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Biochemical Characterization of an Aspartic Protease Inhibitor from Bacillus Licheniformis: Interactions with Hydrolytic Enzymes

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Biochemical Characterization of an Aspartic Protease Inhibitor from Bacillus Licheniformis: Interactions with Hydrolytic Enzymes BIOCHEMICAL CHARACTERIZATION OF AN ASPARTIC PROTEASE INHIBITOR FROM BACILLUS LICHENIFORMIS: INTERACTIONS WITH HYDROLYTIC ENZYMES Thesis submitted to University of Pune For the degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY By Ajit Kumar Division of Biochemical Sciences National Chemical Laboratory Pune – 411008 India March 2007 In Fond Memories of My Father… ii TABLE OF CONTENTS Page No. ACKNOWLEDGMENTS ix CERTIFICATE xi DECLARATION BY THE RESEARCH SCHOLAR xii ABBREVIATIONS xiii ABSTRACT xv LIST OF PUBLICATIONS xxii CONFERENCES /POSTERS/ ABSTRACTS xxiii Chapter I General Introduction 1- 21 The Proteases 1- 21 I. The aspartic proteases 2 i. Pepsin family 3 ii. Retropepsin family 4 iii. Cauliflower mosaic virus proteases family 4 iv. Plant aspartic proteases 4 v. Membrane bound aspartic proteases 5 vi. Putative aspartic proteases 6 II. Proteases inhibitors 8 i. Inhibitors of aspartic proteases 8 a. Proteinaecous inhibitors 9 b. Low-Molecular-Weight Inhibitors 10 ii. HIV-1 proteases inhibitors 12 iii. Renin inhibitors 14 iv. Plasmepsins inhibitors 15 v. Cathepsin D inhibitors 16 vi. Secreted aspartic proteases inhibitors 17 vii) β-Secretase Inhibitors 18 III. Sequence homology 19 iii IV. Inhibitor design and future prospects 20 Chapter II 22 - 62 Biochemical Characterization of an Aspartic Protease Inhibitor from Thermo Tolerant Bacillus licheniformis: Interaction with Pepsin Part I Isolation And Identification Of Thermo Tolerant Bacillus licheniformis Summary 22 Introduction 23 Materials and Methods 24 Materials 24 Isolation and identification of the microorganism 24 Isolation and Sequencing of genomic DNA 24-26 Growth Conditions of the microorganism 26 Results 27 Identification of the microorganism 27 Identification of the microorganism by16SrDNA 27 sequencing Optimum growth conditions for the organism 27 Part II 30- 39 Purification and Characterization of API from Thermo Tolerant Bacillus licheniformis Summary 30 Introduction 31 Materials and Methods 33 Materials 33 Production of the Aspartic Protease Inhibitor 33 Purification of API 34 Biochemical characterization of API 34 Assay for inhibitory activity of API towards pepsin 35 Assay for inhibitory activity of API towards other 35 aspartic proteases iv Temperature and pH stability of API 35 Results 36 Production and Purification of API 36 Biochemical Characterization of API 37 Inhibitory activity of API towards aspartic proteases 39 Part III 40-62 Interaction of API with Pepsin: Implications in the Mechanism of Inactivation Summary 40 Introduction 41 Materials and Methods 46 Assay for inhibitory activity of API towards pepsin 46 Kinetic analysis of API-Pepsin interactions 46-48 Fluorescence analysis 49 Results 50 Determination of Km and Ki 50 Preincubation experiments with API 52 Regeneration of pepsin activity after inhibition 53 Simplified Progress curve analysis for API 54 Effect of API binding on the Fluorescence of pepsin 56 Discussion 60 Chapter III 63-73 Purification and Characterization of an Aspartic Protease from Mid- gut of Plutella Xylostella: Inactivation by API Summary 63 Introduction 64 Materials and Methods 66 Materials 66 Rearing of Plutella xylostella 66 v Preparation of P. xylostella mid-gut homogenate 66 Isolation of Plutella xylostella aspartic protease (PxAP) 66 Enzymes assays 67 Optimum pH and temperature of the purified enzyme 67 Assay for PxAP inhibition by API 68 Determination of Km and Ki 68 Results 69 Detection and Purification of aspartic protease 69 Biochemical properties of API and determine KM and Ki 71 for PxAP Discussion 73 Chapter IV 74-112 Bifunctional Role of API: Structural and Mechanistic Insights into the Inhibition of Chitinase Summary 74 Introduction 75 I. Chitinases 75 i. Classification of chitinases 76 ii. Three dimensional structure of chitinases 77 iii. Catalytic mechanism of family 18 and 19 chitinases 77 iv. Transglycosylation 81 v. Applications of chitinases 81 a. Chitinases in plant defence mechnisms 81 b. Mosquito control 82 c. Chitinase as a target for biopesticides 83 d. Production of chitooligosaccharides 83 e. Single cell protein production 84 f. Application of chitinases in biological research 84 g. Use of chitinase in degradation of fish waste 85 h. Miscellaneous applications 85 v. Cloning and molecular genetics of chtinases 86 vi II. Chitnases inhibitors 87 i. Kinetics and modes of binding of chitinase inhibitors 87 i. The pseudotrisaccharide allosamidin and its 88 derivatives ii. Psammaplins 91 iii. Styloguanidines 92 iv. Cyclic proline-containing dipeptides 92 v. The cyclopentapeptides argifin 93 vi. The cyclopentapeptides argadin 95 vii. Methylxanthines 96 viii. Miscellaneous inhibitors 97 Materials and Methods 98 Materials 98 ChiA inhibition by API 98 Initial kinetic analysis 98-99 Fluorescence analysis 99 Effect of API on the isoindole fluorescence of OPTA- 99 labeled ChiA Circular dichroism analysis 100 Results 101 The determination Km and Ki 101 Time dependent inhibition of ChiA 102 Simplified inhibition progress curve analysis for API 103 Effect of API binding on the fluorescence of chitA 105 Effect of API on the isoindole fluorescence of chiA- 107 OPTA complex Secondary structural analysis of enzyme substrate- 107 inhibitor complexes Discussion 109 vii Chapter V 113-131 Aspartic Protease Inhibitor from Alkalo-Thermophilic Bacillus Sp.: In Vivo and In Vitro Effects on Cuticle Moulting Fluid Enzymes of Helicoverpa Armigera Summary 113 Introduction 114 I. An insite in to insect moulting fluid proteases 114 II. An insite in to Insect chitinases 116 Materials and Methods 121 Materials 121 Purification of ATBI 121 Rearing and bioassays with H. armigera 121 Effect of ATBI on various growth parameters 121 Effect of ATBI on various reproductive parameters 122 Extraction and purification of moulting fluid enzymes 122 Protease activity assay 122 Chitinase assay using colloidal chitin and Ramazol 123 Brilliant Violet dyed chitin Results 125 Purification and biochemical characterization of ATBI 125 In vivo effect of ATBI on Helicoverpa armegera 125 development Extraction and partial purification of enzymes of H. 127 armigera Larvae cuticle Inhibition of moulting fluid proteases activity by specific 127 inhibitors Detection of chitinase activity by plate assay 128 Inhibition of moulting fluid Proteases and chitinase by 128 ATBI Discussion 130 References 132-151 viii ACKNOWLEDGEMENTS I take this opportunity to express my heart-felt gratitude to my guide, Dr. (Mrs.) Mala Rao for her able guidance, unceasing encouragement and sustained interest throughout the course of my work. I consider myself extremely fortunate to have her as my guide. Her integral view on research and her mission for providing 'only high-quality work and not less’ has made a deep impression on me. I owe her lots of gratitude for having me shown this right attitude towards research. Her continued interest and involvement in my work and concern for my welfare have greatly motivated me. I am indebted to her for the valuable suggestions, hints and critical comments in all the time of research and writing of this thesis. I deeply acknowledge the encouragement and support of Dr. Vasanti Deshpande throughout my work. I profoundly thank Prof. Constantinos E. Vorgias (Division of Biochemistry and Molecular Biology, Athens University, Athens, Greece) for providing ChitinaseA during my research. Thanks are due to Dr. M.V. Deshpande and Mahesh Kulye for their help in insect related work. I am highly thankful to Dr. Sushma Gaikwad for her help during the fluorescence spectroscopy experiments and amino acid analysis of the peptide. Dr. Saurav Paul and Akhilesh Tanwar (Physical Chemistry Division) are gratefully acknowledged for their help in structural modeling studies. My seniors, Drs. Sudeep, CV and Jui have in some or the other way helped in tuning my practical hand in the initial stages of my research work. Thank are due to them too. I am thankful to my lab mates, Aarohi, Anamika, Sharmili, Dr. Rachna, Neha, Anish, Vinod, Gyan, Nitin and Nilesh for maintaining a lively environment in the laboratory during the course of my work. I acknowledge Ramakantji and Trehan who were always there whenever required. Indira Mohandasan and Mari are duly acknowledged for their support throughout my tenure at N.C.L. I am also thankful to my friends Atul Thakur, Atul Farenze, Ambrish, Anil, Chetan, Chandrashekar, Feroz, Harish, JP, Manish(s), Nishant, Priyanka, Rohtas, Sagar, Shabab, Shadab, Sajid, Shashidhar, Satya, Sushim, Sameer, Suresh, Santosh, Swati, Kavita, Anil Sharma, Hardev, Kamal, Dharmander, Manas, Jitu Panday, ix Sudheer and Jitu Cheema for their charming company. I would also like to thank my friends Jaswinder, Jaswant, Sukhwinder, Jarnail, Rajesh Nagpal, Mukesh, Manmohan, Ajay, Bansi, Ramavtar and Vinod. Special thanks go to Madan Lal who has a great impact in my life. I have learnt a lot from him and cherish the lighter moments we shared. I am grateful to my neighbors in the village who have helped me a lot during my course of study. I also express my sincere appreciation to my father In-law and Brother In-laws (Anil, Sunil, Pankaj) for their efficient and timely Help. Words fall short to express my feelings towards my uncles Chajju Ram, Subhash, Naresh, Ramniwas and Master Mani Ram. They have made an outstanding contribution towards my outlook to life. They have been always there with me during my ups and downs of life. They always encouraged me and also appreciated my study efforts throughout. I am indeed thankful to my mother and my brother Rajender, who always kept me tension free and encouraged me to complete my Doctorate. I owe my deepest gratitude towards my better half for her eternal support and understanding of my goals and aspirations. Her infallible love and support has always been my strength. Her patience and sacrifice will remain my inspiration throughout my life. Without her help, I would not have been able to complete much of what I have done and become who I am. It would be ungrateful on my part if I thank Saroj in these few words. I am thankful to my son Tejasvi for giving me happiness during the last two years of my studies.
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