ENZYME IMMOBILISATION arun kumar mishra iftm moradabad

8/14/2019 ENZYME IMMOBILISATION arun kumar mishra iftm moradabad

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Applications of EnzymeApplications of Enzyme

ImmobilizationImmobilization

Submitted toSubmitted to

Dr. S.R. HashimDr. S.R. HashimProfessorProfessor

College of pharmacyCollege of pharmacy

IFTM,MoradabadIFTM,Moradabad

Submitted bySubmitted by

Arun kumar MishraArun kumar Mishra

M.Pharm 2M.Pharm 2ndnd semsem

College of pharmacyCollege of pharmacy

IFTM,MoradabadIFTM,Moradabad


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ENZYMEENZYME

IMMOBILISATIONIMMOBILISATION Immobilization of enzyme is a process where an enzymeImmobilization of enzyme is a process where an enzyme

makes use of carrier phase for safe homing.makes use of carrier phase for safe homing.

The use of enzyme in industrial application is limited becauseThe use of enzyme in industrial application is limited because

enzyme are relatively unstable, of high cost isolationenzyme are relatively unstable, of high cost isolation

,purification and recovery of active enzyme from reaction,purification and recovery of active enzyme from reaction

mixture after the completion of catalytic process.mixture after the completion of catalytic process. For soluble enzyme in batch operation is uneconomical asFor soluble enzyme in batch operation is uneconomical as

active enzyme is lost after each reaction.active enzyme is lost after each reaction.

Some of enzyme are rapidly inactivated by heat and becomeSome of enzyme are rapidly inactivated by heat and become

heat stable by attachment to inert polymeric supports.heat stable by attachment to inert polymeric supports.


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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 nding2 ionic b onbing3 co pol ymeri za tion4 poly mer e ntr apme nt5 En caps ula tion6 l i pos ome entr apments


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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 acid6-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 ofrecirculation batch reactor .5%substrate solution hydrolyses to 99%.retention time

required is1.5 hrs.


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ii) Penicillin gii) Penicillin g

Laboratory production from glucose involve the use of conidia,Laboratory production from glucose involve the use of conidia,mycelium and protoplast of penicillium chrysogenum Immoblised withmycelium and protoplast of penicillium chrysogenum Immoblised with

k -caragennen ,and calcium alginate.k -caragennen ,and calcium alginate.A continuous flow bioreactor can be used. Conidia &A continuous flow bioreactor can be used. Conidia & P..chrysogenumP..chrysogenumimmobilized ,with K- carrageen operates for up to 16days.1.2mg\gimmobilized ,with K- carrageen operates for up to 16days.1.2mg\gcells\hr yields can be obtained from glucose.(7.0mg\g glucose ). Thecells\hr yields can be obtained from glucose.(7.0mg\g glucose ). Thehalf life of production is greater than 15 days with a media containhalf life of production is greater than 15 days with a media contain

10.0g of glucose\l.10.0g of glucose\l.

iii) Ampicilline and amoxicilliniii) Ampicilline and amoxicillin

Penicillin amidase fromPenicillin amidase from E.coliE.colientrapped in cellulose triacetate fibreentrapped in cellulose triacetate fibrecancan

be employed in continuous flow reaction to produce ampicilline orbe employed in continuous flow reaction to produce ampicilline or

amoxycilline from 6APA and d -phenylglycine -methyl esteramoxycilline from 6APA and d -phenylglycine -methyl esterrespectively.respectively.

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.


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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 expansionreaction from penicillin g and penicillin v.

CEPHALEXIN

The enzyme from.E. Coli entrapped in cellulose triacetate fibers can beUsed for production of cephalexin and Dphenylglycine methyl ester.


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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 .


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2.PRODUCTION OF STEROIDS

Steroid transformation can be done by biocatalytic conversion cofactoers

are also needed

METHOD 1Synthesis of hydrocartisone and prednisolone by immoblisation with

Polyacrylamide entrapement.

C O

H2C

OH

OH

O

C O

H2C

OH

OH

H2C

C O

H2C

OH

OH

O

HO

11beta hydroxylationcurvularia lunata

dehydrogenation

corynebacterium simplex


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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 acidSynthesised acyleDL amino acids are assymetrycally hydrolysed by aminoacylase

to give L-aminoacid and unhydrolysed acyle D aminoacid.

Immbolized aminoacylase ofAspergillus oryzaeis 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


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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.


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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 fromSecologanin.

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 proinsulineWere 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.


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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 specificMetabolites 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 developedTo 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.


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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.


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REFERENCESREFERENCES

1.1.DIXIT V K,VYAS S P.PHARMACEUTICAL BIOTECHNOLOGY, CBSDIXIT V K,VYAS S P.PHARMACEUTICAL BIOTECHNOLOGY, CBSPUBLISHER AND DISTRIBUTERS,PP.13-119,2002.PUBLISHER AND DISTRIBUTERS,PP.13-119,2002.

2.KORI S.S.,HALKANI M.A.,PHARMACEUTICAL BIOTECHNOLOGY,VALLAB2.KORI S.S.,HALKANI M.A.,PHARMACEUTICAL BIOTECHNOLOGY,VALLAB

PUBLICATION,PP 260-278,1999.PUBLICATION,PP 260-278,1999.


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ENZYME IMMOBILISATION arun kumar mishra iftm moradabad

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