Studies on Production, Characterization and Application Potential of Cholesterol Oxidase from Rhodococcus sp. NCIM 2891 A Thesis Submitted by SERAJ AHMAD For the award of the degree Of Doctor of Philosophy Department of Biosciences & Bioengineering Indian Institute of Technology Guwahati Guwahati-781039, Assam, India October, 2014 Dedicated to My Beloved Parents TH-1373_08610616 Department of Biosciences & Bioengineering Indian Institute of Technology Guwahati Guwahati - 781 039, Assam, INDIA STATEMENT I do hereby declare that the matter incorporated in this thesis is the result of investigations carried out by me in the Department of Biosciences & Bioengineering, Indian Institute of Technology Guwahati, India, under the guidance of Prof. Pranab Goswami. In keeping with the general practice of reporting scientific observations, due acknowledgements have been made wherever the work described is based on the findings of other investigators. October, 2014 Seraj Ahmad TH-1373_08610616 Department of Biosciences & Bioengineering Indian Institute of Technology Guwahati Guwahati - 781 039, Assam, INDIA Dr. Pranab Goswami Tel: +91-(0)361 2582202 (Professor) Fax:+91-(0)361 2582249/2690762 Email: [email protected] Certificate This is to certified that the thesis entitled “Studies on Production, Characterization and Application Potential of Cholesterol Oxidase from Rhodococcus sp. NCIM 2891” that is being submitted by Seraj Ahmad (Roll No. 08610616) for the award of the degree of Doctor of Philosophy is an authentic record of the results obtained from the research work carried out under my supervision in the Department of Biosciences & Bioengineering, Indian Institute of Technology Guwahati, Assam, India. The results incorporated in this thesis have not been submitted to any other University or Institute for the award of any degree. Dr. Pranab Goswami October, 2014 Professor (Research Supervisor) TH-1373_08610616 ACKNOWLEDGEMENT With immense pleasure and sincere gratitude first of all, I would like to acknowledge my thesis supervisor Prof. Pranab Goswami for his valuable support and guidance throughout the years of my research work. I sincerely very thankful to him for giving me an opportunity to work in his lab and for his valuable suggestions. I would also like to thank my Doctoral Committee members, Dr. Biplab Bose, Dr. K. Pakshirajan and Dr. Anil Kumar Saikia for helping me in improving my work with their valuable suggestions. I am very thankful to Indian Institute of Technology Guwahati for giving me the opportunity and platform to carry out my research work. I am also grateful to Dr. Utpal Bora, other faculty members, office staff from Department of Biosciences & Bioengineering for their constant supports and helps. I am also thankful to all staff members of Center for Energy, particularly Lepakshi ba and Huzuri da for their constant help. I thankful to Council of Scientific and Industrial Research (CSIR), India for financial assistances and University Grants Commission (UGC), India for providing Maulana Azad National Fellowship to carry out my research smoothly. I would like to express my gratitude to my colleagues from our research group, Kiran da, Preety ba, Urmila, Madhuri, Reddy, Ankana, Vishwa, Santosh, Mrinal, Priyamda, Babina, Anupa, Sharbani, Priyanki, Naveen, Phurpa and Abdul for their help and suggestions. I am also thankful to my friends Faizi, Haneef, Ejaz, Mohitosh, Manab, Subhmoy, Sudeep, Ziauddin, Samsher and Asif. My heartiest thanks to my best friend Haniyah for her support. Lastly, I want to thank my family for their constant support and believe in me and almighty Allah for giving me the courage to complete my work. Seraj Ahmad October, 2014 i TH-1373_08610616 ABSTRACT Cholesterol oxidase (EC 1.1.3.6) is a flavoenzyme that in most cases catalyses the oxidation of cholesterol to 4-cholesten-3-one (CEO, cholestenone). The product cholestenone has been used as a precursor for the synthesis of hormones and many intermediate steroidal compounds which are eventually used to produce anabolic drugs and contraceptive. Classical (one-variable-at-a-time) and statistical methods (Plackett-Burman and Central composite design) were used to optimize growth medium for the production of cholesterol oxidase from Rhodococcus sp. NCIM 2891. Cholesterol oxidase activities from the classically and statistically optimized media were 0.75 and 3.25 U/ml, respectively. The statistically optimized medium had 4.33- and 9.7-fold higher enzymatic activity than the classically optimized and unoptimized basal medium, respectively. The ratio of enzyme production to cell growth rate was 29-fold higher in our statistically optimized medium than in the basal medium, indicating that the enzyme production could be classified as mixed type of growth. Cell-bound cholesterol oxidase accounted for 90.68±2 % of the total enzymatic activity of the growth medium. Interactions between the cholesterol oxidase-inducing substrate cholesterol and medium growth substrates yeast extract and (NH4)2HPO4 significantly enhanced the production of cell bound cholesterol oxidase. Our results validate the statistical approach as a potential technique for achieving the large-scale production of cell-bound cholesterol oxidase from Rhodococcus sp. NCIM 2891. The cell-bound cholesterol oxidase from the Rhodococcus sp. NCIM 2891 was purified 3-fold by Diethylaminoethyl-Sepharose chromatography. The estimated molecular 2+ mass (SDS-PAGE) and Km of the enzyme were ~ 55.0 kDa and 151 µM, respectively. Fe and Pb2+ at 0.1 mM of each acted as inhibitors, while Ag+, Ca2+, Ni2+ and Zn2+ activated the enzyme at similar concentration. The purified cholesterol oxidase was immobilized on ii TH-1373_08610616 chitosan beads by glutaraldehyde cross-linking reaction and immobilization was confirmed by Fourier Transform Infrared spectroscopy, Scanning Electron Microscopy and Energy Dispersive X-ray analysis. The optimum temperature (45 °C, 5 min) for activity of the enzyme was increased by 5 °C after immobilization. The both free and immobilized cholesterol oxidases were found to be stable in many organic solvents except for acetone. The biotransformation of cholesterol (3.75 mM) with the cholesterol oxidase immobilized beads (3.50 U) leads to ~ 88 % milli molar yield of cholestenone in a reaction time of 9 h at 25 °C. The immobilized enzyme retains ~ 67 % activity even after twelve successive batches of operation. The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone. iii TH-1373_08610616 CONTENTS Title Page No. ACKNOWLEDGEMENTS i ABSTRACT ii CONTENTS iv LIST OF SCHEME viii LIST OF FIGURES ix LIST OF TABLES xi LIST OF ABBREVIATIONS xii LIST OF SYMBOLS xiv 1. Introduction 1 2. Review of Literature 5 2.1 3-Hydroxysteroid dehydrogenase 7 2.2 Cholesterol oxidase 8 2.2.1 Production of cholesterol oxidase 12 2.2.2 Physico-chemical characteristics of cholesterol oxidase 18 2.2.2.1 Substrate specificity and kinetic parameters of cholesterol oxidase 24 2.2.2.2 Effect of pH and temperature on the cholesterol oxidase activity and 24 stability 2.2.2.3 Effect of detergents and solvents on the cholesterol oxidase activity 26 2.2.2.4 Effect of chemicals and metal ions on the cholesterol oxidase activity 30 2.3 Immobilization of cholesterol oxidase 31 2.4 Production of cholestenone 31 iv TH-1373_08610616 2.4.1 Chemical methods 33 2.4.2 Biological methods 33 2.4.2.1 Whole cell based catalysis 33 2.4.2.2 Cholesterol oxidase based catalysis 33 3. Production of Cholesterol Oxidase from Rhodococcus sp. NCIM 2891 35 3.1 Overview 35 3.2 Experimental approaches 36 3.2.1 Microorganism and culture conditions 36 3.2.2 Isolation and assay of enzyme 37 3.2.3 Optimization of medium components by the experimental designs 38 3.3 Results and discussion 40 3.3.1 Optimization of cholesterol oxidase production using classical methods 40 3.3.2 Evaluation of significant components by the Placket-Burman design 42 3.3.3 Optimization of medium components by central composite design 42 3.3.4 Experimental validation of the medium composition optimized by central 43 composite design 3.4 Conclusions 45 3.5 Figures 46 3.6 Tables 49 4. Isolation, Purification and Characterization of the Cholesterol Oxidase from 54 Rhodococcus sp. NCIM 2891 4.1 Overview 54 4.2 Experimental approaches 54 4.2.1 Isolation of cholesterol oxidase from bacterial culture 54 4.2.2 Enzyme assay and protein measurement 55 4.2.3 Enzyme purification and SDS-PAGE analysis 56 v TH-1373_08610616 4.2.4 Effect of pH, temperature and chemicals on cholesterol oxidase activity and 57 stability 4.2.5 Thermo-deactivation studies and estimation of thermodynamic parameters 58 4.3 Results and discussion 59 4.3.1 Isolation and purification of cell-bound cholesterol oxidase 59 4.3.2 Substrate specificity and kinetic parameters of cholesterol oxidase 60 4.3.3 Effect of pH and temperature on the cholesterol oxidase activity and stability 61 4.3.4 Effect of solvents and chemicals on cholesterol oxidase activity and stability 62 4.3.5 Thermo-deactivation studies of cholesterol oxidase 63 4.4 Conclusion 64 4.5 Figures 66 4.6 Tables 72 5. Immobilization and Application of the Cholesterol Oxidase from Rhodococcus 78 sp. NCIM 2891 5.1 Overview 78 5.2 Experimental approaches 79 5.2.1 Enzyme assay and protein measurement 79 5.2.2 Preparation and characterization of cholesterol oxidase immobilized chitosan 80 beads 5.2.3 Effect of pH, temperature and chemicals on cholesterol oxidase activity 83 5.2.4 Storage stability