Enzyme Immobilisation Arun Kumar Mishra Iftm Moradabad

Applications of Enzyme Immobilization

Submitted to Dr. S.R. Hashim Professor College of pharmacy IFTM,Moradabad

Submitted by Arun kumar Mishra M.Pharm 2nd sem College of pharmacy IFTM,Moradabad

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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.

Immobilized enzyme can be reused. process can be repeated continuously and can be radically controlled. Products can be easily separated and enzyme properties can be preserved. METHOD OF IMMOBILISATION: Depending upon physical relationship of catalyst to matrix. Matrix chosen must enhance the operational stability of immobilized enzyme purification. Carrier may be porous or nonporous, matrices with organic natural or synthetic) or Inorganic nature. The catalyst may be covalently bonded to polymer, physically adsorbed onto the Polymercross linked with itself and possibly another inert protein, entrapped inside a polymer matrix or encapsulated in polymer bag. 1 cov alent bo nding 2 ionic b onbing 3 co pol ymeri za tion 4 poly mer e ntr apme nt 5 En caps ula tion 6 lipos ome entr apments

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 K- carrageen 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 be employed in continuous flow reaction to produce ampicilline or amoxycilline from 6APA and d -phenylglycine -methyl ester respectively.

Succinylated penicilline amidase adsorbed on a DEAEsephadexcan be used for the Production of ampicilline with an yield of 67%in stirred batch reactor. b)Cephalosporins Cephalosporin can be obtained by fermentation with cephalosporium acremonium. Cephalosporin amidase from various microorganism can be immobilised by different Methods. Enzymatic deacylation of cephalosporin can be carried out with compounds containing 7ADCA nuclus.The substrate for reaction are usually 7phenylacetamidodesacetoxy cephalosporin Acid and 7phenoxyacetamidodesacetoxy cephalosporin acid obtained by ring expansion reaction from penicillin g and penicillin v. CEPHALEXIN The enzyme from.E. Coli entrapped in cellulose triacetate fibers can be Used for 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 .

2.PRODUCTION OF STEROIDS Steroid transformation can be done by biocatalytic conversion cofactoers are also needed METHOD 1 Synthesis of hydrocartisone and prednisolone by immoblisation with Polyacrylamide entrapement. OH H2C CO OH

11beta hydroxylation curvularia lunata

OH H2C HO

CO OH

O H2C

dehydrogenation corynebacterium simplex

H2C

O

OH CO OH

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.. opti cal res olut ion of D L ami no acid Synthesised acyleDL amino acids are assymetrycally hydrolysed by aminoacylase to give L-aminoacid and unhydrolysed acyle D aminoacid. Immbolized aminoacylase of Aspergillus oryzae is used for continous . optical resolution of DL- amino acid. L-aminoacid crystlises from concentrated effulent. Acyle Daminoacid contained in Mother liquor is recimized by heating and can be used for optical resolution

B…. OPTICALLY ACTIVE AMINOACID. L-aspartic acid Can be produced by fermetation / enzymatic bathe proceses from fumaric acid and ammonia using aspartase. E.coli immobilised with K.Carragennan tearted with glutraldehyde and hexameyhylenediamine . Column packed with immobilsed cells of E.coli with higest prodictivity has half Life period of 680 days at 37. degree. 4. PR OD UC TION OF ACIDS . For improvement of productivty of organic acids immobilised enzyme are used. Process is developed by Acetobecter aceti immbiliesd porous ceramics, Citric acid by A.niger with calciun alginate,lactic acid by L.casei with Polyacrylamide, Lmalic acid by B.flavum with carageenan,12 ketocheno Deoxycholic acid B.fuscum with carageenan,2keto gluconic acid by S.Marcesmens 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.

6.RECENT APPLICATION IN CLINICAL ANALYSIS……. For diagnostic and industrial monitoring programs immobilization is employed . In clinical analysis enzymes are used as reagents to measure specific Metabolites and are measured as such in body fluids and tissues as an Indicator of pathological condition or metabolic disturbances 9. BIOSENSORS enzyme electrodes commercial devices has been developed To measure sugar amino acid urea, cholesterol penicillin. In this nylon coil to which hexokinase G6PD are immobilized. Glucose is estimated by rate of formation of NADH. For this poly aryl amide gel and oxygen sensor are used. 2.LIPID SENSOR.. Cholesterol conc. In serum indicates abnormality of lipid metabolism, Hypertension for this an immobilized enzyme reactor containing cholesterol Oxidases is coupled with ampearometric detector system based on platinum electrode.

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; Enzyme thermistors have been developed in which a substrate is feed to an enzyme ,a product is formed with liberation of heat..A thermistors is used to monitor this Heat of reaction whIch can be co related with concentration of substrate.

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 260-278,1999.