PRODUCTION AND CHARACTERIZATION OF

PROTEASE BY BACILLUS LICHENIFORMIS ON SKIM

LATEX SERUM FORTIFIED MEDIA

BY

VIVI MARDINA

A dissertation submitted in fulfilment of the requirement for

the degree of Master of Science

(Biotechnology Engineering)

Kulliyyah of Engineering

International Islamic University Malaysia

AUGUST 2015

ii

ABSTRACT

Protease from Bacillus licheniformis (ATCC 12759) can be produced using a readily

available agro-industrial residue as potential substrate. Skim latex serum effluent is an

abundant and inexpensive liquid waste from natural rubber industry that provides

various organic compounds for microbial growth. The present study utilized skim latex

effluent as a basal medium to cultivate B. liceniformis for extracellular protease

production. Statistical based experimental designs were adopted to optimize the

physicochemical factors for the maximization of protease production. Screening the

eleven factors such as lactose, galactose, casein, KH2PO4, MgSO4.7H2O, LB broth,

skim latex serum, inoculums size, agitation, initial pH, and temperature for protease

production was performed using Plackett-Burman design prior to optimization. Four

variables (galactose, skim latex serum, agitation and pH) were identified as the most

critical factors and selected for further optimization to enhance protease production

using Face Centered Central Composite Design (FCCCD) under Response Surface

Methodology (RSM). The protease production was found to increase from 2 U/ml to

19.35 U/ml approximately a nine fold increase as compare to the original medium. The

validation of developed model was established to verify the adequacy and accuracy of

the model, and the results showed that predicted value agreed well with experimental

value with error less than 20 %. ANOVA of the quadratic model showed a significant

of the model (p = 0.0002) with high determination coefficient (R2 = 0.9537) indicating

a satisfactory fit of the model with experimental data. Following the optimization

strategy, the sequential purification steps of the optimized media were conducted using

ammonium sulphate precipitation, dialysis and ion exchange chromatography. The

results revealed that the enzyme activity increase to 2.28 fold of purification compare

to the crude enzyme. Assessment of the purified protein by SDS PAGE showed a single

band with molecular mass of about 47 kDa. The enzyme was stable at temperature range

of 35 oC to 65 oC and also at pH 6.0 and 7.0 for 60 min. The stimulatory effects on

protease activity were observed in the presence of Mn2+and Ca2+ , while inhibitory

effects were found in the presence of Cu2+, Zn2+, Mg2+, and EDTA. This indicated that

the produced protease might be a metallo protease. In the case of detergent application,

the enzyme exhibited the stability toward surfactants (Triton X100, Tween 20, SDS),

solvents (acetone, chloroform, hexane and toluene), oxidizing agent (H2O2) and Tesco

Everyday Value® detergent with the residual activity around 80 %. It also demonstrated

the removal activity of blood stain completely with supplementation of the 7 mg/ml

detergent solution. The characteristics of produced protease suggest that it may be used

as a potential additive for detergent formulation as well as laundry detergent and clinical

waste treatment.

iii

ملخص البحث

الم فات باستتتتتتن ا يمك أن يننج Bacillus licheniformis (ATCC 12759) الأنزيم البروتيني المستتتتتتن تجة رخيصتتة كنفايات ا فرة والالمن الستتاة ة النفايات إن صتتا المطاا المودتت د .كمادة ركيزة المناحة والصتتناعية الكا نة و الزراعية

الحالية استتتتتتن راستتتتتتةالنم الجراثيم. في المناستتتتتتبة ل المركبات العضتتتتتت ية التي تن فر فيها الع ي المطاا الطبيعي صتتتتتتناعة خارج الخ ية. لو الأنزيم البروتيني لإنناج B.liceniformisل تتتتتتتتتتتتتتتتتتتتتتتتتت قاع ي غذي ك سط ايات الساة ة ل مطاا المود دالنف

حصتتتتت ف كما .لبروتينياللانزيم إنناج الفيزي كيمياةية ل صتتتتت ى ع الع ا ا لنحستتتتتن الإحصتتتتتاةية النجريبية النصتتتتتا يم اعنم تو LB brothو O2.7H4MgSOو 4PO2KHو الكاستتتتتتتتتتتنو الجا كن زو اللاكن ز ثا ح عدتتتتتتتتتتترالأ الع ا ا

أمري و نيالأنزيم البروتي لإنناج ودرمة الحرارة الأوليةوستتتتترعة الرج ودرمة الحم تتتتتة ادة الن ويح حجمو صتتتتتا المطاا المودتتتتت د أربعة نغيرات وق تم تح ي الأ ثا. كمؤها لفح النحستتتتتتتتتتن Plackett-Burman باستتتتتتتتتتن ا تصتتتتتتتتتتميم هذا الفح

زي لم والتي اخنيرت حوا أهم الع ا ا الحاسمة بإعنبارها درمة الحم تتتتة و وستتتترعة الرج صتتتتا المطاا المودتتتت دالجا كن ز و ) نهجيةفي إطار (FCCCD)تصتتتميم وامهة الن ستتتط المركزي المرك باستتتن ا الأنزيم البروتيني إنناج لنعزيزالنحستتتن الأ ثا

وح ة/ ا مما يوارب 19.35 وح ة/ ا إلى 2إرتفع الأنزيم البروتيني إننامية ووم أن. (RSM)لستتتتطحيةا ا ستتتتنجابة ل نأك النصميم المط ر النحوق صحة أظهرت نناةج عم ية .ق رن بال سظ الغذاةي الأص ي أ عاف ا ننامية اذا تسعة

أظهر نم ذج و .٪20 أقا ع نستتتتتبة خطأ النجريبية الويمة مي ع بدتتتتتكا اتفو الويمة المن قعة أن النصتتتتتميم دقة كفاية و مما ي ى )2R = 0.9537 (عالي تح ي عا ا ع )P = 0.0002(بنستتبة النطبيق عالية ANOVAال رمة الثانية لأ ثا ا ال ستتتتط المغذي ننويةل ننابعة الخط ات النالية بإمراء البياات النجريبية. وكان الخط ة عنم ذج الموب ى ل ع النناستتتت ندتتتتتتتتاا ا نزيم النوي النناةج بإزدياد وكدتتتتتتتتف الكرو ات غرافي.النبادى الأي ني ال ياى و ل ترستتتتتتتتي الأ ني كبرينات باستتتتتتتتن ا

الكن ة الجزيئية واح ة ذات فرقة SDS PAGE ب اسطة البروتن النوي توييم وأظهر .الخا وارنة با نزيم أ عاف 2.28إلىدرمة أيضتتتتتتتتتتتتتتا و درمة ئ ية 65درمة ئ ية إلى 35درمة حرارة تتراوح بن ستتتتتتتتتتتتتتنور في نزيما كانو .كي دالن ن 47ح الي

نغنيز آي ات الم ب م د ندتتتتتتتتتاا الأنزيم البروتيني ع تندتتتتتتتتتيطية آثار وق ل حظ .دقيوة 60لم ة 7.0و 6.0الحم تتتتتتتتتة بن إلى يدير هذا. EDTA و آي ات النحاس والزنك والمغنيسي ب م د كا يرات ثبطةتأث تم العث ر ع في حن والكالسي

عالية ع ا ا الف نح استتتتتتتتتتتنورار ا نزيم أظهر المنظفات نطبيق ع ال في حالة ف زي. بروتينيأنزيم ق يك ن المننج الأنزيم البروتيني أن ا ا عالن ل ي و الو الأستتتتين ن والك روف ر وانكستتتتان ) والمذيباتTriton X100, Tween 20, SDS) الستتتتطحية ندتتااوبنن أيضتتا .٪80ح الي المنبويا نزيم ندتتاا ع ®Tesco Everyday Valueو نظف )2O2H (المؤكستت ة

بالإ كان أن يسن أنه المننج البروتيني خصاة ا نزيمو . المنظف الساةا غ/ ا 7بتت ع إكمانا تما ا ال بوعةإزالة .الإك ينيكية عالجة النفايات و نظفات الغسيا وكذلك المنظفات في إع اد كمادة إ افية

iv

APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms

to acceptable standards of scholarly presentation and is fully adequate, in scope and

quality, as a dissertation for the degree of Master of Science (Biotechnology

Engineering).

Faridah Yusof

Supervisor

Md Zahangir Alam

Co-supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable

standards of scholarly presentation and is fully adequate, in scope and quality, as

dissertation for the degree of Master of Science (Biotechnology Engineering).

Parveen Jamal

Internal Examiner

Dzun Noraini Jimat

Internal Examiner

This dissertation was submitted to the Department of Biotechnology Engineering and

is accepted as a fulfilment of the requirement for the degree of Master of Science

(Biotechnology Engineering).

Faridah Yusof

Head, Department of

Biotechnology Engineering

This dissertation was submitted to the Kulliyyah of Engineering and is accepted as a

fulfilment of the requirement for the degree of Master of Science (Biotechnology

Engineering).

Md. Noor Salleh

Dean, Kulliyyah of Engineering

v

DECLARATION

I hereby declare that this dissertation is the result of my own investigation, except where

otherwise stated. I also declare that it has not been previously or concurrently submitted

as a whole for any other degrees at IIUM or other institutions.

Vivi Mardina

Signature Date

vi

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AN AFFIRMATION OF

FAIR USE OF UNPUBLISHED RESEARCH

Copyright © 2015 International Islamic University Malaysia. All rights reserved.

PRODUCTION AND CHARACTERIZATION OF PROTEASE

BY BACILLUS LICHENIFORMIS ON SKIM LATEX SERUM

FORTIFIED MEDIA

No part of this unpublished research may be reproduced, stored in a retrieval system,

or transmitted, in any form or by any means, electronic, mechanical, photocopying,

recording or otherwise without prior written permission of the copyright holder

except as provided below:

1. Any material contained in or derived this unpublished research may only

be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print

or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in retrieval system

and supply copies of this unpublished research if requested by other

universities and research libraries.

By signing this form, I acknowledged that I have read and understand the IIUM

Intellectual Property Right and Commercialization Policy.

Affirmed by Vivi Mardina

……………………… ……………………

Signature Date

vii

ACKNOWLEDGEMENTS

In the name of Allah, the most merciful and the most compassionate

Alhamdulillah, all praise and thanks to Allah for the successful completion of this

research work. He gave me the health, patience and strength to reach this far.

I would like to express my sincere gratitude to my supervisor, Prof. Dr. Faridah

Yusof for her supports, motivation, encouragement, advice, knowledge, guidance,

untiring assistance and patience throughout the course of this study. I would also like to

thank to my co-supervisor, Prof. Dr. Md. Zahangir Alam for his idea, guidance, sharing

the knowledge and encouragement through the period of the research. My appreciation

is expressed to Prof. Dr. Hamzah Mohd Salleh and Br. Aziz for the permission to excess

their labs and for the chemicals. I am also thankful to Mardec Industrial Latex Sdn. Bhd,

Tapah, Perak for supply of the raw material.

Sincere thanks to my colleague, Johan Ariff Mokhtar for his assistance, valuable

inputs and contribution during the project. My thanks to my lab mates: Nazira, Bala,

Mohd. Ezza Faiz, Nafeesah, Jannah, Jamil, Shima, Ainur, Shah, Safa, Omar, Emi,

Silvia, and Sofiya for their contributions and friendship. Thanks are also extended to

Department technical staff especially Br. Hafizul, Aslan, Haji Sukiman for their

laboratory support.

My deepest gratitude goes to my parents (Marlina and Chairruddin) and my

parent in laws (Sri Hayati and Thohirin) and family as well. Finally, I am grateful to my

husband (Kudam Yusof) for his endless support and unconditional love and also to my

children (Ahmad, Umaimah, Khalid and my baby, Bari) who promote my spirit to

complete my study as soon as possible.

viii

TABLE OF CONTENTS

Abstract................................................................................................................. ii

Abstract in Arabic................................................................................................. iii

Approval Page....................................................................................................... iv

Declaration............................................................................................................ v

Copyright Page...................................................................................................... vi

Acknowledgements............................................................................................... vii

List of Tables........................................................................................................ xii

List of Figures....................................................................................................... xiv

List of Abbreviations............................................................................................ xvii

CHAPTER ONE: INTRODUCTION............................................................... 1

1.1 Background........................................................................................

1.2 Problem Statement.............................................................................

1.3 Research Objectives...........................................................................

1.4 Scope of Research..............................................................................

1.5 Significant of Study...........................................................................

1.6 Research Methodology......................................................................

1.7 Dissertation Organization...................................................................

1

4

6

7

7

7

9

CHAPTER TWO: LITERATURE REVIEW.................................................. 10

2.1 Introduction........................................................................................ 10

2.2 Natural Rubber Latex......................................................................... 10

2.2.1 General Properties of Natural Rubber Latex........................... 15

2.2.2 Composition of Latex.............................................................. 16

2.2.2.1 Rubber Matters.......................................................... 17

2.2.2.1.1 Rubber Particle.......................................... 17

2.2.2.1.2 Lipid.......................................................... 18

2.2.2.1.3 Protein....................................................... 19

2.2.2.2 Non Rubber Matters.................................................. 19

2.2.2.2.1 Latex Serum............................................. 20

2.2.2.2.2 Lutoid....................................................... 21

2.2.2.2.3 Frey-Wyssling......................................... 21

2.2.3 Utilization of Skim Latex Effluent.......................................... 22

2.2.3.1 Useful Biochemical Extraction................................ 22

2.2.3.2 Fertilizer................................................................... 23

2.2.3.3 A Medium for Culturing Fish.................................. 23

2.2.3.4 A Nutrient Media for Cultivating Microorganism... 24

2.3 Proteases: Overview.......................................................................... 25

2.3.1 Source of Proteases................................................................. 26

2.3.1.1 Plant proteases...................................................... 26

2.3.1.2 Animal Proteases.................................................. 26

2.3.1.3 Microbial Proteases.............................................. 27

2.3.1.3.1 Bacteria proteases..................................... 28

2.3.1.3.2 Fungi Proteases......................................... 28

ix

2.3.1.3.3 Viruses Proteases...................................... 29

2.3.2 Classification of Proteases...................................................... 29

2.3.2.1 Nomenclature and Terminology.......................... 29

2.3.2.2 Enzyme Commission Classification.................... 30

2.3.2.3 Exopeptidases....................................................... 31

2.3.2.4 Endopeptidase...................................................... 32

2.3.2.4.1 Serine Proteases........................................ 32

2.3.2.4.2 Aspartic Proteases..................................... 33

2.3.2.4.3 Cysteine Proteases..................................... 33

2.3.2.4.4 Metallo Proteases...................................... 34

2.3.3 Mechanism of Action of Proteases......................................... 35

2.3.3.1 Serine Proteases................................................... 35

2.3.3.2 Aspartic Proteases................................................ 37

2.3.3.3 Cysteine Proteases................................................ 39

2.3.3.4 Metallo Proteases................................................. 40

2.4 Properties of Bacillus licheniformis................................................... 41

2.5 Physicochemical Formulation............................................................ 43

2.5.1 Effect of Nutritional Factors................................................... 44

2.5.1.1 Carbon Source...................................................... 44

2.5.1.2 Nitrogen Source................................................... 45

2.5.1.3 Metal Ions and Salts............................................. 46

2.5.2 Effect of Physicochemical Parameters.................................... 48

2.5.2.1 Temperature......................................................... 48

2.5.2.2 pH......................................................................... 49

2.5.2.3 Agitation and Aeration......................................... 50

2.5.2.4 Inoculums Percentage and Incubation Time........ 51

2.6 Physicochemical Optimization.......................................................... 52

2.6.1 Media Optimization................................................................ 54

2.6.1.1 Synthetic Medium................................................ 55

2.6.1.2 Agricultural Residue............................................ 57

2.7 Purification and Characterization....................................................... 58

2.7.1 Partial Purification Strategies.................................................. 59

2.7.1.1 Concentration of Protein............................................ 59

2.7.1.2 Precipitation............................................................... 59

2.7.1.3 Column Chromatography.......................................... 61

2.7.1.3.1 Ion Exchange Chromatography................ 62

2.7.1.3.2 Gel Filtration Chromatography................ 64

2.7.1.3.3 Hydrophobic Interaction

Chromatograph………………………......

64

2.7.2 Characterization of Proteases Enzyme.................................... 65

2.7.2.1 pH and Temperature............................................. 65

2.7.2.2 Activators and Inhibitors...................................... 66

2.8 Industrial Application of Bacterial Proteases..................................... 67

2.8.1 Detergent Additives................................................................ 67

2.8.2 Leather Industry...................................................................... 69

2.8.3 Food Industry.......................................................................... 70

2.8.4 Silver Recovery....................................................................... 71

2.8.5 Waste Treatment..................................................................... 71

2.8.6 Silk Industry............................................................................ 72

x

2.8.7 Pharmaceutical Industry.......................................................... 73

2.9 Summary............................................................................................ 74

CHAPTER THREE: MATERIALS AND METHODS.................................. 75

3.1. Introduction....................................................................................... 75

3.2. Experimental Materials..................................................................... 75

3.2.1. Raw Material........................................................................ 75

3.2.2. Microorganism and Maintenance of Culture....................... 76

3.2.3. Experimental Apparatus....................................................... 77

3.3. Experimental Methods...................................................................... 77

3.3.1. Pretreatment Skim Latex Effluent........................................ 77

3.3.2. Inoculums Preparation......................................................... 77

3.3.3. Protease Production.............................................................. 78

3.3.4. Protease Activity Assay....................................................... 78

3.3.5. Standard Curve for L-Tyrosine............................................ 79

3.3.6. Estimation of Protein Concentration....................................

3.3.7. The Effect of Skim Latex Effluent on Bacillus licheniformis

Proteases Production......................................

79

80

3.3.8. Statistical Optimization Procedure....................................... 81

3.3.8.1. Plackett-Burman Experimental Design for

Parameter Screening............................................. 81

3.3.8.2. One Factor at a Time (OFAT) Study.................... 83

3.3.8.3. Response Surface Methodology............................ 83

3.3.8.4. Validation of Experimental Model....................... 85

3.3.9. Purification of Protease........................................................ 86

3.3.9.1. Ammonium Sulphate Precipitation....................... 86

3.3.9.2. Ion Exchange Chromatography............................ 86

3.3.10. Characterization of Purified Protease................................... 87

3.3.10.1. Molecular Mass Determination of Protease.......... 87

3.3.10.2. Effect of pH on the Protease Activity................... 88

3.3.10.3. Effect of Temperature on the Protease Activity.... 89

3.3.10.4. Effect of Metal ion on the Protease Activity......... 89

3.3.10.5. Effect of Enzyme Inhibitors on the Protease

Activity..................................................................

89

3.3.10.6. Determination of Enzyme Kinetics....................... 90

3.3.11. Application of Produced Protease in Detergent Industry..... 91

3.3.11.1. Effect of Surfactants on Protease Activity............ 91

3.3.11.2. Effect of Solvents on Protease Activity................ 91

3.3.11.3. Compatibility Study of the Produced Protease

with Commercial Detergent and Removal of

Blood Stain............................................................

91

3.4. Summary..................................................................................................... 92

CHAPTER FOUR: RESULTS AND DISCUSSION................................. 93

4.1. Introduction...................................................................................

4.2. The Effect of Skim Latex Effluent on Bacillus licheniformis

Protease Production.......................................................................

93

94

4.3. Optimization of Skim Latex Serum Medium Using Statistical

Techniques....................................................................................

95

xi

4.3.1. Screening of Physicochemical Factors for Protease

Production Using Plackett-Burman Design......................

4.3.2. One Factor at a Time (OFAT) Study................................

4.3.2.1. Effect of pH on Protease Production.................

4.3.2.2. Effect of Agitation on Protease Production.......

4.3.2.3. Effect of Incubation Periods on Protease

Production..........................................................

4.3.3. Optimization of Physicochemical Factors by Response

Surface Methodology........................................................

4.3.4. Validation of the Experimental Model..............................

96

103

103

104

105

106

113

4.4. Purification of Produced Protease................................................. 114

4.5. Characterization of Produced Protease......................................... 117

4.5.1. Molecular Weight............................................................. 117

4.5.2. pH Optimum and pH Stability of Protease

Activity..............................................................................

118

4.5.3. Temperature Optimum and Thermal Stability of Protease

Activity……..…….…………………………...................

119

4.5.4. Effect of Metal Ions on Protease Activity......................... 122

4.5.5. Effect of Enzyme Inhibitors on Protease Activity............ 124

4.5.6. Kinetic Study of the Produced Protease............................ 126

4.6. Application of Protease in Detergent Industry…….…….............. 128

4.6.1. Effect of Surfactants and Solvents on Protease

Activity..............................................................................

128

4.6.2. Compatibility Study of the Protease with Commercial

Detergent and Removal of Blood Stain............................

130

4.7. Summary....................................................................................... 132

FIVE: CONCLUSION AND RECOMMENDATION.................................... 135

5.1. Conclusion................................................................................... 135

5.2. Recommendation......................................................................... 137

REFERENCES....................................................................................................

LIST OF PUBLICATIONS................................................................................

139

156

APPENDIX 1........................................................................................................ 157

APPENDIX 2........................................................................................................ 158

APPENDIX 3........................................................................................................ 159

APPENDIX 4........................................................................................................ 161

APPENDIX 5........................................................................................................ 163

APPENDIX 6........................................................................................................

APPENDIX 7........................................................................................................

165

166

APPENDIX 8........................................................................................................ 172

xii

LIST OF TABLES

Table No. Page No.

2.1 Composition of field NR latex 12

2.2 Average chemical composition of rubber processing effluent 15

2.3 Characteristic of process effluent from rubber processing with

tolerance limit based on Environmental protection act 1996

15

2.4 The elements of effluent from concentrated and RSS latex 25

2.5 Classification of proteases (peptidases) 31

2.6 Nutrition requirement and their function for growing bacteria 44

2.7 Ion-exchange resins 64

3.1 Mixture compositions of L-tyrosine standard curve 79

3.2 Preparation of BSA standard curve 80

3.3 Comparing study between present and absent of skim latex

serum component in the fermentation culture

81

3.4 Physicochemical components used in Plackett-Burman

design

82

3.5 Experimental Design using Face Centered Central Composite

Design of four independent variables with six center points

85

3.6 Validation of the Experimental Model 86

4.1 Plackett-Burman experimental design for evaluation of 11

factors with actual and coded values for protease production

98

4.2 Ranking of the variables investigated in the Plackett-Burman

design

100

4.3 Analysis of variance for protease production by Bacillus

licheniformis on skim latex serum as the basal media

102

4.4 Face centered central composite design of four independent

variables with their actual value showing the experimental

and predicted response

107

xiii

4.5 Analysis of variance for response surface quadratic model 108

4.6 Experimental and predicted value of protease activity for

FCCCD matrix (Second optimization)

111

4.7 Analysis of variance for response surface quadratic model

(second optimization)

112

4.8 Validation of the experimental model 114

4.9 Purification scheme for B.licheniformis protease 116

4.10 Effect of various metal ions on B.licheniformis protease 123

4.11 Effect of Detergents and Solvents on Protease Activity 129

xiv

LIST OF FIGURES

Figure No. Page No.

1.1 The contribution of proteases in the total enzyme sale

2

1.2 Summary of some keys research methods and their

descriptions

8

2.1 Schematic diagram of raw rubber processing and products

manufacturing

12

2.2 The flow sheet of steps involved in concentration of NRL by

centrifugation

14

2.3 Ultracentrifugation of Hevea brasiliensis latex

16

2.4 Structure of Cis-1,4-polyisoprene

17

2.5 Schematic drawing of a ruber molecule

18

2.6 Structure of alpha-lechitin

19

2.7 Organic non-rubber constituents of latex

20

2.8 Acylation and deacylation of mechanism of action of

chymotrypsin proteases

37

2.9 Mechanism of aspartic proteases

38

2.10 A mechanism for the action of papain

40

2.11 A mechanism for peptide hydrolysis by carboxypeptidase A

41

2.12 Principle of anion exchange separation

63

3.1 An overview of experimental strategies

76

4.1 The comparison study between the present and absent skim

latex serum component on the fermentation culture at fixed

level of galactose, skim latex serum, LB broth, inoculums

size, temperature and incubation period.

94

4.2 Pareto graph showing the main effect result of the 11

components for protease production based on Plackett-

Burman experimental result

100

xv

4.3 Effect of different pH on protease production by B.

licheniformis (ATCC 12759) with constant for other

conditions

104

4.4 Effect of different agitation rate on protease production by

Bacillus licheniformis (ATCC 12759) with constant for other

conditions

105

4.5 Effect of different incubation time on protease activity by

B.licheniformis with constant for other conditions

106

4.6 3D response surface curves showing the effects of: (a) skim

latex serum and galactose, (b) pH and galactose and (c)

agitation and galactose on protease production by

B.licheniformis (ATCC 12759)

110

4.6 Three dimensional graphs showing the interaction between

pH and agitation on protease production by Bacillus

licheniformis (ATCC 12759) at fixed level of galactose, skim

latex serum, LB broth, inoculums and temperature

113

4.8 Chromatography of B.licheniformis protease on DEAE-

sepharose. The column (1.5x10 cm) was equilibrated with

1.5 M Tris-buffer (pH 8) loaded with enzyme preparation and

eluted with a linear gradient (0 up to 1M NaCl) at a flow rate

2 ml/min

116

4.9 SDS-PAGE analysis of collected fraction from ion exchange

chromatography. M: marker, F2-F10: inactive fraction in

first washing, F12-F30: fraction in elution phase, F20 – F26:

fraction with high protease activity

118

4.10 Effect of pH and stability on the enzyme activity. The

maximum activity at pH 7 was taken as a control (100 %),

(*value in figure represented as mean ± SD)

119

4.11 Effect of temperature on B. licheniformis protease activity,

the maximum activity of enzyme at 65 oC was taken as a

control (100 %) (*value in figure represented as mean ± SD)

120

4.12 Effect of temperature on the thermo stability of B.

licehniformis protease

121

4.13 Effect of various protease inhibitors on The protease activity

from B.licheniformis

124

xvi

4.14 Hyperbolic regression of Michaelis-Menten equation for

Bacillus licheniformis protease

126

4.15 Lineweaver-Burk plot for determining of KM and Vmax using

casein as substrate

127

4.16 Compatibility of protease from Bacillus licheniformis

(ATCC 12759) with Tesco Everyday Value® detergent

132

4.17 Application of the enzyme in removal of blood stain. (a) The

stained clothes after drying. (b) The stained clothes after 30

min incubation at 60 oC (1: control, 2: the stained cloth + the

detergent only, 3: the stained cloth + the detergent + the

protease, 4: the stained cloth +enzyme only).

132

xvii

LIST OF ABBREVIATIONS

3D 3 Dimensions

ADS Air dried sheet

ANOVA Analysis of variance

ATCC American Type Culture Collection

BOD Biochemical oxygen demand

BSA Bovine serum albumin

C/N Carbon/Nitrogen

CCD Central composite design

COD Chemical oxygen demand

CV Coefficient variation

CV* Constant viscosity

DCL Dichloroisocoumarin

DEAE Diethylaminoethyl

DFP Diisopropyl fluoro phosphate

DGDG Digalactosyl diglyceride

DOE Department of environment

DPNR Deproteinised natural rubber

DRC Dried rubber content

DTT Dithiothreitol

EC Enzyme commission

EDTA Ethylene diamine tetra acetic acid

ENR Expoxidised natural rubber

ESEM Environmental scanning electron microscope

ESG Esterified sterylglycoside

FCCCD Face centered central composite design

FW Frey-Wyssling

GF Gel filtration

GLA (γ-linoleic acid)

GP General purpose

H2S Hydrogen sulphide

HIC Hydrophobic interaction chromatography

IAA Indole acetic acid

IEC Ion exchange chromatography

IIUM International Islamic University Malaysia

KM Michaelis constant

LB Luria bertani

LBHB Low-barrier hydrogen bond

LNR Liquefied natural rubber

MGDG Monogalactosyl diglyceride

NR Natural rubber

NRSL natural rubber skim latex

NRSP Natural rubber serum powder

OFAT One factor at a time

PBd Plackett-Burman design

xviii

PHA Polyhydroxyalkanoates

PLC Pale latex crepe

PMSF Phenyl methyl sulfonyl fluoride

RRIM Rubber Research Institute of Malaysia

RSM Response surface methodology

RSS Ribbed smoked sheet

SD Standard deviation

SDS Sodium dodecyl sulphate

SDS-PAGE Sodium dodecyl sulphate-Polyacrylamide gel

electrophoresis

SmF Submerged fermentation

SMR Standard Malaysia rubber

SSF Solid state fermentation

STR Standard Thai rubber

TLCK Tosyl-L-lysine chloromethyl ketone

TSR Technically specified rubber

v/v Volume per volume

Vmax Maximum rate of reaction

w/v Weight per volume

1

CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND

Proteases (hydrolyses endopeptidases, EC. 3.4.21-24), are enzyme that catalyze the

breakdown of protein. They represent about 2 % of the total proteins in all organisms

(Polgar, 2005). Principally, they hydrolyze protein via the addition of water across

peptide bonds and catalyze peptide synthesis in solvent or in organic solvent with

limited water content. The distinctive characteristics of proteases such as substrate

specificity, catalytic mechanism, pH and thermo stability, and solvent tolerant convey

them to a crucial position with respect to their applications in both technical and

physiological field (Rhao et al., 1998; Jisha et al., 2013).

According to Li et al. (2012), currently almost 4000 enzymes have been reported

by researchers which are about 158 enzymes for nutritional industry, 64 enzymes for

technical application, 57 enzymes for feedstuff, and 24 enzymes have been applied in

three mentioned sectors. However, the industrial scale production has been satisfied by

only 20 enzymes with 75 % are hydrolytic enzymes. Proteases occupy the second

position after carbohydrases and before lipases in the world enzyme market with high

potential in technical application as well as tools for research and development.

At present, the total industrial enzymes market attained $3.3 billion in 2010 and

is predicted to attain a value of $4.4 billion by 2015. Of these, technical enzyme that is

used in huge amount as crude enzyme has been found its application in textile,

detergent, pulp and paper, and bio-fuels industries. Technical enzyme alone had

revenues approximately $1.2 billion in 2011 and estimated to increase up $1.5 billion

2

in 2015 and $1.7 billion in 2016 respectively (Adrio and Demain, 2014). Of the

industrial enzymes that are dominated by hydrolyses, proteases have been accounted

for 60 % of the global sale of enzymes (Figure 1.1) (Rhao et al., 1998).

Figure 1.1. The contribution of proteases in the total enzyme sale (Rhao et al., 1998).

Proteases have executed a large variety of functions from cell to organism level.

They mediate processes in human body such as coagulation, digestion, activation of

proenzyme and prohormones, apoptosis, and breakdown of intracellular proteins

(Chapman et al., 2001). Their applications against cancer and AIDS were reported by

researchers (Blankenvoorde et al., 2000; Rakashanda et al., 2012; Chanalia et al., 2011).

In addition, proteases have documented their history in food and detergent industries.

Protease from Bacillus licheniformis strain was recorded in the third edition of Food

Chemicals Codex as a source of enzyme involving in food processing (Salleh et al.,

2006). Proteases as detergent additives began in 1913 when Rohm and Hass marketed

‘Burnus’, then was followed by Bio-40, Biotex, Maxatase, Era plus®, Tide®, Dynamo®

and others (Kumar et al., 2008). The new development of proteases in leather industries

was suggested as early as 1910 for de-hairing and bating of hides for substituting toxic

3

chemicals (Gaur and Gupta, 2012). Thus, the vast diversity of proteases has attracted

researches attention in attempts to produce them with the excellent properties for a

specific application.

Among the available sources for proteases, microbial proteases that can be

easily manipulated to improve the desired properties are currently receiving more

attention due to technological and economic reasons. In the technical production,

microbes showed the outstanding properties such as fast growing, simple culturing for

large scale production, and metabolically vigorous to secrete large amount of protein

directly into the fermentation medium that help to simplify the purification steps

(Illanes, 2012). Moreover, in the economic perspective, micro and macro nutrients for

growth of microbial proteases can be provided easily by using low cost media to

enhance the yield (Nadeem et al., 2008). However, since there is no defined medium

established for the excellent protease production by different microbes (Bhunia et al.,

2012), identification of the optimized nutritional and physical parameters has been the

main goal of basic research and industrial application (Saravankumar et al., 2010).

Hence, this strategy is foreseen to reduce the budget of enzyme production by using

abundantly inexpensive raw material such as skim latex effluent.

Skim latex effluent, a liquid waste from rubber factory that is rich in various

organic compounds and potentially environment polluting has been proved to be an

important basal media for various fermentation process (Ishizaki and Fukuoka, 1991;

Mahat and MacRae, 1991; Tri-Panji et al., 1994; Tri-Panji and Suharyanto, 2001,

Kresnawati et al., 2008). The utilization of latex serum as a major component of

microbiological media could produce protease with an alternative low cost media as

well as increase the added value of the effluent. This is due to the fact that serum from

skim latex could replace nitrogen, carbohydrates and minerals (Mg, P, K, and Ca)

4

sources (Tri-Panji and Suharyanto, 2001; Siswanto, 1999). Besides, skim latex serum

has been found to have a remarkable growth-promoting effects for certain bacteria

including Bifidobacterium (Etoh et al., 1999), Spirulina platensis (Tri-Panji and

Suharyanto, 2001), and Rhizopus oligosporus (Nuradibah, 2012). Therefore, this

research effort utilized skim latex serum as a growth medium for Bacillus licheniformis

(ATCC 12759) in protease production by liquid state fermentation.

1.2 PROBLEM STATEMENT

The use of proteases was found in all aspects of human life from detergent to brewing

industries and the highest application of proteases has been scored in laundry detergent

formulation which was account for 25 % of the global enzyme sale (Andrio and Demain,

2014). However, the expansion of proteases in detergent industries was restricted in

supply to those only four major producers of protease in the world; they are Novo

Industries Dermark, Gist-Brocades Netherlands, Genecor International United State and

Miles Laboratories United State (Salleh et al., 2006) and small companies that come

from Japan and China (Li et al., 2012). Malaysia alone was reported as a major importer

of enzymes (protease, lipase, amylases and cellulases) and detergents with increased

consumption rapidly. The imported enzyme by this country causes the cost of detergent

products relatively expensive. Moreover, another obstacle hindering the expansion of

protease is the high production budget (Ibrahim, 2008) that around 40 % of the

production budget depends on the cost of composition medium (Nadeem et al., 2008).

Hence, it is relevant for any countries particularly Malaysia to consider cheap source

material for enzyme production (Ibrahim, 2008). One of them is skim latex that come

from natural rubber (NR) industries.

5

NR industry plays an important role in Malaysia by offering employment

opportunities for more than 68,700 people (Saidur and Mekhilef, 2010) and providing

important raw material for local rubber-based industries. At the same time, it has

produced large quantities of effluent since the production of rubber products from NR

requires huge amount of water for its operation (Smitha et al., 2012; Hien and Thao,

2012; Rosman et al., 2013). The main sources of the effluent in Malaysia have been

identified from latex skim, latex serum, uncoagulated latex and washing from the

various processing stages which generate 20 tons of rubber and 410 tons of the waste

daily (Mohammadi et al., 2010). In particular, it was reported by Atagana et al. (1999)

that Malaysia alone produces up to 205 tons of natural rubber waste serum per day.

Effluent from concentrated latex factory could render environmental impact that include

water and odour pollutions due to a high COD (32,690 mg/l), BOD (13,760 mg/l),

nitrogen (4.620 mg/l) and suspended solid (SS) (42,550 mg/ml) level with acidic pH

(4.8) (Tekasaful and Tekasaful, 1999; Krisnawaty et al., 2008; Arimoro, 2009; Hien and

Thao, 2012).

The effluent has also high level of concentration ammonia (540 mg/ml)

(Tekasaful and Tekasaful, 1999) and sulphate (1500 mg/ml) (Mohammadi et al., 2010)

that cause problem in the biological anaerobic treatment system. The ammonia and

sulphate from the natural rubber process discharge into water body and promote

acidification process as well as inhibit the digestion process by lowering organic

removal efficiency (Veerasamy et al., 2008; Mohammadi et al., 2010; Veerasamy and

Ismail, 2012). These processes are harmful for the life (Atagana et al., 1999; Arimoro,

2009). Another problem related to the ammonia and sulphate solution released into air

is the unpleasant odour especially near the centrifugation area. This has adverse effect

6

on worker’s health as well as the nearby community’s health that may develop the

respiratory system irritant (Tekasaful and Tekasaful, 1999).

Apart from odour and health problems, the cost for discharging the waste is also

expensive. This is because high quantity of water is needed to discharge the waste into

effluent treatment pond in order to meet the requirement that had been stated by

Department of Environment (DOE) on the biological treatment system (Mohammadi et

al., 2010). Besides, large amount of organic matter (95 %) including acetic acid, sugar,

protein, lipid and mineral salt that are present in skim latex, forcing rubber manufactures

to spend a lot of money in waste management and effluent treatment. This massive

waste production requires proper treatment that is efficient, rapid and low cost

technology (Werathirachot et al., 2008; Al Khidir and Zailani, 2009; Mohammadi et al.,

2010; Hien and Thao, 2012).

Therefore, with respect to natural rubber waste serum that contains various

organic compounds, utilization of this effluent as a basal media could be a promising

technology for culturing bacteria using alternative either nitrogen, carbohydrate, and

trace metal source from the liquid waste of latex concentrate as well as minimize

environmental problem by valorization of the effluent.

1.3 RESEARCH OBJECTIVES

The objectives of the research are:

i. To identify the optimized conditions for production of protease using skim

latex serum as a basal media by Response Surface Methodology.

ii. To purify and characterize the produced protease

iii. To apply the produced protease in removal of blood stain with and without

of the commercial detergent for enhancing cleaning process.