Extraction, Purification and Production of Enzymes (Biotechnology) (Polystyrenes, Polypeptides, Polysaccharides, Proteins, Carbon, Propylene Oxide, Vinyl Chloride, Biosensors, Amino Acids, Antibiotics, Acrylamide, Organic Acids, Maltose Syrups, Hollow Fibres, Hollow Fibres, Enzyme Immunoassay (ELA), Enzyme Electrodes, Biocatalysts) www.entrepreneurindia.co
Introduction Industrial biotechnology is the practice of using cells to generate industrially useful products. An enzyme is a protein that catalyzes, or speeds up, a chemical reaction. Enzymes are the focal point of biotechnological processes, without them biotechnology as a subject would not exist. The main advantage of enzymes compared to most other catalysts is their stereo, region and chemo selectivity and specificity. Enzymes are responsible for many essential biochemical reactions in microorganisms, plants, animals, and human beings. Biotechnology processes may have potential in energy production, specifically in the substitution of renewable plant biomass for fossil feedstock. www.entrepreneurindia.co
This will depend on the development of enzymes able to degrade cellulose in plant biomass and designing methods to recycle or dispose of spent biomass. With time, research, and improved protein engineering methods, many enzymes have been genetically modified to be more effective at the desired temperatures, pH, or under other manufacturing conditions typically inhibitory to enzyme activity (e.g. harsh chemicals), making them more suitable and efficient for industrial or home applications. www.entrepreneurindia.co
Enzymes are used in the extraction of natural products, as catalysts in organic chemistry, in clinical analysis, in industrial processes, and so on. The application of enzymes is found in many different fields and it is one of the good sectors to venture. In coming few years it is estimated that world enzyme demand will average annual increases of 6.3 percent. www.entrepreneurindia.co
Market Outlook The global enzymes market is expected to grow on average 4.6 percent through 2020 to $7.2 billion from $5.78 billion in 2015. Global demand for enzymes is forecast to grow on average 4.6 percent through 2020 to $7.2 billion. www.entrepreneurindia.co
This market includes enzymes used in industrial applications (food and beverages, cleaning products, biofuel production, animal feed, and other markets) and specialty applications (research and biotechnology, diagnostics, and biocatalysts). The global enzymes market size was USD 8.18 billion in 2015 and is expected to witness significant growth over the next eight years on account of its increasing application in detergents, pharmaceuticals, and food & beverages. www.entrepreneurindia.co
Enzymes Market www.entrepreneurindia.co
Global Industrial Enzymes Market (US$ Billion) www.entrepreneurindia.co
Table of Contents 1. LARGE-SCALE EXTRACTION AND PURIFICATION OF ENZYMES AND OTHER PROTEINS • Extraction by Chemical Methods • Alkali • Lysozyme and EDTA • Detergents • Cold Shock • Osmotic Shock • Extraction by Physical Methods • Sonication • Freezing and Thawing www.entrepreneurindia.co
• Solid Shear • Grinding or Agitation with Abrasives • Liquid Shear • Isolation and Purification • Nucleic Acid Removal • Cetyltrimethyl Ammonium Bromide • Streptomycin Sulphate • Polyethyleneimine • Nuclease Treatment • Concentration by Precipitation • Ammonium Sulphate • Organic Solvents • High Molecular Weight Polymers www.entrepreneurindia.co
• Concentration by Ultrafiltration • Concentration by Freeze-Drying • Gel Chromatography • Ion Exchange Chromatography • Ion Exchange Resins • Ion Echange Celluloses • Other Ion Exchange Gels • Affinity Chromatography • Coupling Techniques • Non-Specific Adsorbents • Hydroxyapatite • Celite • Hydrophobic Interaction Chromatography www.entrepreneurindia.co
• High Performance Liquid Chromatography • Electrophoretic Techniques • Electrophoresis. • Isoelectric Focusing • Multimembrane Electrodecantation • Chromatofocusing • Aqueous Two-Phase Separation www.entrepreneurindia.co
2. PRINCIPLES OF INDUSTRIAL ENZYMOLOGY: BASIS OF UTZATION OF SOLUBLE AND IMMOBILIZED • ENZYMES IN INDUSTRIAL PROCESSES • Glossary of Symbols • Assay of Enzyme Activity • Cofactors • The Distinctive Features of Enzymes as Catalysts • Enzyme Catalysis • Enzyme Kinetics • The Effect of pH on Enzyme Activity • The Effect of Temperature on Enzyme Activity • Enzyme Inhibition www.entrepreneurindia.co
• The Various Types of Enzymic Catalyst • A Comparison of Enzymes with Chemical Catalysts • A Comparison of Enzymes with Fermentations • Immobilized Biocatalysts • A Comparison of Immobilized Enzymes and Cells • An Assessment of Immobilization Supports and Methods • Characterisation of Immobilized Biocatalysts • Co-Immobilized Enzymes • Two-Phase Reaction • Industrial Enzyme Kinetic • Effects on Equilibria www.entrepreneurindia.co
• Effectiveness Factors • Steady - State Kinetics • Intrinsic Activity of Enzymes - Modifying Factors • Introduction • Diffusional Limitations on the Activity of Immobilized • Biocatalysts • External Diffusional Limitations • Internal Diffusional Restrictions • Regeneration of Cofactors • Biochemical Reactors • Introduction • The Various Types of Biochemical Reactor • Assessment of the Performance of Biochemical Reactors www.entrepreneurindia.co
• Batch Reactors • Continuous Stirred Tank Reactors • Plug-flow Column Reactors (or Tubular Reactors) • Fluidized Bed Reactors • Electrochemical Reactors • Ultrafiltration Reactors • Enzyme Kinetic in Reactors • Inhibition in Enzyme Reactors • The Effect of Non-Ideal Flow on Biochemical • Reactor Performance • Physical Problems in Biochemical Reactor using • Immobilized Biocatalysts www.entrepreneurindia.co
• Abrasion • Compression • Fouling • Microbial Contamination • The Stability of Immobilized Biocatalysts • Introduction • The Stability of Biochemical Reactors Employing • Immobilized Enzymes or Immobilized Cells • Regeneration of Biocatalyst Activities • Constant Productivity with Biocatalyst Reactors • Scale-Up • Discussion www.entrepreneurindia.co
3. PRINCIPLES OF IMMOBILIZATION OF ENZYMES • Classification of Immobilized Enzymes • Techniques of Enzyme Immobilization • Entrapment • Gel Entrapment • Fibre Entrapment • Micro-encapsulation • Carrier Binding • Physical Adsorption • Ionic Binding Method • Chelation or Metal Binding • Covalent Binding • Crosslinking www.entrepreneurindia.co
• Immobilized Soluble Enzymes • Immobilization without Enzyme Derivitization • Immobilization with Enzyme Derivitization • Miscellaneous Methods • Choice of Immobilization Method • Outline of Properties of Immobilized Enzymes • Stability • Kinetic Properties • Outline of Enzyme Reactors • Batch Reactors • Continuous Reactors • Application and Future Trends www.entrepreneurindia.co
• Analytical Applications • Enzyme Electrodes • Automated Analysis • Therapeutic Applications • Enzyme Replacement • Enzyme Therapy • Industrial Applications • Future Trends www.entrepreneurindia.co
4. ENZYMES IN CLINICAL ANALYSIS – PRINCIPLES • Measurment of Substrate Concentration with Enzymes • Principles of Equilibrium Methods • Principles of Kinetic Methods • Comparison of Equilibrium and Kinetic Methods • Common Indicator Species Used in Routine Clinical Analysis • Nicotinamide Adenine Dinucleotides • Oxygen and Hydrogen Peroxide • Measurement of Enzymes • Principles of Enzyme Assay Using Coupled Enzymes • Immobilized Enzymes for Measuring Substrate www.entrepreneurindia.co
• Concentrations • Immobilized Enzyme Reactor Tubes • Bioanalytical Probes • Dry Reagent Chemistry • Enzyme Immunoassay (ELA) • Preparation of Enzyme Labels • Homogeneous EIA • Heterogeneous EIA • Choice of Enzyme Label • Assay in EIA • Simultaneous Assay of Two Haptens • The Future www.entrepreneurindia.co
5. PRACTICAL ASPECTS OF LARGE-SCALE PROTEIN PURIFICATION • Enzyme Inactivation • Containers and Ancillary Equipment • Glass Vessels • Metal Vessels • Plastic Vessels • Liouid Transer • Couplings • Pumps • Bacterial Disruption www.entrepreneurindia.co
• Resuspension • Liquid Shear • Grinding • Centrifugation • Batch Centrifuges • Continuous Flow Centrifuges • Disc Type Centrifuge • Hollow Bowl Centrifuges • Basket Centrifuges • Tangential Flow Filration • Concentration • Ultrafiltration www.entrepreneurindia.co
• Stirred Cells • Thin Channel Systems • Cartridge Membranes • Hollow Fibres • Dialysis • Chromatography • 1 Columns • Gel Chromatography • Ion Exchange Chromatography • Affinity Chromatography www.entrepreneurindia.co
6. THE APPLICATIONS OF ENZYMES IN INDUSTRY • Glossary of Terms • Production of Enzymes • Use of Enzyme - General Comments • The Characteristics of Industrial Enzymes • Soures of Enzymes • The Isolation, Purification and Formulation of Enzymes • Legislation on the Use of Enzymes • Enzyme Manufacturers • Biochemical Applications • Use of Enzymes in Analysis • General www.entrepreneurindia.co
• In Clinical Assays • Medical Uses of Enzymes • The Use of Enzymes as Catalysts in Organic Chemistry • Introduction • Stereospecificity of Enzymes • Prochiral Stereospecificity • Combinations of Stereospecificity • Multiple-Step Reactions • Synthesis of Radioactive Compounds • Restrication Endomucleases • Biochemical Processing • Applications of Enzymes in the Food Industry • Polysaccharide Processing www.entrepreneurindia.co
• Bacterial -amylase • Amyglucosidase (EC 3.2.1.3) • Maltose Syrups • Glucose Isomerase (EC 53.1.5) • Inversion of Sucrose • Sugar Refining • Raffinase • a-amylase • Dextranase (EC3.2.1.11) • Debranching Enzymes • Cyclodextrin Glucosyltransferase and Other Amylases • Cellulase (EC3.2.1.4) • Ethanol Fermentation • Brewing www.entrepreneurindia.co
• Baking • The Dairy Industry • The Dairy Industry • Lactose Hydrolysis • Cheese Manufacture • Coagulation • Flavour Development • Other Applications • Organic Acids • Amino Acids • Introduction • Enzymic Resolution • Enzymic Production of Amino Acids • Antioxident www.entrepreneurindia.co
• Introduction • Glucose Oxidase (EC 1.1.3.4) • Protein Processing • Introduction • The Plastein Reaction • Aspartame • Others • Flavouring Agents • Fruit Processing • Use of Enzymes in the Extraction of Natural Products • Detoxifying Enzymes • Enzyme-Based Detergents • Use of Enzymes as Cleansing Agents • The Leather Industry www.entrepreneurindia.co
• Textiles • Paper Manufacture • Antibiotics • Penicillin Acylase, (EC 3.5.1.11) • Cephalosporins • Miscellaneous uses of Biocatalysts • Conclusion • Note in Proff • Acrylamide • Propylene Oxide • Vinyl Chloride • Biosensors • Amino Acids www.entrepreneurindia.co
7. DATA ON TECHNIQUES OF ENZYME IMMOBILIZATION AND BIOAFFINITY PROCEDURES • Entrapment • Gel Entrapment • Fibre Entrapment • Microencapsulation • Phase Separation Method • Interfacial Polymerization Method • Liquid Surfactant Membrane Method • Liquid Drying Method • Chelation or Metal Binding • Covalent Binding • Diazotization www.entrepreneurindia.co
• Amide Bond Formation • Acid Anhydride Derivatives • Acylazide Derivatives • Cyclic Imidocarbonate Derivatives • Isocyanatc and Isothiocyanate Derivatives • Acyl Chloride Derivatives • Cyclic Carbonate Derivatives • Condensing Reagents • Alkylation and Arylation • Schiff's Base Formation • Ugi Reaction • Amidination Reactions • Thiol-Disulphide Interchange www.entrepreneurindia.co
• Mercury-Enzyme Interactions • -Irradiation Induced Coupling • Matrices for Carrier Binding • Inorganic Supports • Controlled Pore Supports • Other Porous Supports • Non-porous supports • Coupling Reactions for Inorganic Supports • Organic Supports • Polysaccharides • Proteins • Carbon • Polystyrenes • Polyacrylates • Maleic Anhydride Based Copolymers www.entrepreneurindia.co
• Polypeptides • Vinyl and Allyl Polymers • Polyamides • Crosslinking • Immobilized Cells • Entrapment • Physical Adsorption • Chelation or Metal Binding • Covalent Binding • Crosslinking • Other Immobilized Biologically Active Moleccules • Immunoadsorbents • Affinity Chromatography Media www.entrepreneurindia.co
• Immobilized Lactins • Immobilized Amino Ecids and Peptides • Immobilized Carbohydrates • Immobilized Nucleosides, Nucleotides and Nucleic Acids • Immobilized Antibiotics 8. ENZYMES IN CLINICAL ANALYSES – DATA • Substrates Measured Enzymically in Clinical Laboratories • Enzymes Measured by Coupled Enzyme System in • Clinical Laboratories • Immobilized Enzymes for Measuring Substrates • Enzymes Used in Enzyme Immunoassay (EIA) www.entrepreneurindia.co
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