in the
supernatant. In most of the studies, extracellular yeast invertase being glycoprotein
remains soluble in concentrated ammonium sulfate because of its carbohydrate content
(Lehle et al., 1979; Rodriguez et al., 1995). According to Belcarz et al. (2002a) and
Cipollo and Trimble (2002), the extracellular invertase is glycoprotein in nature where as
intracellular invertase is simple protein. The major enzyme fraction was eluted at 0.2 M
NaCI using DEAE-Sephadex column chromatography. After SDS-PAGE, only one
protein band of molecular mass of 110 kDa containing 48 % carbohydrate was found.
Milintawisamai et al. (2007) found extracellular invertase from C. humicolus as the
result from 60-100 % ammonium sulfate saturation followed by DEAE column
chromatography and eluted extracellular protein fraction using 0.3-0.35 M NaCl with
molecular mass of 110 kDa as a single active fraction. In Xanthophyllomyces
dendrorhous invertase was found to be as glycoprotein with molecular mass of 160 kDa
(Linde et al., 2009). The results shows the specific activity of the purified extracellular
invertase estimated to be as 1915 U/mg, which is about 15 fold increase than crude
enzyme with final activity recovery of 38 %. Chan et al. (1991) purified extracellular
invertase in Saccharomyces uvarum by one-step DEAE chromatography with enzyme
recovery of 84 % and 9 fold purification.
The invertase was characterized by optimizing pH, temperature, additives, Km
and Vmax. The optimum pH (5.0) and temperature (60оC) for glycosylated invertase were
recorded. Chavez et al. (1997) obtained invertase from Candida utilis with molecular
weight of 150 kDa at pH of 5.5 and temperature of 60-75оC. The addition of different
additives such as NaCl, KCl, MnCl2, EDTA, BaCl2, MgCl2, CuSO4, HgCl2, CoCl2,
CuCl2, FeSO4, CaCl2 and ZnSO4 on glycosylated invertase was achieved. Of the all,
MgCl2, MnCl2 and CoCl2 was found to be as slightly stimulatory with relative activity
from 102-111 % while remaining caused partially or comletely inhibitory effect on
155
glycosylated invertase. The maximum relative activity (111 %) was exhibited by MnCl2.
Similar findings by Guimaraes et al., (2007) also stimulated invertase activity by
Mn+2(57 %) in Aspergillus ochraceus. In contrast, Rubio et al. (2002) obtained 3-fold
increase in stimulation of glycoprotein invertase activity by adding Mg2+ and Ca2+ ions.
Almost completely enzyme inhibition by HgCl2, CuSO4 and CuCl2 was observed. Fujita
et al. (1990) reported that invertase from Arthrobacter sp. inactivated completely by 1
mM Cu2+, Hg2+, Ag+ and SDS. By using Lineweaver-burk plot, the Km and Vmax values
for sucrose were observed to be as 1.8 mM and 1429 U/ml/min, respectively. Workman
and Day (1983) reported the Km value for sucrose was 13.6 mM in Kluyveromyces
fragilis. The similar finding with the Bhatti et al. (2006) obtained Km value of 3.57 mM
for sucrose in Fusarium solani. Hernalsteens and Maugeri (2008) gave the Km (13.4 g/l)
and Vmax (21 μmol/ml/min) for sucrose by invertase in Candida sp.
As intracellular invertase enzyme was found to be in two forms (L and S- form).
The procedure of ammonium sulfate was also used for the separation of two forms of
intracellular enzyme. Carlson et al. (1983) and Batista et al. (2004) reported the presence
of two forms of invertase (glycosylated and non-glycosylated) in S. cerevisiae. The L-
form of invertase being glycoprotein was purified by two-step of purification as applied
for extracellular invertase whereas S-form of invertase was purified by three-steps. S-
form being non-glycosylated was recovered from ammonium sulfate saturation (20-85
%) in the precipitated form. The active fraction of S-form was eluted at 0.3 M NaCl
using DEAE-Sephadex. After Gel-filteration, a single band of 55 kDa free of
carbohydrate was found. Kern et al. (1992) found intracellular invertase in yeast at NaCl
(0.15-0.3 M) by using Q-Sepharose and found it of 115 kDa. Similar finding by Trimble
and Maley (1977) who obtained non-glycosylated invertase primarily composed of two
subunits (60 kDa) in S. cerevisiae. The specific activity of the purified intracellular S-
form and L-form of invertase were estimated to be as 1670 U/mg, 1964 U/mg and fold
purification of 13, 19 with recovery of 13 % and 32 %, respectively. The optimum pH 5
but temperature (50 оC) was found to be optimal. The carbohydrate free invertase is
prone to aggregation in the denatured state at high temperature; the protein moieties of
cytoplasmic non-glycosylated and external invertase were identical (Schulke and
Schmid, 1988). The relationship between these two enzymic forms suggested as
precursor-product by Rodriguez et al. (1978).
The effect of different additives as mentioned above on relative activity of non-
glycosylated invertase was found to be same but with overall slight decrease. In contrast
156
with glycosylated invertase, the nonglycosylated intracellular invertase was found be
sensitive towards proteolytic attack (Williams et al., 1985). The Km and Vmax values for
intracellular non-glycosylated were found to be as 1.2 mM and 909 U/ml/min,
respectively. Similar findings by Belcarz et al. (2002a), obtained Km values against
sucrose for S (slow) and F (fast) forms of invertase in Candida utilis as 2 and 1.5 mM,
respectively. Buttner et al. (1990) determined Km value (71-83 mM) for sucrose in
Trichosporon adeninovorans for two internal invertases.
157
CONCLUSION
The use of the invertase in food and pharmaceutical industry has been increasing with
growing demands of population. Microbial source is preferred over plant and animal
sources. In this context, Saccharomyces cerevisiae was used as organism of choice for
the production of invertase. The parent culture (IS-66) was isolated from various soil and
fruit samples and improved through mutagenesis (NA and EMS) and screening. The
potent mutant strain (EMS-42) gave maximum extracellular production of enzyme (34.2
U/ml), which was about 6 fold higher than wild-culture (5.6 U/ml). The mutant strain
EMS-42 was cultured on the medium containing 2-deoxy-D-glucose (2dg) and thus high
yielding stable colonies were obtained at 0.04 mg/ml. The optimization of the culture
conditions were carried out in shake flask and fermentor for mutant strain of EMS-42.
The over all increase in enzyme production in shake flask and fermentor was found to be
as 9 and 14 fold, respectively. The intracellular enzyme activity (57 U/ml) was also
noticed in the mutant cells of EMS-42 grown in the fermentor. After optimizing
sonication parameters, the maximum specific activity (106 U/mg of protein) for
intracellular invertase was achieved. The whole cells of S. cerevisiae EMS-42 were
immobilized into calcium alginate beads for the production of inverted syrup with
maximum sucrose hydrolysis (76.3 %) under optimum conditions. A 15 fold purification
of extracellular invertase with recovery of 38 % was achieved. The molecular weight of
glycosylated invertase was found to be as 110 kDa with 48 % carbohydrate content
whereas 55 kDa for intracellular non-glycosylated invertase was recorded.
158
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