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Molecular and Functional Aspects of Hydrolyases / Inhibitors with Emphasis on Aspartic Protease Inhibitor

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Molecular and Functional Aspects of Hydrolyases / Inhibitors with Emphasis on Aspartic Protease Inhibitor MOLECULAR AND FUNCTIONAL ASPECTS OF HYDROLYASES / INHIBITORS WITH EMPHASIS ON ASPARTIC PROTEASE INHIBITOR Thesis submitted to University of Pune For the degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY By Vishnu Menon Division of Biochemical Sciences National Chemical Laboratory Pune – 411008 India January 2012 Dedicated to my mentor & my family ‘The whole of science is nothing more than a refinement of everyday thinking’ Albert Einstein TABLE OF CONTENTS ACKNOWLEDGMENTS (i) CERTIFICATE (iii) DECLARATION BY THE RESEARCH SCHOLAR (iv) ABBREVIATIONS (v) ABSTRACT (vii) LIST OF PUBLICATIONS (xv) CONFERENCES /POSTERS/ ABSTRACTS (xvii) Chapter 1 General Introduction 1-72 Introduction 3 Enzyme classification 3 Significance of proteases 4 Occurrence of proteases 8 Classification of proteases 8-13 Serine proteases 10 Thiol/ Cysteine proteases 10-11 Metalloproteases 11 Glutamic acid proteases 11-12 Threonine proteases 12 Aspartic proteases 13 Occurrence 13-14 Crystallographic studies 15 Mechanism of action 16 Classification 17-24 Pepsin family 17-18 Retropepsin family 18-19 Cauliflower mosaic virus proteases family 19-20 Membrane bound aspartic proteases 20-21 GxGD aspartic proteases 20-21 Putative aspartic proteases 22-23 Plant aspartic proteases 23-24 Proteases inhibitors 24-25 Classification of Proteases inhibitors 25-29 Based on mechanism of inhibition 27 Based on physiological relevance 27-28 Aspartic protease inhibitor 30-54 Mechanism of inhibition 30-31 Classification 31-32 Proteinaceous Inhibitor 31-32 Low-Molecular-Weight Inhibitors 32-34 Inhibitors of pepsin 34-36 Inhibitors of cathepsin D 36-39 Inhibitors of HIV protease 40-43 Inhibitors of renin 43-46 Inhibitors of BACE 46-48 Inhibitors of γ-secretase 48-50 Inhibitors of Plasmepsins 50-52 Inhibitors of secretory aspartic proteases (SAPs) 52-54 Future perspective for inhibitor design 54-57 Bibliography 59-72 Chapter 2 Isolation and identification of microbes for the production of aspartic protease inhibitor 73-103 Summary 75 Introduction 76-77 Experimental procedures 78-87 Results 88-100 Isolation and identification of actinomycetes 88-89 Morphological and biochemical identification 90-91 Identification of isolate by 16S rDNA sequencing 90-91 Production of aspartic protease inhibitor from Streptomyces sp MBR04 91-95 Isolation and identification of the fungal isolate 96-98 Morphological characteristics 96 Identification of isolate by ITS sequencing 97-98 Production of aspartic protease inhibitor from Penicillium sp VM24 98-100 Discussion 101 Bibliography 102-103 Chapter 3 Purification and characterization of aspartic protease inhibitors from Streptomyces sp MBR04 and Penicillium sp VM24 104-126 Summary 106 Introduction 107 Experimental procedures 108-113 Results 114-124 Purification of inhibitor from Streptomyces sp MBR04 114-115 Biochemical characterization 115-118 Purification of inhibitor from Penicillium sp VM24 19-120 Biochemical characterization 120-124 Discussion 125 Bibliography 126 Chapter 4 Mechanistic and structural insights into the inhibition of therapeutically significant aspartic protease by inhibitor from Streptomyces sp MBR04 127-190 Part A Kinetic interactions of pepsin with the inhibitor: Correlation to inhibitor induced conformational changes 129-157 Summary 130 Introduction 131-135 Experimental procedures 136-140 Results 141-153 Kinetic analysis of the inhibition of pepsin 141-146 Thermodynamics of inhibitor binding to pepsin 147-148 Fluorescence analysis of pepsin-inhibitor interactions 148-151 Circular dichroism analysis of pepsin-APD-inhibitor complex 151-152 Chemical modification of inhibitor 152-153 Discussion 154-157 Part B Slow-tight binding inhibition of Cathepsin D: Implications in mechanism of inactivation 158-190 Summary 159 Introduction 160-163 Experimental procedures 164-168 Results 169-178 Inhibitor progress curve analysis 169-172 Thermodynamics of inhibitor binding to human cathepsin D 172-173 Fluorometric analysis of cathepsin D-inhibitor interactions 174-177 Circular dichroism analysis 177-178 Discussion 179-185 Bibliography 186-190 Chapter 5 Aspartic protease inhibitor from Penicillium sp VM24 as antifungal peptide: Implications in combating mycotic infections 191-222 Summary 193 Introduction 194-200 Experimental procedures 201-203 Results 204-215 Antifungal activity of the inhibitor from Penicillium sp VM24 204-210 Aspartic protease and xyloglucanase in growth inhibition 211-212 Chemical modification of the inhibitor and assessment of its antifungal potency 212-215 Discussion 216-218 Bibliography 219-222 Chapter 6 Bifunctional role of aspartic protease inhibitor from Penicillium sp VM24: inactivation of aspartic protease and xyloglucanase 223-285 Part A Interactions of a protease inhibitor with an aspartic protease from Aspergillus sp 225-245 Summary 226 Introduction 227-228 Experimental procedures 229-232 Results 233-242 Kinetic analysis of the inhibition of fungal aspartic protease 233-234 Substrate kinetics 235 Progress curve analysis 235-240 Fluoremetric analysis 240-241 Circular dichroism analysis 241-242 Discussion 243-245 Part B Slow-tight binding inhibition of xyloglucanase from Thermomonospora sp by an aspartic protease inhibitor 246-285 Summary 247 Introduction 248-250 Experimental procedures 251-257 Results 258-274 Purification of xyloglucanase 258 Kinetic analysis of the inhibition of xyloglucanase 259-265 Fluorescence studies on enzyme-inhibitor interactions 265-269 Circular dichroism analysis of enzyme-substrate-inhibitor 269-270 Inactivation of xyloglucanase by TNBS, PHMB and DEP 270-273 Effect of inhibitor on the isoindole fluorescence 273-274 Discussion 275-281 Bibliography 282-285 Chapter 7 Functional aspects of hydrolyases 286-308 Summary 288 Introduction 289-290 Experimental procedures 291-294 Results 295-304 Acid hydrolysis of TKP 295-296 Enzymatic hydrolysis 297-300 Effect of surfactants on GXG hydrolysis 301-302 Fermentation of acid and enzymatic hydrolysates 302-303 Immobilization and reuse of D hansenii 304 Discussion 305-306 Bibliography 307-308 ACKNOWLEDGEMENTS I would like to connote my whole-hearted gratitude and indebtedness to my mentor Dr Mala Rao, for introducing me to the mesmerizing dominion of Biochemistry and Microbial Biotechnology. I consider myself extremely fortunate to have her as my mentor who gave me the freedom to explore on my own, and owe her lots of gratitude for guidance to recover when my steps faltered. I sincerely thank her for her excellent guidance, teaching, encouragement, benign attention, and for helping me out in most stressful times, both scientific and personal, which helped me to think positively and remain optimistic. Her scientific temperament, innovative approach, dedication towards her profession and down to earth nature has inspired me the most. I hope that I could be as lively, enthusiastic and energetic as her and to someday be able to command an audience as well as she can. Although this exaltation is insufficient, I preserve an everlasting gratitude for her. With much appreciation, I would like to mention the role of Dr Yogesh Souche and Girish Kulkarni for their help in my research works on DNA sequencing and molecular identification of the microbes. I am also grateful to Dr’s Sushama Gaikwad and Mahesh Kulkarni for their support in HPLC and fluorescence studies. Special thanks to Dr Asmita Prabhune for introducing me to NCL family. I profoundly thank Dr Barry MacCleary (Megazyme International Ireland Ltd) and Mr A. Agarwal (Simosis International Ltd.) for kindly providing xyloglucan from tamarind seeds and tamarind kernel powder respectively during my research. I also thank Mrs Snehal More for her help and support. I would like to thank Dipali for her encouragement, valuable and timely help and the lighter moments shared. I am indeed thankful to her for helping me in the purification of xyloglucanase and gel electrophoresis. I find words inadequate to express the appreciation of my seniors Dr’s Vinod and Ajit for their constructive comments and suggestions, ceaseless inspiration, advice and timely help throughout my work. Special thanks are to i my seniors Dr’s Anish, Sharmili, Aarohi, CV, Shiv, Ashwini, Sachin, Sharath, Sridevi, Atul for their help and inspiration. I fall short of words to express my feelings towards my all time friends, Rupesh and Pramod. They have made an outstanding impact on my outlook towards life and science. They have been there all the time with me in my ups and downs. Thanks are due to my labmates Gyan, Vaibhav, Harish, Jayshree, Siddharth, Malathi, Ishwar, Dhananjay, Suresh, Avinash, Yashwant, Pushpa, Raj, Padma, Aparna, Nupur, Asmita, Nilesh, Tanaji, Mehraj, for the cordial and friendly atmosphere in the lab. Special thanks to all my FB and Orkut friends. Will always cherish the moments shared with you all. I thank Mrs. Indira Mohandas and Mr. Ramakant Lambhar for their ready and unpretentious help whenever required, even in times of need. I am thankful to Mr. Trehan and Mr. Jagtap for their technical assistance for instrumentation and other chores. I thank the Director, National Chemical Laboratory for making the facilities available for my work. I acknowledge the financial assistance by CSIR, New Delhi in the form of Junior and Senior research fellowship during the tenure of my work. I owe my deepest gratitude to my parents, brothers, sisters, BIL’s, SIL’s, my lovable nephews and nieces for their eternal support and understanding of my goals and aspirations. Without them, I would not have been able to complete much of what I have
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