PRODUCTION AND CHARACTERIZATION OF PROTEASE BY BACILLUS LICHENIFORMIS ON SKIM LATEX SERUM FORTIFIED MEDIA BY VIVI MARDINA A dissertation submitted in

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

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 (R 2 = 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 o C to 65 o C and also at ph 6.0 and 7.0 for 60 min. The stimulatory effects on protease activity were observed in the presence of Mn 2+ and Ca 2+, while inhibitory effects were found in the presence of Cu 2+, Zn 2+, Mg 2+, 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. ii

ملخص البحث األنزمي الربوتيين املستتتتتتن )12759 Bacillus licheniformis (ATCC ميك أن يننج ابستتتتتتن ا الزراعية والصتناعية الكا نة و املناحةكمادة ركيزة. إن صتا املطاا املودت د صتتتتتتناعة املطاا الطبيعي اليت تن فر فيها الع ي النفاايت الساة ة ل مطاا املود دك سط قاع ي غذي امل فات النفاايت الستاة ة املن فرة والرخيصتةكنفاايت اجتة املركبات العضتتتتتت ية املناستتتتتتبة لنم اجلراثيم. يف ال راستتتتتتة احلالية استتتتتتن اعنم ت النصتتتتتا يم النجريبية اإلحصتتتتتاةية لنحستتتتتن الع ا ا الفيزيكيمياةية ل صتتتتت ى ع الع ا ا األح عدتتتتتتتتتتتر صتتتتا املطاا املودتتتت د و حجم هذا الفح ثا الالكن ز ل تتتتتتتتتتتتتتتتتتتتتتتتتت B.liceniformis إلنناج األنزمي الربوتيين خارج اخل ية. لو إنناج لالنزمي الربوتيين. كما فحصتتتتت واجلا كن ز و الكاستتتتتتتتتتتن و KH 2 PO 4 و MgSO 4.7H 2 O و LB broth و ادة الن ويح وستتتترعة الرج ودرمة احلم تتتتة األولية ودرمة احلرارة إلنناج األنزمي الربوتيين ابستتتتتتتتتتن ا تصتتتتتتتتتتميم Plackett-Burmanكمؤها لفح النحستتتتتتتتتتن األ ثا. وق مت حت ي )اجلا كن ز و صتتتا املطاا املودتتت د وستتترعة الرج النحستتتن األ ثا لنعزيز إنناج األنزمي الربوتيين ابستتتن ا ودرمة احلم تتتة وأمري أربعة نغريات إبعنبارها أهم الع ا ا احلامسة واليت اخنريت حوا ملزي تصتتتميم وامهة الن ستتتط املركزي املرك ا ستتتنجابة الستتتطحية.(RSM) ووم أن إننامية األنزمي الربوتيين إرتفع تسعة أ عاف ا ننامية اذا ق رن ابل سظ الغذاةي األص ي. أظهرت نناةج عم ية النحوق كفاية ودقة النصتتتتتميم ال رمة الثانية ANOVA ع النناستتتت ابستتتتتتتتن ا أن الويمة املن قعة اتفو املوب ى ل نم ذج كربينات األ ني بنستتبة بدتتتتتكا مي ع الويمة النجريبية النطبيق عالية 0.0002) = (P ع (FCCCD) يف إطار نهجية 2 وح ة/ ا إىل 19.35 وح ة/ ا مما يوارب ع نستتتتتبة خطأ أقا عا ا حت ي ع البياات النجريبية. وكان اخلط ة النالية إبمراء اخلط ات ل رتستتتتتتتتي ال ايى والنبادى األي ين الكرو ات غرايف. وكدتتتتتتتتف صحة النصميم املط ر ل نأك 20.و أظهر من ذج عايل (0.9537 = 2 R) مما ي ى إىل 2.28 أ عاف ح ايل 47 كي دالن ن. احلم تتتتتتتتتة بن 6.0 والكالسي وارنة اب نزمي اخلا. وأظهر توييم الربوتن النوي ب اسطة وكان ا يف حن مت العث ر ع ننابعة لننوية ال ستتتتط املغذي األ ثا النناةج إبزدايد ندتتتتتتتتاا ا نزمي النوي SDS PAGE فرقة واح ة ذات الكن ة اجلزيئية نزمي ستتتتتتتتتتتتتنور يف درمة حرارة ترتاوح بن 35 درمة ئ ية إىل 65 درمة ئ ية و 7.0 مل ة 60 دقيوة.وق ل حظ أتثريات ثبطة ب م دكا آاثر تندتتتتتتتتتيطية ع أن األنزمي الربوتيين املننج ق يك ن أنزمي بروتيين ف زي. يف حالة النطبيق ع الستتتتطحية SDS( Triton X100, Tween 20, املؤكستت ة ) 2 (H 2 O و نظف Tesco Everyday Value متا ا إزالة بوعة ال ع إكمانا بتت 7 غ/ ا كمادة إ افية يف إع اد املنظفات وكذلك املنظف الساةا. و آي ات النحاس والزنك واملغنيسي املنظفات وأيضتتتتتتتتتتتتتتا درمة ندتتتتتتتتتاا األنزمي الربوتيين ب م د آي ات املنغنيز وEDTA. يدري هذا إىل أظهر ا نزمي استتتتتتتتتتتنورار حن واملذيبات )األستتتتين ن والك روف ر وانكستتتتان ع ا ا الفعالية والن ل ي و الع ا ا ع ندتتاا ا نزمي املنبوي ح ايل 80. وبن أيضتتا ندتتاا خصاة نظفات الغسيا و عاجلة النفاايت اإلك ينيكية. ا نزمي الربوتيين املننج أنه ابإل كان أن يسن iii

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 iv

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 v

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 vi

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. vii

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 1.2 Problem Statement... 4 1.3 Research Objectives... 6 1.4 Scope of Research... 7 1.5 Significant of Study... 7 1.6 Research Methodology... 7 1.7 Dissertation Organization... 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 viii

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 ix

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... 79 3.3.7. The Effect of Skim Latex Effluent on Bacillus licheniformis Proteases Production... 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... 93 4.2. The Effect of Skim Latex Effluent on Bacillus licheniformis Protease Production... 94 4.3. Optimization of Skim Latex Serum Medium Using Statistical Techniques... 95 x

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... 165 APPENDIX 7... 166 APPENDIX 8... 172 xi

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 3.4 Physicochemical components used in Plackett-Burman design 3.5 Experimental Design using Face Centered Central Composite Design of four independent variables with six center points 81 82 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 4.2 Ranking of the variables investigated in the Plackett-Burman design 4.3 Analysis of variance for protease production by Bacillus licheniformis on skim latex serum as the basal media 4.4 Face centered central composite design of four independent variables with their actual value showing the experimental and predicted response 98 100 102 107 xii

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) 4.7 Analysis of variance for response surface quadratic model (second optimization) 111 112 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 xiii

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 2.1 Schematic diagram of raw rubber processing and products manufacturing 2.2 The flow sheet of steps involved in concentration of NRL by centrifugation 8 12 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. 4.2 Pareto graph showing the main effect result of the 11 components for protease production based on Plackett- Burman experimental result 94 100 xiv

4.3 Effect of different ph on protease production by B. licheniformis (ATCC 12759) with constant for other conditions 4.4 Effect of different agitation rate on protease production by Bacillus licheniformis (ATCC 12759) with constant for other conditions 4.5 Effect of different incubation time on protease activity by B.licheniformis with constant for other conditions 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) 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 4.8 Chromatography of B.licheniformis protease on DEAEsepharose. 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 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 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) 4.11 Effect of temperature on B. licheniformis protease activity, the maximum activity of enzyme at 65 o C was taken as a control (100 %) (*value in figure represented as mean ± SD) 4.12 Effect of temperature on the thermo stability of B. licehniformis protease 4.13 Effect of various protease inhibitors on The protease activity from B.licheniformis 104 105 106 110 113 116 118 119 120 121 124 xv

4.14 Hyperbolic regression of Michaelis-Menten equation for Bacillus licheniformis protease 4.15 Lineweaver-Burk plot for determining of KM and Vmax using casein as substrate 4.16 Compatibility of protease from Bacillus licheniformis (ATCC 12759) with Tesco Everyday Value detergent 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 o C (1: control, 2: the stained cloth + the detergent only, 3: the stained cloth + the detergent + the protease, 4: the stained cloth +enzyme only). 126 127 132 132 xvi

LIST OF ABBREVIATIONS 3D ADS ANOVA ATCC BOD BSA C/N CCD COD CV CV* DCL DEAE DFP DGDG DOE DPNR DRC DTT EC EDTA ENR ESEM ESG FCCCD FW GF GLA GP H2S HIC IAA IEC IIUM KM LB LBHB LNR MGDG NR NRSL NRSP OFAT PBd 3 Dimensions Air dried sheet Analysis of variance American Type Culture Collection Biochemical oxygen demand Bovine serum albumin Carbon/Nitrogen Central composite design Chemical oxygen demand Coefficient variation Constant viscosity Dichloroisocoumarin Diethylaminoethyl Diisopropyl fluoro phosphate Digalactosyl diglyceride Department of environment Deproteinised natural rubber Dried rubber content Dithiothreitol Enzyme commission Ethylene diamine tetra acetic acid Expoxidised natural rubber Environmental scanning electron microscope Esterified sterylglycoside Face centered central composite design Frey-Wyssling Gel filtration (γ-linoleic acid) General purpose Hydrogen sulphide Hydrophobic interaction chromatography Indole acetic acid Ion exchange chromatography International Islamic University Malaysia Michaelis constant Luria bertani Low-barrier hydrogen bond Liquefied natural rubber Monogalactosyl diglyceride Natural rubber natural rubber skim latex Natural rubber serum powder One factor at a time Plackett-Burman design xvii

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 xviii

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 1

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 2

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) 3

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. 4

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 5

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. 6