Luận văn bioprospecting of haloarchaea for the development and expression of potent novel antioxidant enzymes

BIOPROSPECTING OF HALOARCHAEA FOR THE DEVELOPMENT AND EXPRESSION OF POTENT NOVEL ANTIOXIDANT ENZYMES A THESIS Submitted by S MURUGAN (Reg No 8177) BIOCHEMISTRY in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY MANONMANIAM SUNDARANAR UNIVERSITY TIRUNELVELI - 627 012 APRIL - 2018 MANONMANIAM SUNDARANAR UNIVERSITY TIRUNELVELI - 627 012 CERTIFICATE The research work embodied in the present thesis entitled “BIOPROSPECTING OF HALOARCHAEA FOR THE PRODUCTION AND EXPRESSION OF POTENT NOVEL ANTIOXIDANT ENZYMES” has been carried out in the Department of Biochemistry of ManonmaniamSundaranaruniversity in Tirunelveli The work reported herein is original and does not form part of any other thesis or dissertation on the basis of which a degree or award was conferred on an earlier occasion or to any other scholar I understand the University’s policy on plagiarism and declare that the thesis and publications are my own work, except where specifically acknowledged and has not been copied from other sources or been previously submitted for award or assessment MURUGAN S RESEARCH SCHOLAR Dr R.D STEVENS JONES, Dr K.R.T ASHA, Co- SUPERVISOR, SUPERVISOR, Associate professor, Assistant Professor Department of Zoology, Department of Biochemistry Scott Christian college, Govt Arts College, Nagercoil 629 003 Paramakudi -623701 Place : Date : i ACKNOWLEDGEMENT I am extremely thankful to GOD ALMIGHTY for giving me strength, health and support for successfully completing this work at the right time I wish to record my deep sense of gratitude and profound thanks to my research supervisor Dr K.R.T ASHA, Assistant Professor, Department of Biochemistry, Government Arts College, Paramakudi, Ramanathapuram District, Tamil Nadu,for her keen interest, inspiring guidance, constant encouragement with my work during all stages, to bring this thesis into fruition I am extremely indebted to my joint supervisor Dr STEVENS JONES, Professor Department of Zoology, Scott Christian College (Autonomous) for his valuable suggestions and support during the course of my research work I express my sincere thanks to Dr Pamaela Oliver, Department of English, Lekshmipuram College of Arts and Science, Neyyoor, Dr A RamalingomPillai, Department of Chemistry, Lekshmipuram College of Arts and Science, Neyyoor for their valuable encouragement throughout the study I am extremely indebted to Dr A Tamil Selvi, Dr C Sunithra, Dr P NandhaKumari, Department of Zoology, Lekshmipuram College of Arts and Science, Neyyoor,Dr N.C.J PackiyaLekshmi, Department of Microbiology, Udhaya College of Arts and Science, Vellamadi, for theirvaluable suggestions and support during the course of my research work I wish to express my heartfelt thanks to The Management, Principal and the Staff members (Teaching and Non-Teaching) of Biochemistry Department of Lekshmipuram College of Arts and Science, Neyyoor I am deeply indebted to my beloved Parents, Wife, Children and my family members for their moral support and constant encouragement at every stage of this work S MURUGAN iv LIST OF CONTENTS Page No GENERAL INTRODUCTION WITH REVIEW OF LITERATURE CHAPTER - I 2.0 ISOLATION AND CHARACTERIZATION OF HALOARCHAEAL STRAIN 14 2.1 INTRODUCTION 2.2 MATERIALS AND METHODS 14 16 2.2.1 Sampling site 2.2.2 Collection of samples 2.2.3 Isolation and screening of Haloarchaea 2.2.4 Screening of SOD activity 2.2.5 Screening of catalase activity 2.2.6 Biochemical characterization 2.2.7 Determination ofhalophily 2.2.8 Determination of temperature in the growth of isolate 2.2.9 Determination of optimum pH 2.2.10 Determination of growth curve and generation time 2.2.11 Growth of isolate in different agars 2.2.12 Growth in various carbon sources 2.2.13 Growth in various N2sources 2.2.14 Growth in various inorganic sources 2.2.15 Growth in various organic solvents 2.2.16 Media formulation 2.2.17 Antimicrobial analysis 2.2.18 16SrRNA Sequencing 2.3 RESULTS 16 16 17 17 17 18 21 21 22 22 22 23 23 23 24 24 24 25 27 2.3.1 Description of sampling site 2.3.2 Physico-chemical analysis 2.3.3 Isolation and screening 2.3.4 Screening of SOD producing Halobacterium 2.3.5 Screening of catalase producing strain 2.3.6 Biochemical characterization of isolate 2.3.7 Determination of halophily 2.3.8 Effect of temperature in the growth of isolate 2.3.9 Effect of pH in the growth of strain 2.3.10 Growth curve and generation time 2.3.11 Growth in different agars 2.3.12 Growth in various carbon sources 2.3.13 Effect of various carbon sources v 27 29 31 33 33 33 34 36 38 40 41 42 43 2.3.14 Growth in various N2 sources 2.3.15 Effect of N2 sources 2.3.16 Effect of inorganic salts in the growth 2.3.17 Growth of isolate in various organic solvents 2.3.18 Effect of organic solvents 2.3.19 Antimicrobial Assay 2.3.20 16SrRNA sequence analysis 45 46 49 50 51 52 55 2.4 DISCUSSION 2.5 CONCLUSION 59 67 CHAPTER II 3.0 PURIFICATION AND CHARACTERIZATION OF ANTIOXIDANTENZYMES 69 3.1 INTRODUCTION 3.2 MATERIALS AND METHODS 69 73 3.2.1 Maintenance of Halobacterial culture 3.2.2 SOD production 73 73 3.2.2.1 Media for SOD production 3.2.2.2 Preparation of enzyme source 3.2.2.3 Enzyme assay 73 73 74 3.2.3 Estimation of protein 3.2.4 Purification 74 74 3.2.4.1 Precipitation by solvent 3.2.4.2 Desalting of protein 3.2.4.3 Purification by chromatography 74 75 75 3.2.5 Characterization of enzyme activity 75 3.2.5.1 Effect of pH 3.2.5.2 Effect of temperature 3.2.5.3 Effect of enzyme concentration 3.2.5.4 Effect of substrate concentration 3.2.5.5 Effect of metal ions 3.2.5.6 Effect of various buffers 3.2.5.7 Effect of inhibitors 76 76 76 76 77 77 77 3.2.6 Catalase production 78 3.2.6.1 Media formulation for catalase activity 3.2.6.2 Preparation of enzyme source 3.2.6.3 Estimation of catalase by dichromoacetic acid method 3.2.7 Estimation of protein 3.2.8 Purification of catalase 78 78 78 79 79 3.2.8.1 Precipitation by solvent vi 79 3.2.8.2 Purification by chromatography 3.2.9 Characterization of catalase 3.2.9.1 Effect of pH 3.2.9.2 Effect of Temperature 3.2.9.3 Effect of enzyme concentration 3.2.9.4 Effect of substrate concentration 3.2.9.5 Effect of metal ions 3.2.9.6 Effect of various buffers 3.2.9.7 Effect of inhibitors 3.3 RESULTS 79 81 81 81 82 82 82 83 83 83 3.3.1 Enzyme production in optimized medium 3.3.2 Extraction and purification of SOD 3.3.3 Characterization of enzyme 84 84 85 3.3.3.1 Effect of pH on SOD 3.3.3.2 Effect of temperature 3.3.3.3 Effect of enzyme concentration in SOD from purified sample 89 3.3.3.4 Effect of substrate concentration in SOD activity 3.3.3.5 Effect of NaCl 3.3.3.6 Effect of metal ions in SOD Activity 3.3.3.7 Activity of SOD in various buffers 3.3.3.8 Effect of inhibitors in SOD activity 89 90 91 94 97 3.3.4 Catalase production 85 87 99 3.3.4.1 Enzyme production in optimized medium 3.3.4.2 Purification of catalase 3.3.4.3 Activity of catalase 3.3.4.4 Characterization of enzyme 3.3.4.4.1 Effect of pH 3.3.4.4.2 Effect of temperature 3.3.4.4.3 Effect of enzyme concentration 3.3.4.4.4 Effect of substrate concentration 3.3.4.4.5 Effect of NaCl 3.3.4.4.6 Effect of metal ions 3.3.4.4.7 Activity of catalase in various buffers 3.3.4.4.8 Effect of inhibitors in catalase activity 3.4 DISCUSSION 3.5 CONCLUSION 99 100 101 101 101 103 104 105 106 107 110 113 115 120 CHAPTER – III 4.0 MOLECULAR STUDIES OF ANTIOXIDANT ENZYMES 4.1 INTRODUCTION 4.2 MATERIALS AND METHODS vii 122 122 127 4.2.1 Sample preparation 4.2.2 SDS-PAGE Electrophoresis 4.2.3 Protein identification and mass spectroscopy 4.2.4 Gene ontology by DAVID software and Protein interaction by STRING 127 127 128 129 4.3 RESULTS 4.4 DISCUSSION 4.5 CONCLUSION 129 141 142 CHAPTER - IV 5.0 BIOACTIVITY/BIOMEDICAL APPLICATIONS OF ANTIOXIDANT ENZYMES 143 5.1 INTRODUCTION 5.2 MATERIALS AND METHODS 143 145 5.2.1 Determination of invitroantiproliferative effect of SOD on culturedHeLa cell lines, A-549, MCF-7, HepG2 andHt-29 cell lines 145 5.2.2 Determination of invitro cardiac protective activity of SOD and catalase on cultured H9C2 cell lines 5.3 RESULTS 146 147 5.3.1 Determination of antiproliferative effect of SOD on culturedHeLa cell line, A549, MCF-7, HepG2 andHt-29 cell lines 5.3.1.1 Antiproliferative effect of SOD on cultured HeLa cell line 5.3.1.2 Invitroantiproliferative effect of SOD against cultured MCF-7 cell lines 5.3.1.3 Determination of invitroantiproliferative effect of SOD on cultured A-549 cell line 152 5.3.1.4 Determination of invitroantiproliferative effect of SOD on cultured HT-29 cell line 154 5.3.1.5 Determination of invitroantiproliferative effect of SOD on cultured Hep-G2 cell line 5.3.2 Determination of invitroantiproliferative effect of catalase on cultured HeLa cell line, A-549, MCF-7, HepG2 andHt-29 cell lines 5.3.2.1.Determination of invitroantiproliferative effect of catalase on cultured HeLa cell lines 5.3.2.2 Determination of invitroantiproliferative effect of catalase on cultured A-549cell lines 5.3.2.3 Determination of invitroantiproliferative effect of catalase on cultured MCF-7 cell lines viii 147 147 150 156 158 158 160 162 5.3.2.4.Determination of invitroantiproliferative effect of catalase on cultured HepG2 cell lines 5.3.2.5 Determination of invitroantiproliferative effect of catalase on cultured HT-29 cell lines 166 5.3.3 Determination of invitro cardiac protective effect of SOD on cultured H9C2 cell lines 5.3.4.Determination of invitro cardiac protective effect of catalase on cultured H9C2 cells 5.4 DISCUSSION 5.5 CONCLUSION 164 168 170 172 177 SUMMARY AND CONCLUSION 178 REFERENCES 183 LIST OF PUBLICATIONS ix LIST OF TABLES Table No Title Page No 2.1 Physicochemical characteristics of solar saltern located at Puthalam near Kanyakumari coast of India 31 2.2 Activity of SOD in different strains 33 2.3 Activity of catalase in different strains 33 2.4 Characterization of isolated strain 34 2.5 Effect of NaCl 35 2.6 Effect of temperature in the growth of isolate 37 2.7 Effect of pH in the growth of strain 39 2.8 Growth curve of isolate 40 2.9 Growth of isolate in different agars 42 2.10 Growth of isolate in various carbon sources 43 2.11 Effect of carbon sources in the growth of isolate 44 2.12 Growth of isolate in various N2 sources 46 2.13 Effect of N2 sources in the growth of isolate 47 2.14 Effect of inorganic salts in the growth of isolate 49 2.15 Growth of isolate in various organic solvents 50 2.16 Effect of organic solvents in the growth of isolate 51 2.17 Antimicrobial activity of isolated strain 53 3.1 Activity of SOD in optimized medium 84 3.2 Activity of SOD in ice cold ethanol solvent fraction 85 3.3 Purification of SOD 85 3.4 Effect of substrate concentration in SOD activity 90 3.5 Activity of SOD in control 91 x Table No Title Page No 3.6 Activity of catalase in control and optimized medium 100 3.7 Purification of catalase showing fractions 100 3.8 Activity of catalase in culture 101 3.9 Effect of substrate concentration in catalase activity 105 3.10 Activity of catalase in control 107 4.1 Mass Spectrum Analysis of Chromatogram view Band ID-I and II 132 4.2 Mass spectrum analysis of Chromatogram view Band ID-3 and 136 5.1 Growth inhibitory effect of SOD on HeLa cell line 148 5.2 Antiproliferative effect of SOD against cultured MCF-7 cell lines 150 5.3 Antiproliferative effect of SOD on cultured A-549 cell line 152 5.4 Antiproliferative effect of SOD on cultured HT-29 cell line 154 5.5 Antiproliferative effect of SOD on cultured Hep-G2 cell line 156 5.6 Antiproliferative effect of catalase on cultured HeLa cell lines 158 5.7 Antiproliferative effect of catalase on cultured A549cell lines 160 5.8 Antiproliferative effect of catalase on cultured MCF-7 cell lines 162 5.9 Antiproliferative effect of catalase on cultured HepG2 cell lines 164 5.10 Antiproliferative effect of catalase on cultured HT-29 cell lines 166 5.11 Cardiac protective effect of SOD on cultured H9C2 cell lines 168 5.12 Cardiac protective effect of catalase on cultured H9C2 cells 170 xi Litzner, BR, 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CERTIFICATE The research work embodied in the present thesis entitled ? ?BIOPROSPECTING OF HALOARCHAEA FOR THE PRODUCTION AND EXPRESSION OF POTENT NOVEL ANTIOXIDANT ENZYMES? ?? has been carried out in the. .. fermentation The bacterial isolate was inoculated in the TSI agar slant by stabbing the butt down to the bottom and then streaking the surface of the slant, incubated at 37˚C for – 12 days The result... 37˚C for – 12 days After the incubation, a few drops of (0.2 ml) Kovac’s reagent was added into the tubes and the result was observed The development of bright red colour at the interface of reagent