DEVELOPMENT OF BIOPROCESSING TECHNIQUE FOR THE PRODUCTION AND PURIFICATION OF
PHYTATE-DEGRADING ENZYME FROM MALAYSIAN SOIL BACTERIA
BY
ANIS SHOBIRIN MEOR HUSSIN
INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA
2008
DEVELOPMENT OF BIOPROCESSING TECHNIQUE FOR THE PRODUCTION AND PURIFICATION OF
PHYTATE-DEGRADING ENZYME FROM MALAYSIAN SOIL BACTERIA
BY
ANIS SHOBIRIN MEOR HUSSIN
A thesis submitted in fulfilment of the requirement for the degree of PhD in Engineering (Biotechnology)
Kulliyyah of Engineering International Islamic University
Malaysia
FEBRUARY 2008
ii
ABSTRACT
Due to several biological characteristics, bacterial phytate-degrading enzymes have considerable potential in commercial and environmental applications. Until now, there is no single phytase that is able to meet the diverse needs for all commercial and environmental applications. Phytase enzyme preparations have a wide range of applications in animal and human nutrition as well as bio fertilizer. Bacteria strains were isolated from Malaysian maize fields and roots, and screened for phytate- degrading enzyme activity. The production and purification of extra-cellular phytate- degrading enzyme from the most potential strain as phytase producer, Enterobacter sakazakiii ASUIA279 was optimized using response surface methodology (RSM) with full-factorial faced centered central composite design (FCCCD). An extra- cellular phytate-degrading enzyme synthesized by E. sakazakii ASUIA279 was purified to homogeneity using FPLC anion exchange chromatography and gel filtration. The arrangement of the bacterial isolates according to their ability to produce extra-cellular phytate-degrading enzymes were Enterobacter sakazakii ASUIA279 > Pantoea stewartii ASUIA271 > Bacillus cereus ASUIA260. The optimum combination of cultivation conditions for maximum phytate-degrading enzyme production were determined at incubation temperature which is 39.7 ºC, initial pH 7.1, rice bran percentage at 13.6 %, 320 rpm of agitation and 0 vvm of aeration. The optimum combinations of experimental conditions in ion exchange chromatography were at pH 5.1 and 47 mM of sodium acetate buffer. Its molecular mass was estimated to be 43 kDa by SDS-PAGE. The Machaelis constant (KM) and turnover number (kcat ) for sodium phytate at pH 5.0 and 50 °C were calculated from the Lineweaver-Burk plot to 760 µM and 4.14 s-1, respectively. Optimal activity was determined at pH 4.5 and 50 ± 5 °C. The enzyme was strongly inhibited by Fe3+, Cu2+, Zn2+, molybdate, vanadate, fluoride and phosphate (1 mM). In this study, an extra- cellular phytate-degrading enzyme with promising properties as an animal feed additives or soil nutrient enhancement produced by E. sakazakii ASUIA279, a newly isolated bacteria strain was obtained. The phytate-degrading enzyme synthesis by these bacteria was triggered by the high content of organic phytate in the rice bran.
Rice bran could be utilized and developed as a media for the production of this enzyme.
iii
ثحبلا صخلم
وذ ربتعت اھنإف تيتيافلا ايريتكب للحت نم ةجتانلا تاميزنلال ةديدعلا ةيجولويبلا صاوخلل رظنلاب ةديدع ةيئيبو ةيداصتقا تاقيبطت .
ةھجاومل لباقلا سيتيافلا نم عون يأ دجويلا ، نلاا ىتح
ةيئيبلاو ةيداصتقلاا تاقيبطتلا لكل ةددعتملا تاجاحلا .
فلا ميزنأ ريضحت ةعساو تاقيبطت هل سيتيا
يجولويب دامسك كلذكو ،ناويحلاو ناسنلإا ةيذغت يف .
ةرذلا لوقح نم ايريتكبلا راتوأ لصف مت
طشنلا ميزنلاا ىلع لوصحلل اھفينصتو ،ايزيلام يف روذجلاو .
تيتيافلا نم ميزنلاا ةيقنتو جاتنإ
رتكابورتنإ نم ناك سيتيافلا جتنمل ةيعون ىلعأ نم ديجلا يولخلا ييكازاكاس
) يأ يآ وي سأ يأ
279 ( ةقيرطب هل ةجيتن ىصقأ ىلع لوصحلا متو )
مأ سأ رآ (
لماوعو )
يد يس يس يس فأ .(
زاھجب هتيقنت متو )
أ فأ يس يب ل (
لجلا ةرتلف عم .
ةيلضفأ بسح ةلوصفملا ايريتكبلا بيترت نإ
يتلآاك ناك ديجلا ميزنلاا جاتنإ :
ييكازاكاس رتكابورتنإ )
يآ وي سأ يأ يأ
279 ( يوتناب ،
يتراويتس )
يأ يآ وي سأ يأ 271
( سويريس سوليساب مث )
يأ يآ وي سأ يأ 260
.(
تناكو
ةناضح ةجرد يف يھ ميزنلال جاتنإ نسحأ ىلع لوصحلل فورظ ىصقأ 39.7
،ةيوئم ةجرد
ةيلوأ ةيضماح 7.1
زرلا ةلاخن ةبسن ، 13.6
% ةعرسب طلخو ، 320
ديوزت نودبو ةقيقدلاب ةرود
ءاوھلل . و ةيضماح ةجرد يھ ينويلاا لدابتلا فاركوتامورك يف فورظ ىصقأ تناك 5.1
و 47
مويدوصلا تلاخ نم رلاوم يلم .
يئيزجلا نزولا نيمخت مت 43
ةينقت مادختساب نوتلادوليك )
سأ
سأ يد -
جيب .(
سيليكام تباث باسح مت )
مأ يك ( نارودلا ددعو )
تاك يك (
مويدوصلا تيتيافل
ةيضماح دنع 5.0
ارحلاو ةر 50 رفييو نيلا ةقلاع نم ةيوئم ةجرد -
ىلا كروب 760
و رلاوموركيام 4.14
ةبلاس ةيناث .
ةيضماح دنع هيلع لوصحلا مت طاشن ىصقأ 4.5
ةرارحو 50
+ 5 ةيوئم ةجرد .
،نيصراخلا ،ساحنلا ،كيديدحلا نويآ ةطساوب ميزنلاا طاشن ليلقت مت
تافسوفلاو ديارولفلا ،تيدانافلا ،تيدبيلوملا )
1 م رلاوم يل .(
ىلع لوصحلا مت ،ةساردلا هذھ يف
ةيذغت نيسحتل وأ يناويحلا ءاذغلل ةفاضم ةدامك ةرظتنملا تافصاوملا عم ديجلا يولخلا ميزنلاا ييكازاكاس رتكابورتنإ ةطساوب ةبرتلا )
يأ يآ وي سأ يأ 279
( ةديدج ةيعون ىلع لوصحلا متو ،
ةلوصفملا ايريتكبلا راتوأ نم .
تملا ميزنلاا نإ يلاعلا ىوتحملا ببسب ناك ايريتكبلا هذھ نم نوك
زرلا ةلاخن يف دوجوملا يوضعلا تيتيافلل .
جاتنلا طسوك اھنيسحتوزرلا ةلاخن مادختسإ نكمم
ميزنلاا
.
iv
APPROVAL PAGE
The thesis of Anis Shobirin Meor Hussin has been approved by the following:
Abd-El Aziem Farouk Gad Supervisor
Abdul Manaf Ali Co-Supervisor
Mohammed Ismail Abdul Karim Co-Supervisor
Hamzah Mohd Salleh Co-Supervisor
Suleyman Aremu Muyibi Internal Examiner
Isam Yassin M. Qudsieh Internal Examiner
v Aini Ideris External Examiner
Nasr Eldin Ibrahim Ahmed Chairman
vi
DECLARATION
I hereby declare that this dissertation is the result of my own investigations, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degree at IIUM or other institutions.
Anis Shobirin Meor Hussin
Signature……… Date………
vii
INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA
DECLARATION OF COPYRIGHT AND
AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH
Copyright © 2008 by Anis Shobirin Meor Hussin. All rights reserved.
BIOPROCESS DEVELOPMENT OF PRODUCTION AND PURIFICATION OF PHYTATE-DEGRADING ENZYME FROM
MALAYSIAN SOIL BACTERIA
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 as provided below.
1. Any materials contained in or derived from 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 a retrieval system and supply copies of this unpublished research if requested by other universities and research libraries.
Affirmed by Anis Shobirin Meor Hussin
………. ……….
Signature Date
viii
To my husband Azmi Alias for his patience, endurance and understanding through out this project and my children Muhammad Aniq, Aliah Batrisyia
and Muhammad Aqil
ix
ACKNOWLEDGEMENTS
All praise and thanks to Allah s.w.t for all His Mercy and Blessings for me to complete my PhD thesis. I would like to express my sincerest gratitude to my supervisor, Prof. Dr. Abd-Elaziem Farouk Gad, for his idea, guidance, assistance, and patience, my co-supervisors Prof. Dr. Abdul Manaf Ali, Prof. Dr. Mohammad Ismail Abdul Karim and Assoc. Prof. Dr. Hamzah Mohd Salleh, and my guest advisor Prof.
Dr. Ralf Greiner for their advice and co-operation throughout my period of study.
Beside that, my sincere thanks are due to Prof. Dr. Suleyman Aremu Muyibi and Assoc. Prof Dr Faridah Yusof for their support and the valuable discussion we used to have throughout. I would also like to express my warmest appreciation to my dear husband and adorable children for their patience and continued support throughout this time. Their loves have made it possible for me to continue my studies until now. I would also like to thank my parent, in laws, siblings and all my friends, in and outside of the university, for their care and support. Sincerely thanks to my friends, Rohani Salleh, Haslinda Hasim, Mohamed Faizal Batcha, Mohamed Faizal Ghouse and Shareef Mohideen Ismail for their help and kind friendship. Last but not least, I would like to thank all IIUM staff, direct and indirect helped me to complete this thesis.
x
TABLE OF CONTENTS
Abstract………..……… ii
Abstract in Arabic………..……… iii
Approval Page……….………….. iv
Declaration Page……….……….. vi
Copyright Page……….……... vii
Dedication………..…..….. viii
Acknowledgements………..………….. ix
Table of Contents………..……... x
List of Tables…... xiv
List of Figures………...………. xvi
List of Abbreviations………..……..…. xx
CHAPTER ONE: INTRODUCTION……… 1
1.1 Overview………... 1
1.2 Problem Statement……….………... 3
1.3 The Significance of the Study………..……… 4
1.4 Research Objectives………...………... 5
1.5 Research Methodology ...………..………...6
1.6 Scope of Research…………..……….. 7
1.7 Thesis Organisation………. 8
CHAPTER TWO: LITERATURE REVIEWS………..…….………. 9
2.1 Overview………... 9
2.2 Phytate………...…..…..……….……..9
2.2.1 Unfavourable Effects of Phytate…………...……….. 13
2.2.2 Favourable Effects of Phytate………...…..……….. 15
2.2.3 Rice Bran……….…...………. 16
2.3 Phytate-degrading Enzyme………..…………..………... 17
2.4 The Occurrence of Phytate-degrading Enzyme…………..………..…... 19
2.4.1 Phytate-degrading Enzyme from Plant………..………. 20
2.4.2 Phytate-degrading Enzyme from Animal……..………. 20
2.4.3 Phytate-degrading Enzyme from Micro-organisms.……... 21
2.5 Bacterial Phytate-degrading Enzyme Formation………..………... 24
2.5.1 In vivo Function of Phytate-degrading Enzyme…..……… 25
2.6 Bioprocess of Production and Purification For Phytate-degrading Enzyme from Micro-organisms……….……….. 28
2.6.1 Phytate-degrading Enzyme Production……….………. 28
2.6.1.1 Physical Parameters……….………... 28
2.6.1.2 Nutritional Parameters……….………29
2.6.2 Phytate-degrading Enzyme Purification.……… 31
2.6.2.1 Ion-exchange Chromatography………... 34
2.7 Characterization of Phytate-degrading Enzyme.………. 35
2.7.1 Physical Properties……….…. …... 35
2.7.1.1Temperature……….…... 35
xi
2.7.1.2 pH………... 36
2.7.1.3 Effect of Metal Ions……… 37
2.7.1.4 Effect of Substrate………... 39
2.7.1.5 Thermostability………..………. 40
2.7.2 Molecular Properties………..…………..……... 41
2.7.2.1 Molecular Weight………..………. 41
2.8 Application of Phytate-degrading Enzyme………….……….……... 42
2.8.1 Animal Feed Industry……….……….………... 42
2.8.2 Food Industry……….………. 43
2.8.3 Potential in Aquaculture……….……...………. 44
2.8.4 Soil Nutrient Enhancement...……….………. 44
2.8.5 Semisynthesis of Peroxidase……….. 45
2.9 About the Present Study………... 46
3.0 Summary……….. 46
CHAPTER THREE: MATERIALS AND METHODS………... 47
3.1 Overview………. 47
3.2 Isolation and Screening of Bacterial Phytate-degrading Enzymes from Malaysian Maize Fields and Roots……….…..47
3.1.1 Chemicals and Media……….………. 47
3.1.2 Collection of Samples……….……….……... 48
3.1.4 Storage Procedure for Bacterial Isolates.……….…….. 49
3.1.5 Screening of Phytate-degrading Enzymes.………...……... 49
3.1.6 Sample Preparation for Enzyme Assays.………...…….. 49
3.1.7 Standard Phytate-degrading Enzyme Assay………... 50
3.3 Evaluation and Identification of Potential Phytate-degrading Enzyme Producing Bacteria from Malaysian Maize Fields and Roots………..……. 51
3.3.1 Bacteria Culture and Media……….…... 51
3.3.2 Cultivation Media………….……….…...……….. 51
3.3.3 Induction Study………... 51
3.3.4 Species Identification………...…... 52
3.3.4.1 Phenotypic Technique………...…... 52
3.3.4.2 Genotypic Technique………... 52
3.4 The Behavioural Study of Phytate-degrading Enzyme Production Produced by Newly Isolated Bacteria from Malaysian Maize Root……….... 53
3.4.1 Bacterial Strains………... 53
3.4.2 Behavioural Study in Media with Different Concentration of Rice Bran ……….……... 54
3.4.3 Behavioural Study in Phytate Media and Phosphate-limited Media……….…... 54
3.4.4 Behavioural Study in Different Temperature and pH……... 55
3.4.5 Bacteriological Analysis……….……… 55
3.5 Optimization of Cultivation Conditions for the Production of Phytate-degrading Enzyme by Enterobacter sakazakii ASUIA279 Newly Isolated Bacteria from Malaysian Maize Root…….…….……... 56
3.5.1 Bacterial Strain……….………..….…………56
3.5.2 Experimental Design……….……….………. 56
xii
3.6 Optimization of Experimental Conditions in Ion Exchange Chromatography for the Purification of Phytate-degrading Enzymes Produced by Enterobacter sakazakii ASUIA279
Newly Isolated Bacteria from Malaysian Maize Root………...…. 59
3.6.1 Bacterial Strains and Chemicals………....……… 59
3.6.2 Production of Phytate-degrading Enzyme………...……… 60
3.6.3 Enzyme Extraction………..……… 60
3.6.4 CM-Fast Flow Sepharose Chromatography………... 60
3.6.5 Experimental Design………..……… 61
3.7 Purification and Properties of A Phytate-degrading Enzyme Produced by Enterobacter sakazakii ASUIA279 Newly Isolated Bacteria from Malaysian Maize Root……….…… 63
3.7.1 Bacterial Strain and Chemicals………..……… 63
3.7.2 Enzyme Extraction……….……… 63
3.7.3 Purification of Phytate-Degrading Enzyme……….……….. 63
3.7.3.1 CM-Fast Flow Sepharose Chromatography……….. 63
3.7.3.2 Sephacryl S-200 HR Chromatography……….. 64
3.7.4 Protein Determination……….……… 64
3.7.5 Molecular Weight Determination………... 64
3.7.6 Phytate-degrading Enzyme Properties Study………. 64
3.7.6.1 Substrate Selectivity……… 64
3.7.6.2 pH Optimum and Stability………..………….……... 65
3.7.6.3 Temperature Optimum and Stability…………...………... 65
3.7.6.4 Cations and Potential Inhibitors ……….………… 65
3.8 Statistical Analysis…………...………..……. 66
3.9 Summary……….…… 66
CHAPTER FOUR: ANALYSIS OF RESULTS AND DISCUSSION……….. 67
4.1 Overview……….…….... 67
4.2 Isolation and Screening of Bacterial Phytate-degrading Enzymes from Malaysian Maize Fields and Roots………. 67
4.2.1 Discussion……….. 71
4.3 Evaluation and Identification of Potential Phytate-degrading Enzyme Producing Bacteria from Malaysian Maize Fields and Roots………. 73
4.3.1 Effect of Cultivation Media……… 73
4.3.2 Induction Study……….. 79
4.3.3 Species Identification………... 81
4.3.3.1 Phenotypic Technique……… 81
4.3.3.1 Genotypic Technique………... 84
4.3.4 Discussion………...………… 85
4.4 The Behavioural Study of Phytate-degrading Enzyme Production Produced by Newly Isolated Bacteria from Malaysian Maize Root………..…….. 88
4.4.1 Behavioural Study in Different Concentration of Rice Bran Media………...……… 88
4.4.2 Behavioural Study in Phytate Media and Phosphate-limited Media………..……... 95
4.4.3 Behavioural Study at Different Temperature and pH…...……… 96
xiii
4.4.4 Discussion………..……… 98
4.5 Optimization of Cultivation Conditions for the Production of Phytate-degrading Enzyme from Enterobacter sakazakii ASUIA279 Newly Isolated Bacteria from Malaysian Maize Root………..……..……... 102
4.5.1 Discussion……….………….. 112
4.6 Optimization of Experimental Conditions in Ion Exchange Chromatography for the Purification of Phytate-degrading Enzymes Produced by Enterobacter sakazakii ASUIA279 Newly Isolated Bacteria from Malaysian Maize Root……….……... 115
4.6.1 Discussion……….……... 121
4.7 Purification and Properties of a Phytate-degrading Enzyme Produced by Enterobacter sakazakii ASUIA279 Newly Isolated Bacteria from Malaysian Maize Root……….……. 123
4.7.1 Purification of the Phytate-degrading Enzyme………….……….. 123
4.7.2 Molecular Properties……….……….. 127
4.7.3 Enzymatic Properties……….. 128
4.7.3.1Substrate Selectivity……….128
4.7.3.2 pH Optimum and pH Stability………...………. 131
4.7.3.3 Temperature Optimum and Thermal Stability……... 133
4.7.3.4 Effect of Cations and Potential Inhibitors... ……... 135
4.7.4 Discussion……….….………. 137
4.8 Summary………...………….………….. 140
CHAPTER FIVE: CONCLUSION AND RECOMMENDATION….………… 143
5.1 Overview……….……. 143
5.2 Conclusion……….…….……. 143
5.3 Contribution………..….…... 145
5.4 Future Work………..………... 146
BIBLIOGRAPHY………...…...148
APPENDIX A……….………..…... 168
APPENDIX B……….. 169
APPENDIX C……….. 172
APPENDIX D……….. 178
APPENDIX E………... 184
APPENDIX F………... 185
APPENDIX G……….. 186
APPENDIX H... 187
APPENDIX I... 197
APPENDIX J……… 198
xiv
LIST OF TABLES
Table No. Page No.
2.1 Total phosphate and phytate phosphate in common poultry
feedstuffs 10
2.2 Different methods for purifying phytate-degrading enzyme
from microorganism. 33
2.3 Physico-chemical properties of purified phytate-degrading
enzymes 38
3.1 4 X5 factorial arrangement of treatment 54 3.2 Experimental design of three independent variable in
step-one 57
3.3 Experimental design of two independent variable in
step-two 57
3.4 Experimental range and levels of the independent variables
For phytate-degrading enzyme production 59
3.5 Experimental design of three independent variable
For ion exchange chromatography 62
3.6 Experimental range and levels of the independent variables
For phytate-degrading enzyme purification 62 4.1 Production of bacterial phytate-degrading enzymes in different
media after 2 days and 5 days of cultivation 77 4.2 Phenotypic profile of the 30 bacterial strains 82 4.3 Species identification of 30 bacterial isolates using
phenotypic method 83
4.4 Species identification by phenotypic and genotypic methods 85 4.5a Experimental design of three independent variables in
step-one showing experimental response 104 4.5b Experimental design of two independent variables in
step-two showing experimental response 104
xv
4.6a The least-squares fit and parameter estimates (significance of
regression coefficient) for step-one 106 4.6b The least-squares fit and parameter estimates
(significance of regression coefficient) for step-two 106 4.7a ANOVA of the experiment in step-one 107 4.7b ANOVA of the experiment in step-two 108 4.8a Experimental design of three independent variables
in step-one showing experimental and predicted responses 109 4.8b Experimental design of two independent variables
in step-two showing experimental and predicted responses 110 4.9 Experimental (validation studies) and predicted results
(from Response Surface Model) of phytate-degrading enzyme
activity (U/ml) under optimized cultivation condition 112 4.10 Experimental design of three independent variables showing
experimental response for phytate-degrading enzyme
purification 116
4.11 The least-squares fit and parameter estimates (significance
of regression coefficient) 117 4.12 ANOVA of the experiment conditions 118 4.13 Experimental design of three independent variable showing
experimental and predicted responses 119 4.14 Experimental (validation studies) and predicted results
(from Response Surface Model) of phytate-degrading enzyme
activity (U/ml) under optimized experimental conditions 121 4.15 Purification scheme for the phytate-degrading enzyme from
Enterobacter sakazakii ASUIA279 127 4.16 Kinetic constants for the hydrolysis of phosphorylated compounds
by the phytate-degrading enzyme from Enterobacter sakazakii
ASUIA279 at temperature 50 °C and pH 5 130
xvi
LIST OF FIGURES
Figure No.
Page No.
2.1 Biochemical pathway of phytate biosynthesis in plant 11
2.2 Illustration of the complexes formation of phytate with protein
and mineral 14
2.3 Rice bran 17
2.4 Reaction catalysed by the phytate-degrading enzyme (phytase) 18 4.1 Maize (Zea mays) plantation in Seremban, Negeri Sembilan
(2 month old) 68
4.2 Maize (Zea mays) root was harvested from maize plantation in
Seremban, Negeri Sembilan. 68
4.3a Screening for phytate-degrading enzyme activity of bacteria
isolated from halosphere zone of maize plantation 69 4.3b Screening for phytate-degrading enzyme activity of bacteria
isolated from rhizosphere zone of maize plantation 70 4.3c Screening for phytate-degrading enzyme activity of bacteria
isolated from endophyte zone of maize plantation 70 4.4a Phytate-degrading enzyme activity bacterial strains
cultivated in different types of media after 5 days
cultivation (Origin: R- rhizosphere, E- endophyte). Error bars
shows mean standard deviation 74
4.4b Phytate-degrading enzyme activity of bacterial strains cultivated in different types of media after 5 days
cultivation (Origin: R- rhizosphere, E- endophyte). Error bars
shows mean standard deviation 75
4.4c Phytate-degrading enzyme activity bacterial strains cultivated in different types of media after 5 days
cultivation (Origin: R- rhizosphere, E- endophyte). Error bars
shows mean standard deviation 76
4.5 Phytate-degrading enzyme activities of 30 strains of soil bacteria after 5 days cultivation in LB and LB + 5% rice bran media.
Error bars show mean standard deviation 80
xvii
4.6 PCR products of partial amplication of 16 rRNA from
Primer (A) PF3, PR4, and (B) PF5, PR6. Lane: 1, ASUIA279;
2, ASUIA271; 3, ASUIA260; 4, ASUIA243; 5, ASUIA138;
6, ASUIA30; M, 1 kb Ladder; B, Negative Control 84 4.7 Colonies morphology of Bacillus cereus ASUIA260 on LB
agar plate 88
4.8 Colonies morphology of Pantoea stewartii ASUIA271 on LB
agar plate 89
4.9 Colonies morphology of Enterobacter sakazakii ASUIA279 on
LB agar plate 89
4.10 Phytate-degrading enzymes produced by the bacterial strains in different media (DW5- distilled water + 7.5 % Rice Bran, LB5-LB + 7.5 % rice bran, PFE5- PFE + 7.5 % rice bran, PMM5- PMM + 7.5 % rice bran). (E. sakazakii ASUIA279 - after 5 days; P. stewartii ASUIA271 and B. cereus ASUIA260
- after 3 days). Error bars show mean ± standard deviation. 91 4.11a Secretion of phytate-degrading enzyme by
Enterobacter sakazakii ASUIA279 in LB with different
percentage of rice bran 91
4.11b Secretion of phytate-degrading enzyme by Panteoa stewartii
ASUIA271 in LB with different percentage of rice bran 92 4.11c Secretion of phytate-degrading enzyme by Bacillus cereus
ASUIA279 in PFE with different percentage of rice bran 92 4.12a The biomass and the phytate degrading enzyme produced
of Bacillus cereus ASUIA260 in LB and LB + 7.5 % rice bran 93 4.12b The biomass and the phytate degrading enzyme
produced of Pantoea stewartii ASUIA271 in LB and
LB + 7.5 % rice bran 94
4.12c The biomass and the phytate degrading enzyme produced of Enterobacter sakazakii ASUIA279 in LB and
LB + 7.5 % rice bran 94
4.13 Comparison of phytate-degrading enzyme production of the bacterial strains in LB and LB + 0.1% Sodium phytate.
Error bars show mean ± standard deviation 95 4.14 Comparison of phytate-degrading enzyme production by the
bacterial strains in LB and Low Phosphate media.
Error bars show mean ± standard deviation 96
xviii
4.15 Phytate-degrading enzymes secreted by three bacterial strains cultivated at different incubation temperature.
Error bars show mean ± standard deviation. 97 4.16 Phytate-degrading enzymes secreted by three bacterial strains
cultivated in media with different pH. Error bars show
mean ± standard deviation. 97
4.17 Bioreactor Systems (Biostat® B-DCU) 102 4.18 Contour plot of phytate-degrading enzyme production
from Enterobacter sakazakii ASUIA279 showing
interaction between incubation temperature and initial pH 111 4.19 Contour plot of phytate-degrading enzyme activity showing
interaction between pH and concentration of buffer
during purification using ion-exchange chromatography 120 4.20 CM- Fast Flow Sepharose in 10/10 column attached to
ActaPrime Machine (GE Healthcare, Piscataway, USA) 124 4.21 Sephacryl S-200 HR in 16/100 column attached to
ActaPrime Machine (GE Healthcare, Piscataway, USA) 124 4.22 The chromatographic profiles for ion exchange chromatography
(CM Sepharose FF) derived from ActaPrime Software 125 4.23 The chromatographic profiles for gel filtration (Sephacryl
S200 HR) derived from ActaPrime Software 126 4.24 Ten percent SDS-PAGE of a preparation of the phytate-degrading
enzyme from Enterobacter sakazakii ASUIA279 stained Coomassie Blue. Lane 1: Low Range SDS-PAGE Molecular Weight Standards, Lane 2: Crude Enzyme, Lane 3: Enzymes after Ammonium sulphate precipitation, Lane 4: Enzymes after ion exchange chromatography,
Lane 5: Enzyme after gel filtration 128 4.25 The profile of phytate-degrading enzyme from Enterobacter
sakazakii ASUIA279 at different pH. The enzyme was
incubated at 50 °C 131
4.26 Effect of pH on the stability of phytate-degrading enzyme from
Enterobacter sakazakii ASUIA279 within 10 days 132 4.27 The phytate-degrading enzyme activity profile of Enterobacter
sakazakii ASUIA 279 at different temperature. The enzyme was
incubated in 100 mM sodium acetate buffer at pH 4.5 133
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4.28 Effects of the temperature on the stability of phytate-degrading
enzyme from Enterobacter sakazakii ASUIA279 134 4.29 Phytate-degrading enzyme activity of Enterobacter sakazakii
ASUIA279 at different temperature and pH 135
4.30 Effects of cations and potential inhibitors on enzyme activity 136
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LIST OF ABBREVIATIONS
µg microgram
µgg-l microgram per gram
µmol micromole
A.D.P. adenosine-5’- diphosphate A.M.P. adenosine-5’- monophosphat A.P.I. Analytical Profile Index
A.T.P. adenosine-5’-triphosphate B.L.A.S.T. Basic Local Alignment and Search Tool
bp base pairs
C.F.U. Colony Forming Unit C.F.U./ml Colony Forming Unit per mililiter
C.M. Carboxymethyl
Da Dalton
D.E.A.E. Diethylaminoethyl E.D.T.A. ethylenediamine tetraacetate
F.F. Fast Flow
F.P.L.C. fast protein liquid chromatography g l-1 gram per liter
g gram
G.T.P. guanosine-5’- triphospahte
h hour
H.R. high resolution
kbp kilo base pairs kcat Catalytic Constant
kDa kilo Dalton
KM Mechalis Constant
L Liter
L.B. Luria Bertani
M Molar
mg milligram
min minute
ml millilitre
M.L.B. Modified Luria Bertani
mm millimetre
mM millimolar
M.P.F.E. Modified Peptone-Meat Extract-Soil Extract M.P.M.M. Modified Phytase Modified Media
N Normality
N.A. no activity
N.A.D.P. nicotinamide adenine dinucleotide phosphate N.C.B.I. National Centre of Biotechnology Information
nm nanometer
P Probability
P.C.R. Polymerase Chain Reaction
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P.F.E. Peptone-Meat Extract-Soil Extract P.M.M . Phytase Modified Media
rpm revolution per min s-1 per second
s-1M-1 per second per Molar
S.D.S-P.A.G.E. sodium dodecyl sulphates- polyacrylamide gel electrophoresis
sec second
T T-test value
Tris tris (hydroxymethyl) aminomethane
U Unit
Ukg- Unit per kilogram Umg-1 Unit per milligram Uml-1 Unit per millilitre v/v volume per volume Vmax maximum velocity
vvm volume per volume per min w/v weight per volume
w/w weight per weight
1
CHAPTER ONE INTRODUCTION
1.1 OVERVIEW
During the last 20 years, phytate-degrading enzymes had attracted considerable attention from both scientists and entrepreneurs in the areas of nutrition, environmental protection and biotechnology. Phytate-degrading enzymes (myo- inositol hexakisphosphate phosphohydrolase), is also known as phytases, a special class of phosphomonoesterases (myo-inositol hexakisphosphate 3-phosphorylase EC 3.1.3.8 and myo-inositol hexakisphosphate 6-phosphorylase, EC 3.1.3.26), are capable of initiating the stepwise release of myo-inositol and phosphoric acid in a manner forming myo-inositol phosphate intermediates from phytate [myo-inositol (1, 2, 3, 4, 5, 6) hexakisphosphate] (Koneitzny and Greiner, 2002).
Phytate is an abundant plant constituent comprising 1 to 5 % (w/w) of edible legumes, cereals, oil seeds, pollens and nuts (Cheryan, 1980). Most plants origin food contain 50 to 80 % of their total phosphorus as phytate (Harland and Morris, 1995).
Rice contains about 60 % phytate phosphorus of its total phosphorus (Tyagi, Tyagi and Verma, 1998). Phytate content in the rice bran is 3.65 % (Ravindran, Ravindran, and Sivalogan, 1994). Phytate chelates essential minerals, binds to amino acids and proteins as well as inhibits digestive enzymes and decreases the nutritive value of food (Cheryan, 1980; Reddy, Pierson, Sathe and Salunkhe, 1989; Wodzinski and Ullah, 1996; Dvorkova, 1998). Phytate-bound phosphorus is poorly utilized by single- stomached animals, due to insufficient phytate-degrading activity in the gut (Taylor,
2
1965; Nelson, 1967; Ravindran, Bryden and Kornegay, 1995; Fandrejewski, Raj, Weremko and Zebrowska, 1997).
Therefore, hydrolysis of phytate is desirable to release valuable nutrients for beneficial utilization. The addition of phytate-degrading enzyme can improve the nutritional value of plant-based foods by enhancing protein digestibility and mineral availability through phytate hydrolysis during digestion in stomach or during food processing (Reddy et al., 1989; Sandberg and Andlid, 2002). Recently, addition of phytate-degrading enzyme has been seen as a way to reduce the level of phosphate pollution in areas of intensive animal production. Inorganic phosphate supplementation in the diets for single-stomached animal can be obviated by including adequate amounts of phytate-degrading enzyme and as a result, the faecal phosphate excretion of these animals may be reduced up to 50 % (Walz and Pallauf, 2002). In addition, a biotechnological application of phytate-degrading enzyme in the food area was taken into consideration (Konietzny and Greiner, 2003). With the addition of phytate-degrading enzymes, the nutritional value of plant-based food can be improved by enhancing protein digestibility and mineral availability through phytate hydrolysis during digestion in the stomach or during food processing (Reddy et al., 1989;
Sandberg and Andlid, 2002). Since certain myo-inositol phosphates have been proposed to have novel metabolic effects (Ohkawa, Ebisuno, Kitagawa, Morimoto, Miyazaki and Yasukawa, 1984; Potter, 1995; Vucenik and Shamsuddin, 2003), phytate-degrading enzymes may also find application in food processing to produce functional food (Konietzny and Greiner, 2003).
Phytate-degrading enzymes can be derived from a number of sources including plants, animal tissues and microorganisms (Konietzny and Greiner, 2002; Vohra and Satyanarayana, 2003). However, most of the scientific work has been done on
