Applications of Enzyme

Immobilization

Prepared by

1.Amrita Mishra 2.Arun kumar Mishra

Lecturer[Pharmacognosy] Lecturer[Pharm. Chemistry]
Pharmacy College, Azamgarh Pharmacy College, Azamgarh
UP iNDIA 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 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
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 11beta hydroxylation OH
OH
curvularia lunata H2C
CO
HO OH

H2C

dehydrogenation
corynebacterium simplex

OH
H2C
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 260-
278,1999.