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Studies on Novel Oxalate Oxidase Produced by an Endophytic Bacterium Ochrobactrum Intermedium Cl6

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Studies on Novel Oxalate Oxidase Produced by an Endophytic Bacterium Ochrobactrum Intermedium Cl6 STUDIES ON NOVEL OXALATE OXIDASE PRODUCED BY AN ENDOPHYTIC BACTERIUM OCHROBACTRUM INTERMEDIUM CL6 Thesis Submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY By KUNAL KUMAR DEPARTMENT OF CHEMICAL ENGINEERING NATIONAL INSTITUTE OF TECHNOLOGY KARNATAKA SURATHKAL, MANGALORE – 575 025 APRIL, 2017 D E C L A R A T I O N I hereby declare that the Research Thesis entitled “Studies on novel Oxalate Oxidase produced by an endophytic bacterium Ochrobactrum intermedium CL6”which is being submitted to the National Institute of Technology Karnataka, Surathkal in partial fulfillment of the requirements for the award of the Degree of Doctor of Philosophy in the Department of Chemical Engineering, is a bonafide report of the research work carried out by me. The material contained in this Research Thesis has not been submitted to any University or Institution for the award of any degree. Place: NITK, Surathkal Name: KUNAL KUMAR Date: Register Number: 121160CH12F01 Department of Chemical Engineering CERTIFICATE This is to certify that the Research Thesis entitled “Studies on novel Oxalate Oxidase produced by an endophytic bacterium Ochrobactrum intermedium CL6” submitted by Mr. Kunal Kumar (Register Number: 121160CH12F01) as the record of the research work carried out by him, is accepted as the Research Thesis submission in partial fulfillment of the requirements for the award of degree of Doctor of Philosophy. Research Guide Dr. Prasanna B.D Assistant Professor Dept. of Chemical Engineering NITK, Surathkal Chairman -DRPC ACKNOWLEDGEMENT Pursuing the path of this doctoral study has been a very insightful phase in the journey of my life. This wonderful experience would not have been complete without the contribution and support of several people, whom I shall remain indebted to. At the outset, I would like to express my sincerest gratitude to my research guide Dr. Prasanna B.D for his perpetual guidance, friendly support and constructive criticism, which were essential in the fulfillment of this task. His perceptive thoughts have inspired me to develop as a whole on the professional and personal life front. His understanding and personal guidance have provided a good basis for the present research. The hard question raised by him has encouraged me to widen my research from various perspectives. He has been a great source of inspiration to me. I am greatly thankful to the RPAC committee members Dr. I. Regupathi for invaluable advice and feedback which enabled me to speed up my research work and Dr. B. Ramachandra Bhat for his insightful comments and advice at different stages of my research, which were inspiring and they helped me work in a coherent fashion. My humble gratitude to the Director, NITK, present and former Head of the Department of Chemical Engineering, NITK, Surathkal Dr. Raj Mohan B, Dr. M.B Saidutta and Dr. Vidya Shetty K for providing me the necessary facilities and support during my research work. I take this opportunity to thank all teaching staff, Department of Chemical Engineering, NITK, Surathkal, for their valuable suggestions and support throughout this work. I take this opportunity to express my sincere thanks to Mr. Sadashiva, for gladly helping in the handling of laboratory equipments and chemicals, Mrs. Shashikala for all the official documentation, Mrs. Thrithila Shetty for her patience and support in giving consumables required for the research work, Mrs. Prema, Mrs. Sunitha, Mrs. Leela, Late Mr. Keshava Shettigar, Mr. Ananda Devadiga, Mr. Mahadeva, Mr. Harish Shetty, Mr. Suresh and Mr. Ramesh and all other non-teaching staff members of Department of Chemical Engineering for their kind helpful suggestions and timely maintenance of the laboratory equipments. I sincerely thank to NITK for providing me financial support in the form of fellowship. I am grateful to all my former and current research colleagues and student colleagues for providing a stimulating and fun-filled environment during my period of stay in NITK, and all those who have helped me during my dissertation work and have been involved directly or indirectly in this endeavor. I specially thank my dear friends Ms. Manjula, Ms. Vaisali, Ms. Charanya, Ms. Anusha, Ms. Swapnali, Ms. Anushree, Ms. Shwetashree, Ms. Aishwarya, Ms. Pooja, Mr. Basavraj, Mr. Shivananth, Mr. Pragadeesh, Mr. Harsha, Mr. Vishnu, Mr. Ramana Reddy, Mr. Vijay, Mr. Arun Augustin, Mr. Manoj, Dr. Supriyo Basak, Dr. Subha K who stood behind me during my tough times professionally and personally. I also thank all the members of Voice club of NITK for helping me to walk on the path of spiritual wisdom. Finally, I would like to thank God for giving me forbearance, perseverance and immense energy to accomplish this research work and pursue my dream. I wish to place on record my deepest gratitude to my beloved parents, Mr. Nirod Baran Paul and Mrs. Nandita Paul; my brother, Mr. Sourav Kumar Paul and my sister, Ms. Suhita Paul for their constant support, love and motivation in this journey of life. Kunal Kumar DEDICATED TO MY FAMILY ABSTRACT Oxalate Oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate Oxidase (OxO) finds application in clinical assay for oxalate in blood and urine, apart from several potential industrial applications. This research work is about isolation of endophytic bacteria from the tubers of the plants Colocasia esculenta, Beta vulgaris, Ipomoea batatas and peel of Musa paradisiaca fruit for screening of OxO producing strain. A total of 49 endophytes were isolated and out of which, 4 were OxO producing strains. Based upon the OxO activity produced in nutrient medium, one strain CL6 isolated from tubers of C. esculenta was selected and identified as Ochrobactrum intermedium by 16 S rDNA sequencing. The effect of media components and process variables on the growth kinetics of the O. intermedium CL6 and on the production of the enzyme OxO showed that production is inducible and requires manganese ions in the medium, and very low fill-up volume is beneficial. Characterization of the partially purified OxO revealed many intriguing characteristics. The enzyme is thermostable and remains active for 6 h in the temperature range of 4-80°C. This enzyme is the only known OxO which did not show substrate inhibition up to a substrate concentration of 50 mM. The enzymatic activity was not adversely affected by most of the metal ions and biochemical agents (K+, Na+, Zn2+, Fe3+, Mn2+, Mg2+, Glucose, Urea, and Lactate). The enzyme appears to be a metalloprotein stimulated by Ca2+ and Fe2+. It’s Km and kcat for oxalate was found to be 0.45mM and 85 s-1 respectively. Chemical modification of OxO revealed that cysteine, carboxylates, histidine and tryptophan residues are part of the active site. A two-fold increase in Oxalate Oxidase activity was observed when histidine residues were modified with 15mM diethylpyrocarbonate. Application studies on the development of a novel enzymatic treatment method to reduce total oxalate content of the extracted starch from Taro flour resulted in 97% reduction and this treatment did not alter any of the desirable physico-chemical properties of the starch. Keywords: Oxalate Oxidase, Endophytes, Ochrobactrum intermedium, Thermostable, Taro, Starch i CONTENTS PAGE NO. ABSTRACT i CONTENTS ii LIST OF FIGURES x LIST OF TABLES xiii ABBREVIATIONS xv NOMENCLATURE xvi CHAPTER TITLE 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 8 2.1 Oxalate rich plants 8 2.2 Role of oxalates in plants 10 2.3 Occurrence of Oxalate Oxidase in different organisms 11 2.4 Application of Oxalate Oxidase 14 2.5 Endophytes 14 2.6 Plant – Endophyte interactions 16 2.7 Purification of Enzymes 17 2.7.1 Protein Purification scheme 21 2.8 Characterization of enzymes 21 2.8.1 Influence of the pH on enzyme assays 21 2.8.2 Dependence of the enzyme activity on the 22 temperature 2.8.3 Metal ions in biological catalysis 22 2.8.4 Kinetic Properties 23 2.9 Purification of Oxalate Oxidase 24 ii 2.10 Kinetic and Biochemical Characterization of Oxalate 25 Oxidase 2.11 Chemical Modification of amino acid residues of 27 enzymes 2.12 Mechanism of Oxalate Oxidase catalysis 29 2.13 Starch and its industrial application 31 2.13.1 Starch from Taro 34 2.13.2 Anti-nutritional factors present in food crops 37 2.13.3 Strategies to reduce oxalate content in Taro 38 flour 3 ISOLATION AND SCREENING OF ENDOPHYTES 44 FOR OXALATE OXIDASE PRODUCTION AND MEDIA AND PROCESS OPTIMIZATION FOR ENHANCED YIELD BY ONE FACTOR APPROACH 3.1 Materials and Methods 44 3.1.1 Selection of plants 44 3.1.2 Isolation of endophytes 45 3.1.3 Screening of Oxalate Oxidase positive 45 endophytes 3.1.4 Maintenance and preservation methods 45 3.1.5 Physical and molecular characterization of 47 the positive strain. 3.1.6 Inoculum medium 47 3.1.7 Media design for production of Oxalate 47 Oxidase 3.1.8 Enzyme Assay procedure. 47 3.1.8.1 Principle 47 3.1.8.2 Reagents 48 3.1.8.3 Procedure 48 iii 3.1.9 Effect of nutritional parameters on 49 Oxalate Oxidase production 3.1.9.1 Effect of different carbon sources 49 3.1.9.2 Effect of different nitrogen sources 49 3.1.9.3 Effect of Mn2+ ions 50 3.1.10 One factor study of Non-nutritional 50 parameters 3.1.10.1 Temperature: 50 3.1.10.2 pH 50 3.1.10.3 Fill up volume 50 3.1.10.4 Agitation speed 50 3.1.10.5 Inoculum age 51 3.2 Results and Discussions 51 3.2.1 Isolation of endophytes 51 3.2.2 Physical and molecular characterization 55 3.2.3 Effect of nutritional parameters 62 3.2.3.1
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