FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION:
"MACROPOROUS POLYVINYL ACETATE COPOLYMER BEADS FOR
LIPASE IMMOBILIZATION AND METHOD OF PREPARATION OF THE
POLYMER BEADS THEREOF"
2. APPLICANT:
(a) NAME: FERMENTA BIOTECH LIMITED
(b) NATIONALITY: Indian Company incorporated under the Companies
Act, 1956
(c) ADDRESS: 'DIL' Complex, Ghodbunder Road, Majiwada,
Thane (West) - 400610, Maharashtra, India.
3.PREAMBLE TO THE DESCRIPTION:
The following specification particularly describes the invention and the manner
in which it is to be formed:

TECHNICAL FIELD OF THE INVENTION:
The present invention relates to macroporous polyvinyl acetate copolymer beads, directly used to immobilize the lipase without any functional modification. The present invention also relates to the process for preparation of said copolymer beads.
BACKGROUND AND PRIOR ART:
Enzyme immobilization is an important aspect of industrial biocatalysis and the success of any enzyme biocatalysis significantly depends on the type of immobilization. There are several types of immobilization and there is no one generalized technique or platforms which will fit for all the enzymes.
Epoxy activated supports such as Eupergit C and Sepabeads are widely used and convenient systems for covalent binding of enzymes. They bind to enzymes via reaction of its oxirane moieties with amino, hydroxyl or sulfhydryl groups of enzymes depending on pH of the buffer used to form covalent bonds which have long-term stability within pH range of 1 to 12. However, epoxy activated systems have inherent deficiency of deactivating the enzyme. Hence, it becomes imperative to look at new platforms for enzyme immobilization.
One of the important enzyme used in biocatalysis is Candida Antarctica Lipase B (CAL B) which needs a good immobilization support for its wide spread commercial utility. Cal-B was physically immobilized onto Lewatit VP OC 1600 (supplied by Bayer) that consists of poly(methyl methacrylate-co-divinylbenzene) and has average values of particle size, surface area and pore diameter of 315-1000 µm, 130 m2/g and 150°A respectively. This immobilized form of Cal-B, known as Novozym 435 is commercially available (Chen 2008).
Numerous publications document the utility of Novozym 435 as an extraordinary catalyst for enzymatic transformations. However, Chen et al. found that Novozym 435 suffers from physical desorption or leaching of Cal-B during reactions (unpublished results). This underlines the need to develop immobilized Cal-B catalysts that overcome shortcoming of Novozym 435 (Chen 2008).

Polomo et al. immobilized Cal-B on various activated carriers including octyl sepabeads, PEI-agarose, glyoxyl-agarose, glutaraldehyde-agarose and Eupergit-Cu. By using Cal-B covalently immobilized onto the glutaraldehyde derivative, the (S)-ester and (R)-acid of (RS)-2-butyroyI-2-phenylacetic acid were obtained with high enantioselectivity (E >400) (Polomo, 2002).
Chen et al. reported covalent immobilization of Cal-b lipase onto epoxy-activated macroporous poly(methyl methacrylate) amberzyme beads (235|jm particle size, 220°A pore size). Activity of Cal-B immobilized on Amberzyme and Lewatit VP OC 1600 (carrier of Novozym 435) was assayed for e-caprolactone ring opening polymerization. The % conversion of e-caprolactone using same amount of enzyme catalyzed by Amberzyme and Lewatit VP OC 1600 for 20 mins was 7, 16 % respectively.
Nemanja Miletic et al. reported immobilization of Cal-B lipase onto polystyrene nanoparticles. Activity was assayed by hydrolysis of p-nitrophenol acetate. Cal-B immobilized on PS nanoparticles performs much higher activity (4422.7 nmol pNP/ min/mg Cal-B) than crude enzyme powder and Novozyme 435 (2396.0 and 3795.0 nmol pNP /min/mg of Cal-B respectively).
Torres et al. reported immobilization of Cal-B onto polypropylene (Accurel EP 100) with tributyrin hydrolysis activity of 2310 U/g biocatalyst and transesterification activity of 29.4 U/g biocatalyst.
Quite a lot of work has been done on divinyl benzene (DVB) and polystyrene polymer resins in combination with other monomers like ethylene glycol methacrylate, glycidly methacrylate, allyl glycidyl methacrylate styreneetc.
An article titled "Synthesis of Hydroxylated Macroporous Polymer Beads for Microwave-Assisted Desorption of Nonpolar Volatile Organic Compounds" by Mi-Lim Hwang et al., published in Bull. Korean Chem. Soc. 2010, Vol. 31, No. 8, pp. 2395-2398. The said article discloses the preparation of macroporous polymer beads via suspension polymerization of a mixture of divinylbenzene and vinyl acetate in the presence of toluene and isooctane as pore-forming agents. The resulting polymer beads were

hydrolyzed in an aqueous KOH solution to give polymer beads bearing hydroxyl groups.
An article titled "The Study on Synthesis and Structure Properties of Vinyl Acetate-Divinylbenzene Macroporous Copolymers" by Wu Xiangdong et al., published in 1994. In said abstract of the article a series of macroporous vinyl acetate-divinyl benzene copolymer beads were synthesized with ethyl acetate and n-heptane as diluent by suspension copolymerization.
An article titled "Monodisperse porous poly(vinyl acetate-co-divinylbenzene) particles by single-stage seeded polymerization: a packing material for reversed phase HPLC" by Caglayan B et al., published in J Sep Sci. 2006 May; 29(7):936-44. The said article discloses a single-stage swelling and polymerization method proposed for the synthesis of monodisperse porous polyvinyl acetate-co-divinylbenzene) [poly(VAc-co-DVB)] particles with different VAc/DVB feed ratios.
An article titled "Immobilization of Lipase on Various Acrylic Copolymers" by Jolanta Bryjak et al., published in Chemical Engineering Journal Volume 65, Issue 3, August 1997, Pages 249-256. The said article discloses that polymer carriers with various degrees of hydrophobicity, porosity, and polarity were studied for immobilization of lipase from Candida rugosa.
European Patent No. 0320023(B1), titled "Macroporous polymeric membranes, their preparation and their use for polymer separation", filed on 12/12/1988, published on 21/09/1994 of Ceskoslovenska Akademie Ved. The said EP'023 claims a macroporous polymeric membranes for the separation of macromolecular substances consisting of a cross-linked polymer or copolymer on the basis of one or more monovinylic monomers and one or more cross-linking agents, and having a globular microstructure.
One of the interesting polymer systems for enzyme immobilization is Poly vinyl alcohol based polymer beads. Poly(vinyl alcohol), PVA is the most frequently used material in embolization of tumors, aneurisms and arteriovenous malformations due to its low toxicity, good biocompatibility, elasticity, high compressibility, in addition to good chemical resistance to acids, bases and detergents. PVA based polymer systems also finds

application in the enzyme immobilization. For example, PVA based Lentikats are used for immobilizing various biomolecules ranging from whole cell to purified enzymes (Parascandola 2005). However, PVA beads cannot be prepared by direct polymerization due to the tautomerism of vinyl alcohol monomer. Hence, it is typically synthesized by saponification of poly (vinyl acetate) PVAc beads produced by polymerization of vinyl acetate (VAc) by suspension polymerization.
Jianguo et al. reported the use of Poly (vinyl acetate-co-divinyl benzene) beads after saponification with methanol solution containing 3% NaOH for immobilization of penicillin G acylase. They reported the use of activators such as p-sulfato-ethylsulfonyl aniline (SESA), glutaraldehyde and p-benzoquinone to covalently attach penicillin acylase to poly (vinyl alcohol) beads (Jianguo 2001).
Guo Z. et al., synthesized magnetic Poly (vinylacetate-co-divinyl benzene) beads by copolymerization of vinyl acetate and divinyl benzene encapsulating nanomer-sized magnetite (Fe304) and these magnetic microspheres were used to immobilize Candida cylindracea lipase (Guo 2003).
In view of the above prior art, the polyvinyl acetate beads for immobilization of enzymes with functional modification is known in the art. But there is a still need to develop a better polymer beads that can have good hydrophobicity, porosity, binding efficacy towards various enzymes. Hence, the object of the present invention is to evaluate the possibility of using the PVAc based copolymer beads directly for enzyme immobilization preferably, Cal-B lipase, without any additional functional modification and comparing the same with commercially available polymer supports having modifications like saponification and crossl inking.
SUMMARY OF THE INVENTION:
In accordance with the above object, the present invention provides macroporous polymer beads with vinyl functionality, used directly to immobilize the enzyme (e.g. Cal B lipase) without the need for any functional modification.

In an aspect, the present invention discloses polyvinyl acetate copolymers cross linked with divinyl benzene (DVB) and Ethylene glycol dimethaceylate (EGDM), with varying cross link density and the type and quantity of porogens, by using suspension polymerization technique.
In another aspect, the copolymer thus produced is directly used in enzyme immobilization without any additional functional modification with good enzyme binding and expression. The resultant polymer beads have varying degree of hydrophobicity, porosity, binding efficiency, enzyme expression towards Cal-B enzyme.
In another aspect, the present invention provides a method for immobilizing Cal-B lipase, characterized by adsorption of lipase onto macroporous poly (vinyl-co-divinyl benzene) copolymers.
DETAILED DESCRIPTION OF THE INVENTION:
The invention will now be described in detail in connection .with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.
The present invention discloses macroporous polymer beads with vinyl functionality, directly used to immobilize the enzyme, preferably Cal B lipase, without the need for any functional modification.
In one preferred embodiment, the present invention discloses Immobilized lipase onto macroporous polyvinyl acetate beads comprising vinyl acetate along with at least one hydrophobic cross linker selected from divinyl benzene (DVB) and Ethylene glycol dimethaceylate (EGDM) either alone or in combination.
In another preferred embodiment, the present invention discloses the process of immobilization of lipase onto macroporous vinyl beads comprising of following steps: a) contacting the support with the aqueous solution of the lipase containing Iso-propyl alcohol and glycerol to obtain a supported lipase;

b) washing and drying of supported lipase of step (a) to obtain immobilized lipase;
and
c) evaluating immobilized lipase of step (b) using hydrolysis of Tributyrin
The support used in the above process is selected from Poly (vinyl acetate-co-divinylbenzene) copolymers or Poly (vinyl acetate-co-ethylene glycol dimethacrylate) copolymers or Polyvinyl acetate-co-divinyl benzene + ethylene glycol dimethacrylate) copolymers.
The contact time between aqueous lipase solution and support is atleast 24 hrs.
The average pore diameter of immobilized lipase onto macroporous Polyvinyl acetate beads is in the range of 70 nm to 115 nm with stable activity.
The lipase of the present invention is obtained from recombinant Candida antarctica lipase B expressed in methylotrophic yeast, Pichia pastoris. The lipase concentration is based on activity loading of 10000 units per gram dry support.
The tributyrin hydrolysis activity of different biocatalysts varies from 1635 U/g dry to 2647 U/g dry.
The percentage protein binding in different biocatalysts varies from 68% to 74% with activity expression varies from 18% to 31%.
In another preferred embodiment, the present invention describes the process of preparation of macroporous vinyl beads comprising of following steps:
a) polymerizing vinyl acetate with single hydrophobic cross linker, in the presence of a polymerization initiator, a suspension stabililizer in aqueous media and a pore generating organic solvent selected from aromatic/aliphatic group, under stirring with a special "Inverted Umbrella" type stirrer with 8 glass rods, at a temperature range of 60- 70 °C for 6 hrs.; and
b) isolating the polymer beads thus formed.

The hydrophobic cross linker is selected from Divinyl benzene (DVB) and Ethylene glycol dimethacrylate (EGDM). The said hydrophobic crosslinker is used in the range of 28.33% to 73.33%. (Weight percentage with reference to the total monomer weight) and the cross-link density varies in the range of 25% to 200%.
The polymerization initiator is selected from benzoyl peroxide, azo bis isobutyronitrile, methyl ethyl ketone peroxide. The said polymerization initiator is used in the range of 15% to 20% (Weight percentage with reference to the total monomer weight).
The suspension stabilizer is selected from poly vinyl pyrolidone, poly vinyl alcohol, poly acrylic acid and the like. The said suspension stabilizer is used in the range of 22% to 28 % (Weight percentage with reference to the total monomer weight).
The pore generating organic solvent (Porogen) is selected from a group of higher aliphatic or cyclic alcohol such as lauryl alcohol, octanol and cyclohexanol. The said pore generating solvent is used in the range of 10% to 15%. The pore generating solvent is used in the ratio of 1.7 to 2.2 times with reference to the total weight of monomers.
The present invention discloses the compositions of macroporous polyvinyl acetate copolymer beads comprising vinyl acetate (VAc) cross-linked with at least one hydrophobic cross linker selected from divinyl benzene (DVB) and Ethylene glycol dimethacrylate (EGDM) using suitable pore generating organic solvent selected from a group of higher aliphatic or cyclic alcohol such as lauryl alcohol, octanol and cyclohexanol.
In one preferred embodiment, the present invention provides the compositions of Poly (VAc-co-DVB) copolymers comprising vinyl acetate (VAc) crosslinked with divinyl benzene (DVB) using suitable porogen selected from cyclohexanol or octanol or lauryl alcohol.
Accordingly, the Poly (VAc-co-DVB) copolymers are prepared by suspension polymerization includes the following steps:-

a) mixing divinyl benzene, vinyl acetate and suitable porogen followed by stirring with 250 ml distilled water at 300 rpm in an inert atmosphere of nitrogen;
b) polymerizing the mixture of step (a) using Polyvinyl pyrollidone and benzoyl peroxide for 6 hrs. at 70°C to obtain macroporous beads;
c) filtering and washing the beads obtained in step (b) with water and soaking in methanol overnight followed by vacuum filtration and vacuum drying at 40°C.
In another preferred embodiment, the present invention provides the compositions of PoIy(VAc-co-EGDM) copolymers comprising vinyl acetate (VAc) crosslinked with Ethylene glycol dimethacrylate (DVB) using suitable porogen selected from cyclohexanol or lauryl alcohol or octanof.
Accordingly, the Poly (VAc-co-EGDM) copolymers are prepared by suspension polymerization includes the following steps:-
a) mixing Ethylene glycol dimethacrylate, Vinyl acetate and suitable porogen followed by stirring with 250 ml distilled water at 300 rpm in an inert atmosphere of nitrogen;
b) polymerizing the mixture of step (a) using Polyvinyl pyrollidone and benzoyl peroxide for 6 hrs. at 70°C to obtain macroporous beads;
c) filtering and washing the beads obtained in step (b) with water and soaking in methanol overnight followed by vacuum filtration and vacuum drying at 40°C.
In another preferred embodiment, the present invention provides the compositions of Poly (VAc-co-EGDM-co-DVB) copolymers comprising vinyl acetate (VAc) crosslinked with Ethylene glycol dimethacrylate (EGDM) and divinyl benzene (DVB) using suitable porogen selected from cyclohexanol or lauryl alcohol or octanol.
Accordingly, the Poly (VAc-co-EGDM-co-DVB) copolymers are prepared by suspension polymerization includes the following steps:-
a) mixing Ethylene glycol dimethacrylate, divinyl benzene, Vinyl acetate and
suitable porogen followed by stirring with 250 ml distilled water at 300 rpm in
an inert atmosphere of nitrogen;

b) polymerizing the mixture of step (a) using Polyvinyl pyrollidone and benzoyl peroxide for 6 hrs. at 70°C to obtain macroporous beads;
c) filtering and washing the beads obtained in step (b) with water and soaking in methanol overnight followed by vacuum filtration and vacuum drying at 40°C.
The macroporous beads were subjected to Particle size distribution and porosity measurements by using laser diffraction analyzer (Particle size analyser, HELOS HI004) and mercury intrusion porosimeter (Fisons Instruments Pascal 140/240 porosimeter) respectively to evaluate the particle size, pore volume, pore diameter and BET (Brunaer-Emmett-Teller) surface area.
The particle size distribution of the beads of example 1 to 24 is in range from 100 microns to 500 microns, with pore volumes ranging between 0.917 MLg1 and 1.646 MLg-1 and BET (Brunaer-Emmett-Teller) surface area ranging from 89.655 m2/g to 204.45 m2/g.
The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.
EXAMPLES:
Example 1: Poly (VAc-co-DVB) copolymers with varing cross-link density using cyclohexanol as a porogen
a) Polymers were prepared by suspension polymerization technique as per following compositions: Compositions of Poly (VAc-co-DVB) copolymers with varing cross-link density

Polymer No.
Vinyl acetate (VAc) Divinyl benzene (DVB) % CLD Total weight grams in
Weight in grams Weight in grams
Example 1 21.5 8.5 25 30
Example 2 17.5 12.5 50 30
Example 3 13.5 16.5 75 30
Example 4 12.0 18.0 100 30
Example 5 10.0 20.0 150 30
Example 6 8.0 22.0 200 30
In the inert atmosphere of Nitrogen, required quantity of Divinyl benzene, and Vinyl acetate, 30 grams of cyclohexanol are stirred with 250 ml distilled water at 300 rpm and polymerized using 8.0 grams of Polyvinyl pyrollidone and 5.5 grams of benzoyl peroxide for 6 hrs at 70 deg C. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and soaked in methanol overnight, followed by vacuum filtration and vacuum drying at 40 deg C.
b) Immobilization of Candida antarctica lipase B onto Poly (VAc-co-DVB) copolymers with varing cross-link density.
Cal-B lipase enzyme produced by recombinant Candida antarctica was isolated directly after centrifugation of fermentation broth. The enzyme solution with enzyme activity 1350 TBU per ml and specific activity of 265.75 TBU per mg protein was taken for immobilization.
Depending on enzyme activity loading of 10000 TBU per gram dry polymer, required quantity of enzyme was mixed with 25% (v/v) Iso-propyl alcohol and 12.5 % (v/v) glycerol. Polymer beads were added into the enzyme solution and stirred for 24 hrs at 25 deg C. After 24 hrs, the immobilized beads were filtered off, washed with Isopropyl alcohol and then air dried.

c) Activity measurements
The hydrolytic activity of immobilized biocatalysts was determined by Tributyrin hydrolysis assay using pH stat (Spectalab, India, AT 38C) and compared with a control catalyst prepared by immobilizing Cal-B lipase onto Lewatit VP OC 1600. Activity was expressed in TBU unit. One TBU unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of 1 umol of free fatty acid from 10 % (v/v) tributyrin in the milieu of lOOmM sodium phosphate buffer at pH 7.5 at temperature 40°C.

Lewatit VP OC 1600 Example 1 Example 2 Example 3 Example 4 Example 5 Example 6
TBU activity
(U/g) 1804 984 1873 2051 1373 1486 1392
TBU
Activity (U/gdry) 2405.33 1782.61 2647.35 2610.41 1755.31 1744.13 1635.53
% protein binding 70.80 74.8 74.15 78.0 68.84 74.97 72.26
%
Activity
binding 98.78 91.75 95.76 96.15 92.81 95.45 91.43
% activity expression 25.82 22.66 31.76 27.31 18.31 19.13 18.71
Example 2: Poly (VAc-co-DVB) copolymers with varing cross-link density using lauryl alcohol or octanol as a porogen
Polymers were prepared by suspension polymerization technique as per following
compositions:
Composition of Poly (VAc-co-DVB) copolymers with varing cross-link density

Polymer No.
Vinyl acetate (VAc) Divinyl benzene (DVB) % CLD Total weight grams in
Weight in grams Weight in grams
Example 7 21.5 8.5 25 30
Example 8 17.5 12.5 50 30
Example 9 13.5 16.5 75 30
Example 10 12.0 18.0 100 30
Example 11 10.0 20.0 150 30
Example 12 8.0 22.0 200 30
In the inert atmosphere of Nitrogen, required quantity of Divinyl benzene, and Vinyl acetate, 30 grams of lauryi alcohol or 30 gms of octanol are stirred with 250 ml distilled water at 300 rpm and polymerized using 8.0 grams of Polyvinyl pyrollidone and 5.5 grams of benzoyl peroxide for 6hrs at 70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and soaked in methanol overnight, followed by vacuum filtration and vacuum drying at 40 deg C.
Example 3: Poly (VAc-co-EGDM) copolymers with varing cross-link density using cyclohexanol or lauryi alcohol or octanol as a porogen
Polymers were prepared by suspension polymerization technique as per following
compositions:
Composition of Poly (VAc-co-EGDM) copolymers with varing cross-link density

Polymer No. Vinyl acetate (VAc) Ethylene glycol dimethacrylate (EGDM) % CLD Total weight grams in
Weight in grams Weight in grams
Example 13 19.0 11.0 25 30
Example 14 13.9 16.1 50 30
Example 15 11.0 19.0 75 30
Example 16 9.1 21.0 100 30
Example 17 6.7 23.2 150 30
Example 18 5.3 24.7 200 30

In the inert atmosphere of Nitrogen, required quantity of Ethylene glycol dimethacrylate and Vinyl acetate, 30 grams of cyclohexanol or lauryl alcohol or octanol are stirred with 250 ml distilled water at 300 rpm and polymerized using 8.0 grams of Polyvinyl pyrollidone and 5.5 grams of benzoyl pefoxide for 6hrs at 70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and soaked in methanol overnight, followed by vacuum filtration and vacuum drying at 40 degC.
Example 4: Poly (VAc-co-EGDM-co-DVB) copolymers with varing cross-link density using cyclohexanol or lauryl alcohol or octanol as a porogen
Polymers were prepared by suspension polymerization technique as per following
compositions:
Composition of Poly (VAc-co-EGDM-cO-DVB) copolymers with varing cross-link
density

Polymer No. Vinyl acetate (VAc) Ethylene glycol dimethacrylate (EGDM) Divinyl
benzene
(DVB) % CLD Total weight in grams
Weight in grams Weight in grams Weight in grams
Example 19 18.9 6.6 4.5 30 30
Example 20 12 8.5 9.5 80 30
Example 21 9.5 10 10.5 120 30
Example 22 7.9 U 11.1 150 30
Example 23 7.0 13.5 9.5 175 30
Example 24 6.5 13.5 13.5 190 30
In the inert atmosphere of Nitrogen, required quantity of Ethylene glycol dimethacrylate, divinyl benzene and Vinyl acetate, 30 grams of cyclohexanol or lauryl alcohol or octanol are stirred with 250 ml distilled water at 300 rpm and polymerized using 8.0 grams of Polyvinyl pyrollidone and 5.5 grams of benzoyl peroxide for 6hrs at 70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and soaked in methanol overnight, followed by vacuum filtration and vacuum drying at 40 deg C.

Industrial applicability:
1. Vinyl group based polymers without modification is not reported earlier and the efficiency of enzyme binding is better than the commercially available polymer supports (e.g. Lewatit VP OC 1600)
2. The binding of enzyme can be done directly on supports, without any modification of the support, which is needed in case of other polymers as per the literature.
3. This system is not reported elsewhere for the immobilization of enzyme directly and also with better efficiency.

WE CLAIM,
1. Immobilized lipase onto macroporous polyvinyl acetate beads having average pore diameter in the range of 70 nm to 115 nm with stable activity comprising vinyl acetate along with at least one hydrophobic cross linker selected from Divinyl benzene or Ethylene glycol dimethacrylate either alone or in combination.
2. Immobilized lipase onto macroporous polyvinyl acetate beads according to claim 1, wherein the said hydrophobic cross-linker is present in an amount ranges from 28.33 % to 73.33%.
3. Immobilized lipase onto macroporous polyvinyl acetate beads according to claim 1, wherein the particle size distribution of the said beads ranging from 100 microns to 500 microns, with pore volumes ranging between 0.917 MLg-1 and 1.646 MLg-1and BET (Brunaer-Emmett-Teller) surface area ranging from 89.655 m2/g to 204.45 m2/g.
4. Immobilized lipase onto macroporous polyvinyl acetate beads according to any of the preceding claims, wherein said lipase is selected from the fermentation broth of recombinant Candida antarctica lipase B expressed in methylotropic yeast Pichia pastoris.
5. A process for preparation of Immobilized lipase onto macroporous polyvinyl acetate beads comprising the steps of;
a) contacting the support with the aqueous solution of the lipase containing Iso-
propyl alcohol and glycerol to provide a supported lipase;
b) washing of supported lipase and drying the supported lipase to obtain said
immobilized lipase; and
c) evaluating immobilized lipase of step (b) using hydrolysis of Tributyrin.
6. The process for preparation of Immobilized lipase onto macroporous polyvinyl
acetate beads according to claim 5, wherein the said support is selected from Poly
(vinyl acetate-co-divinylbenzene) copolymers or Poly (vinyl acetate-co-ethylene
glycol dimethacrylate) copolymers or Poly (vinyl acetate-co-divinyl benzene +
ethylene glycol dimethacrylate) copolymers.

7. The process for preparation of Immobilized lipase onto macroporous polyvinyl acetate beads according to claim 5, wherein tributyrin hydrolysis activity of different biocatalysts varies from 1635 U/g dry to 2647 U/g dry.
8. A process of preparation of macroporous polyvinyl acetate beads according to claim 1 comprising of;

a) polymerizing vinyl acetate with single hydrophobic cross linker, in the presence of a polymerization initiator, a suspension stabililizer in aqueous media and a pore generating organic solvent selected from aromatic/aliphatic group, under stirring with a special "Inverted Umbrella" type stirrer with 8 glass rods, at a temperature range of 60-70 °C for 6 hrs.; and
b) isolating the polymer beads thus formed.

9. The process of preparation of macroporous polyvinyl acetate beads according to claim 8, wherein said polymerization initiator is selected from benzoyl peroxide, azo bis isobutyronitrile and methyl ethyl ketone peroxide.
10. The process of preparation of macroporous polyvinyl acetate beads according to. claim 8, wherein said suspension stabililizer is selected from poly vinyl pyrolidone, poly vinyl alchohol, and poly acrylic acid.
11. The process of preparation of macroporous polyvinyl acetate beads according to claim 8, wherein said pore generating organic solvent is selected from a group of higher aliphatic or cyclic alchohol such as lauryl alcohol, octanol and cyclohexanol.