Production, Purification and Characterization of Psychrotolerant Alkaline Lipase and Protease and Their Application in Detergent Formulation
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Production, Purification and Characterization of Psychrotolerant Alkaline Lipase and Protease and their Application in Detergent Formulation By Yasir Ali Registration # 8-FBAS/PHDBT/S-14 Department of Biological Sciences Faculty of Basic and Applied Sciences International Islamic University Islamabad, Pakistan 2014-2019 Production, Purification and Characterization of Psychrotolerant Alkaline Lipase and Protease and their Application in Detergent Formulation Submitted By Yasir Ali 08-FBAS/PHDBT/S-14 Supervised By Dr. Bashir Ahmad Assistant Professor Department of Biological Sciences, FBAS, IIUI Department of Biological Sciences Faculty of Basic and Applied Sciences International Islamic University Islamabad, Pakistan 2014-2019 A thesis submitted to the Department of Biological Sciences, International Islamic University, Islamabad in the partial fulfillment of the requirements of the degree of Doctor of Philosophy in Biotechnology DEDICATION This doctoral dissertation is dedicated to my beloved parents whose constant prayers, support and guidance enabled me to achieve this milestone DECLARATION It is hereby declared that the work present in this doctoral dissertation is my own effort except where otherwise acknowledged and that the dissertation is my own composition. No part of the dissertation has been previously presented for any other degree. Date ______________________ Yasir Ali TABLE OF CONTENTS CHAPTER TITLE PAGE TABLE OF CONTENTS I LIST OF FIGURES IX LIST OF TABLES XII LIST OF ABBREVIATIONS XIII ACKNOWLEDGMENT XV ABSTRACT XVI 1. INTRODUCTION 1-18 1.1. Microbes Producing Cold-Active Proteases 2 1.2. Catalytic mechanism 7 1.3. Classification of Proteases 7 1.4. Mechanism and Structure of lipase 11 1.5. Bacterial Lipases 14 1.6. Goals and objectives 17 1.7. Problem Statement, Research Gap, and Research Hypothesis 18 2. REVIEW OF LITERATURE 19-63 2.1. Psychrotrophic Bacteria: Sources, Isolation and Characterization 20 2.2. Ice cabins and freezers 20 2.3. Unfrozen soils 20 2.4. Clouds 20 2.5. Water and snow 21 2.6. Permafrost soils and ice 22 2.7. Extraterrestrial sources 22 2.8. Glaciers 22 2.9. Polar glaciers 23 2.10. Non-Polar Glaciers 25 2.11. Metabolism and adaptations for cold environment 27 2.12. Microbes Producing Cold-Active Proteases 28 2.13. Classification of Proteases 29 2.14. Isolation of Alkaline protease producing microorganism 36 2.15. Properties of alkaline proteases 36 I 2.16. Production and optimization of psychrotrophic alkaline protease 39 2.17. Purification and characterization of thermolabile alkaline protease 41 2.18. Cloning and Expression of Cold-Active Proteases 44 2.19. Application of cold active alkaline protease 46 2.20. Cold active lipases 47 2.21. Sources of cold active lipases 48 2.22. Fermentation conditions for cold active lipase production 51 2.23. Purification and characterization of cold active lipases 54 2.24. Biotechnological approaches in cold active lipase 59 2.25. Industrial applications of cold active lipases 62 3. MATERIAL AND METHODS 64-102 3.1. Isolation and identification of extracellular cold active protease from psychrotrophic Stenotrophomonas sp. PAK-01 64 3.1.1. Samples collections 64 3.1.2. Sampling, Isolation and screening of thermolabile lipase producing bacteria 64 3.1.3. Genomic bacterial DNA Extract 68 3.1.4. Amplification and sequencing of 16S ribosomal RNA gene 68 3.1.5. Sequencing and analysis of Phylogeny 68 3.1.6. Sequence submission to Genbank and Accession Numbers 69 3.1.7. Molecular Cloning and Expression of alkaline thermolabile protease gene in BL21 cells 70 3.1.7.1. Microorganisms 70 3.1.7.2. Cloning of the thermolabile alkaline protease gene 70 3.1.7.3. Sequence analysis of nucleotide 71 3.1.7.4. Enzyme and protein assays 71 II 3.1.7.5. Expression and purification Analysis of Pry 71 3.1.8. Production optimization of extracellular Thermolabile alkaline protease from Stenotrophomonas sp. 73 3.1.8.1. Fermentation of crude protease 73 3.1.8.2. Inoculum preparation 73 3.1.8.3. Fermentation for Crude Enzyme Production 74 3.1.8.4. Selection of Media for Protease Production 75 3.1.8.5. Quantitative estimation of protease 79 3.1.8.6. Production of thermolabile protease enzyme 79 3.1.8.7. Enzyme and protein assays 80 3.1.8.8. Protein estimation 81 3.1.8.9. Determination of specific activity (Unit/mg) 82 3.1.8.10. Data analyses 82 3.1.9. Purification and characterization of alkaline and cold active protease from psychrotropic bacterium Stenotrophomonas sp. PAK-01 for detergent Formulation 83 3.1.9.1. Material and methods 83 3.1.9.2. Microorganisms 83 3.1.9.3. Production of thermolabile crude enzyme 83 3.1.9.4. Purification of alkaline protease 83 3.1.9.5. Enzyme and protein assays 84 3.1.9.6. SDS electrophoresis 84 3.1.9.7. Characterization of the alkaline protease 85 3.1.9.7.1. Temperature influence on protease molecules hydrolysis and stability 85 3.1.9.7.2. Effect of pH and various metal Ions, surfactants and oxidants on purified enzymatic hydrolysis and stability 85 III 3.1.9.7.3. Inhibitors and organic solvents effect 86 3.1.9.7.4. Km, Vmax and Kcat analyses 86 3.1.10. Stenotrophomonas sp. PAK-01 thermolabile alkaline protease as a laundry additive 88 3.1.10.1. Stability of Thermolabile protease in commercial Detergents 88 3.1.10.2. Oxidizing and bleach result on thermolabile protease 88 3.1.10.3. Washing analysis 88 3.2. Isolation and identification of extracellular cold active lipase from psychrotrophic Pseudomonas peli PAK-03 89 3.2.1. Sampling and screening of thermolabile lipase secreting bacteria 89 3.2.2. Isolation of genomic DNA 89 3.2.2.1. Amplification and nucleotide sequencing of 16S ribosomal RNA gene 89 3.2.2.2. Construction and analysis of Phylogeny 90 3.2.3. Molecular cloning, expression and characterization of organic solvent tolerant alkaline lipase gene from pseudomonas peli PAK03 (MH338242) 91 3.2.3.1. Cloning of the thermolabile alkaline lipase gene 91 3.2.3.2. Sequence analysis of nucleotide 91 3.2.3.3. Expression and purification Analysis of YLip 92 3.2.3.4. Thermolabile recombinant lipase assay 92 3.2.4. Production of extracellular thermolabile alkaline lipase from psychrotrophic pseudomonas peli 93 3.2.4.1. Production of thermolabile lipase enzyme 93 3.2.4.2. Lipolytic assay 94 3.2.5. Purification and Characterization of solvent stable thermolabile lipase from Psychrotrophic Pseudomonas peli PAK-03 95 IV 3.2.5.1. Microorganisms 95 3.2.5.2. Production of thermolabile lipase enzyme 95 3.2.5.3. Purification of thermolabile alkaline protease 95 3.2.5.4. Characterization of Purified Alkaline Lipase 96 3.2.5.4.1. Temperature influence on lipase 96 3.2.5.4.2. Effect of pH on purified lipase 96 3.2.5.4.3. Effect of various metal ions 97 3.2.5.4.4. organic solvents effect 97 3.2.5.4.5. Km, Vmax and Kcat analyses 97 3.2.6. Alkaline Lipase from Psychrotrophic Pseudomonas peli for Detergent Additive Capability 99 3.2.6.1. Microorganisms, Chemicals, and Reagents 99 3.2.6.2. Lipase Production and Purification 99 3.2.6.3. Thermolabile lipase assay 99 3.2.6.4. Alkaline Lipase Compatibility with anionic Surfactants and Detergents 99 3.2.6.5. Alkaline Lipase Compatibility with Oxidizing compound 100 3.2.6.6. Olive Oil muddied Cotton Cloth Formulation 100 3.2.6.7. Formulation of Washing solution 100 3.2.6.8. Washing analysis 101 4. RESULTS 103-213 4.1. Isolation and identification of extracellular thermolabile protease producing psychrotrophic PAK-01 103 4.1.1. Sampling 103 4.1.2. Isolation of bacteria from samples obtained from glaciers 103 4.1.3. Psychrotrophic Alkaline protease producing bacteria 103 4.1.4. Identification of potential isolate and phylogenetic Analysis 104 4.1.5. Phylogenetic studies of glacial strains 107 V 4.2. Molecular Cloning and Expression in Escherichia coli BL21(lDE3) 112 4.2.1. Protease nucleotide sequence and proteomic study 112 4.2.2. Expression and purification of protease gene PrY 117 4.3. Production optimization of extracellular thermolabile alkaline protease from Stenotrophomonas sp. 120 4.3.1. Psychrotrophic protease producing bacteria 120 4.3.2. Tyrosine Standard Curve 121 4.3.3. Optimization production of cold active alkaline protease 122 4.4. Characterization of alkaline and cold active protease enzyme from psychrotropic bacterium Stenotrophomonas sp. PAK-01 for detergent formulation 130 4.4.1. Enzyme purification 130 4.4.2. pH and temperature effect 136 4.4.3. Metal ions effect 140 4.4.4. Surfactants and oxidants effect 142 4.4.5. Inhibitors effect 142 4.4.6. Organic solvents effect 146 4.4.7. Determination of kinetic parameters 148 4.5. Stenotrophomonas sp. PAK01 thermolabile alkaline protease as laundry additive 153 4.5.1. Stability of Protease in Detergents/Surfactants 153 4.5.2. Bleach and Oxidizing Agent 157 4.5.3. Thermolabile protease enzyme effect on proteinous pigment Removal 159 4.6. Production of Extracellular Psychrotrophic Alkaline Lipase from Pseudomonas peli strain PAK-03 162 4.6.1. Isolation and phylogenetic analysis 162 4.6.2. Phylogenetic Evaluation of Strain PAK-03 162 4.7. Molecular Cloning and Expression in Escherichia coli of Pseudomonas peli lipase gene 164 4.7.1. Cloning and sequence of YLip gene analysis 164 4.7.2. Examining the expression and purification 166 VI 4.7.3. Thermal stability of purified lipase enzyme 170 4.7.4. Organic solvents, metal ions and detergents effect on recombinant lipase 172 4.8.