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Selective Extraction and Partial Purification of Biomolecules Using Reverse Micelle Based System

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Selective Extraction and Partial Purification of Biomolecules Using Reverse Micelle Based System SELECTIVE EXTRACTION AND PARTIAL PURIFICATION OF BIOMOLECULES USING REVERSE MICELLE BASED SYSTEM A THESIS SUBMITTED TO UNIVERSITY OF PUNE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CHEMICAL ENGINEERING BY RAGHAVENDRA P. GAIKAIWARI UNDER THE GUIDANCE OF Dr. B.D.KULKARNI DISTINGUISHED SCIENTIST CHEMICAL ENGINEERING AND PROCESS DEVELOPMENT DIVISION NATIONAL CHEMICAL LABORATORY PUNE – 411008 (INDIA) APRIL 2011 CONTENTS Page No. List of Tables List of Figures Certificate Declaration Acknowledgement List of abbreviations Abstract CHAPTER 1 Introduction and Review of Literature Part A Review of biomolecules 2 1.1 Solanesol 2 1.1.1 Uses of solanesol 2 1.1.2 Methods for recovery/ extraction of solanesol 3 1.2 Lycopene 6 1.2.1 Uses of lycopene 8 1.2.2 Methods for manufacturing/extraction of lycopene 8 1.3 11 Hydroxy canrenone (11 OHC) 16 1.3.1 Characteristics of 11 OHC 17 1.3.2 Biotransformation 17 1.4 Lipase 20 1.4.1 Applications of lipases 20 1.4.2 Purification of lipases 22 1.5 Tannase 22 1.5.1 Applications of tannase 23 1.5.2 Purification of tannase 24 PART B Microemulsions – structure, transport phenomenon and 25 applications 1.6 Microemulsions – general introduction 25 1.6.1 Surface active agents (surfactants) 26 1.6.2 Micellar structure and properties 27 1.6.3 Micelles and reverse micelles 30 1.6.4 Mass transport and solubilization phenomena in micelle 31 1.6.5 Molecular interactions and surfactant adsorption 33 1.6.6. Microemulsion phase behavior 34 1.6.7 Applications of microemulsions and micelles 35 1.6.8 Role of reverse micelle in protein extraction 38 1.7 References 42 CHAPTER 2 Extraction of solanesol from tobacco dust using microemulsion/reverse micelle 2.1 Introduction 52 2.2 Materials and Methods 53 2.2.1 Materials 53 2.2.2 Methods 53 2.2.2.1 Extraction of solanesol 53 2.2.2.2 Saponification of n-hexane extract 54 2.2.2.3 Extraction of solanesol by using reverse micelle system 54 2.2.2.4 Determination of total solid content 55 2.2.2.5 HPLC analysis of solanesol 55 2.2.2.6 TLC analysis of solanesol 55 2.2.2.7 Presentation of observations 56 PART A Extraction of solanesol without surfactant 57 2.3 Results and discussion 57 2.3.1 Extraction of solanesol by leaching of tobacco dust 57 2.3.2 Effect of temperature 58 2.3.3 Effect of solvent to tobacco dust (feed) ratio 60 2.3.4 Saponification of crude solanesol 62 2.3.3.1 Effect of temperature on saponification 62 2.3.3.2 Effect of solvent (n-propanol) to crude solanesol ratio on 63 saponification PART B Extraction of solanesol with surfactant 65 2.4 Results and discussion 65 2.4.1 Effect of AOT and its concentration 65 2.4.2 Effect of SDS and its concentration 66 2.4.3 Effect of CTAB and its concentration 67 2.4.4 Selection of surfactant 70 2.4.5 Effect of different process parameters on solanesol 70 extraction using CTAB 2.54.5.1 Effect of contact time on solanesol extraction 70 2.4.5.2 Effect of temperature on solanesol extraction 71 2.4.5.3 Effect of solvent to tobacco dust ratio on solanesol 72 extraction 2.5 Conclusion 74 2.6 Process economics considerations 76 2.7 References 77 CHAPTER 3 Extraction of lycopene from tomato paste using microemulsion/reverse micelle 3.1 Introduction 79 3.2 Materials and Methods 80 3.2.1 Materials 80 3.2.2 Methods 80 3.2.2.1 Extraction of lycopene 80 3.2.2.2 Saponification of n-hexane extract 81 3.2.2.3 Extraction of lycopene by using reverse micelle system 81 3.2.2.4 Determination of total solid content 82 3.2.2.5 HPLC analysis of lycopene 82 3.2.2.6 TLC analysis of lycopene 83 3.2.2.7 Presentation of observations 83 PART A Extraction of lycopene without surfactant 83 3.3 Results and discussion 83 3.3.1 Extraction of lycopene by leaching of tomato paste 83 PART B Extraction of lycopene with surfactant 84 3.4 Results and discussion 84 3.4.1 Effect of AOT and its concentration 84 3.4.2 Effect of CTAB and its concentration 86 3.4.3 Effect of SDS and its concentration 87 3.4.4 Effect of significant parameters in presence of surfactant 88 (AOT and CTAB) 3.4.4.1 Effect of contact time for lycopene extraction using CTAB 88 and AOT 3.4.4.2 Effect of temperature for lycopene extraction using CTAB 88 3.4.4.3 Effect of solvent to tomato paste ratio for lycopene 90 extraction using CTAB 3.4.4.4 Effect of temperature on lycopene extraction using AOT 91 3.4.4.5 Effect of solvent to tomato paste ratio for lycopene 93 extraction using AOT 3.5 Conclusion 94 3.6 Process economics considerations 96 3.7 References 97 CHAPTER 4 Extraction and enrichment of 11α-hydroxy canrenone (11 OHC) from fermentation broth using microemulsion/reverse micelle 4.1 Introduction 99 4.2 Materials and methods 100 4.2.1 Materials 100 4.2.2 Methods 100 4.2.2.1 Organism 100 4.2.2.2 Production of A. ochraceous mycelial mass 100 4.2.2.3 Biotransformation of canrenone 100 4.2.2.4 Downstream procedure to recover 11 OHC 101 4.2.2.5 Extraction of 11 OHC by using reverse micelle system 101 4.2.2.6 Analytical procedures 101 PART A Extraction of 11 OHC without surfactant 103 4.3 Results and discussion 103 4.3.1 Extraction of 11 OHC from the feed broth by ethyl acetate 103 4.3.2 Effect of temperature 104 4.3.3 Effect of solvent to feed broth ratio (v/v) 105 PART B Extraction of 11 OHC using surfactant 109 4.4 Results and discussion 109 4.4.1 Effect of T80 and its concentration 109 4.4.2 Effect of CTAB and its concentration 109 4.4.3 Effect of SDS and its concentration 109 4.4.4 Selection of surfactant 110 4.4.5 Effect of contact time for extraction of 11 OHC 112 4.4.6 Effect of other significant parameters in presence of 112 surfactant 4.4.7 Effect of temperature 112 4.4.8 Effect of solvent to feed broth ratio 115 4.5 Conclusion 117 4.6 Process economics considerations 118 4.7 References 119 CHAPTER 5 Extraction and purification of lipase from fermented broth using reverse micelle 5.1 Introduction 120 5.2 Materials and methods 121 5.2.1 Materials 121 5.2.2 Methods 121 5.2.2.1 Organism 121 5.2.2.2 Production of extracellular crude lipase 121 5.2.2.3 Purification of lipase 122 5.2.2.4 Purification of lipase by reverse micelle system 122 5.2.2.5 Enzyme assay 123 5.2.2.6 Estimation of protein 124 5.2.2.7 Polyacrylamide gel electrophoresis (PAGE) 124 5.2.2.8 Staining of proteins 124 5.3 Results and discussion 124 5.3.1 Purification of lipase by conventional method 124 5.3.2 Purification of lipase by reverse micellar system 126 5.3.2.1 Effect of various parameters on forward extraction 126 5.3.2.1 Effect of surfactant on forward extraction 126 5.3.2.1.2 Effect of contact time on forward extraction 127 5.3.2.1.3 Effect of AOT concentration on forward extraction 129 5.3.2.1.4 Effect of pH of fermentation broth 130 5.3.2.1.5 Effect of crude broth: organic phase ratio (Vaq:VRM) on 133 forward extraction 5.3.2.2 Effect of various parameters on backward extraction 135 5.3.2.2.1 Effect of NaCl concentration on backward extraction 135 5.3.2.2.2 Effect of pH on backward extraction 139 5.3.2.2.3 Effect of Vaq:VRM on backward extraction 141 5.3.2.2.4 Effect of contact time on backward extraction 142 5.4 Conclusion 143 5.5 References 145 CHAPTER 6 Extraction and purification of tannase using reverse micelle 6.1 Introduction 148 6.2 Materials and methods 148 6.2.1 Materials 148 6.2.2 Methods 149 6.2.2.1 Organism 149 6.2.2.2 Production of intracellular crude tannase 149 6.2.2.3 Purification of tannase 149 6.2.2.4 Purification of tannase by reverse micelle system 150 6.2.2.5 Enzyme assay 151 6.2.2.6 Estimation of protein 151 6.2.2.7 Polyacrylamide gel electrophoresis (PAGE) 151 6.2.2.8 Staining of proteins 151 6.3 Results and discussion 152 6.3.1 Purification of tannase by conventional method 152 6.3.2 Purification of tannase by reverse micellar system 153 6.3.2.1 Effect various parameters on forward extraction of tannase 153 6.3.2.1.1 Effect of surfactant 153 6.3.2.1.2 Effect of contact time 154 6.3.2.1.3 Effect of CTAB concentration 156 6.3.2.1.4 Effect of pH 158 6.3.2.1.5 Effect of crude enzyme: organic phase (Vaq:VRM) ratio 160 6.3.2.2 Effect of various parameters on backward extraction of 161 tannase 6.3.2.2.1 Effect of pH of stripping solution 161 6.3.2.2.2 Effect of NaCl extraction 162 6.3.2.2.3 Effect of Vaq:VRM on backward extraction of tannase and its 164 correlation with extraction contact time 6.4 Conclusion 170 6.5 References 172 CHAPTER 7 Summary and Conclusion 174 List of publications/ patents/ presentations/ technology 183 transfer 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