Extraction, Purification and Production of Enzymes ...... This will depend on the development of enzymes able to degrade cellulose in plant biomass and designing methods to recycle

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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)

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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.



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.



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.



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.



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.



Enzymes Market



Global Industrial Enzymes Market (US$ Billion)



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



• 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



• 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



• High Performance Liquid Chromatography• Electrophoretic Techniques• Electrophoresis. • Isoelectric Focusing• Multimembrane Electrodecantation• Chromatofocusing• Aqueous Two-Phase Separation



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



• 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



• 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



• 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



• 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



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



• 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



• Analytical Applications• Enzyme Electrodes• Automated Analysis• Therapeutic Applications• Enzyme Replacement• Enzyme Therapy• Industrial Applications• Future Trends



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



• 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



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



• Resuspension• Liquid Shear• Grinding• Centrifugation• Batch Centrifuges• Continuous Flow Centrifuges• Disc Type Centrifuge• Hollow Bowl Centrifuges• Basket Centrifuges• Tangential Flow Filration• Concentration• Ultrafiltration



• Stirred Cells• Thin Channel Systems• Cartridge Membranes• Hollow Fibres• Dialysis• Chromatography• 1 Columns• Gel Chromatography• Ion Exchange Chromatography• Affinity Chromatography



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



• 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



• 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



• 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



• 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



• 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



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



• 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



• 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



• 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



• 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)


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Niir Project Consultancy Services (NPCS) can provide Process Technology Book on

Extraction, Purification and Production of Enzymes (Biotechnology)

(Polystyrenes, Polypeptides, Polysaccharides, Proteins, Carbon, PropyleneOxide, Vinyl Chloride, Biosensors, Amino Acids, Antibiotics, Acrylamide,Organic Acids, Maltose Syrups, Hollow Fibres, Hollow Fibres, EnzymeImmunoassay (ELA), Enzyme Electrodes, Biocatalysts)

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Extraction, Purification and Production of Enzymes ...... This will depend on the development of enzymes able to degrade cellulose in plant biomass and designing methods to recycle