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Vidhi A.

Modh
Assistant professor at ssscp ,zundal
DEFINATION
Immobilization is define as the imprisonment of cell or enzyme in a
distinct support or matrix.

 Microbial enzymes are most extensively employed in the food


and beverage industries across the globe to meet the ever
increasing demand for nutritionally superb and high-value
products.
 Importantly, the predominant utilization of the enzymes in an
industrial environment has been drastically restrained and
limited by virtue of the fact that a large number of enzymes are
not only relatively unstable but also involve exhorbitantly high-
cost of isolation, purification, and recovery of ‘active enzymes’
from the reaction mixtures after the due completion of the
ensuing catalytic on-going process.
 In actual practice, the soluble enzymes engaged in ‘batch
operations’ is found to be not-so-economical due to the
fact that the active enzyme is virtually lost (not recovered)
after each viable reaction.
 Therefore, in order to combat and overcome such a non-
productive, economically not feasible,and deleterious
effect the enzymes have been ultimately immobilized ; and
the process is termed as
 enzyme immobilization. Hence, it may be defined as —
‘confining the enzyme molecules to a distinct phase from
the one wherein the substrates and the products are
present’.
Advantages of Enzyme Immobilization
 quite expensive and also having the unique ability to be used
repeatedly only in a situation when these may be recovered
completely from the accomplished reaction mixtures. In true
sense, immobilization distinctly and specifically allows their
repeated usage by virtue of the fact that such enzyme
preparation may be separated conveniently from the reaction
system involved.
 Importantly, the final desired product should be readily from the
enzyme. It goes a long way in affecting reduction and saving
upon the cost of ‘downstream processing’ of the ensuing end-
product.
 Non-aqueous systems (i.e., using organic solvents exclusively)
are found to be fairly compatible with the immobilized enzymes
particularly, and this may be regarded to be extremely
desirable in certain typical and specific instances.
 Immobilized enzymes may be used predominenty in
most continuous production systems ; and, of course,
this not absolutely feasible and possible with the ‘free-
enzymes’
 Immobilized enzymes, a few selected ones, may exhibit
thermostability of the highest order, viz.,the free-enzyme
glucose isomerase usually gets denatured only at 45°C
in solution ; however, when immobilized suitably the
enzyme is found to be stable enough upto 1 year at
65°C.
 Importantly, the ultimate recovery of ‘immobilized
enzyme’ would drastically minimise the high effluent
disposable problems (which is quite acute in several
fermentation industries).
 Immobilized enzymes may be employed at a much
higher concentration range in comparison to the
corresponding free enzyme.
Disadvantages
 High cost for the isolation,purification and recovery of active
enzyme
 Industrial appication are limited and only very few indusries
are using immobilized enzyme .
 Catalytic properties of some enzyme are reduced or completely
lost after their immobilizaton on support or carrier
 Some enzymes become unstable after immobilization.
 Enzyme are inactivated by the heat generated in the system.
Carrier Matrices
The substances that are solely employed for the immobilization
of enzymes are known as carrier matrices e.g.,inorganic
materials (salts), and inert polymers.
characteristic features,
(a) cost effectiveness,
(b) inertness,
(c) reasonable physical strength,
(d) adequate stability,
(e) regenerability
(f) enhancement in specificity of enzyme,
(g) reduction in product inhibition,
(h) a possible shift in optimum pH for enzyme activity to the
desired value for the process, and
(i) appreciable reduction in non-specific adsorption and microbial
contamination.
METHODS OF IMMOBILIZATION
 The immobilization methods are of four distinct types
depending solely upon the physical relationship of
the catalyst being used to the carrier matrix, such as
(a) adsorption
(b) covalent bonding ;
(c) entrapment
(d) membrane confinement
Adsorption Method
 simplest and oldest method.
 It was first used by Nelson and Griffin in 1916
 when they immobilized Invertase on an activated charcoal. Enzyme
is adsorbed on the physical outer surface of the support.
 Adsorption of an enzyme may be accomplished by allowing the
contact of the enzyme and the polymer support either by
percolating* the enzyme via a packed bed, tube, membrane formed
from a support material or in a stirred bioreactor.
 In other words, in the particular instance of adsorption, the enzyme
molecules evidently get adhered to the surface of a carrier matrix on
account of the spectacular combination of hydrophobic effects and
the critical formation of several salt-linkages per enzyme molecule.
Carriers used in adsorption can be

Mineral based support : Aluminum oxide or


clay, alginate beads
 Organic Bimolecular based support : Starch ,
Cellulose
 Modified ion exchange resin : Sepharose
Methodology
 The actual methodology involved in the adsorption of
enzymes to the matrices is quite simple, easy, and
employed largely. The appropriate enzyme is adequately
mixed with a right adsorbent usually under appropriate pH
parameters as well as the desired ionic strength.
 After incubation for a stipulated duration, the carrier
matrix is washed thoroughly to get rid of the entire
unabsorbed enzyme molecules, whereby the ‘immobilized
enzyme’ is ready for actual usage.
 Interestingly, this specific method invariably gives rise to
a high loading (nearly 1 g enzyme per g matrix) of the
enzyme.
 Methods for adsorbing enzymes on carrier surface:

1. Static Method: In this method the carrier is just dipped and left in
the enzyme solution without any stirring.
2. Dynamic Method: In this method carrier is placed in the enzyme
solution and stirring is maintained to load the enzyme on carrier
surface.
3. Reactor loading Method: In this method the carrier is placed in
reactor and enzyme solution is loaded with continuous stirring.
4. Electrode position Method: In this method carrier is put in
vicinity of an electrode. Now the enzymes migrate to carrier in
presence of electric current.
Advantages
 No pore diffusion limitation.
 Easy to carry out
 No reagent are required
 Minimum activation steps involved
 Comparatively cheap method
 Less distruptive to enzyme
disadvantages
 Desorption of enzymes from the carrier
 Efficiency is less
Covalent bondind
 This method involves the formation of covalent bonds
between the chemical groups in enzyme and to the
chemical groups on the support or carrier.
 Widely used method.
 Hydroxyl group and amino groups of support or enzyme
form covalent bonds more easily.
 The formation of colvalent bond usually takes place
particularly with the side chains of amino acids present in
the enzyme ; however, their actual strength of reactivity
being exclusively linked to the status of ‘charge present in
them as given below :
 — S– > — SH > — O– > — NH2 > — COO– > — OH >> —
NH3
 Thus, the various functional moieties mostly present
in enzyme which actively take part in the formation of
the numerous viable chemical bonds are : sulphide,
sulphhydril, oxide, amino, carboxyl,hydroxyl,
ammonium, imino, amide, methylthiol, guanidyl,
imidazole, and phenol ring.
 The covalent bonding of an enzyme may be accomplished
either by activating the polymer with a reactive moiety (i.e.,
copolymerization with ethylene, anhydride of maleic acid) or by
effectively employing the bifunctional reagent to serve as a bridge
between the two entities : enzyme and polymer, whereby 3D-
network may be obtained by cross-linking with low molecular
weight bifunctional agent(s).
 In doing so, the enzyme invariably may get inactivated because
the reactions normally engage a functional moiety strategically
located at the ‘active site’ of the enzyme. Thus, the overall net
effect being the substantial loss of enzymatic activity.
 Importantly, such an overwhelmingly loss in the enzymatic
activity may be overcome by judiciously carrying out the
‘enzyme immobilization’ either in the presence of a
competitive inhibitor or an enzyme substrate.
Carriers or support commonly
used for covalent bond
 Carbohydrates:- eg.cellulose, DEAD cellulose, agarose
 Synthetic agents:-eg. Polyacrylamide
 Protein carriers:- collagen, gelatin
 Amino group bearing carriers:- eg.amino benzyl
cellulose
 Inorganic carriers:porous glass,silica
 Cyanogen bromide: eg.agarose and CN Br sepharose
Methods of covalent bonding
1. Diazoation:- bonding between amino group of
support and tyrosil or histidine group of enzyme.
2. Peptide bond: bonding between amino or carboxyl
group of the support and that of the enzyme.
3. Poly functional reagents:use of bi-functional or
multifunctional reagent (glutaraldehyde) which
forms covalent bonds between the amino group of
the support and amino group of the enzyme.
advantages
 Adsorption of enzymes to the carrier matrices is quite
easy and convenient, and hence used extensively.
 Covalent bonding attachment is not reversed by pH,
ionic strength or substrate.
 Relatively broader spectrum of bonding reactions, and
of matrices with functional moieties capable of either
having covalent bondage or prone to be activated to
yield such groups renders this method into a highly
acceptable one.
disadvantages
 Chemical modifiaction of enzymeleading to the lose of
functional conformation of enzyme.
 Enzyme inactivation by change in the conformation
when undergoes reactions at the active site.
Entrapment

 In this method enzymes are physiacally entrapped


inside a porous matrix.
 Bonds involved in stabilizing the enzyme to the matrix
may be covalent or non-covalent.
 The matrix used will be a water soluble polymer.
 The form and nature of matrix varis with diffent
enzyme.
 Agar-agar and carrageenam have comparatively large
pore sizes.
 Examplles of matrixes used.
 Polyacrylamide gels
 Cellulose triacetate
 Agar
 Gelatin
 Carrageenam
 Alginate
Methods of entrapment
 Inclusion in the gels:-enzymes trapped inside the gels.
 Inclusion in fibers:enzymes supported on fibers made
of martix material.
 Inclusion in microcapsule: enzymes entrapped in
microcapsulesformed by monomer mixtressuch as
polyammine and calcium alginate.
Methodology
 The enzyme(s) may be dissolved in a solution of the
polymer’s precursors.
 Polymers may be selected from a variety of materials e.g.,
natural gels (e.g., cellulose triacetate, alginate, agar,
gelatin) ; synthetic gels e.g., polyacrylamide gels.
 In order to check and prevent the possible leakage of the
low molecular weight enzymes from the body of the gel,
the average pore size of the gel must be maintained as large
as possible.
 Efforts should be geared into action to practically contain
two important aspects in ‘entrapment’ process, namely :
 (a) excessive diffusion limitation, and
 (b) variability of pore size
Advatages
 Fast method of immobilization.
 Cheap (low cost matrixes available)
 Easy to practice at small scale
 Mild condition are required
 Less chance of contaminational changes in enzyme.
Disadvatages
 Leakage of enzyme
 Pore diffusion limitation
 Chance of microbial contamination
 Not much success in industrial process.