PERSPECTIVES ON LIPASEENZYME TECHNOLOGY -- CONTENTS -- ACKNOWLEDGMENTS -- BIOTECHNOLOGY:THE SCIENCE AND ORIGIN OF LIPASE -- 1.1. INTRODUCTION -- 1.2. LIPASES: AN INTRODUCTION -- 1.3. LIPASES: THE STRUCTURE -- 1.4. LIPASES: SPECIFICITY -- 1.5. LIPASES: KINETICS -- 1.6. LIPASES: ACTIVITY DETERMINATIONS -- 1.7. LIPASES: TAXONOMY -- 1.8. LIPASES: SOURCES -- 1.9. LIPASES: APPLICATIONS -- CONCLUSION -- ISOLATION STRATEGIES OF MICROBIAL LIPASES -- 2.1. INTRODUCTION -- 2.2. SAMPLING -- 2.2.1. Simple Sampling -- 2.2.2. Enrichment Culture Technique -- 2.3. SCREENING METHODS FOR MICROBIAL ISOLATES -- 2.3.1. Lipase Plate Assays -- 2.3.2. Lipase Production in SmF -- 2.3.3. Environmental Screening -- 2.4. IDENTIFICATION OF THE MICROBIAL ISOLATE -- LIPASE PRODUCTION: DEFINING THE MEDIA -- 3.1. INTRODUCTION -- 3.2. FERMENTATION MEDIA -- 3.3. MEDIA OPTIMIZATION WITH NUTRITIONAL FACTORS -- 3.4. MODES OF FERMENTATION PROCESS -- 3.4.1. Batch Culture -- 3.4.2. Synchronous Culture -- 3.4.3. Fed-batch Culture -- 3.4.4. Continuous Culture -- 3.5. GOOD MANUFACTURING PRACTICE (GMP) -- LIPASE PURIFICATION AND CHARACTERIZATION -- 4.1. INTRODUCTION -- 4.2. PRESERVING ENZYME ACTIVITY -- 4.3. PROTEIN CONTENT -- 4.3.1. Spectrophotometry -- 4.3.2. Lowry's Method -- 4.3.3. Bradford's Dye-binding Method -- 4.3.4. Bicinchoninic Acid (BCA) Method -- 4.4. CONCENTRATION OF PROTEIN -- 4.4.1. Ultrafiltration -- 4.4.2. Addition of a Dry Matrix Polymer -- 4.4.3. Ammonium Sulfate Precipitation -- 4.4.4. Precipitation by Organic Solvents -- 4.4.5. Precipitation with PEG -- 4.5. PURIFICATION BY COLUMN CHROMATOGRAPHY -- 4.5.1. Ion-exchange Chromatography -- 4.5.2. Gel Filtration Chromatography -- 4.5.3. Hydrophobic Interaction Chromatography -- 4.5.4. Affinity Chromatography -- 4.5.5. HPLC and FPLC -- 4.6. PLANNING A LIPASE PURIFICATION STRATEGYBY AFFINITY CHROMATOGRAPHY.

Ammonium Sulfate Precipitation -- Packing of Affinity Matrix -- Sample Loading -- Purification of Lipase -- Elution of Lipase -- 4.7. POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) -- 4.7.1. SDS-PAGE -- 4.7.2. Native PAGE -- 4.7.3. Staining of Gels -- 4.8. BIOCHEMICAL CHARACTERIZATION -- 4.8.1. Thermostability -- 4.8.2. Substrate Specificity -- 4.8.3. Fatty Acid Specificity -- 4.8.4. Positional Specificity -- 4.8.5. Electrophoretic Mobility -- 4.8.6. Structural Features -- 4.8.7. Chemical Modification -- LIPASE STRUCTURE -- 5.1. INTRODUCTION -- 5.2. STRUCTURE ANALYSIS -- 5.2.1. Circular Dichroism -- 5.2.2. Fluorescence Spectroscopy -- 5.3. PROTEIN ENGINEERING -- 5.4. LIPASE STRUCTURE -- 5.4.1. Structure of RmL -- 5.4.2. Open Conformation of Pseudomonas Lipase -- Alpha/beta Hydrolase Fold -- Active Site Cleft -- Oxyanion Hole -- 5.4.3. Disulfide Bond in Pseudomonas aeruginosa Lipase -- 5.4.4. Structural Features of Penicillium expansum Lipase -- MOLECULAR CLONING AND OVEREXPRESSION -- 6.1. INTRODUCTION -- 6.2. VECTORS -- 6.3. TECHNIQUES IN GENE CLONING -- 6.3.1. Isolation of Plasmid DNA -- 6.3.2. Restriction Enzyme Digestion -- 6.3.3. Ligation -- 6.3.4. Transformation -- Transformation in Bacteria -- Transformation in Yeast -- 6.4. PCR AND RT-PCR -- 6.4.1. Primers -- 6.4.2. Taq Polymerase -- 6.4.3. PCR Cycle -- 6.4.4. Factors Affecting PCR -- Denaturing Temperature and Time -- Annealing Temperature and Primer Design -- Primer Length -- Elongation Temperature and Time -- Cycle Number -- Helix Destabilizers/Additives -- Optimization -- 6.4.5. PCR Protocol -- 6.4.6. Reverse Transcription -- 6.4.7. RT-PCR Protocol -- 6.4.8. PCR Methods -- Nested PCR -- Hotstart PCR -- Inverse PCR -- Multiplex PCR -- Quantitative PCR -- PCR Mutagenesis -- 6.5. TECHNICAL TIPS -- 6.6. A CLONING STRATEGY FOR LIPASE BY RT-PCR CLONING.

6.6.1. Calculation of Concentrations for RT-PCR Components -- 6.6.2. RT-PCR Protocol -- Positive Control Reaction -- Negative Control Reaction -- Positive Control Protocol -- 6.6.3. Vector Digestion -- 6.6.4. Ligation -- 6.6.5. Transformation -- BIOINFORMATICS -- 7.1. COMPUTING IN BIOINFORMATICS -- 7.1.1. Internet -- 7.1.2. HTTP and URL -- 7.1.3. Softwares -- 7.2. DNA AND PROTEIN INFORMATION RESOURCES -- 7.2.1. NCBI -- 7.2.2. EMBL -- 7.2.3. DDBJ -- 7.2.4. PIR -- 7.2.5. MIPS -- 7.2.6. SWISS-PROT -- 7.3. DATABASE STRUCTURE AND ANALYSIS -- 7.3.1. BLAST -- 7.3.2. Clustal W -- 7.3.3. DOMO -- 7.3.4. PROF-PAT -- 7.4. LIPASE ENGINEERING DATABASE (LED) -- LIPASES: BIOCATALYSTS FOR THE FUTURE -- 8.1. INTRODUCTION -- 8.2. LIPASE BIOCATALYSIS -- 8.3. LIPASE CATALYSIS IN ORGANIC SOLVENTS -- 8.4. APPLICATIONS OF LIPASE CATALYSIS -- 8.4.1. Lipases in Oleochemical Processing -- Lipase-catalysed Hydrolysis of Oils and Fats -- Lipase-Catalysed Esterification -- Lipase-catalysed Transesterification -- Lipase-catalysed Interesterification -- 8.4.2. Lipases in Detergent Formulations -- 8.4.3. Applications of Lipase in 'Green Chemicals' -- 8.5. MECHANISMS OF LIPASE CATALYSIS -- 8.6. INTERACTIONS IN LIPASE CATALYSIS -- 8.7. LIPASE PROTEIN DESIGN FOR ENANTIOSELECTIVITY -- 8.7.1. Functional Expression of Isoenzymes -- 8.7.2. Rational Computer-guided Protein Design -- 8.7.3. Directed Evolution -- EXPERIMENTS -- I. SPECTROPHOTOMETRIC LIPASE ASSAY -- Reagents -- Substrate -- Buffer -- 6 N HCl -- Isooctane -- Copper Reagent -- Protocol -- Standard -- Calculation -- Lipase Unit Definition -- II. PROTEIN ESTIMATION BY BRADFORD'S METHOD -- Reagents -- Bradford's Reagent (5X) -- Protocol -- Standard -- III. RECOVERY OF PROTEINS IN PRESENCEOF LIPIDS AND DETERGENTS -- IV. PROTEIN DETECTION IN POLYACRYLAMIDEGELS WITHOUT STAINING -- V. STAINING OF GLYCOPROTEINS.

VI. ISOLATION OF TOTAL RNA -- VII. FORMALDEHYDE GEL ELECTROPHORESIS OF TOTAL RNA -- Reagents -- 5X Gel Running Buffer (MOPS Buffer) -- Loading Buffer (100 μl) -- Loading Dye -- DEPC-treated Water -- Protocol -- VIII. VECTOR DEPHOSPHORYLATION -- Calf Intestinal Alkaline Phosphatase (CIAP) -- Vector DNA -- Reaction Mixture -- CIAP Stop Buffer -- Ethanol Precipitation -- Reaction Protocol -- IX. TRANSFORMATION BY PEG METHOD -- X. PREPARATION OF SURFACTANT-COATED LIPASE -- Preparation of Surfactant-Modified Lipase by Basheer et al. (1995) -- Preparation of Surfactant-coated Lipase by Kamiya et al. (1995) -- APPENDIX -- I. PREPARATION OF BUFFERS (BUFFER CHART) -- A. 0.1 M Acetate Buffer -- B. 0.1 M Phosphate Buffer -- C. 0.1 M Tris.HCl Buffer -- II. AMMONIUM SULFATE PRECIPITATION TABLE -- III. SDS-PAGE -- A. Trichloroacetic Acid (TCA) Precipitation -- Protocol -- B. Troubleshooting -- Gels Fail to Polymerize -- Electrophoresis Time is too Long -- IV. MATRICES FOR CHROMATOGRAPHY -- A. Gel Filtration Media -- B. Ion-exchange Resins -- C. Affinity Matrices -- V. THE GENETIC CODE -- VI. ENZYMES IN MOLECULAR CLONING -- Alkaline Phosphatase -- DNA Ligase (Phage T4) -- DNA Polymerase I -- Exonuclease III -- Mung Bean Nuclease -- Nuclease S1 -- Polynucleotide Kinase -- Restriction Enzymes -- Reverse Transcriptase -- RNase A -- RNase H -- T7, T3 and SP6 RNA Polymerases -- Taq DNA Polymerase -- Terminal Transferase -- VII. RESTRICTION ENZYMES -- A. Diluent Buffers for Restriction Enzymes -- B. Tips to Use Restriction Enzymes -- VIII. NUCLEIC ACID CONVERSIONS -- A. Spectrophotometric Conversions -- B. Nucleotide MWs -- C. Molar Conversions -- IX. NUCLEOTIDE AND PROTEIN DATABASES -- A. Primary Nucleotide Sequence Databases -- B. Secondary Nucleotide Sequence Databases -- C. Protein Sequence Databases -- D. Sequence Motif Databases.

E. Macromolecular 3-D structure Database -- F. General Databases -- X. GENBANK FORMAT FOR NUCLEOTIDE SEQUENCES -- XI. BIOINFORMATICS TOOLS FOR PROTEIN STRUCTURE PREDICTION -- XII. RELATED JOURNALS -- ABBREVIATIONS -- REFERENCES -- I. INTERNET -- II. BOOKS -- III. JOURNAL ARTICLES -- INDEX.