Applications of Enzyme Immobilization

Prepared by
1.Amrita Mishra Lecturer[Pharmacognosy] Pharmacy College, Azamgarh UP iNDIA 2.Arun kumar Mishra Lecturer[Pharm. Chemistry] Pharmacy College, Azamgarh UP India

ENZYME IMMOBILISATION Immobilization of enzyme is a process where
an enzyme makes use of carrier phase for safe homing. The use of enzyme in industrial application is limited because enzyme are relatively unstable, of high cost isolation ,purification and recovery of active enzyme from reaction mixture after the completion of catalytic process. For soluble enzyme in batch operation is uneconomical as active enzyme is lost after each reaction. Some of enzyme are rapidly inactivated by heat and become heat stable by attachment to inert polymeric supports.

lized enzyme can be reused. process can be repeated continuously n be radically controlled.

s can be easily separated and enzyme properties can be preserved.

D OF IMMOBILISATION:

ding upon physical relationship of catalyst to matrix. chosen must enhance the operational stability of immobilized enzyme ation.

may be porous or nonporous, matrices with organic natural or synthetic) or ic nature. alyst may be covalently bonded to polymer, physically adsorbed onto the rcross linked with itself and possibly another inert protein, entrapped inside mer matrix or encapsulated in polymer bag.

lent bonding bonbing olymerization mer entrapment psulation ome entrapments

APPLICATION OF ENZYME IMMOBILISATION
Immobilization is beneficial for economic purpose and reduces the overall cost. • PHARMACEUTICAL APPLICATION: Immobilized biocatalyst are useful for pilot plant and fpr laboratory set up 1.Production of antibiotics a) PENICILLINES: i. 6 Aminopenicillanic acid 6-APA can be industrially produced on large scale from penicillin g or penicillin v by deacylation with penicillin amidase these antibiotics are produced by fermentation. a).Partially purified amidase from E.coli trapped into cellulose triacetate fibers can be used for production of 6APAfrom penicillin G,in a column as a part of recirculation batch reactor .5%substrate solution hydrolyses to 99%.retention time required is1.5 hrs.

Penicillin amidase immobilised by covalent binding to amberlite XDA-7 with glutaraldehyde,by physical absorption to bentonite,or by ionic binding to DEAE-sephadex and by covalent binding to a copolymer of acrylamide and maleic anhydride can be used for production of 6 APA from penicillin v.

ii) Penicillin g Laboratory production from glucose involve the use of conidia, mycelium and protoplast of penicillium chrysogenum Immoblised with k -caragennen ,and calcium alginate. A continuous flow bioreactor can be used. Conidia & P..chrysogenum immobilized ,with Kcarrageen operates for up to 16days.1.2mg\g cells\hr yields can be obtained from glucose.(7.0mg\g glucose ). The half life of production is greater than 15 days with a media contain 10.0g of glucose\l. iii) Ampicilline and amoxicillin Penicillin amidase from E.coli entrapped in cellulose triacetate fibre can

ted penicilline amidase adsorbed on a DEAEsephadexcan be used for the n of ampicilline with an yield of 67%in stirred batch reactor.

osporins porin can be obtained by fermentation with cephalosporium acremonium porin amidase from various microorganism can be immobilised by differe

c deacylation of cephalosporin can be carried out with compounds conta e substrate for reaction are usually 7phenylacetamidodesacetoxy cepha 7phenoxyacetamidodesacetoxy cephalosporin acid obtained by ring exp rom penicillin g and penicillin v.

XIN

me from.E. Coli entrapped in cellulose triacetate fibers can be production of cephalexin and Dphenylglycine methyl ester.

Immobilised acetone dried whole cells of Achromobacter spp adsorbed on a DEAE Cellulose or hydroxylapatite can also be used. c)BACITRACIN Cells of bacillus spp ,producing bacitarcin immobilised in polyacrylamide gel lattice can Be employed in batch and continous culture system. D)TYLOSIN AND NIKKOMYCIN Macrolide antibiotics tylosin and nikkomycin can be produced by living cells of streptomycin spp Immobilised with calcium alginate .

DUCTION OF STEROIDS transformation can be done by biocatalytic conversion cofactoers o needed

D1 sis of hydrocartisone and prednisolone by immoblisation with ylamide entrapement. OH
H2C CO OH

11beta hydroxylation curvularia lunata
HO

OH H2C CO OH

O H2C

dehydrogenation corynebacterium simplex

H2C

OH CO OH

O

METHOD 2. Whole cells of C.simplex entraped in collegen membrane can also be used for production of prednisolone from hydrocortisone. METHOD 3. The two step tranformation (cortexolone) to its dehydro 11-hydroxy derivative) Can also be performed by combined used of C.lunata mycelia and immobilized arthobector simplex. 3.PRODUCTION OF AMINO ACID:Many large scale process using immobilized enzyme are in operation. Amino acid production by enzymatic resolution using amino acids acylase and fructose syrups form glucose isomerase are few among them. A.. optical resolution of DL amino acid Synthesised acyleDL amino acids are assymetrycally hydrolysed by aminoacylase to give L-aminoacid and unhydrolysed acyle D aminoacid.

ALLY ACTIVE AMINOACID. cid oduced by fermetation / enzymatic bathe proceses from id and ammonia using aspartase. E.coli immobilised with nnan tearted with glutraldehyde and hexameyhylenediamine . acked with immobilsed cells of E.coli with higest prodictivity has half of 680 days at 37. degree.

CTION OF ACIDS. vement of productivty of organic acids immobilised enzyme are used. developed by Acetobecter aceti immbiliesd porous ceramics, by A.niger with calciun alginate,lactic acid by L.casei with mide, Lmalic acid by B.flavum with carageenan,12 ketocheno ic acid B.fuscum with carageenan,2keto gluconic acid by mens with colagen.

5.OTHER ORGANIC COMPOUNDS. • Coenzyme A…. It is produced from pentothenic acid, l cystein , and ATP can be carried out with Bravibacterium ammoniagenes cells immobilised on Polyacryleamind gel. • Athraquinones Denovo synthsis of anthraquninoes can be performed by Immobilised plants cells of Morindacitrifolia with calcium alginate from Secologanin. iii. Azmalicine isomers. Plant cells catharnthus roesus immobilised with Calcium alginate were used for synthsis of Azmalicine isomers from tryptamine iv. Prostglandine. Using arachidonic acid and ram seminal microsomes Immobilisd on photocrose linkable resin. v. Proinsuline B.subtiles cells carrying plasmide encoding for rat proinsuline Were immbilised in agrose beeds. vi. FAD(Flavine adenine dinucletide). With FMD and ATP by whole cells of orthobecteroxydans. vii. Pyridoxile 5phaphate it is porduced with highenzyme activity formpyridoxine 5-phaphate by whole cells P.fluorescnes immblised on film of polyvinyl alcohal Crosed linked with tetraethyl silicates.

ENT APPLICATION IN CLINICAL ANALYSIS…….

gnostic and industrial monitoring programs immobilization is yed . cal analysis enzymes are used as reagents to measure specific olites and are measured as such in body fluids and tissues as an tor of pathological condition or metabolic disturbances

SENSORS enzyme electrodes mercial devices has been developed easure sugar amino acid urea, cholesterol penicillin. In this nylon coil ch hexokinase G6PD are immobilized. Glucose is estimated by rate of tion of NADH. For this poly aryl amide gel and oxygen sensor are used.

D SENSOR.. sterol conc. In serum indicates abnormality of lipid metabolism, tension for this an immobilized enzyme reactor containing cholesterol ses is coupled with ampearometric detector system based on platinum ode.

3. L LACTATE DEHYRO GENASE… The amperometric determination of pyruvate can also be carried with pyruvate Sensor for analyzing LDH level. In consisted of pyruvate oxydase and an Oxygen electrode. 4. ELISA.. •It rapidly replaces RIA. For hormones, by coupiling their catalytic ability specific immunoglobulin. In ELISA enzyme activity lable tagged on to antigen antibody molecules. In this plastic tube coated known quantity of antigen to Which is added the sample antigen and a known quantity of enzyme labeled Antibody. •The enzyme can be linked to proteins that bind spcifically to various subclass Of immunoglobulin G. this enzyme protein reagent can employed to bind to Any bound immunoglobulin instead of enzyme labeled immunoglobins. Enzymes used in such a way includes peroxidase,alkaline phosphatase, and beta Galactosidases. 5. ENZYME THERMISORTS;

REFERENCES
1.DIXIT V K,VYAS S P.PHARMACEUTICAL

BIOTECHNOLOGY, CBS PUBLISHER AND DISTRIBUTERS,PP.13-119,2002. 2.KORI S.S.,HALKANI M.A.,PHARMACEUTICAL BIOTECHNOLOGY,VALLAB PUBLICATION,PP 260278,1999.

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