EXTRACELLULAR PECTINASE PRODUCTION BY Aspergillus ...

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EXTRACELLULAR PECTINASE PRODUCTION BY Aspergillus niger HFM 8 THROUGH SOLID SUBSTRATE FERMENTATION USING POMELO PEELS AS A SUBSTRATE NOR HAWANI BINTI SALIKIN UNIVERSITI SAINS MALAYSIA 2014

Transcript of EXTRACELLULAR PECTINASE PRODUCTION BY Aspergillus ...

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EXTRACELLULAR PECTINASE PRODUCTION

BY Aspergillus niger HFM 8 THROUGH SOLID

SUBSTRATE FERMENTATION USING

POMELO PEELS AS A SUBSTRATE

NOR HAWANI BINTI SALIKIN

UNIVERSITI SAINS MALAYSIA

2014

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EXTRACELLULAR PECTINASE PRODUCTION BY Aspergillus niger

HFM 8 THROUGH SOLID SUBSTRATE FERMENTATION USING

POMELO PEELS AS A SUBSTRATE

by

NOR HAWANI BINTI SALIKIN

Thesis submitted in fulfilment of the requirement for the degree of Master

of Science

August 2014

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ACKNOWLEDGEMENT

By The Name of Allah The Al-Mighty

First of all, Alhamdulillah, the most sincere gratitude to Allah The Al-

Mighty. Only with His blessing and endless help, this research could be successfully

completed even though life is sometime harsh and wretched. Think positively,

willing to face the saddle and only with His permission everything is going

smoothly.

A big appreciation goes to my inspiring supervisor, Professor Darah Binti

Ibrahim. The quality of her supervision is the best. Thank you for the precious

advice, guidance and motivation that brace my enthusiasm in accomplishing this

research. Thank you indeed.

The deepest gratitude also goes to Associate Professor Dr. Rosma Binti

Ahmad for her precious motivations and indulgence which instigate me to do the

best. Also for Puan Noor Hasmah Binti Idris and Puan Nazariah binti Noordin from

School of Industrial Technology who are very supportive and thoughtful. Really

appreciate all of your kindness.

My fully appreciation is also dedicated to Universiti Sains Malaysia and

Malaysia Ministry Of Higher Education for the splendid opportunity given to me in

pursuing this Master degree with fully financial support under the scheme of ASTS

USM or SLAB. I also would like to thank all of the lecturers and staffs in School Of

Biological Science especially in the Microscopy Unit for their support and help.

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Also, my full appreciation is devoted to my beloved parents, En. Salikin Bin

Haji Ihsan and Puan Marinah Binti Bibit who are inspiring me to be a positive-

minded individual and always standing beside me in every moment of my life. Also

to my beloved husband, En. Hasrul Hafizan Bin Rahmatullah, thank you very much

for your understanding and endless encouragement. Indeed, this victory is a reflect to

their blessing, love and endless prayer that I received from them. To my lovely kids;

Nur Iman Batrisya and Fawwaz Ziqri, both of you are heartening my life and

fortifying me to struggle.

My special thanks also goes to my understanding and happy family of IBRL

(Industrial Biotechnology Research Laboratory) especially Dr. Lim Sheh Hong (Post

Doctorate), Dr. Lee Chee Keong, Syarifah, Lee Kok Chang, Tong Woei Yen, Nisha,

Hareetharan, Anuradha, Taufiq, Norhaida, Tisha, Afifah, Chong Chai Lee, Nor

Azlinah, Husna, Yu, Angeline, Olivia, Mat and Gee who have been sharing the joys

and tears together. The moments we share are definitely unforgettable.

My apologies and request of forgiveness for those names never mentioned. I

would have never been able to complete this project without all of the blessings and

support from you. Again, thank you all for making this as a reality.

Wassalam

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TABLE OF CONTENTS

Page

ACKNOWLEDGEMENT ii

TABLE OF CONTENTS iv

LIST OF TABLES xiv

LIST OF FIGURES xvi

LIST OF PLATES xix

LIST OF SYMBOLS AND ABBREVIATION xxi

ABSTRAK xxiii

ABSTRACT xxv

CHAPTER ONE : INTRODUCTION

1.1 General introduction on pectinase research 1

1.2 Rational of study 4

1.3 Objective of research 5

CHAPTER TWO : LITERATURE REVIEW

2.1 Growth of agricultural industry worldwide 6

2.2 Agro-industrial residues as a detrimental issue 7

2.3 Utilization of agro industrial residues in pectinase production 10

2.4 Pomelo peel as a potential substrate for pectinase production 13

2.5 Pectic substances 15

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2.5.1 Classification of pectic substances 17

2.5.1.1 Protopectin 17

2.5.1.2 Pectic acid 18

2.5.1.3 Pectinic acid 18

2.5.1.4 Pectin 19

2.5.2 Structure of pectin 21

2.5.2.1 Primary structure of pectin 21

2.5.2.2 Secondary structure of pectin 22

2.6 Pectinase 23

2.7 Classification of pectinase enzyme 26

2.7.1 Polygalacturonase (PG) 26

2.7.2 Polymethylgalacturonase (PMG) 27

2.7.3 Protopectinase 27

2.7.4 Pectate lyases (PGL) 28

2.7.5 Pectin lyases (PL) 28

2.7.6 Pectin methyl esterase (PME) 29

2.7.7 Rhamnogalacturonan rhamnohydrolases 30

2.7.8 Rhamnogalacturonan acetylesterases 30

2.8 Microorganisms as potential pectinase producer 33

2.8.1 Fungi as a potential pectinase producer 34

2.8.2 Bacteria as a potential pectinase producer 36

2.8.3 Yeasts as a potential pectinase producer 37

2.9 Biotechnological application of microbial pectinase 41

2.9.1 Acidic pectinase 41

2.9.1.1 Fruit juices extraction and clarification 41

2.9.1.2 Industrial wines making and manufacturing 44

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2.9.1.3 Peel oil extraction 45

2.9.1.4 Preparation of dried animal feed from

citrus fruits 45

2.9.2 Alkaline pectinase 46

2.9.2.1 Retting and degumming of plant fiber 46

2.9.2.2 Pectic wastewater treatment 47

2.9.2.3 Vegetable oil extraction 48

2.9.2.4 Coffee and tea fermentation 49

2.10 General introduction on solid substrate fermentation (SSF) 50

2.10.2 Fungal interaction in solid substrate fermentation (SSF) 55

2.10.3 General considerations on enzyme production through

solid substrate fermentation (SSF) 59

2.10.3.1 Selection of substrate 59

2.10.3.2 Screening of potential microorganism 60

2.10.3.3 Physical parameters governing the

enzyme production 62

2.10.3.4 Chemical parameters governing the enzyme

Production 68

2.11 Concluding remarks 72

CHAPTER 3 : MATERIALS AND METHODS

3.1 Microorganisms 73

3.2 Preparation of inoculums 73

3.3 Preparation of substrate 74

3.4 Primary screening for potential fungal pectinase producer 74

3.5 Secondary screening for potential fungal pectinase producer 75

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3.6 Extraction of crude pectinase 76

3.7 Identification of isolate HFM 8 as a potential fungal pectinase producer 76

3.7.1 Macroscopic observation of fungal isolate HFM 8

on different agar media 76

3.7.2 Microscopic observation on fungal isolate HFM 8

under light microscope 77

3.7.3 Microscopic observation on fungal isolate HFM 8

under scanning electrone microscope (SEM) 77

3.7.4 Identification of fungal isolate HFM 8

through molecular approach 78

3.7.4.1 Extraction of DNA 78

3.7.4.2 Polymerase chain reaction (PCR) amplification 80

3.7.4.3 Purification of PCR product 81

3.8 Analytical methods 82

3.8.1 Determination of pectinase activity 82

3.8.2 Determination of fungal growth 83

3.8.3 Determination of total protein concentration 84

3.9 Improvement of cultural conditions for pectinase production

by Aspergillus niger HFM 8 through a flask system 85

3.9.1 Improvement of physical conditions 85

3.9.1.1 Initial profiles of pectinase production and fungal

growth of Aspergillus niger HFM 8 before the

improvement of physical conditions 86

3.9.1.2 Effect of different particle size of substrate 87

3.9.1.3 Effect of different initial moisture content 87

3.9.1.4 Effect of different incubation temperature 88

3.9.1.5 Effect of different initial pH of moisturizing agent 88

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3.9.1.6 Effect of different size of inoculum 89

3.9.1.7 Effect of mixing frequency 90

3.9.1.8 Effect of different extracting solvent during

enzyme recovery 90

3.9.1.9 Profile of pectinase production and fungal

growth of Aspergillus niger HFM 8

after physical improvement 91

3.9.2 Improvement of chemical conditions 91

3.9.2.1 Effect of different carbon source supplementation 92

3.9.2.2 Effect of different nitrogen source supplementation 92

3.9.2.3 Effect of different concentrations of the selected

nitrogen source supplementation 93

3.9.2.4 Profile of pectinase production and fungal

growth of Aspergillus niger HFM 8

after physical and chemical improvement 93

3.10 Improvement of pectinase production and fungal growth

of Aspergillus niger HFM 8 in a tray system 94

3.10.1 Initial profiles of pectinase production and growth

of Aspergillus niger HFM 8 under different substrate thickness 94

3.10.2 Effect of different initial moisture content 95

3.10.3 Effect of different inoculum size 95

3.10.4 Effect of different mixing frequency 96

3.10.5 Profiles of pectinase production and growth of

Aspergillus niger HFM 8 in a tray system after the

improvement of physical conditions 96

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3.11 Statistical analysis 97

3.12 Purification of pectinase 97

3.12.1 Ultrafiltration 97

3.12.2 Anion exchange column chromatography 98

3.12.2.1 Determination of appropriate pH for chromatography 98

3.12.2.2 Determination of elution condition for chromatography 99

3.12.2.3 Fraction collection through anion exchange

column chromatography 99

3.12.3 Gel filtration 100

3.13 Pectinase purification analysis with Sodium Dodecyl

Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 101

3.14 Silver staining 102

3.15 Determination of molecular weight 103

3.16 Characterization of purified pectinase 104

3.16.1 Determination of optimum temperature 104

3.16.2 Temperature stability of purified pectinase 104

3.16.3 Optimal pH determination 105

3.16.4 pH stability of purified pectinase 105

3.16.5 Substrate specificity of purified pectinase 106

3.16.6 Effect of different reagents and metal ions on

purified pectinase activity 106

CHAPTER 4 : RESULTS

4.1 Primary screening of potential fungal pectinase producer 107

4.2 Secondary screening of potential fungal pectinase producer 113

4.3 Identification of isolate HFM 8 as a potential fungal pectinase producer 116

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4.3.1 Macroscopic observation of fungal isolate HFM 8

on different agar media 116

4.3.2 Microscopic observation of fungal isolate HFM 8

through Light Microscope (LM) 126

4.3.3 Microscopic observation on fungal isolate HFM 8 under

Scanning Electrone Microscope (SEM) 129

4.3.4 Identification of fungal isolate HFM 8 through molecular approach 132

4.4 Improvement of cultural conditions for extracellular pectinase

production by Aspergillus niger HFM 8 through a flask system 135

4.4.1 Initial profile before the improvement of physical conditions 135

4.4.2 Improvement of physical condition for extracellular

pectinase production by Aspergillus niger HFM 8 139

4.4.2.1 Effect of different size of substrate 139

4.4.2.2 Effect of different initial moisture content 141

4.4.2.3 Effect of different incubation temperature 143

4.4.2.4 Effect of different initial pH 145

4.4.2.5 Effect of different inoculum size 147

4.4.2.6 Effect of mixing frequency 149

4.4.2.7 Effect of different extracting solvent during

the enzyme recovery 151

4.4.3 Profile of pectinase activity and growth of Aspergillus niger

HFM 8 after the improvement of physical conditions 153

4.4.4 Improvement of chemical condition for extracellular

pectinase production by Aspergillus niger HFM 8 156

4.4.4.1 Effect of external carbon source supplementation 156

4.4.4.2 Effect of external nitrogen source supplementation 159

A

C

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4.4.4.3 Effect of different concentration of urea supplementation 161

4.4.5 Profile of pectinase activity and fungal growth of

Aspergillus niger HFM 8 after chemical improvement

of cultural condition 163

4.4.6 Comparison of pectinase activity and growth of

Aspergillus niger HFM 8 before and after the improvement

of physical and chemical conditions 166

4.5 Improvement of physical cultural conditions for extracellular

pectinase production by Aspergillus niger HFM 8 through

a tray system 169

4.5.1 Initial profile in different substrate thickness before the

improvement of physical conditions 169

4.5.2 Improvement of physical conditions for extracellular

pectinase production through a tray system 173

4.5.2.1 Effect of initial moisture content 173

4.5.2.2 Effect of inoculum size 175

4.5.2.3 Effect of mixing frequency 177

4.5.3 Final profile after the improvement of physical conditions

in a tray system 179

4.6 Purification of pectinase 183

4.7 Molecular weight determination of purified pectinase 188

4.8 Characterization of purified pectinase 191

4.8.1 Determination of optimal temperature 191

4.8.2 Stability of purified pectinase in different temperature 192

4.8.3 Determination of optimal pH 194

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4.8.4 Stability of purified pectinase in different pH 195

4.8.5 Determination of substrate specificity 197

4.8.6 Effects of reagents and metal ions 198

CHAPTER 5 : DISCUSSION

5.1 The feasibility of pomelo peel as a cheap substrate for fungal

pectinase production through solid substrate fermentation (SSF) 200

5.2 Screening of potential fungal pectinase producer 202

5.2.1 Primary screening of potential fungal pectinase producer 202

5.2.2 Secondary screening of potential fungal pectinase producer 204

5.3 Identification of fungal isolate HFM 8 207

5.4 Improvement of physicochemical conditions for pectinase

production through solid substrate fermentation employing

a shake flask system 209

5.4.1 Improvement of physical cultural conditions 209

5.4.2 Improvement of chemical cultural conditions 222

5.5 Improvement of pectinase activity by Aspergillus niger

HFM 8 under physicochemical optimized conditions 228

5.6 Large scale pectinase production employing a tray system 230

5.7 Improvement of pectinase activity by Aspergillus niger

HFM 8 under physicochemical optimized conditions employing

a tray system 240

5.8 Purification of pectinase and molecular weight determination 241

5.9 Characterization of the purified pectinase 248

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CHAPTER 6 : CONCLUSION AND RECOMMENDATION

6.1 Conclusion 255

6.2 Future recommendation 257

REFFERENCES 258

APPENDICES

Appendix A : Standard curve for pectinase activity determination

at 520 nm based on Galacturonic acid as a reducing sugar

Appendix B : Standard curve of glucosamine content for fungal growth

determination at 530 nm

Appendix C : Standard curve for protein determination based on Bovine

Serum Albumin (BSA) at 750 nm

Appendix D : Preparation of SDS-PAGE reagents

Appendix E : The consensus sequence of Aspergillus niger HFM 8

LIST OF PUBLICATIONS

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LIST OF TABLES

Page

Table 2.1 Agricultural waste generation in Asia 8

Table 2.2 Consumption of assorted agro industrial residues in pectinase

production through solid substrate fermentation (SSF) 11-12

Table 2.3 Percentage of pectin in different fruits and vegetables 20

Table 2.4 Classification of pectinase enzyme 31

Table 2.5 Acidic and alkaline microbial pectinase producer 39-40

Table 2.6 Contribution of solid substrate fermentation towards

economical sector 51

Table 2.7 Application of microorganisms in solid substrate

fermentation process 56

Table 4.1 Diameter of hydrolysis zone and fungal colony on pectinase

screening agar medium after 24 hours incubation period 108-109

Table 4.2 Diameter of hydrolysis zone and fungal colony on pectinase

screening agar medium after 48 hours of incubation period 110-111

Table 4.3 Pectinase activity and growth of 43 fungal isolates on

pomelo peel substrate through solid substrate fermentation 114-115

Table 4.4 Macroscopic features of fungal isolate HFM 8 cultivated

on different agar medium 117-119

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Table 4.5 Review on comparison of pectinase activity and growth of

Aspergillus niger HFM 8 in non-improved and improved

cultural conditions 168

Table 4.6 Comparison of the highest pectinase activity and growth of

Aspergillus niger HFM 8 depicted under the cultivation in

different substrate thickness 172

Table 4.7 Summary of non-improved and improved physical condition

in cultivation system employing a tray system 182

Table 4.8 Summary of pectinase enzyme purification 187

Table 4.9 The effects of reagents and metal ions towards purified pectinase 199

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LIST OF FIGURES

Page

Figure 2.1 Primary cell wall structure in most flowering plants 16

Figure 2.2 Primary structure of pectin 21

Figure 2.3 Secondary structure of pectin 22

Figure 2.4 Pectinase reaction against pectic substances 32

Figure 2.5 Pectinase application in the production of apple juice 43

Figure 2.6 Model or schematic drawing of fungal growth in solid

substrate fermentation system 58

Figure 4.1 Initial profile of pectinase activity and growth of Aspergillus

niger HFM 8 before physical condition improvement 136

Figure 4.2 Effect of different size of substrate on pectinase activity and

growth of Aspergillus niger HFM 8 140

Figure 4.3 Effect of different initial moisture content on pectinase

activity and growth of Aspergillus niger HFM 8 142

Figure 4.4 Effect of different incubation temperature on pectinase

activity and growth of Aspergillus niger HFM 8 144

Figure 4.5 Effect of different initial pH on pectinase activity and

growth of Aspergillus niger HFM 8 146

Figure 4.6 Effect of different inoculum size on pectinase activity

and growth of Aspergillus niger HFM 8 148

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Figure 4.7 Effect of different mixing frequency on pectinase activity

and growth of Aspergillus niger HFM 8 150

Figure 4.8 Effect of different extracting solvent consumption on

pectinase activity and growth of Aspergillus niger HFM 8 152

Figure 4.9 Profile of pectinase activity and growth of Aspergillus niger

HFM 8 after the improvement of physical condition 154

Figure 4.10 Effect of supplementation of different carbon source on

pectinase activity and growth of Aspergillus niger HFM 8 157

Figure 4.11 Effect of supplementation of different nitrogen source on

pectinase activity and growth of Aspergillus niger HFM 8 160

Figure 4.12 Effect of urea supplementation in different concentration on

pectinase activity and growth of Aspergillus niger HFM 8 162

Figure 4.13 Profile of pectinase activity and growth of Aspergillus niger

HFM 8 after the improvement of chemical condition 164

Figure 4.14 Comparison of pectinase activity and growth of Aspergillus

niger HFM 8 before and after the improvement of

physical and chemical conditions in solid substrate

fermentation through a flask system 167

Figure 4.15 Initial profile of pectinase activity and growth of Aspergillus

niger HFM 8 in tray system with different substrate thickness 170

Figure 4.16 Effect of different initial moisture content on pectinase

activity and growth of Aspergillus niger HFM 8 in a tray system 174

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Figure 4.17 Effect of different inoculum size on pectinase activity and

growth of Aspergillus niger HFM 8 in a tray system 176

Figure 4.18 Effect of different mixing frequency on pectinase activity and

growth of Aspergillus niger HFM 8 in a tray system 178

Figure 4.19 Final profile of pectinase activity and growth of Aspergillus

niger HFM 8 in a tray system after the improvement

of physical conditions 180

Figure 4.20 Elution profile of pectinase produced by Aspergillus niger

HFM 8 in initial stage of purification employing anion

exchange column chromatography (DEAE Sephadex) 184

Figure 4.21 Elution profile of pectinase produced by Aspergillus niger

HFM 8 in second stage of purification employing

gel filtration column chromatography (Sephadex G-100) 185

Figure 4.22 Molecular weight determination of purified pectinase using

SDS-PAGE 190

Figure 4.23 The effect of different temperatures on the purified

pectinase activity 191

Figure 4.24 Stability of the purified pectinase in different temperatures 193

Figure 4.25 The effect of different pH on the purified pectinase activity 194

Figure 4.26 Stability of the purified pectinase in different pH conditions 196

Figure 4.27 Substrate specificity of the purified pectinase 197

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LIST OF PLATES

Page

Plate 4.1 Hydrolysis zone formation 112

Plate 4.2 Morphology of isolate HFM 8 on Potato Dextrose Agar (PDA)

plates after seven days of incubation period 120

Plate 4.3 Morphology of isolate HFM 8 on Oatmeal Agar (OA) plates

after seven days of incubation period 121

Plate 4.4 Morphology of isolate HFM 8 on Czapex Yeast Agar (CYA)

plates after seven days of incubation period 122

Plate 4.5 Morphology of isolate HFM 8 on Creatine Sucrose Agar (CREA)

plates after seven days of incubation period 123

Plate 4.6 Morphology of isolate HFM 8 on Malt Extract Agar (MEA)

plates after seven days of incubation period 124

Plate 4.7 Morphology of isolate HFM 8 on Yeast Extract Agar (YES)

plates after seven days of incubation period 125

Plate 4.8 Microscopic features of the Fungal Isolate HFM 8 under

a light microscope observation 127 - 128

Plate 4.9 SEM micrographs of the fungal isolate HFM 8

microscopic features 130 - 131

Plate 4.10 Purified PCR product of Aspergillus niger HFM 8 133

Plate 4.11 Top 16 blast hit sequence of sample HFM 8 through

BLAST 2.2.26+ Search Tool database 134

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Plate 4.12 Growth of Aspergillus niger HFM 8 on pomelo peel

substrate 138

Plate 4.13 SDS-PAGE of purified pectinase produced by

Aspergillus niger HFM 8 with 12.5% polyacrylamide 189

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LIST OF SYMBOLS AND ABBREVIATION

% Percent

± Plus minus

oC Degree Celcius

α Alpha

β Beta

® Registered Trade Mark

µ Micro

∆ Delta

A Absorbance

g Gram

L Litre

mg Milligram

ml Millilitre

mm Milimeter

cm Centimeter

µg Microgram

µmol Micromol

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M Molar

nm Nanometer

v/v Volume over volume

w/v Weight over volume

U Unit of activity

U/ml Unit of activity per millilitre enzyme solution

U/g Unit of activity per gram substrate

V Volt

g/L Gram per litre

g/mol Gram per mol

bp Base pair

Rf Relative mobility

kDa Kilo Dalton

SDS Sodium dodecyl sulphate

SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis

SEM Scanning Electron Microscope

CMC Carboxymethylcellulose

rpm Revolution per minute

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PENGHASILAN PEKTINASE EKSTRASEL OLEH Aspergillus niger HFM 8

MELALUI FERMENTASI SUBSTRAT PEPEJAL MENGGUNAKAN KULIT

LIMAU BALI SEBAGAI SUBSTRAT

ABSTRAK

Penimbunan kulit limau bali (Citrus grandis) sebagai sisa industri tani setelah

pemprosesan buah-buahan sewajarnya dieksploitasi sepenuhnya dengan bantuan

mikroorganisma untuk menghasilkan produk lain yang mempunyai nilai komersil

seperti enzim. Kulit limau bali telah dibuktikan mengandungi pektin pada kadar yang

tinggi dan berperanan sebagai penggalak untuk penghasilan pektinase oleh

mikroorganisma melalui kaedah fermentasi substrat pepejal (SSF). Pencilan HFM 8

telah dipilih sebagai kulat penghasil pektinase yang terbaik dengan aktiviti

maksimum sebanyak 112.79 ± 17.2 U/g substrat setelah dua kali proses penyaringan

melibatkan penyaringan primer dan penyaringan sekunder. Pencilan ini dikenalpasti

sebagai Aspergillus niger HFM 8 setelah melakukan proses identifikasi berdasarkan

sifat-sifat koloni dan juga secara molekul. Penambahbaikan keadaan fizikal dan

kimia adalah ditekankan dalam kajian ini dengan objektif untuk meningkatkan

produktiviti pektinase oleh mikroorganisma tersebut. Hasilnya, peningkatan

pektinase sebanyak 48.82% telah berjaya dihasilkan oleh Aspergillus niger HFM 8

apabila ditumbuhkan di dalam sistem fermentasi keadaan pepejal menggunakan

kelalang di dalam keadaan fizikal dan kimia yang telah dioptimumkan iaitu dengan

menggunakan substrat bersaiz 0.75 mm, kadar kelembapan sebanyak 60% (i/b)

(menggunakan air suling steril pada pH 5.0), saiz inokulum sebanyak 1 x 104

bilangan spora /ml, pengeraman pada suhu bilik (30 ± 2oC), penambahan sumber

nitrogen iaitu 0.1% (b/b) urea, pengeraman tanpa kesan pengadukan dan penggunaan

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larutan penimbal asetat (pH 4.5, 0.1 M) untuk pengekstrakan pektinase. Penghasilan

pektinase oleh Aspergillus niger HFM 8 telah dilakukan pada skala besar dengan

menggunakan sistem dulang. Dalam keadaan fizikal yang telah dioptimumkan iaitu

dengan ketebalan substrat sebanyak 1.0 cm bersamaan 60 g serbuk kulit limau bali

(saiz 0.75 mm), 40% (i/b) kelembapan (air suling steril pada pH 5.0), saiz inokulum

sebanyak 1 x 106 bilangan spora/ml, pengeraman pada suhu bilik (30 ± 2

oC),

penambahan 0.1% (b/b) urea sebagai sumber nitrogen tambahan, kesan pengadukan

sekali pada setiap 24 jam dan penggunaan larutan penimbal asetat (pH 4.5, 0.1M)

untuk pengekstrakan pektinase, didapati produktiviti pektinase telah meningkat

sebanyak 23.00%. Secara perbandingan, penghasilan enzim pektinase telah

meningkat sebanyak 191.41% daripada pemfermentasian di dalam sistem kelalang ke

sistem dulang yang membuktikan potensi Aspergillus niger HFM 8 untuk

menghasilkan lebih banyak pektinase pada skala industri. Penulenan enzim

seterusnya dijalankan dengan melaksanakan kaedah kromatografi pada dua peringkat

melibatkan penukaran ion (DEAE Sephadex) dikuti penapisan gel (Sephadex G-

100). Prosedur SDS-PAGE mengesahkan bahawa berat molekul pektinase tulen ini

adalah 55.37 kDa. Enzim ini bertindak-balas secara optimum pada 40oC dan pH 4.0

di samping stabil pada suhu 35oC ke 45

oC dalam julat pH 3.5 ke pH 4.0. Pektinase

tulen ini didapati bertindak-balas sangat spesifik terhadap pektin tanpa melibatkan

peranan agen pelarut atau ion logam untuk aktiviti yang optimum. Sebaliknya, FeCl3

dan AgNO3 didapati telah merencatkan tindak-balas pektinase. Secara jelasnya, kulit

limau bali terbukti dapat diaplikasikan untuk penghasilan pektinase secara komersil

melalui kaedah fermentasi substrat pepejal di mana enzim pektinase yang dihasilkan

berpotensi untuk digunakan dalam industri pengekstrakan dan penjernihan jus buah-

buahan.

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EXTRACELLULAR PECTINASE PRODUCTION BY Aspergillus niger

HFM 8 THROUGH SOLID SUBSTRATE FERMENTATION USING

POMELO PEELS AS A SUBSTRATE

ABSTRACT

The accumulation of pomelo peels (Citrus grandis) as the agro-industrial

residues after the fruit processing and manufacturing should be maximally exploited

for the microbial bioconversion of the detrimental wastes into a value-added by

product such as enzymes. Pomelo peels are authenticated as rich in pectin, acting as

the main inducer for pectinase enzyme production by microorganism through solid

substrate fermentation system (SSF). Isolate HFM 8 revealed as the best fungal

pectinase producer after the implementation of double steps screening processes

(primary and secondary screenings) with maximal activity of 112.79 ± 17.2 U/g

substrate. Based on the phenotypic features and molecular identification, this novel

isolate was denoted as Aspergillus niger HFM 8. The improvement of

physicochemical cultural conditions was emphasized in this current research with an

attempt to enhance the pectinase productivity by the microorganism. An

augmentation of 48.82% on pectinase yield by Aspergillus niger HFM 8 in a shake

flask system was recorded after being cultivated in an enhanced physicochemical

condition constituted of 0.75 mm particle size of substrate, 60% (v/w) of initial

moisture (sterilized distilled water, pH 5.0), inoculum size of 1 x 104 spores/ml,

incubation in room temperature (30 ± 2oC), inclusion of 0.1% (w/w) urea as the

external nitrogen source, no mixing effect (static condition during fermentation) and

utilization of acetate buffer (pH 4.5, 0.1 M) as the extracting solvent during the

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enzyme recovery. A large scale pectinase production by Aspergillus niger HFM 8

was then conducted employing a tray system. Under the optimized physical

conditions comprised of 1.0 cm substrate depth equalized to 60 g pomelo peels

substrate (0.75 mm of particle size), 40% (v/w) of initial moisture (sterilized distilled

water, pH 5.0), inoculum size of 1 x 106 spores/ml, incubation in room temperature

(30 ± 2oC), inclusion of 0.1% (w/w) urea as the external nitrogen source, inclusion of

mixing effect once for every 24 hours interval and utilization of acetate buffer (pH

4.5, 0.1 M) as the extracting solvent during the enzyme recovery, an amount of

23.00% increment on pectinase productivity was detected. Comparatively, the fungal

pectinase production boosted up to 191.41% of increment from a shake flask system

to a tray system indicating the potentiality of Aspergillus niger HFM 8 to be

industrially employed particularly in pectinase production. Further purification of the

crude pectinase was conducted by practising double steps chromatography

constituted of anion exchange (DEAE Sephadex) followed by gel filtration

(Sephadex G-100). SDS-PAGE procedure determined the molecular weight of the

purified pectinase as 55.37 kDa. This enzyme was found optimal at 40oC and pH 4.0

whereby the catalytic reaction was stable between 35oC to 45

oC and in condition of

pH 3.5 to pH 4.0. The purified pectinase was highly specific on pectin as the

substrate whilst no solvents or metal ions found enhancing the catalytic reaction.

Inversely, FeCl3 and AgNO3 prohibit the pectinase reaction. Apparently, pomelo

peels are markedly applicable for commercial pectinase production employing a

solid substrate fermentation system whereby the produced enzyme is highly

applicable to be incorporated into the fruit juice extraction and clarification process.

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CHAPTER 1

INTRODUCTION

1.1 General introduction on pectinase research

Pectinase is one of the forthcoming enzymes at the moment which constitutes

25% of the global food enzyme market (Jayani et al., 2005). This group of

heterogenous enzymes catalyze the degradation of pectic substances in plant

structures such as pectin, pectic acid or oligo-D-galacturonate in different mode of

actions including transelimination and hydrolysis (Favela-Torres et al., 2006; Jayani

et al., 2010 ; Heidar et al., 2011). In nature, pectinase is mainly found in plant tissue

to facilitate the cell wall extension and softening some tissues during the maturation

of part of the plant such as fruits. In addition, pectinase also catalyze the

decomposition of plant residues and contribute to the carbon cycle and ecosystem

stabilization. During the phytopathogenic process, pectinase is secreted copiously by

microorganisms to infect the host plant (Siddiqui et al., 2012). For industrial sector,

pectinase is vastly applied in fruit juice extraction and clarification, textile and fiber

processing and manufacturing, coffee and tea fermentations, industrial pectic

wastewater treatment, paper making, purification of plant virus, poultry feed

production and oil extraction (Saito et al., 2004; Sharma and Satyanarayana, 2006;

Siddiqui et al., 2012). Furthermore, this novel enzyme is also utilized in the

extraction of specific plant bio-compounds from raspberry residues, tomatoes, grape

skin and also apple skin (Neagu et al., 2012). Undeniably, pectinase is a profitable

enzyme which has enormous significant contributions to the mankind.

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In order to accomplish the increasing worldwide demand, industrial scale

production of pectinase enzyme has been generated either through solid substrate

fermentation (SSF) or submerged fermentation (SmF) (Neagu et al., 2012). It has

been proven that pectinase is an inducible enzyme specifically with the visibility of

pectic substances as the substrate (Reddy and Sreeramulu, 2012). In solid substrate

fermentation, the microorganisms grow together with the metabolites production

occur on the surface of the solid substrate in near absence of free water (low water

activity) (Heidar et al., 2011) whilst in submerged fermentation, the microorganisms

grow in free flowing liquid substrate such as broth and the metabolites are secreted

into the liquid medium (Subramaniyam and Vimala, 2012). Comparatively, in

producing bioactive compound such as pectinase enzyme, fungi is more applicable in

solid substrate fermentation rather than submerged fermentation which is more

suitable for bacteria and yeast due to the high water activity (aw). Low water activity

in solid substrate fermentation generally mimicing the nature environment favoured

by fungi to grow and produce various metabolites. Furthermore, the fungal

metabolism is enhanced since the bacterial contamination is prohibited due to the

low water activity in solid substrate fermentation (Favela-Torres et al., 2006).

Several studies conducted have significantly proven that the volume of

pectinase yielded through solid substrate fermentation is higher compared to

submerged fermentation (Favela-Torres et al., 2006). Moreover, the cost of

production through solid substrate fermentation is cheaper due to the exploitation of

agro-industrial residues as substrates and the pectinase generated has better

physicochemical properties, contradictory to submerged fermentation. In fact, the

application of agro wastes as the main carbon source in solid substrate fermentation

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is an effective solution in solving detrimental problem arises due to the waste

disposal management. For example, every year in Egypt, large amount of orange

peels are generated as residues after the oranges canning process by the

manufacturing factories (Abdel-Mohsen, 1996). According to Mamma et al. (2008),

about 35 000 tons (dry weight) of citrus peels are generated every year in Greece. In

Spain, around 250 millions kilograms of grape pomace which consists of seed, stem

and skin are produced by the wine cellars (Chatterjee et al., 2013). While in

Karnataka, India, approximately, 3.1 tons/hectare husk of pulses and 14.3

tons/hectare deseeded sunflower head are being generated every year respectively

after the processing of sunflower oilseed. All of these agro-industrial residues are

rich in pectin and applicable as the pectin-containing substrate to be utilized in the

microbial pectinase production through solid substrate fermentation. In Malaysia,

approximately 1.2 million tonnes of agro-wastes are being accumulated each year,

especially from the palm oil and oil palm industries.

In order to produce pectinase enzyme, different types of microorganism

including fungi, bacteria and yeasts are incorporated into the fermentation system.

According to Favela-Torres et al. (2006), approximately 30 genera of filamentous

fungi, bacteria and yeast which are frequently employed in producing pectinase such

as Erwinia, Rhizopus, Penicillium, Fusarium, Aspergillus, Bacillus, Saccharomyces

and Kluyveromyces. These microorganisms must be non-pathogenic, non-toxic and

generally do not produce antibiotics which are the important characteristics of GRAS

(Generally regarded as safe) microorganism. One of the most frequently utilized

microorganisms in fungal pectinase production is Aspergillus niger which is

proclaimed possessing the GRAS status (Reddy and Sreeramulu, 2012).

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Technically, commercial enzyme with enviable biochemical and physico-

chemical properties with low cost production has been the focus of researchers

currently (Silva et al., 2002). It is undeniable that the utilization of agro industrial

residues as pectin-rich substrate in pectinase production through solid substrate

fermentation is promising a low cost and eco-friendly enzyme industry (Suresh and

Viruthagiri, 2010). However, further research is crucial in screening the best

substrate to be incorporated into the fermentation system and its suitability for the

microbial growth and enzyme production.

1.2 Rational of study

Pomelo (Citrus grandis) is massively cultivated in Asia especially in

Malaysia, China, Southern Japan, Vietnam, Indonesia and Thailand (Hameed et al.,

2007). Commonly in Malaysia, during the trimming stage, some of the immature

pomelo fruits are removed to improve the quality of the others and disposed to the

environment subsequently. This scenario will only lead to the detrimental pollution

problem to the community (Saikaew et al., 2009). Evidenced by research conducted

by Norziah et al. (2000) and Burana-Osot et al. (2010), pomelo peel is rich in pectin

and applicable in pectinase production through solid substrate fermentation

employing fungal strains. Since the biotechnological research on pectinase

production using pomelo peel is still scanty (Foo and Hameed, 2011), this vigorous

study is implemented to maximize its potential as the main substrate in fermentation

system to generate commercialized pectinase enzyme employing local fungal isolate.

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1.3 Objective of research

With an aim to use pomelo peels as a substrate for pectinase production by a

local fungal isolate via solid substrate fermentation system, these objectives are

employed;

i. To identify and screen the best fungal pectinase producer based on its

capability to produce extracellular pectinase qualitatively and

quantitatively.

ii. To optimize the physical and chemical conditions for a maximal

pectinase production by the selected fungal isolate in a shake flask system

and a tray system.

iii. To purify and characterize the purified pectinase

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CHAPTER 2

LITERATURE REVIEW

2.1 Growth of agricultural industry worldwide

With an aim to stimulate the agricultural growth, combating poverty and

hunger for human worldwide and at the same time to encourage environmental

sustainability, agricultural sector is believed as the efficient tool.

In United States Of America, agriculture is the major industry with 2.2

millions farms which dominate an area of 922 million acres in 2007. Corn, tomatoes,

potatoes, peanuts and sunflower seeds are among the major crops being cultivated.

Meanwhile, in China, over 300 millions farmers are employed in agricultural

industry which endowed this country as the first place in worldwide farm output.

Primarily, China is the main producer of rice, wheat, sorghum, potatoes, peanuts, tea,

millet, barley, oilseed and cotton. Besides, India ranks as the second largest

contributor of farm output after China. According to FAO (Food And Agriculture

Organization) 2010 Worldwide Statistics, India is the largest supplier of many fresh

fruits and vegetables, spices, milk, jute, millet and castor oil seeds. India is also the

second largest producer of wheat and rice and also ranked as one of the five largest

contributors of 80% of agricultural products including coffee and cotton in 2010

(FAO, 2012).

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The same scenario is also taking place in Malaysia where agricultural sector

plays an imperative role to the economy, providing 16% of employment to the local

citizen. Rubber, palm oil and cocoa are among the foremost commercial crops being

cultivated locally. In 1999, about 10.55 million matric tons of palm oil was generated

which ranked Malaysia as the world‟s largest producer. Besides, paddy, banana,

coconut, durian, pineapple, rambutan and other local fruits and vegetables are also

cultivated for domestic market (Encyclopedia Of The Nation, 2013).

In future, it is assumed that the expansion of agricultural sector will be

boosting up throughout the year in order to accomplish the worldwide population‟s

demand. Nonetheless, the disposal management of agro-industrial residues remains

as problematic issue to be solved by the developing countries.

2.2 Agro-industrial residues as a detrimental issue

Despite of soaring development depicted by the global agricultural sector

yearly, the accumulation of assorted disposed agro-industrial residues remain a major

concern to the public and the ecosystem as well. According to Agamuthu (2009),

around 998 million tonnes of agricultural wastes were produced around the world.

While in Malaysia, approximately 1.2 million tonnes of agro wastes were being

disposed every year. Table 2.1 shows the agricultural wastes generated in Asian

countries as reported by Agamuthu (2009). The table also demonstrates the predicted

increasing numbers of agricultural wastes in the year of 2025.

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Table 2.1 : Agricultural waste generation in Asia

Country In Asia

Agricultural Waste

Generation

(kg/capita/day)

Projected Agricultural

Waste Generation In

2025 (kg/capita/day)

Brunei

0.099

0.143

Cambodia 0.078 0.165

Indonesia 0.114 0.150

Laos 0.083 0.135

Malaysia 0.122 0.210

Myanmar 0.068 0.128

Philipines 0.078 0.120

Singapore 0.165 0.165

Thailand 0.096 0.225

Vietnam 0.092 0.150

Nepal 0.060 0.090

Bangladesh 0.040 0.090

Adapted from : Agamuthu (2009)

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Commonly, for agro-industrial waste management in developing countries,

crop residues, animal garbages, forest litter and grasses are being burnt as fuel which

leads to problematic air pollution. In general, the main source for these horrendous

agro industrial wastes comes from the processed animal products and crops.

Brewer‟s waste, maize milling by-product, molasses, oilseed cakes and bagasse are

among the common wastes produced by the agro-industrial processing sector. While

for the crop residues such as straw, stem, peel, shell, leaves, husk, seed, pulp, lint and

also stubble which are derived from cereals (which include rice, maize or corn,

millet, barley, wheat and sorghum), legumes (soy bean and tomatoes), jute,

groundnut, cotton, coffee, cocoa, tea, olive, fruits (banana, mango, cashew) and palm

oil are also defined as agricultural waste (Pandey et al., 2009).

Undeniably, managing agro-industrial waste as one of the solid waste is a big

challenge for a developing country around the world. Without an effective step, the

accumulation of these residues will only lead to the public health hazard and

detrimental pollution to the environment (Hwa, 2007). Apparently, converting the

„waste‟ into „wealth‟ through the utilization of agro-industrial residues in profitable

enzyme production is one of the practical steps in encountering such problem.

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2.3 Utilization of agro industrial residues in pectinase production

Abundance of agro-industrial waste as by-product in agricultural sector

(around 3.5 billion tonnes/year) is promising a low cost enzyme yield especially in

microbial pectinase production employing solid substrate fermentation. These

renewable residues can be utilized as source of energy for microbial growth during

fermentation and also as carbon supply during the synthesis of biomass and other

products (Jacob, 2009). In addition, these agro industrial residues also serve as an

anchorage for the cells and provide sufficient nutrients for the microbial growth.

However, for certain nutrients that are available in sub-optimal concentration or even

absent, the substrate should be supplemented externally (Pandey et al., 1999). In

consideration of agro-industrial residues‟ suitability and availability to be exploited

as the substrate in solid substrate fermentation, the problem regarding the pollution

as a result to its accumulation and disposal method should be overcome effectively

(Hoondal et al., 2002).

In nature, variety of substrates come from agro-industrial wastes which are

applicable in producing profitable value added products especially pectinase enzyme

through solid substrate fermentation. However, the number and type of potential

residues varies between the crops and districts in the world (Cohen and Hadar, 2001).

Some potentially utilized residues in pectinase production through solid substrate

fermentation using various microorganisms are recorded in Table 2.2

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Table 2.2 Consumption of assorted agro industrial residues in pectinase production through solid substrate fermentation (SSF)

Agro Industrial Residue Microorganism Reference

Sugar cane bagasse Aspergillus niger Jacob (2009)

Aspergillus awamori Suresh and Viruthagiri (2011)

Penicillium sp. Jacob (2009)

Aspergillus niger CH4 Solis-Pereyra et al. (1996)

Moniliella sp. SB9 Martin et al. (2004)

Penicillium sp EGC5 Martin et al. (2004)

Monosodium glutamate waste water Aspergillus niger Bai et al. (2004)

Sugar beet pulp Aspergillus niger Bai et al. (2004)

Bacillus gibsonii S2 Li et al. (2005)

Penicillium oxalicum Neagu et al. (2012)

Wheat bran Aspergillus carbonarius Jacob (2009)

Aspergillus niger Castilho et al. (2000) ; Heidar et al. (2011)

Aspergillus sojae Heerd et al. (2012)

Thermoascus aurantiacus Jacob (2009)

Bacillus sp. DT7 Kashyap et al. (2003)

Penicillium sp. Jacob (2009) ; Heidar et al. (2011)

Streptomyces sp. RCK-SC Jacob (2009)

Penicillium viridicatum RFC3 Silva et al. (2005)

Streptomyces lydicus Jacob (2009)

Bacillus pumilus dcsr1 Sharma and Satyanarayana (2012)

Alternaria alternata Faten and Abeer (2013)

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Table 2.2 Continue

Agro Industrial Residue Microorganism Reference

Rice bran Aspergillus niger Suresh and Viruthagiri (2011)

Aspergillus awamori Suresh et al. (2009)

Orange bagasse Thermoascus aurantiacus Jacob (2009)

Penicillium viridicatum RFC3 Silva et al. (2005)

Moniliella sp. SB9 Martin et al. (2004)

Penicillium sp EGC5 Martin et al. (2004)

Apple bagasse Penicillium sp Heidar et al. (2011)

Aspergillus niger Heidar et al. (2011)

Orange pulp Fusarium moniliforme Jacob (2009)

Orange peel Aspergillus niger Vasanthi and Meenakshisundaram (2012)

Coffee pulp Aspergillus niger Loera et al. (1999)

Aspergillus niger C28B25 Antier et al. (1993)

Aspergillus niger V22 B35 Boccas et al. (1994)

Coffe mucilage Erwinia herbicola Avallone et al. (2002)

Lactobacillus brevis Avallone et al. (2002)

Grape pomace Aspergillus awamori Botella et al. (2005)

Grape peel Saccharomyces cerevisiae CECT 11783 Arévalo-Villena et al. (2011)

Soy bran Aspergillus niger Castilho et al. (2000)

Citrus peel Aspergillus niger Dhillon et al. (2004)

Citrus waste Aspergillus foetidus Garzỏn (1992)

Aspergillus niger F3 Rodríguez-Fernàndez et al. (2012)

Corn cobs Alternaria alternata Faten and Abeer (2013)

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2.4 Pomelo peel as a potential substrate for pectinase production

Pomelo or its scientific name Citrus grandis (better known in Malaysia as

limau bali or limau kedang) is the largest citrus fruit with average diameter of 30

centimetres and sometime weighs until 10 kilograms. Generally, it possesses very

thick rind with soft texture but yet still easy to peel off (Hameed et al., 2007). This

citrus fruit is also known as “shaddock” and believed as an ancient species to the

genus of Citrus. It is classified in family of Rutaceae, sub-family Aurantioidae, tribe

Citreae and sub-tribe Citrinae (Paudyal and Haq, 2008).

Pomelo is cultivated immensely in Malaysia, Indonesia, Thailand, China, Fiji,

India and Japan. Currently, this large citrus fruit is also grown widely in Caribbean

and United States Of America especially in California and Florida. It is believed that

pomelo or “shaddock” was introduced to the West Indies (Barbados) from the Dutch

East Indies by Captain Shaddock and latterly being spread to the other tropical

countries (Hameed et al., 2007).

Commonly, the juicy flesh of pomelo which is rich in Vitamin C, B1, B2,

B12 and protein is eaten raw or being extracted for commercial purpose. Meanwhile,

its thick rind is utilized widely in sweet manufacturing industry (covered with sugar)

and also in marmalade production (Hameed et al., 2007). The flowers, seeds and

fruits are also employed in medication and the flower itself is applied in perfumery

extraction. Meanwhile, the thick peel is extracted as the source of pectin and dietary

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fiber (Saikaew et al., 2009 ; Burana-Osot et al., 2010). According to the statistics

provided by FAO (Food And Agriculture Organization), the total production of

pomelo and grapefruit amounted about 5.05 million tonnes which constituted 6.2%

of citrus fruit production worldwide in 1994 while in Thailand itself, 11% of its

exported fruit comes from pomelo (Paudyal and Haq, 2008).

In Malaysia, pomelo is principally cultivated in Johor, Kedah, Melaka and

Perak with approximately 1895 hectares of pomelo are cultivated commercially (Foo

and Hameed, 2011). Generally, pomelo is produced throughout the year with its peak

seasons between January to February and also from August to September

respectively. In order to ensure the well growth and quality assertion of certain

amount of pomelos, fruits that are unsuitable to be commercialized in fresh market

and also the immature fruits are eradicated from the trees (Norziah et al., 2000). Due

to the high consumption of pomelo in food manufacturing industry together with the

disposal of immature fruits during the trimming stage, this circumstance leads to a

severe pollution to the community and the environment as well (Hameed et al.,

2007).

As an efficient solution in managing the waste, pomelo peel as part of the

fruit could be employed as a low cost substrate in producing pectinolytic enzymes as

a value added by-product through solid substrate fermentation. According to Norziah

et al. (2000), pomelo peel is a good source of pectin which 20.8% of its total

extraction is constituted by pectin after being extracted through acetone precipitation

utilizing sodium hexametaphosphate. Yuxiang et al. (2012) also reported that

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20.98% of pomelo peel was constituted of pectin after being extracted under the

optimum condition determined by Response Surface Methodology (RSM). In

addition, the raw material for the production of commercialized pectin nowadays is

mainly contributed by residues from fruit juice manufacturing such as citrus fruits

and apple pomace (Hoondal et al., 2002). This chemical component plays a vital role

in inducing the pectinase yielded by microorganisms during the fermentation period

(Jacob, 2009; Reddy and Sreeramulu, 2012). However, the exact potential of pomelo

peel as the raw material in pectinase production through microbial fermentation is

still yet undiscovered since less research was reported by the previous studies.

2.5 Pectic substances

Pectic substances are a complex, high molecular weight, acidic and

negatively charged biomacromolecule (Jayani et al., 2005; Bhardwaj, 2010) which

constituted of 17 different monosaccharides and at least seven different

polysaccharides (Jacob, 2009). In plant cell wall, pectic substances and

hemicelluloses act as the cementing agent for celluloses microfibrils in order to

provide strength and stability to the structure of cell wall (Geetha et al., 2012). The

structure of primary cell wall in most flowering plant is displayed in Figure 2.1.

Generally, pectic substances are ubiquitous to Plant Kingdom and copiously found in

the lamella between the primary cell walls of adjacent young plant cells in form of

calcium pectate and magnesium pectate which are responsible for the cell cohesion

(Jayani et al., 2005). In young and developing cell wall, pectic substances are

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synthesized in the Golgi apparatus from UDP-D-galacturonic acid during the early

stage of plant growth (Hoondal et al., 2002).

Figure 2.1 Primary cell wall structure in most flowering plants

(Adapted from Parenicova, 2000)

The cellulose microfibrils are interweaved with xyloglucan polymers, and this

structure is entrenched in a matrix of pectic polysaccharides, polygalacturonic acid

(PGA) and rhamnogalacturonan (RG).

Cellulose microfibrils

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2.5.1 Classification of pectic substances

According to Kashyap et al. (2001), pectic substances are classified based on

the modification type of its backbone chain. Hence, different type of pectinolytic

enzymes will react accordingly to this variety of pectic substances depending on its

specificity and mode of action (Yadav et al., 2009). The details of pectic substances

are further elaborated in the following description.

2.5.1.1 Protopectin

Protopectin is the “parents” of the pectic substances. The hydrolysis of

protopectin yield pectin or pectinic acid (Kashyap et al., 2001; Jayani et al., 2005).

This kind of pectic substances is a water–insoluble component and mainly found in

the plant tissue predominantly in the middle lamella between the plant cells. The

insolubility of this protopectin is due to its large molecular weight, salt bonding

between the carboxyl group and also the ester bond structure between the carboxylic

group of protopectin or the hydroxyl group of other constituents in the cell wall

(Hoondal et al., 2002).

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2.5.1.2 Pectic acid

According to Jayani et al. (2005), the soluble polymer of galacturonans and

basically free from the methoxyl groups is called as pectic acid. The salt of pectic

acid which is either neutral or acidic is known as pectates (Kashyap et al., 2001;

Hoondal et al., 2002).

2.5.1.3 Pectinic acid

Pectinic acid is composed of polygalacturonan chain that consists of more

than 75% of methylated galacturonate units. The salt of pectinic acid which is either

neutral or acidic is known as pectinates (Jayani et al., 2005). Pectinic acid alone has

a typical property in forming gel with acid or sugar under suitable condition. In

addition, gel can also be formed with certain metallic ion or other compounds such as

calcium salts if the pectinic acid is low in methoxyl content (Hoondal et al., 2002).

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2.5.1.4 Pectin

Pectin is also known as polymethyl galacturonate. It is a soluble polymeric

material which confers rigidity to the cell wall structure after binding together with

cellulose (Jayani et al., 2005). Generally, 75% of the carboxyl groups of the

galacturonate monomer are esterified with methanol (Hoondal et al., 2002).

Previous research had reported that pectin is copiously found in plants

including fruits and vegetables (Bhardwaj, 2010; Chaudhri and Suneetha, 2012).

Approximately, 0.5% to 4% of total plant fresh weight was constituted of pectin

(Jayani et al., 2005). According to Jacob (2009), pectic substances play an

imperative role in the plant cell growth and cell differentiation during the

development of the plant tissue. New materials will be laid down and the old

materials will be removed or degraded by the enzymes. While in an immature fruit,

the pectin binds to the cellulose microfibrils which contribute to the stability and

rigidity of the cell wall. However, during the ripening stage, the main backbone

chain or the side chain of pectin is changing due to the naturally occurring

pectinolytic enzymes reaction (Martin et al., 2004). Consequently, the rigid cell wall

is softened since the pectin becomes more soluble resulting from the natural chain

modification when the fruit turns to ripe (Kashyap et al., 2001). The estimation of

pectin content in assorted fruits and vegetables are different to each other as

described in Table 2.3.

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Table 2.3 : Percentage of pectin in different fruits and vegetables

Fruit or vegetable Tissue Pectic substances (%)

Apple Fresh 0.6 – 1.6

Banana Fresh 0.7 – 1.2

Peach Fresh 0.1 – 0.9

Strawberry Fresh 0.6 – 0.7

Cherry Fresh 0.2 – 0.5

Pea Fresh 0.9 – 1.4

Carrot Dry matter 6.9 -8.6

Orange pulp Dry matter 12.4 – 28.0

Potato Dry matter 1.8 – 3.3

Tomato Dry matter 2.4 – 4.6

Sugar beet pulp Dry matter 10.0 – 30.0

(Adapted from Jayani et al., 2005)

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2.5.2 Structure of pectin

2.5.2.1 Primary structure of pectin

According to Chaudhry and Suneetha (2012), chemically, pectic substances

are a complex colloidal acid polysaccharides with a long unbranched galacturonic

acid backbone chain and linked together by α, 1-4 linkages (Figure 2.2). This is the

basic unit of pectic substances which is also known as homogalacturonan (HG).

Galacturonic acid which is the monomer of this polysaccharide (Reddy and

Sreeramulu, 2012) can be methyl esterified at C-6 and the hydroxyl group at C-2 or

C-3 can be acetylated.

Figure 2.2 : Primary structure of pectin (adapted from Jacob, 2009)

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2.5.2.2 Secondary structure of pectin

The secondary structure of pectin (Figure 2.3) is composed of two main

regions which are “smooth region” (homogalacturonan) and “hairy regions”

(rhamnogalacturonan I and II) (Pedrolli et al., 2009). Homogalacturonan constitutes

the backbone chain of pectin, composed of α, 1-4 linked D-galacturonic acid

residues. Rhamnogalacturonan I is a highly branched region which comprised of a

huge number of side chain α, 1-2 linked L-rhamnopyranose residues. Meanwhile,

the more complex Rhamnogalacturonan II is predominantly found in primary cell

wall of plants and plays a significant role as a signal molecule in the development of

plant cell wall. This biomacromolecule form a very flexible, extended and curve

(worm like) structure in solution (Yadav et al., 2009)

Figure 2.3 : Secondary structure of pectin (adapted from Yadav et al. (2009)

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2.6 Pectinase

Pectinase is a kind of heterogeneous enzyme and occasionally known as

pectic enzyme or pectinolytic enzyme. This enzymatic constituent exhibits pectin-

containing substances degradation which is mainly found in plant structures. This

inducible enzyme can be produced extensively either through solid substrate

fermentation (SSF) or submerged fermentation (SmF) (Solis-Pereyra et al., 1993;

Maldonado and Saad, 1998; Geetha et al., 2012) employing various species of

saprophytic microorganism including fungi, bacteria and yeast (Jacob, 2009; Janani

et al., 2011).

Historically, in 1930, the profitable potential of pectinase enzyme was

principally applied in the home made preparation of fruit juices and wines. Thirty

years later, in 1960s, the understanding of chemical nature and enzymatic reaction

catalysed by pectinase in plant tissue was basically improved. With this awareness,

research on the application of pectinase enzyme for commercial purpose was

implemented extensively which in turn, endowed pectinase as one of the impending

enzymes in commercial sector nowadays. Recent statistics on industrial enzyme sales

worldwide in 1995 noted that $75 millions of total sales came from pectinase enzyme

(Kashyap et al., 2001). Meanwhile, according to Jayani et al. (2005), almost 25% of

global food enzyme sales was contributed by pectinase enzyme currently.

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In nature, pectinase is produced by plants, insects, nematodes, protozoan

(Pedrolli et al., 2009) and also by saprophytic microorganism including bacteria,

fungi, actinomycetes and yeast (Jacob, 2009; Bhardwaj, 2010). Inside the plant

tissue, this pectinolytic enzyme plays an essential role in supporting the extension of

cell wall, facilitating the penetration of pollen tube (Blanco et al., 1999) and

softening the plant tissue in mature or ripening fruits through the alteration of pectin

backbone chain (Jayani et al., 2005). Oranges and tomatoes are believed as plants

that possess considerable amount of pectinase enzyme inside the plant tissues.

Besides, pectinase is also found in insect such as the rice weevil (Sitophilus

oryzae). The endosymbiotic bacteria inside the body of this insect are assumed as the

genetic source of pectinase which plays a significant role in plant – insect interaction.

The rice weevil feeds on seeds of cereals containing pectin. Thus, pectinase in this

insect is functioning as an exclusive digestive system to degrade the pectin (Shen et

al., 1996).

In the other hand, microbial pectinase yielded by bacteria, fungi and yeast is

also important especially during the phytopathologic process, plant and microbe

symbiosis interaction and also in the natural decomposition process of decaying plant

residues. Ultimately, the decomposition of these plant materials, catalyzed by

microbial pectinase contributes to the natural carbon cycle in the ecosystem (Pedrolli

et al., 2009).