enzyme immobilization

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NATIONAL COLLEGE OF PHARMACYDEPT. OF PHARMACOGNOSYSHIMOGA -577201 An evaluation Seminar on Subject : Medicinal Plant Biotechnology Topic: Enzyme Immobilization: Techniques and Application Presented By: Neelanjan Chatterjee (I M.Pharm)Under the guidence of:Dr. G. Narayana Murthy Department of Pharmacognosy NCP, Shimoga.2010

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Enzymes: Enzymes are defined as soluble, colloidal, organic catalysts which are produced by living cells but are capable of acting independently of the cells. In 1873 Berzelius recognized ferments catalyses chemical reactions Later it was coined Enzymes (in side the yeast) 1897 Edword Buchner extracted enzymes from yeast that catalyzes sugars into alcohol

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Immobilized Enzyme Systems: Enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities and which can be used repeatedly and continuously Retrospection: In 1916 Nelson and Griffin discovered that Invertase enzyme showes same activity when absorbed on a solid media ( charcoal, aluminium hydroxide) at the bottom of the reaction vessel Oreste .J. Lantero invented a method for the immobilization of an enzyme producing microorganism by introducing an aqueous solution containing bacterium cells into polyethyleneimine and adding gluteraldehyde and chitosan(US Patent-4,760,024, 1988)

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Tosa et al invented a method of immobilization of enzymes in microbial cells using k-carrageenan as matrix. Gelatin in k-Carrageenan is a new polymer for the immobilization Pietro crimonesi has invented a method of enzyme immobilization by adding a suspension of polysaccharide in aqueous media containing vinyl monomer and an enzyme, then adding ferric salt as catalyst and irradiating the mixture with UV light that lead to polymerization to form a copolymer of polysaccharide having enzymatic activity(USPatent-4,338,401. 1982) Ephraim et al has reported a new carrier Eupergit© for the immobilization of enzymes of industrial potential. It is a carrier consisting nanopore beads of immobilized enzymes

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Types of enzyme immobilization: 1. On surface immobilization: Covalent coupling with polymers Functional group containing polymers Gluteraldehyde containing protein coupling Polysaccharide as solid support Complexation and chelation 2.With support immobilization Inclusion in gels Inclusion in fibers Inclusion in microcapsules

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Covalent coupling: The protein functional groups can be used for the covalent coupling NH2- lysine COOH- α and β Aspertic acid, Glutamic acid OH- Phenol ring on tyrosine SH- Cysteines Polymeric support s which are widely used: Hydroxyl groups of polysaccharide, PVA, Poly methyl acetate Amino ethyl coated polysaccharides, silica gels Aldehyde and acetal groups of polymers Amide groups of polypeptides

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Hydroxyl group containing polymers:

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Gluteraldehyde based protein coupling : Bi functionality of gluteraldehyde can be used for the formation of covalent bond

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Polysaccharides as solid support: Naturally occurring polysaccharides like cellulose containing 1,4 linked β-D glucose, agarose containing 1,3 linked β-D glucose are derivatized and used as solid support for enzyme immobilization.

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Adsorption method: Various sold supports like aluminium, amberlite, bentonite, CMC, silica gels are available to adsorb the enzyme molecule and immobilize depending on various forces like vanDer Walls force and hydrogen boding

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Complexation and chelation methods: In this process chelation and complexation of transition metals are used to immobilize enzymes on solid support. Titanium or zirconium is widely preferred for their non toxic nature.

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With support immobilization: Here enzymes are physically entrapped in a polymer matrix, gels or capsules (micro encapsulation) The support should have very small size pores which facilitates the movement of substrate inside the compartment Inclusion in gels: Poly acrylamide gels Poly vinyl alcohol gels Inclusion in fibers: Cellulose and Poly acrylamide gels Inclusion in micro capsules: polyamine, Polybasic acid chloride monomers

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Inclusion in gels and fibers Inclusion in Microencapsulation

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Examples of Immobilized enzymes by entrapment:

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Applications of immobilized enzymes: Production of antibiotics- immobilized penicillin amidase used for production of Penicillin G, Amoxicillin and ampicillin Production of steroids- immobilized cells of Cornybacterium simplex is used to convert hydrocortisone and prednisolone from cortesolone Production of amino acids- β tyrosinase used for the production of l-dopa Production of organic compounds- Propiniobacterium produce vit B 12 Catharanthus roseus produce ajmalcine Digitalis lanata produce digitoxin

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Clinical applications- Glucokinase as glucose sensor Cholesterol oxidase as lipid sensor Industrial applications-

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References: Pharmaceutical Biotechnology: Vyaas SP and Dixit VK (page-13-159) A Textbook of Biotechnology: Dubey RC (page 238-240) Molecular Biology and Biotechnology: Kumar HD(page-394-404) Elements of Biotechnology: Gupta PK (Page-417-433) Concepts of Biotechnology: Balasubramaniyan D et al (page-114-134) http//:www.cheric.org/ippage/ipdata/2004/05/file/e200405-1101.pdf http//:www.scribd.com/doc/14657530/applications-0f-enzyme-immobilization.pdf http://www.youtube.com/watch?v=XYmioTsy4Cc http://www.youtube.com/watch?v=gV_oIuQEmGE

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Thank you…

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