Title of Invention

OXIRANE MACRO-POROUS BEADS FOR ENZYME IMMOBILIZATION AND PROCESS THEREOF

Abstract This invention discloses stable, highly porous and oxirane macro-porous beads with high enzyme expression abilities based on Glycidyl methacrylate- Ethylene glycol dimethacrylate-Divinyl benzene co-polymers and a novel process for the preparation of the said macro-porous polymer beads involving suspension polymerization technique. The present invention further provides a special type of agitator used for the preparation of uniform and stable macro porous polymer beads.
Full Text FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rulel3)
1. TITLE OF THE INVENTION:
"Stable, highly porous oxirane beads for enzyme immobilization and process of preparation thereof"
2. APPLICANT (S)
(a) NAME: FERMENTA BIOTECH LIMITED
(b) NATIONALITY: Indian Company incorporated under the Indian
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 performed.


Technical Field:
The present invention relates to highly stable highly porous, oxirane macroporous polymer beads with high enzyme expression abilities based on Divinyl benzene-Glycidyl methacrylate-Ethylene glycol dimethacrylate co-polymers and a novel process for the preparation of the said macroporous polymer beads involving suspension polymerization technique. The present invention further provides a special type of agitator used for the preparation of uniform and stable macroporous polymer beads.
Background and prior art:
Enzymes are protein molecules which accelerates the chemical reactions of living cells. Enzymes are obtained from bacteria, plant or any other natural source. Enzyme catalysis is a commonly followed technique in both aqueous and non-aqueous phase reactions. The success of any enzymatic process depends very much on the stability in variety of conditions, solvent systems and most importantly re-cyclability. However, the enzymes are often lost when the desired product is isolated. The enzymes being in solution with the reactant or product are difficult to separate. The enzymes once used can be reused provided they are bound on a suitable network of matrix or on support. An immobilized enzyme is an enzyme that is physically attached to a particular solid support.
Immobilized enzymes offer great advantages in terms of better operational stability, easy removal from the reaction media by simple filtration and cost advantages, without altering the quality of the process or the product. One of the classical mechanisms for immobilizing enzymes on an insoluble carrier is the reaction of an active hydrogen-bearing group, e.g., an alcohol, amine or mercaptan group on the enzyme with an oxirane, or epoxy ring. Such oxirane based supports are widely used and commercially exploited. The use of polymers or structural matrices for immobilization of enzymes is well known, for different supports based on nylon, polyacrylonitrile, cellulose and acrylate polymers.

US Patent no 3887432 discloses the use of ethylene-maleic anhydride co-polymers for the immobilization of Penicillin deacylase enzyme. US patent no 6280983 discloses the use of gelatin in various concentrations for immobilizing enzymes.
US4070348 discloses the use of trimethylopropane methacrylate co-polymers in the immobilization of Penicillin g acylase.
US4363888 discloses a process for the preparation of rigid beads, of cross-linked polyester resin by forming oil-in-water or a water-in-oil-in-water emulsion. The beads formed are solid or vesiculated.
US4607057 discloses a process for the preparation of beads of copolymers bearing reactive groups, by polymerization in suspension in a non-aqueous medium.
US 6930133 discloses low density polymeric beads made by polymerizing monomeric building blocks into large crosslinked polymer molecules in porous, spherical bead form by droplet or suspension polymerization in the presence of a porogen. The low density macroreticluar polymer beads are prepared using Divinylbenzene - styrene and trimethylopropane trimethacrylate co-polymers.
Suspension polymerization, a widely used technique for the preparation of reactive beaded polymers was developed by Hoffman and Delbruch in 1909.
The conventional process of suspension polymerization uses single cross linker with epoxy monomers like Glycidyl methacrylate to make macroporous, oxirane activated polymer beads for enzyme immobilization. However, this approach has limitations in achieving very high selective surface area and porosity of oxirane beads. Under these conditions of single cross linker systems, excess addition of pore generating solvents would eventually shrink the size of the polymer bead due to poor or limited space for networking or compromise the stability of the polymer.
However, there are various disadvantages associated with the commercially available Oxirane beads like less porosity, operational stability, enzyme expression, substrate

selectivity, enzyme selectivity. Hence, there is always a need to develop new polymer beads useful in specific enzyme immobilization.
The inventors of the present invention have tried to overcome the disadvantages associated with the commercially available oxiranes by providing novel and improved oxirane beads useful in enzyme immobilization, especially in Lipases, Penicillin acylase and Polyphenol oxidases.
Object of the Invention:
The main object of the present invention is to provide stable, highly porous, oxirane macroporous polymer beads with high enzyme expression abilities based on Divinyi benzene-Glycidyl methacrylate -Ethylene glycol dimethacrylate co-polymers.
The macroporous beads prepared by the process of this invention are based on the a triple monomer system namely, Divinylbenzene, Glycidyl methacrylate and Ethylene glycol dimethacrylate and meets the requirements of an ideal supports such as high mechanical strength, chemical and temperature stability, large surface area, appropriate pore size and its distribution, selectivity to a particular enzyme.
Another object of the present invention is to provide a novel process for the preparation of the said macroporous polymer beads involving suspension polymerization technique.
It is yet another object of the present invention to provide a special type of agitator which enables gentle mixing to produce high proportion of uniformly sized, stable polymer beads.
Further object of the invention is to evaluate the macroporous polymer beads for porosity and surface area by Nitrogen absorption isotherm and Mercury porosimetry.
It is yet further object of the present invention to evaluate the binding of macroporous polymer beads on various enzymes of commercial importance like Penicillin G acylase, Alkaline protease and Poly phenol oxidase.

Summary of the Invention:
In accordance with the above objectives, the present invention discloses stable, highly porous, oxirane macroporous polymer beads with high enzyme expression abilities based on triple monomer system namely, Glycidyl methacrylate-Ethylene glycol dimethacrylate-Divinylbenzene co-polymers and a novel process for the preparation of the said macroporous polymer beads involving suspension polymerization technique. The monomers as per the present invention are used in specific and suitable volume fraction and the speed of the agitator is suitably controlled to produce polymer beads in the desired range of 150-250microns with specific surface area varying from 200 - 300 m2/ gram. Commercially available macroporous polymer beads for enzyme immobilization have much lower specific surface area (BET) (e.g.Toyopearl AF -Tresyl 650M - 42m2/g, Sepabeads EC EP 3 - 43 m2/g,Sepabeads EC EP 5- 9 m2/g, Eupergit C- 4.5 m2/g, EupergitC250L-49.3m2/g).
In the present invention the oxirane beads with varying porosity, oxirane content and specific surface area are made. This diversity of oxirane activated polymers with varying degree of hydrophobicity, porosity, specific surface area and binding efficiency are useful in immobilizing various enzymes, especially enzymes like Acylases, Proteases, Esterase, Lipases, Oxidases etc.
Macroporous oxirane beads are prepared by suspension polymerization. In suspension polymerization the initiator is soluble in the monomer phase, which is dispersed into the dispersion medium (usually water) to form droplets. Polymerization proceeds in the droplet phase, and occurs by a free radical mechanism. The size distribution of the initial suspension droplets and size of the polymer beads that are formed is dependent upon the balance between droplet break-up and droplet coalescence, which in turn controlled by the type and speed of the agitator used, the volume fraction of the monomer phase, and the type and concentration of stabilizer used. The polymer beads may be made porous by the inclusion of an inert diluent (or porogen) to the monomer phase, which may be extracted after polymerization.

According to the feature of the invention, use of double crosslinkers has helped to greatly achieve high selective surface areas upto 5 times of related commercial polymer beads with high operational and mechanical stability.
Apart from providing physical advantage, this novel approach has given rise to new polymers with more efficient binding and expression of enzymes like Penicillin acylase, Esterase, Protease, Polyphenol oxidases.
Accordingly, stable, highly porous, oxirane macro-porous beads for enzyme immobilization prepared by a suspension polymerization comprising:
a) polymerizing Glycidyl methacrylate with double crosslinkers, selected from at least at least one hydrophilic cross linker and one 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 of60-70°Cfor4hrs;and
b) isolating the polymer beads thus formed.
The present invention further provides a novel special type of agitator, which facilitates the uniform mixing. Further the invention provides an evaluation studies of these stable macroporous polymer beads for binding on various enzymes like Penicillin G acylase, Alkaline protease and Poly phenol oxidase.
Description of the Invention:
The present invention further discloses the process for the preparation of stable, highly porous, oxirane macro-porous beads comprising suspension polymerization. The highly stable and macro-porous polymer beads based on triple monomer system of the present invention are prepared by polymerizing Glycidyl methacrylate with double crosslinkers, namely the hydrophobic, divinyl benzene monomer having excellent cross linker properties along with one or more hydrophilic cross linkers such as aliphatic, hydrophilic monomers selected from a group of Ethylene glycol dimethacrylate and the like.

The use of double cross linkers surprisingly increased selective surface area with by increased porosity by effectively using the available pore generating solvents. Therefore, the inventors of the present invention made use of combination of Divinyl benzene, Ethylene Glycol Dimethaerylate and Glycidy! methacrylate thereby increasing the porosity atleast two to five fold and also retaining the mechanical and operational stability.
The use of other, additional monomers like acrylonitrile, methyl methacrylate along with the above polymer system provides overall mechanical stability of the polymer beads thereby providing the advantages of temperature stability.
According to the preferred embodiment, stable, highly porous, oxirane macro-porous beads for enzyme immobilization prepared by a suspension polymerization comprising:
a) polymerizing Glycidyl methacrylate with double crosslinkers, selected from at
least at least one hydrophilic cross linker and one 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 0C for 4 hrs; and
b) isolating the polymer beads thus formed.
The hydrophilic cross linkers is an aliphatic monomer selected from Ethylene glycol dimethacrylate, pentaerythritol triacrylate. The hydrophobic crosslinker is selected from the group consisting of divinyl benzene, trivinyl benzene, divinylnapthalene and the like.
The pore generating solvent is selected from a group consisting of higher aliphatic or cyclic alcohol such as lauryl alcohol, dodecanol and cyclohexanol.
The suspension stabilizer is selected from the group consisting of poly vinyl pyrolidone, poly vinyl alchohol, poly acrylic acid and the like.

The polymerization initiator is selected from benzoyl peroxide, azo bis isobutyronitrile, methyl ethyl ketone peroxide.
The volume fraction of monomer vary between the range of 0.3 to 0.7, preferably 0.4-0.65 with respect to organic phase.
The particle size distribution of the beads ranges from 150 microns to 250 microns, with pore volumes of 0.77 to 1.22 cm2/g and BET surface area ranging from 58 - 300 m2/g.
The beads thus prepared by the present invention are further used for enzyme immobilization, wherein the enzymes are selected from the group of Alkaline Protease, Poly Phenol Oxidase and Penicillin G Acylase and the like.
The polymer beads obtained according to the present invention are highly porous and stable. This is provided by the use of, double crosslinkers in specific and suitable volume fraction of monomer phase, specific ratio of pore generating solvent as well as a special type of agitator disclosed as per the present invention. The novel special type of agitator is an "inverted Umbrella" type agitator made of 6- 8 glass rods. This tapering agitator has broader base which is half of the diameter of the vessel and the narrower base which is quarter of the diameter of the vessel. This special type of agitator is made of six to eight rods. Use of the special agitator facilitates gentle mixing and hence the bead size is relatively stable than in a Rushton type of agitator, where the mixing is quite vigourous and hence the formation of finer particles are more.
The novel special type of agitator gives very uniform and smooth mixing, leading to the formation of bead particles within the desired range of 150- 250 microns.
Reaction conditions:
All reactions are performed by using a one liter, jacketed cylindrical glass reactor end-fitted with 3 ports. All the reactions are carried out in the presence of the Nitrogen blanketing. The volume fraction of the monomer was varied between 0.3- 0.7, more precisely between 0.4- 0.65 with reference to the organic phase. The hydrophilic cross linker is 35 to 78 weight %, while the hydrophobic crosslinker is between 8 to 12 weight

%on the total monomer weight, with specific ratio ranging 0.1 to 0.2 between the cross linkers. The pore generating solvent is in the ratio of 1.7 to 2.2 times the total weight of monomers, more specifically.
The polymerization stabilizer used in the process is between 1 to 1.8 weight % to the aqueous layer. The ratio of aqueous and organic phase varied between 1 to 1.5 times of volume. The RPM is varied to optimize the right particle size distribution, The macroporous beads obtained as per the present invention are useful in various enzyme immobilization. The enzymes which can be immobilized using the macroporous polymer beads include Penicillin G acylase, Alkaline protease and Poly phenol oxidase The present invention is more specifically explained by following examples. However, it should be understood that that the scope of the present invention is not limited by the examples in any manner. It will be appreciated by any person skilled in this art that the present invention includes the following examples and further can be modified and altered within the technical concept of the present invention.
EXAMPLES
Example 1:
In the inert atmosphere of Nitrogen,12.8 grams of Divinyl benzene, 11.5 grams Glycidyl methacrylate, 77.5 grams Ethylene glycol dimeth aery late, 200 grams of cyclohexanol are stirred with 500 ml distilled water at 300 rpm and polymerized using 7 grams of Polyvinyl pyrrolidone and 1.6 grams of Azobisisobutyronitrile for 4 hrs at 65 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 95 grams.
Example 2
In the inert atmosphere of Nitrogen,12.8 grams of Divinyl benzene, 11.5 grams Glycidyl methacrylate, 77.5 grams Ethylene glycol dimethacrylate, 180 grams of lauryl alchohol are stirred with 500 ml distilled water at 400 rpm and polymerized using 7 grams of Polyvinyl pyrrolidone and 1.6 grams of Azobisisobutyronitrile for 4 hrs at 65 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed

with water and distilled in methanol by using soxlet apparatus. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 90-95 grams.
Example 3
In the inert atmosphere of Nitrogen, 12.8 grams of Divinyl benzene, 11.5 grams Glycidyl methacrylate, 77.5 grams Ethylene glycol dimethacrylate, 150 grams of dodecanol are stirred with 500 ml distilled water at 450 rpm and polymerized using 7 grams of Polyvinyl pyrrolidone and 1.6 grams of Azobisisobutyronitrile for 4 hrs at 65 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 85-88 grams.
Example 4
In the inert atmosphere of Nitrogen, 12.8 grams of Divinyl benzene, 11.5 grams Glycidyl methacrylate, 77.5 grams Ethylene glycol dimethacrylate, 150 grams of dodecanol are stirred with 500 ml distilled water at 450 rpm and polymerized using 7 grams of Polyvinyl pyrrolidone and 1.6 grams of Azobisisobutyronitrile for 4 hrs at 65 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 80-84 grams.
Example 5
In the inert atmosphere of Nitrogen, 12.5 grams of Divinyl benzene, 25.9 grams Glycidyl methacrylate, 30.8 grams Ethylene glycol dimethacrylate,36.5 grams Acrylonitrile, 150 grams of dodecanol are stirred with 500 ml distilled water at 350 rpm and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.5 grams of Azobisisobutyronitrile for 4 hrs at 65 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 85-88 grams.

Example 6
In the inert atmosphere of Nitrogen,12.5 grams of Di vinyl benzene, 25.9 grams Glycidyl methacrylate, 30.8 grams Ethylene glycol dimethacrylate,36.5 grams Acrylonitrile, 200 grams of cyclohexanol are stirred with 500 ml distilled water at 300 rpm and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.5 grams of Azobisisobutyronitrile for 4 hrs at 65 deg C. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 88- 90 grams.
Example 7
In the inert atmosphere of Nitrogen, 12.5 grams of Divinyl benzene, 25.9 grams Glycidyl methacrylate, 30.8 grams Ethylene glycol dimethacrylate,36.5 grams Acrylonitrile, 180 grams of laurylalchohol are stirred with 500 ml distilled water at 400 rpm and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.5 grams of Azobisisobutyronitrile for 4 hrs at 65 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 88- 90 grams.
Example 8
In the inert atmosphere of Nitrogen, 15.4 grams of Divinyl benzene, 53.8 grams Glycidyl methacrylate, 3635 grams Acrylonitrile, 200 grams of cyclohexanol are stirred with 500 mi distilled water at 300 rpm and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.6 grams of Azobisisobutyronitrile for 4 hrs at 65 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 88- 90 grams.
Example 9
In the inert atmosphere of Nitrogen, 15.4 grams of Divinyl benzene, 53.8 grams Glycidyl
methacrylate, 36.35 grams Acrylonitrile, 140 grams of dodecanol are stirred with 500 ml

distilled water at 300 rpm and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.6 grams of Azobisisobutyronitrile for 3 hrs at 65-70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 80 grams.
Example 10
In the inert atmosphere of Nitrogen,15.4 grams of Divinyl benzene, 53.8 grams Glycidyl methacrylate, 36.35 grams Acrylonitrile, 140 grams of dodecanol are stirred with 500 ml distilled water at 300 rpm and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.6 grams of Azobisisobutyronitrile for 3 hrs at 65-70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 80-82 grams.
Example 11
In the inert atmosphere of Nitrogen,12.5 grams of Divinyl benzene, 45.8 grams Glycidyl methacrylate, 10.6 grams Acrylonitrtle,35.9 grams Ethylene dimethacrylate,160 grams of cyclohexanol are stirred with 500 ml distilled water at 300 rpm and polymerized using 7 grams of Polyvinyl pyrrolidone and 1.6 grams of azobisisobutyronitrile for 3 hrs at 65-70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs, After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 87-90 grams.
Example 12
In the inert atmosphere of Nitrogen,12.5 grams of Divinyl benzene, 45.8 grams Glycidyl methacrylate, 10.6 grams Acrylonitrile,35.9 grams Ethylene dimethacrylate, 140 grams of laurylalchohol are stirred with 500 ml distilled water at 300 rpm and polymerized using 7 grams of Polyvinyl pyrrolidone and 1.6 grams of azobisisobutyronitrile for 3 hrs at 65-70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs.

After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 85-87 grams.
Example 13
In the inert atmosphere of Nitrogen, 12.5 grams of Divinyl benzene, 45.8 grams Glycidyl methacryiate, 10.6 grams AcryIonitriIe,35.9 grams Ethylene dimeth aery late, 150 grams of dodecano are stirred with 500 ml distilled water at 300 rpm and polymerized using 7 grams of Polyvinyl pyrroiidone and 1.6 grams of azobisisobutyronitrite for 3 hrs at 65-70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 80-85 grams.
Example 14
In the inert atmosphere of Nitrogen, 15.5 grams of Divinyl benzene, 40.8 grams Glycidyl methacryiate, 50.4 grams Acrylonitrile, 150 grams of cyclohexanol are stirred with 500 ml distilled water at 300 rpm and polymerized using 7 grams of Polyvinyl pyrroiidone and 1.6 grams of azobisisobutyronitrile for 3 hrs at 65-70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 90-92 grams.
Example 15
In the inert atmosphere of Nitrogen, 15.5 grams of Divinyl benzene, 40.8 grams Glycidyl methacryiate, 50.4 grams Acrylonitrile, 180 grams of laurylalchohol are stirred with 500 ml distilled water at 300 rpm and polymerized using 7 grams of Polyvinyl pyrroiidone and 1.6 grams of azobisisobutyronitrile for 3 hrs at 65-70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 87-90 grams

Example 16
In the inert atmosphere of Nitrogen, 15.5 grams of Divinyl benzene, 40.8 grams Glycidyl methacrylate, 50.4 grams Acrylonitrile, 200 grams of laurylalchohol are stirred with 500 ml distilled water at 400 rpm and polymerized using 7 grams of Polyvinyl pyrrolidone and 1.6 grams of azobisisobutyronitrile for 3 hrs at 65-70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 78-84 grams
Example 17
In the inert atmosphere of Nitrogen, 15.5 grams of Divinyl benzene, 40.8 grams Glycidyl methacrylate, 50.4 grams Acrylonitrile, 200 grams of laurylalchohol are stirred with 500 ml distilled water at 400 rpm and polymerized using 7 grams of Polyvinyl pyrrolidone and 1.6 grams of azobisisobutyronitrile for 3 hrs at 65-70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 78-84 grams.
Example 18
In the inert atmosphere of Nitrogen, 18.6 grams of Divinyl benzene, 56.2 grams Glycidyl methacrylate, 33.7 grams Acrylonitrile, 200 grams of cyclohexanol are stirred with 500 ml distilled water at 400 rpm and polymerized using 7 grams of Polyvinyl pyrrolidone and 1.6 grams of azobisisobutyronitrile for 4 hrs at 65-70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 84-86 grams
Example 19
In the inert atmosphere of Nitrogen, 18.6 grams of Divinyl benzene, 56.2 grams Glycidyl methacrylate, 33.7 grams Acrylonitrile, 150 grams of dodecanol are stirred with 500 ml distilled water at 400 rpm and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.6 grams of azobisisobutyronitrile for 4 hrs at 65-70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in

methanol by using soxiet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 86-88 grams.
Example 20
In the inert atmosphere of Nitrogen, 23.5 grams of Divinyl benzene, 61.5 grams Glycidyl methacrylate, 100 grams of cyclohexanol are stirred with 500 ml distilled water at 400 rpm and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.6 grams of azobisisobutyronitrile for 4 hrs at 65-70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxiet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 90-92 grams
Example 21
In the inert atmosphere of Nitrogen, 23.5 grams of Divinyl benzene, 61.5 grams Glycidyl methacrylate, 140 grams of dodecanol are stirred with 500 ml distilled water at 400 rpm and polymerized using 6 grams of Polyvinyl pyrrolidoneand 1.6 grams of azobisisobutyronitrile for 4 hrs at 65-70 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxiet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 85-90 grams.
Example 22
In the inert atmosphere of Nitrogen, 23.5 grams of Divinyl benzene, 61.5 grams Glycidyl methacrylate, 100 grams of cyclohexanol are stirred with 500 ml distilled water at 400 rpm and polymerized using 6 grams of Polyvinyl pyrrolidoneand 1.6 grams of azobisisobutyronitrile for 4 hrs at 68-75 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxiet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 90-92 grams
Example 23
In the inert atmosphere of Nitrogen, 23.5 grams of Divinyl benzene, 61.5 grams Glycidyl
methacrylate, 150 grams of dodecanol are stirred with 500 ml distilled water at 400 rpm

and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.6 grams of azobisisobutyronitrile for 4 hrs at 68-75 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 63-65 grams.
Example 24
In the inert atmosphere of Nitrogen, 12.5 grams of Divinyl benzene, 25.8 grams Glycidyl methacrylate, 68.48 grams of Ethylene glycol dimethacrylate,150 grams of cyclohexanol are stirred with 480 ml distilled water at 300 rpm and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.6 grams of azobisisobutyronitrile for 4 hrs at 68-75 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 85-90 grams.
Example 25
In the inert atmosphere of Nitrogen, 12.5 grams of Divinyl benzene, 46.4 grams Glycidyl methacrylate, 150 grams of cyclohexanol are stirred with 500 ml distilled water at 300 rpm and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.6 grams of azobisisobutyronitrile for 4 hrs at 68-75 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 45-48 grams
Example 26
In the inert atmosphere of Nitrogen, 13.9 grams of Divinyl benzene, 50.5 grams Glycidyl methacrylate, 43.4 grams Ethylene glycol dimethacrylate, 150 grams of cyclohexanol are stirred with 500 ml distilled water at 300 rpm and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.6 grams of azobisisobutyronitrile for 4 hrs at 68-75 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After

distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 80-82 grams
Example 27
In the inert atmosphere of Nitrogen, 13.9 grams of Diviny] benzene, 50.5 grams Glycidyl methacrylate, 43.4 grams Ethylene glycol dimethacrylate, 200 grams of dodecanol are stirred with 500 ml distilled water at 300 rpm and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.6 grams of azobisisobutyronitrile for 4 hrs at 68-75 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 83-86 grams
Example 28
In the inert atmosphere of Nitrogen, 18.6 grams of Divinyl benzene, 50.5 grams Glycidyl methacrylate, 8.6 grams Ethylene glycol dimethacrylate, 30.7 grams acrylonitrile, 200 grams of cyclohexanol are stirred with 500 ml distilled water at 300 rpm and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.6 grams of azobisisobutyronitrile for 4 hrs at 68-75 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 86-89 grams.
Example 29
In the inert atmosphere of Nitrogen, 18.6 grams of Divinyl benzene, 50.5 grams Glycidyl methacrylate, 8.6 grams Ethylene glycol dimethacrylate, 30.7 grams acrylonitrile, 200 grams of dodecanol are stirred with 500 ml distilled water at 300 rpm and polymerized using 6 grams of Polyvinyl pyrrolidone and 1.6 grams of azobisisobutyronitrile for 4 hrs at 68-75 deg c. The macroporous beads thus formed at the end of the reaction were vacuum filtered, washed with water and distilled in methanol by using soxlet apparatus for 10 hrs. After distillation, the beads are filtered and dried in vacuum oven at 40 deg C. The yield obtained was 85-87 grams.

Evaluation Study of the polymer beads.
Porosity and surface area of the polymers were measured by Nitrogen absorbtion isotherm and Mercury porosimetry.
The physical properties of the polymer beads are as shown in Table 1.

Table 1

Enzyme binding and stability studies were done as per specific protocols for each enzyme.
Enzyme immobilization and screening: Beaded polymer supports formed by the above examples were evaluated for binding various enzymes of commercial importance. The enzymes selected are Penicillin G acylase,( EC 3.5.1.11.) Alkaline protease and Poly phenol oxidase (EC 1.14.18.1).
1) Immobilization of Penicillin G acylase:
Penicillin G acylase produced by recombinant E.coli REIII (pKa 18) was isolated by cell
lysis followed by chromatographic purification. The enzyme solution with enzyme

activity 200 IU per ml and specific activity of 20 IU per mg was taken for immobilization with different enzyme loading per gram of the polymer.
Clear enzyme solution was buffered up to 1 Molar, preferably between 0.6- 0.8 Molar with Phosphate buffer. Keeping the partition coefficient constant at 1: 4, polymer beads were added into the buffer solution and stirred for 48 hrs at 25 deg C. After 48 hrs, the polymers with enzyme solution were further incubated without shaking. Finally, the immobilized beads were filtered off and washed thoroughly before analysis. Results of Penicillin G acylase enzyme immobilization on polymer beads are as shown in Table 2.
Table 2


Immobilized polymers showed high expression and high stability, especially at high temperature. Simulative stability studies were carried out at 50 Deg C for repeated cycles and the polymers were evaluated.
2) Immobilization of Polyphenol oxidase:
Crude preparation of Polyphenol oxidase ( EC 1.14.18.1) from white button mushroom Agartcus bisporus was immobilized on the beaded polymers. Polyphenol oxidase has immense commercial value in phenol degradation, synthesis of DOPA intermediates, yet obtained from cheap source.
White button mushroom was purchased from weikfield (commercially available) and freezed at -20°C for three hours. After complete chilling, mushroom was crushed in chilled acetone (enzyme activator). Acetone was filtered off. The remaining Cake was dried at room temperature for 20 minutes and kept in chilled water over night. After overnight incubation the extract was collected. This extract was kept in sucrose (60%) for dialysis, which resulted in four fold concentration.
After concentrating the enzyme solution was brought to 0.3 Molar by addition of 0.5M Phosphate buffer, pH 6.5.Then this solution was added to 3 g dry polymer beads and kept on shaker at 4°C for 2 hours. After that the flasks were kept at 4°C over night. Next day beads were washed with 0.5 M phosphate buffer thrice and stored at 4°C. Polyphenol oxidase immobilized enzymes were assayed for enzyme activity and the resvhs are as shown in Table 3.
Table 3
Sr. Polymer Enzyme Protein Protein Activity per\ctivity per Enzyme
No. sample load per oad per g Expression gram wetgrarn dry expression
gram ot( mg) % weight weight (%)
I support IU (I.u) (I.U)
1 Example 1 2000 78.64 96.85 265.21 740.6 37.03
2 Example 5 2000 78.64 96.67 233.19 680.6 34.03
3 Example26 2000 78.64 97.02 269.6 824.7 | 41-24

3) Immobilization of Alkaline protease:
Commercial preparation of alkaline protease (ALCALASE ™) from Novozymes was used to immobilize in the beaded polymers. Apart from the conventional use in detergents, this enzyme is being used in enantioselective reactions involving peptides and amides. However, immobilized forms of this enzyme are not yet commercially available.
Commercial preparation of protease (ALCALASE™) obtained from NOVOZYMES was dialyzed against 50 mM phosphate buffer pH 7.5 and finally buffered with 0.1M Sodium phosphate buffer pH 7.5 before immobilization. Dry the beads mixed with buffered enzyme solution were shaker incubated for 24 hrs at 28-30 deg C. Immobilized beads were washed with buffer and filtered before enzyme assay.
Protease (Alcalase) enzyme immobilization results on polymer beads are as shown in Table 4.


It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

We Claim,
1. Stable, highly porous, oxirane macro-porous beads for enzyme immobilization prepared by a suspension polymerization comprising:
a) polymerizing Glycidyl methacrylate with double crosslinkers, selected from at
least at least one hydrophilic cross linker and one 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 4 hrs; and
b) isolating the polymer beads thus formed.
2) The stable oxirane macro-porous beads as claimed in claim 1, where in the hydrophilic cross linkers is an aliphatic monomer selected from Ethylene glycol dimethacrylate, pentaerythritol triacrylate
3) The stable oxirane macro-porous beads as claimed in claim 1, wherein the hydrophobic cross-linker is selected from the group consisting of divinyl benzene, trivinyl benzene, divinylnapthalene and the like.
4) The stable oxirane macro-porous beads as claimed in claim 1, wherein the hydrophilic cross linker is used in 35% to 78 weight percentage with reference to the total monomer weight.
5) The stable oxirane macro-porous beads as claimed in claim 1, wherein the hydrophobic crosslinker is used in 8- 12 % weight percentage with reference to the total monomer weight.
6) The stable oxirane macro-porous beads as claimed in claim 1, wherein the ratio of hydrophilic and hydrophobic cross linkers ranging between 0.1 to 0.2.
7) The stable oxirane macro-porous beads as claimed in claim 1, wherein the pore generating solvent is used in the ratio of 1.7 to 2.2 times with reference to the total weight of monomers.
8) The stable oxirane macro-porous beads as claimed in claim 1, wherein the pore generating solvent is from a group of higher aliphatic or cyclic alchohol such as lauryl alcohol, dodecanol and cyclohexanol.

9) The stable oxirane macro-porous beads as claimed in claim 1, wherein the suspension stabilizer is selected from poly vinyl pyrolidone, poly vinyl alchohol, poly acrylic acid and the like.
10) The stable oxirane macro-porous beads as claimed in claim 1 , wherein the polymerization initiator is selected from benzoyl peroxide, azo bis isobutyronitrile, methyl ethyl ketone peroxide.
1 l)The stable oxirane macro-porous beads as claimed in claim 1, wherein the volume fraction of monomer vary between the range of 0.3 to 0.7, preferably 0.4-0.65 with respect to organic phase.
12) The stable oxirane macro-porous beads as claimed in claim 1, wherein the particle size distribution of the beads ranges from 150 microns to 250 microns, with pore volumes 0.77 to 1.22 cm2/g and BET (Brunaer-Emmett-Teller) surface area ranging from58-300m2/g.
13) The stable oxirane macro-porous e beads as claimed in claim 1, further bound thereto the enzymes selected from the group of Alkaline Protease, Poly Phenol Oxidase and Penicillin G Acylase and the like.
Dated this 6,h day of July 2006


Documents:

1066-mum-2006-abstract(6-7-2007).doc

1066-mum-2006-abstract(6-7-2007).pdf

1066-MUM-2006-ABSTRACT(GRANTED)-(28-4-2011).pdf

1066-MUM-2006-CANCELLED PAGES(12-4-2011).pdf

1066-mum-2006-claims(6-7-2007).doc

1066-mum-2006-claims(6-7-2007).pdf

1066-MUM-2006-CLAIMS(AMENDED)-(12-4-2011).pdf

1066-MUM-2006-CLAIMS(AMENDED)-(14-7-2010).pdf

1066-MUM-2006-CLAIMS(AMENDED)-(30-11-2010).pdf

1066-MUM-2006-CLAIMS(GRANTED)-(28-4-2011).pdf

1066-MUM-2006-CLAIMS(MARKED COPY)-(12-4-2011).pdf

1066-MUM-2006-CLAIMS(MARKED COPY)-(30-11-2010).pdf

1066-MUM-2006-CORRESPONDENCE(25-07-2008).pdf

1066-MUM-2006-CORRESPONDENCE(6-7-2006).pdf

1066-mum-2006-correspondence(6-7-2007).pdf

1066-MUM-2006-CORRESPONDENCE(IPO)-(28-4-2011).pdf

1066-mum-2006-correspondence-others.pdf

1066-mum-2006-description(complete)-(6-7-2007).pdf

1066-MUM-2006-DESCRIPTION(GRANTED)-(28-4-2011).pdf

1066-mum-2006-description(provisional).pdf

1066-MUM-2006-FORM 1(12-4-2011).pdf

1066-MUM-2006-FORM 1(14-7-2010).pdf

1066-MUM-2006-FORM 1(19-7-2006).pdf

1066-MUM-2006-FORM 1(6-7-2006).pdf

1066-MUM-2006-FORM 18(25-07-2008).pdf

1066-mum-2006-form 2(6-7-2007).doc

1066-mum-2006-form 2(6-7-2007).pdf

1066-MUM-2006-FORM 2(GRANTED)-(28-4-2011).pdf

1066-MUM-2006-FORM 2(TITLE PAGE)-(12-4-2011).pdf

1066-mum-2006-form 2(title page)-(6-7-2007).pdf

1066-MUM-2006-FORM 2(TITLE PAGE)-(COMPLETE)-(6-7-2007).pdf

1066-MUM-2006-FORM 2(TITLE PAGE)-(GRANTED)-(28-4-2011).pdf

1066-MUM-2006-FORM 2(TITLE PAGE)-(PROVISIONAL)-(6-7-2006).pdf

1066-mum-2006-form 2.doc

1066-MUM-2006-FORM 26(12-4-2011).pdf

1066-mum-2006-form 26(19-7-2006).pdf

1066-mum-2006-form 5(6-7-2007).pdf

1066-mum-2006-form-1.pdf

1066-mum-2006-form-2.pdf

1066-mum-2006-form-3.pdf

1066-MUM-2006-MARKED COPY(30-11-2010).pdf

1066-MUM-2006-OTHER DOCUMENTS(30-11-2010).pdf

1066-MUM-2006-REPLY TO EXAMINATION REPORT(14-7-2010).pdf

1066-MUM-2006-REPLY TO EXAMINATION REPORT(30-11-2010).pdf

1066-MUM-2006-REPLY TO HEARING(12-4-2011).pdf


Patent Number 247630
Indian Patent Application Number 1066/MUM/2006
PG Journal Number 17/2011
Publication Date 29-Apr-2011
Grant Date 28-Apr-2011
Date of Filing 06-Jul-2006
Name of Patentee FERMENTA BIOTECH LIMITED
Applicant Address 'DIL' COMPLEX, GHODBUNDER ROAD, MAJIWADA, THANE (WEST)-400610,
Inventors:
# Inventor's Name Inventor's Address
1 DATLA, ANUPAMA 'DIL' COMPLEX, GHODBUNDER ROAD, MAJIWADA, THANE (WEST)-400610,
2 VYASARAYANI, RAJASEKAR WILLIAMS 'DIL'Complex,Ghodbunder Road, Majiwada,Thane(West)-400 610, Maharashtra,India
3 ZAMBRE, SUJATA YOGESH 'DIL'Complex,Ghodbunder Road, Majiwada,Thane(West)-400 610, Maharashtra,India
4 ASHER, TRUPTI KRISHAKANT 'DIL'Complex,Ghodbunder Road, Majiwada,Thane(West)-400 610, Maharashtra,India
5 SHARMA, NIKUNJ KUMAR 'DIL'Complex,Ghodbunder Road, Majiwada,Thane(West)-400 610, Maharashtra,India
PCT International Classification Number C12N11/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA