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Vinod kumar 636109881033 3/8/2013 2ENZYMES: ENZYMES Complex organic catalysts that is produced by all the living cells but capable of acting independently of the cells that produce them In the organism they regulate the metabolic process Properties: Enzymes are soluble Colloidal organic catalysts formed by living cells Specific in action Protein in nature Thermolabile in nature 3/8/2013 3Enzyme activity: : Enzyme activity: These are characterized by the speed of the reaction they catalysed and their sensitivity to environmental conditions. They enhance the rate of chemical reaction by lowering the activation energy. 3/8/2013 4Eg: : Eg : 3/8/2013 5PRODUCTION OF ENZYMES: PRODUCTION OF ENZYMES 3/8/2013 6Advantages: Advantages Growth of microbes is very fast & they can be grown on medium containing cheap raw material The genetic engineering & manipulations of microbial cells are possible in laboratory conditions Large amounts of enzymes can be produced from microbes In animals/plants mitochondria’s contain a large no. of enzymes Eg : glutamate dehydrogenase 3/8/2013 7PowerPoint Presentation: The granular microsomes , lisosome’s and ribosome’s also contain imp. Enzymes Ribosome’s are the sites of protein bio- synthesis Lysosome’s contain many hydrolases Soluble portion of the cytoplasm contains enzymes responsible for glycolysis In bacterial cell some structures of plant and animal cells are not present Out side the nucleus, the cell is filled with a granular cytoplasm Enzymes are present in granular cytoplasm (or) adsorbed on the membrane 3/8/2013 8Selection of micro – organisms: Selection of micro – organisms The micro- organisms should produce extra cellular enzymes, because their isolation & separation is simple & less expensive The organism must give high yields of enzymes Fermentation time should be less Strain must not produce by products which all inhibit the growth of microbes 3/8/2013 9PowerPoint Presentation: Organism should be non- pathogenic Organism must grow on cheap raw material It must adjust themselves to the physical and chemical properties of the culture medium such as temp., p H and the availability of substrate 3/8/2013 10Fermentation media: Fermentation media The production media should contain sources for carbon, nitrogen, energy, minerals, macro nutrients, micro nutrients& growth factors in the case of auxotropic micro- organisms. CONSTITUENTS VARIOUS SOURCES Carbon and Energy Cereal meal, Soyabean meal, Potato starch, Wheat Nitrogen Fish meal, Gelatin, Casein Substances and trace elements Yeast extract, Corn steep liquor, Plant oil 3/8/2013 11Productivity process: Productivity process Industrial process is carried out by following processes Submerged culture Surface culture (or) solid substrate culture Deep bed cultures Extraction of enzymes 3/8/2013 12SUBMERGED CULTURE: SUBMERGED CULTURE In this the production takes place in mechanically stirred bio reactors of capacities 20,000 to 1,00,000 like batch fermentation. Fermentation lasts for 50 to 200 hours depending upon the enzyme & micro organism used . This operation is carried out in 2 stages: In which high conc. Of cell mass is first produced Then the production of the enzymes can be carried out in a second reactor under different conditions . 3/8/2013 13For optimizing the production: For optimizing the production Effect of nutrient media P H , temp. Partial pressure of O 2 Aeration must be taken into account 3/8/2013 14Surface culture (or) solid substrate culture: Surface culture (or) solid substrate culture Micro organisms are cultivated on solid substrate enriched with a high conc. Of nutrients & having a large surface area This method is adopted for the extraction of enzymes from fungi, such as penicillium , aspergillus This cultivation is done by using 2 process: Drum process Horizontally rotating drums are used Tray process the fungi are cultivated in trays of size 2*40 cm.the micro organisms are inoculated in the form of spores and then the trays are incubated in A/C rooms. 3/8/2013 15Deep bed cultures: Deep bed cultures Microbes arte cultivated in rectangular vessels of dimensions 18*200 inch The nutrient medium is added into vessels into a height of 2 feet The medium is sterilized & inoculated with organism Temperature of the culture vessel is maintained to enhance the growth of organism 3/8/2013 16Extraction of enzymes: Extraction of enzymes Extraction refers to the liberation of enzymes from cells or cell constituents 3/8/2013 17Disruption by microbial cells: Disruption by microbial cells 3/8/2013 18Disruption by chemical methods: Disruption by chemical methods Surfactants Enzymatic lysis Cold and osmotic shock Alkali treatment 3/8/2013 19Surfactants: Surfactants Used to lyse microbial cells Have the capability to solubilize the microbial cell wall Eg : sodium dodecyl sulphate Triton X- 100 3/8/2013 20Enzymatic lysis: Enzymatic lysis Auto lysis is most commonly used for extraction of a intracellular enzymes A suspension of cell is maintained at elevated temp. (23 to 37 0 c) for several hours After cooling, this cell extract is collected by centrifugation This method is used in the extraction of trans aldolase from frozen candida utiles invertase from baker’s yeast 3/8/2013 21PowerPoint Presentation: Drawback: - Risk of thermal denaturation of enzymes 3/8/2013 22Cold and osmotic shock: Cold and osmotic shock Rapid reduction temperature from normal growth temp. to 0 o c results in loss of viability in microbes 3/8/2013 23Steps involved in Osmotic shock: Steps involved in Osmotic shock Washing of bacteria to remove the growth medium by a buffer solution Resuspending the washed cells in a 20% buffered sucrose solution Removal of cells by centrifugation to form paste Paste is then suspended in water and a sudden increase in osmotic pressure inside the cells cause the release of cell constituents 3/8/2013 24Alkali treatment: Alkali treatment A sudden increase in P H will cause the lysis of bacterial cell wall This procedure is used in the large scale extraction of L- Asparginase from Erwinica Chrysantheme 3/8/2013 25Disruption by physical methods: Disruption by physical methods Sonication Freezing and thawing Solid & liquid shear Concussion 3/8/2013 26Sonication & Freezing and thawing: Sonication & Freezing and thawing Ultra sonic waves are used frequently for cell lysis Similar to cold and osmotic shock 3/8/2013 27Solid & liquid shear: Solid & liquid shear Frozen (-20 0 c) microbial paste is placed in a cylindrical hole (0.1 to 1mm in diameter) in a metal block & a tight plunger is then inserted 1000 & 4000 ATP is applied at the top of this plunger 3/8/2013 28PowerPoint Presentation: In liquid shear Microbial suspension is passed through a narrow orifice at pressure upto 40,000 psi Cells undergo disruption due to extreme shear during parsase through the orifice 3/8/2013 29Concussion: Concussion Microbial cells can be disrupted by bombardment with hard particles such as glass beads. 3/8/2013 30ALPHA AMYLASE (DIASTASE): ALPHA AMYLASE (DIASTASE) SOURCE: Aspergillus spp. A.Oryzae (solid state fermentation) A.Niger (submerged fermentation) Medium: A carefull balance of carbohydrate and nitrogen ingredients of the medium is vital. Nitrogen concentration should be higher for growth of the organism It should contain some proteinaceous compounds also 3/8/2013 31PowerPoint Presentation: P H : Neutral P H should be maintained Temperature: - The incubation should be carried @ 25 – 35 0 c for 3-4 days 3/8/2013 32Conti…: Conti… Procedure: Wheat bran is the basic component which is spread relatively thin in trays Moistened with water or dil.acid Sterilized, inoculated with spores and allowed to grow After growth the harvest is dried @ 50 0 c or less and then ground to liberate amylase 3/8/2013 33PowerPoint Presentation: Use: Manufacture of maltose rich syrups Foodstuff’s industry Digestive enzymes 3/8/2013 34FUNGAL AMYLASE: FUNGAL AMYLASE SOURCE: A.Oryzae is universally used 3/8/2013 35IMMOBILIZATION OF ENZYME: IMMOBILIZATION OF ENZYME Defined as confining the enzyme molecules to a distinct phase from the one in which the substrate & the products are present. The material used for immobilization of enzymes, called as “Carrier Matrices ” 3/8/2013 36Methods: Methods adsorption covalent bonding entrapment membrane confinement 3/8/2013 37Adsorption: A dsorption Enzyme molecule adhere to the surface of the carrier matrix due to the combination of hydrophobic effects Ion exchange matrices Porous carbon Clays Glasses Produces high loading 3/8/2013 38Covalent bonding: Covalent bonding Enzyme molecules are attached to the carrier matrix by formation of covalent bonds As a result, the strength of binding is very strong, there is no enzyme loss during process Covalent bond formation occurs with the side chains of amino acids of the enzyme Commonly used matrices : Agaross Cellulose 3/8/2013 39Entrapment : Entrapment Enzyme molecules are entraped within suitable gels or fibres There may/ may not be covalent bond formation b/w E AND M 3/8/2013 40MEMBRANE CONFINEMENT: MEMBRANE CONFINEMENT Enzyme molecules, usually in aqueous solution may be confined within a semi – permeable membrane which allows a free movement in either direction to the substrates & products But doesn’t permit the enzyme molecules to escape 3/8/2013 41Advantages : Advantages As Enzymes are costly items, immobilization permits their repeated use Product is readily freed from the enzyme Immobilized enzymes can be used in non-aqueous system Continuous production systems can be used, which is not possible with free enzymes 3/8/2013 42Disadvantages: Disadvantages Immobilization means additional cost Immobilization often adversely effects the stability & activity of enzymes This approach can not be used when one of the substrate is insoluble 3/8/2013 43 QUESTIONS: QUESTIONS ??? 3/8/2013 44 THANK YOU: THANK YOU 3/8/2013 45 You do not have the permission to view this presentation. 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