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 Epoxy Reactive Terpolymer Beads For The Immobilization Of
Penicillin G Acylase And Its Use In The Synthesis Of Semi-Synthetic β-Lactam
Antibiotics And Process For Preparation 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 a custom designed, macroporous terpolymer beads for the immobilization of Penicillin G acylase (PGA) to form rigid and stable enzyme catalyst and its subsequent application in enzymatic synthesis of semi-synthetic p-lactam antibiotics.
BACKGROUND AND PRIOR ART:
Semi-synthetic p-lactam antibiotics due to better efficacy and broad spectrum action have captured drug market attention and commercial importance. Due to the growing environmental concern and need for eco-efficient processes, industrial synthesis of semisynthetic antibiotics by enzymatic route is preferred over chemical route due to obvious advantages of enzymatic reaction being stereo specific, not requiring protection/ deprotection of side groups or use of organic solvents. In addition, enzymatic reaction does not need extreme temperature or pressure during reaction, forming less by products apart from being environment friendly.
Semi-synthetic antibiotics are synthesized by enzyme catalysed acylation of p-lactam nucleus with amino acid derivative by either thermodynamically or kinetically controlled process. In the earlier case, yields are dependent on thermodynamic equilibrium constant and presently on industrial scale, there is not much of success in thermodynamic coupling due to use of solvents or metal ions which may affect the enzyme (US Patent 5268271', R. V.Ulijn et al.,20022)
Enzymatic synthesis of antibiotics is influenced by various process parameters like pH, temperature, concentration of activated acyl donor or p-lactam nucleus, relative substrate concentration, use of solvents and enzyme type and immobilization system for enzyme (Alemzadeh et al, 20103). The effects of organic solvents on the PGA catalyzed, kinetically controlled synthesis of semi-synthetic antibiotics have been examined in various water-solvent mixtures (Chan B Park, 2000 4). One of the most significant parameter is the enzyme catalyst and support used for immobilization which remarkably influences the course of reaction. Also for kinetically controlled synthesis, yield depends

on balance between synthesis of product (synthetic activity), hydrolysis of activated acyl donor (AAD) (esterase activity) and hydrolysis of product synthesized previously, (Amidase activity) represented by S/H ratio in enzyme kinetics. Obviously design of enzyme catalyst and the enzymatic process is to favour the synthetic process and to reduce hydrolysis of the AAD and the final antibiotic, to achieve maximum product yields. In addition, most of the antibiotic synthesis leads to precipitation of the final product and the hydrolyzed side chain, thus the reaction is precipitate driven or suspension based reaction leading to physical attrition of the immobilized enzyme in the presence of the thick slurry like reaction mass. So in addition to process improvement efforts, there is an imperative need to design a suitable enzyme catalyst with good mechanical stability.
The chemical synthesis of active pharmaceutical ingredients is known for having a high E-factor (ratio of the mass of waste per unit of product). In this context, the enzymatic synthesis is considered particularly 'eco-friendly'. The main enzyme exploited to develop green processes for the production of p-lactam antibiotics is PGA. PGA is a relevant industrial enzyme for the large-scale production of 6-aminopenicilIanic acid (6APA) and 7-amino-3-deacetoxicephalosporanic acid (7ADCA), both being key precursors for the synthesis of p-lactam antibiotics. These intermediates are mostly produced from penicillin G (Pen G) and cephalosporin G using immobilized PGA. Biotechnological applications of PGA has emerged as viable alternative to traditional chemical procedures for manufacture of p-lactam antibiotics, intermediates, small peptides, and pure isomers from racemate mixture. PGA catalysts have also been evaluated for the synthesis of second generation p-lactam antibiotics from 6-APA and 7-ADCA like amoxicillin and cephalexin, these being an interesting application of this hydrolytic enzyme in reactions of synthesis.
Besides amide hydrolysis, PGA is able to catalyze the reverse reaction as, for instance, in the N-acylation reactions of p-lactam core. This process finds a large industrial application to prepare semi-synthetic penicillins and cephalosporins (Youshko et al, 20025).
Hence, with over a decade of holygrail on enzymatic synthesis of antibiotics, use. of immobilized PGA has been successfully implemented at various scales, pioneered by

DSM followed by several players. One of the main issue in the enzymatic synthesis of semi-synthetic penicilins and cephalosporins is the suspension nature of the reaction leading to critical diffusional limitation as well as physical attrition of the enzyme carrier. Hence, it assumes importance to study various immobilization systems which can be used in the above mentioned application.
One of the most successful industrial processes using immobilized PGA enzyme as a catalyst is the preparation of p-lactam building blocks namely, 6-APA, 7-ADCA. The emerging new applications include synthesis of structural analogs of penicillins and cephalosporins, synthesis of antibiotics by replacing hazardous chemical process (e.g. amoxicillin and cephalexin).
For the successful implementation of an enzyme based technology, it is imperative to have the enzyme immobilized on a suitable platform, with overall process and economic advantage. Immobilization of enzyme by definition means "fixing the enzyme or any biomolecule on a fixed support so that the desired reaction is carried out". There are several advantages of using immobilized enzymes as compared to using the soluble counter parts, the primary advantage being enzyme reusability with the aim of reducing the production cost by efficient recycling and control of the process. In addition, the immobilized enzyme can be handled easily and after the reaction, the product of interest can be separated easily. Enzyme immobilization also enhances overall operational stability of the enzyme.
The technology for PGA immobilization has been improved greatly over the years and is still evolving. For instance, PGA is covalently bound on various supports such as micro particulate and monolithic silica supports, poly (vinyl acetate-co-divinyl benzene) beads (Jianguo et ah, 2001 6), grafted nylon membranes , hydrophobic acrylic carriers ( Bryjak and Trochimczuk, 2006 7), use of macroporous co-polymers of epoxy supports (Xue et al, 2006 8), magnetic hydroxyl particles (Wang et ah., 2007 9), use of activated chitosan for immobilization of permeabilized E.coli cells having PGA (Bagherinejad et ah, 2012 10), use of magnetic nano particles (Huocong et ah, 2012 11), surface immobilization of PGA on PVC membrane (Eldin et ah, 2012 12), to mention the few.

Among various immobilization methods studied in literature, covalent binding on solid supports (beaded polymer) has proved to be more efficient at industrial scale. However, with the growing demand and application portfolio for immobilized enzymes, there is a need for developing more efficient immobilized enzymes. Carrier support and method for immobilization not only influences operational stability and reusability but also catalytic efficiency, kinetic properties, thermo stability, tolerance to reaction conditions like pH, temperature and solvents. As discussed earlier, one of the major drawback for the industrial implementation of the enzymatic synthesis of p-lactam antibiotics is the limited yield of the product, due to undesirable hydrolytic reactions. Since enzymatic synthesis of p-lactam antibiotics invariable leads to precipitation of the product and the by-product during the reaction, the immobilized enzyme catalyst undergoes severe attrition as well as diffusional limitation. One of the latest approach to partially circumvent the first drawback was reported by reducing the water activity in the medium by using ionic liquids where in the immobilized PGA showed very high selectivity towards amoxicillin synthesis (Pereira et ah., 2012 ,3). Hence this process may not be industrially viable. For the second draw back as discussed above, one of the solution is to prepare highly stable acrylic carriers which can withstand the physical abrasion and also reduces the diffusional limitation. PGA from Arthrobacter viscosus immobilized on hydrophobic acrylic epoxy-supports (Eupergit C) during N -acylation of 7-mmocephalosporanic acid acted as a poor matrix and showed low turnover rate.(Terrani M et.aI-2007 l4, US 7,264,943 B2.-2007 15) PGA entrapped in hydrophilic gel supports acrylamide N,N-methylene bis acrylamide (EP 2173892 2011 l6) and Polyvinyl alcohol-gelatin. (PCT/IN2008/00769 l7) used in suspension reaction of enzymatic acylation have disadvantage of reduced operational stability, increased swell volume and filtration time thereby affecting process cost. Also in spite of high specific activity achieved in gel immobilized enzyme, gels being more hydrophilic drives the reaction more towards hydrolysis of ester and product than synthesis, thus reducing product yield. US patent 6218138B1, 2007 reported EupergitR C (copolymer of methacrylamide, N, N, methylene bis acrylamide, glycidyl methacrylate and allyl glycidyl ether) as support for covalent immobilization of PGA from E.coli and suitability of immobilized enzyme for reusability for 5 cycles in synthesis of p-lactam antibiotics. Additionally, multipoint covalent attachment has been proven fruitful for increasing enzyme stability as reported by several authors (Mateo et al, 2005 18, Sheldon, 2007 19).

Most of the immobilized enzyme catalyst which are commercially exploited, were beaded polymer systems prepared by suspension polymerization. Protein and enzyme immobilization on non-porous microspheres on polystyrene reported for immobilization of p-lactamase and Concavalin A by covalent binding of enzyme which were found suitable for column reactor. Macroporous poly(glycidyl methacrylate-co-divinyl benzene) polymer particles for immobilization of p-galactosidase from Aspergillus oryzae is reported , where epoxy groups on surface covalently bind the enzyme and divinyl benzene as cross linking agent alters the pH stability, thermal and operational stability.
Adsorption induced denaturation of alpha-chymotrypsin on poly (allyl glycidyl-co-ethylene dimethacrylate) due to polymer hydrophocity (Lahari et al 2010 20).
Poly (acrylonitrile-co-glycidyl methacrylate) nanofibrous mats used for immobilization of Candida antarctica Lipase was reported for improving storage stability and better reusability. (Tianhe et al,2011 21).
Synthetic polymer resins, both porous and nonporous generated by free radical polymerization are widely popular as stable and inert supports for binding different enzymes including PGA. The present invention is focused on using new combinations of monomers to develop new polymer systems based on suspension polymerization.
It is obvious from the reports that immobilization of PGA on either hydrophilic or hydrophobic supports renders it less productive in enzymatic acylation reaction. Hence there is need to design a support with a balance of hydrophobicity and hydrophilicity which is not only suitable for immobilization but also influences kinetic and catalytic properties of PGA to make it more selective for the synthetic reaction over hydrolysis in enzymatic acylation.
Most of the porous polymer resins or acrylic epoxy supports synthesized by suspension polymerization are polymer or copolymers as beads or membrane used for adsorption or covalent binding of various enzymes. In view of prior art, the present invention attempts to design more robust acrylic epoxy supports which are not only suitable for

immobilization but are also selective for synthesis and recyclability in suspension reaction.
SUMMARY OF THE INVENTION:
In accordance with the above, the present invention discloses macroporous terpolymer systems with three monomers which are polymerized by free radical suspension polymerization to form macroporous beads.
In this aspect, terpolymer systems which are used to prepare macroporous beads consist of monomer with epoxy functional group, cross linker monomer and acrylonitrile with a porogenic diluent, polymerization initiator and stabilizer, by suspension polymerization.
The acrylonitrile as one of the monomer serves as rigid backbone for aligning the epoxy groups. Similar use of acrylonitrile was reported in modification of epoxy resin using reactive liquid rubber, (N. Chikhi et al, 2002 22 and J Lopez et al„ 2001 23). However, there are few reports of using acrylonitrile in beaded polymer supports for enzyme immobilization and to best of knowledge, there are no reports of using acrylonitrile copolymers or terpolymers for the purpose of synthesis of semi-synthetic penicillins and semi-synthetic cephalosporins. Hence present invention wherein the acrylonitrile-epoxy group cross linked with different crosslinking density in terpolymer series form macroporous beads of physical properties serve as suitable supports favouring PGA immobilization to form immobilized enzyme catalyst.
In another aspect the immobilized enzyme catalyst by virtue of use of terpolymer support shows maximum enzyme binding and expressed activity of enzyme rendering it suitable to achieve conversion of substrate to product in desired time.
In another aspect, significant content of acrylonitrile in terpolymer imparts rigidity, strength and hydrophobic-hydrophilic balance to the macroporous beads preventing breakage of beads and rendering reusability of immobilized enzyme catalyst in suspension reaction.

BRIEF DESCRIPTION OF FIGURES:
Figure 1 (A) to Figure 3 (A): Scanning Electron Microscope (SEM) micrographs of
macroporous beads as per examples.
Figure 1 (A): SEM micrograph of macroporous beads from Example-3
Figure 1 (B): SEM micrograph of macroporous beads from Example-3
Figure 2 (A): SEM micrograph of macroporous beads from Example-8
Figure 2 (B): SEM micrograph of macroporous beads from Example-8
Figure 3 (A): SEM micrograph of macroporous beads from Example-3 (A)
Figure 4 (A): Time course profile of % 7-ADCA conversion in CEX synthesis with
immobilized enzyme catalyst prepared in Example 1 to Example 5.
Figure 4 (B): Time course profile of % PGME hydrolysis in CEX synthesis with
immobilized enzyme catalyst prepared in Example 1 to Example 5.
Figure 5 (A): Time course profile of % 7-ADCA conversion in CEX synthesis with
immobilized enzyme catalyst prepared in Example 6 to Example 10.
Figure 5 (B): Time course profile of % PGME hydrolysis in CEX synthesis with
immobilized enzyme catalyst prepared in Example 6 to Example 10.
Figure 6 (A): Time course profile of % 7-APCA conversion in CPZL synthesis with
immobilized enzyme catalyst prepared in Example 6 to Example 10.
Figure 6 (B): Time course profile of % HPGME hydrolysis in CPZL synthesis with
immobilized enzyme catalyst prepared in Example 6 to Example 10.
Figure 7 (A): Time course profile of % 7-ACCA conversion in CCL synthesis with
immobilized enzyme catalyst prepared in Example 1 to Example 5.
Figure 7 (B): Time course profile of % PGME hydrolysis in CPZL synthesis with
immobilized enzyme catalyst prepared in Example 1 to Example 5.
Figure 8 (A): Time course profile of % 7-ADCA conversion in CDL synthesis with
immobilized enzyme catalyst prepared in Example 3(A), Example 3 (B)> Example 4(A),
Example 4 (B), Example 8(B), and Example 9 (B).
Figure 8 (B): Time course profile of % HPGME hydrolysis in CDL synthesis with
immobilized enzyme catalyst prepared in Example 3(A), Example 3 (B), Example 4(A),
Example 4 (B), Example 8(B), and Example 9 (B).

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.
Abbreviations:
PGA : Penicillin G acylase
Pen G : Penicillin G
6-APA : 6-Aminopenicillinic acid
7-ADCA : 7-Aminodesacetoxycephalosporanic acid
7-ACA : 7-Aminocephalosporanic acid
7-ACCA : 7-amino-3-chloro-3-cepham cephalosporanic acid
7-APCA : 7-amino-3-propylene-3-cepham cephalosporanic acid
HPGME : D (-)-4-hydroxy phenyl glycine methyl ester hydrochloride
AMOX : Amoxicillin
CEX : Cephalexin
HPLC : High-performance liquid chromatography
PGME : D (-)-phenyl glycine methyl ester hydrochloride
CDL : Cefadroxil
CPZL : Cefprozil
CCL : Cefaclor
AGE : Allyl glycidyl ether
GMA : Glycidyl methacrylate
EGDM : Ethylene glycol dimethacrylate
DVB : Divinyl benzene
MMA : Methyl methacrylate
EMA : Ethyl methacrylate
AN : Acrylonitrile
nm : Nano meter
mL : Milli liter
M : Molar
min : Minute

mM : Milli Molar
μl : Micro liter
cm3/g : centimeter cube per gram
m2/g. : square meter per gram
°C : degree Celsius
The present invention discloses the terpolymer system forming macroporous beads and immobilization of PGA to form efficient enzyme biocatalyst. Further the immobilized enzyme catalyst find application in enzymatic acylation for synthesis of semi-synthetic (3-lactam antibiotics.
The terpolymer systems which are used to prepare macroporous beads consist of monomer with epoxy functional group, cross linker monomer and acrylonitrile with a porogenic diluent, polymerization initiator and stabilizer, by suspension polymerization
The .terpolymer systems prepared by suspension polymerization have range of macroporous beads with effective crosslinking and epoxy groups on a rigid acrylic backbone. Thus the resulting macroporous beads with variable crosslinking density have balance of hydrophobicity and hydrophilicity making it suitable for PGA binding and significant expression of synthetic activity over hydrolysis and reusability in suspension reaction.
In one embodiment, terpolymer systems includes monomer (Ml) containing epoxy functional groups for covalent enzyme binding and are capable of free radical polymerization comprising either glycidyl methacrylate (GMA), or allyl glycidyl ether (AGE) alone or in combination. The concentration of said Ml used is between 15-60% of total weight of the monomers.
In embodiment, included is monomer (M2) comprising of hydrophobic cross linker used in variable concentration and are capable of free radical polymerization selected from divinylbenzene (DVB) or ethylene glycol dimethacrylate (EGDM) used alone or in combination. The concentration of said M2 used is 20-55% of total weight of the monomers.

Crosslinking of polymer refers to chemical reaction with specific chemical to form covalent bond or ionic bonds with other monomer components of the reaction mixture to alter the mechanical properties like viscosities and strength of the resulting polymer. The chemical which serves as crosslinker may be monomer forming part of the reaction mixture and itself undergoes polymerization. The concentration of crosslinker is significant to obtain the desired property in the final product and is generally considered w.r.t to concentration of other components in reaction mixure.
The crosslinking density of the terpolymer is calculated by the molar % of M2 added with respect to total molar % of Ml and M2 where in the concentration of M3 is constant. The crosslinking density in terpolymer series varies between 25% and 200%.
In embodiment, monomer (M3) serving as backbone and capable of free radical polymerization to form terpolymer includes either acrylonitrile (AN) or methyl methacrylate (MMA) or ethyl methacrylate (EMA). The concentration of said M3 used is 20-55%) of total weight of the monomers
The porogenic diluent is selected from a group of higher aliphatic or cyclic alcohol such as Iauryl alcohol, octanol and cyclohexanol or combination of these solvents. The weight of porogenic diluents varies up to 1.2 -1.5 times with respect to total weight of the monomers.
The initiator is selected from benzoyl peroxide, azobisisobutyronitrile, methyl ethyl ketone peroxide. The weight of initiator used is up to 4% with respect to total weight of the monomers.
The suspension stabilizer is selected from poly vinyl pyrollidone, poly vinyl alcohol, poly acrylic acid and the like. The weight of stabilizer used is up to 8%) with respect to total weight of the monomers.
The present invention also provides a series of terpolymer system in the form of macroporous beads consist of poly(AGE-ter-EGDM-ter-AN) and poly(GMA-ter-DVB-ter-AN).

The macroporous beads were subjected to particle size distribution and porosity measurements by using laser diffraction analyzer (Particle size analyser, HELOS H1004) 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.
In another embodiment, the immobilized enzyme catalyst based on macroporous beads have particle size between 200-500 microns, average pore diameter between 30-120 nm, pore volume between 0.6-1.2 cm3/g and surface area between 50 m2/g-110 m2/g.
The said enzyme in the embodiment immobilized on macroporous beads from terpolymer systems include PGA or alpha amino ester hydrolase to form immobilized enzyme catalyst.
The said PGA is from Achromobacter sp. 7394 cloned in E.coli RE3 bearing the plasmid pKXIPl and used as purified enzyme.
The bound enzyme in immobilized enzyme catalyst is quantified in terms of protein binding determined by Bradford method and activity.
The said activity is expressed as U/g wet of biocatalyst catalyzing hydrolysis of 25 mM of Pen G K in 50 mM sodium phosphate buffer pH 8.0, at 37°C to release phenyl acetic acid, neutralized with 0.1 N sodium hydroxide in the milieu of first order kinetics.
The immobilized enzyme catalyst from different terpolymer series show 60-90% protein binding and 15-40% enzyme expression and the total activity binding up to 97.7%. In another embodiment wherein the said immobilized enzyme catalyst find application in reaction for enzymatic synthesis of semi-synthetic p-lactams includes the group of semisynthetic penicillin and semi-synthetic cephalosporin antibiotics namely Amoxicillin (AMOX), Ampicillin (AMP), Cephalexin (CEX), Cefadroxil (CDL), Cefaclor (CCL), and Cefprozil (CPZL).
In another embodiment, the present invention discloses a process for synthesis of semi-

synthetic p-lactam antibiotics using immobilized enzyme catalyst comprises;
a) reacting P-lactam nucleus with activated acyl donor in the molar ratio of 1:1.2 to 1:1.5;
b) adding immobilized enzyme catalyst in the ratio of 250U/g of activity per gram of p-lactam nucleus;
c) conversion of (3-lactam nucleus achieved between 74-86 % in 120 minutes for 10 repeated cycles;
d) reusing immobilized enzyme catalyst for 10 repeated cycles in suspension reaction containing substrates and
e) precipitating product as semi-synthetic p-lactam antibiotic.
The activity of immobilized enzyme catalyst used in synthesis of semi-synthetic p-lactam antibiotics remained unchanged after 10 repeated cycles.
The substrates in said reaction wherein in the said immobilized enzyme catalyst catalyses the acylation to synthesize semi-synthetic p-lactam antibiotic are p-lactam nucleus selected from 6-aminopenicillanic acid (6-APA), 7-desacetoxycephalosporanic acid (7ADCA), 7-aminocephalosporanic acid (7-ACA), 7-Amino-3-Chloro-3-Cephem-4-Carboxylicacid (7ACCA), and 7-Amino-3-[(Z)-propen-l-yl] -3-cephem-4-carboxylic acid (7APCA) and activated acyl donor selected from ester or amide of hydroxyphenyl glycine (HPGME) or phenylglycine (PGME).
The concentration of p-lactam nucleus in the said reaction varies between 230 to 240mM and concentration of activated acyl donor varies from 320-340mM.
The said reaction mixture consists of p-lactam nucleus and activated acyl donor in molar ratio of 1:1.2 - 1:1.5. The weight of immobilized enzyme catalyst is added equivalent to 250 U/g of activity per gram of P-lactam nucleus. The concentration of substrates and immobilized enzyme catalyst in water varies from 8-10% (w/v). The pH of reaction is maintained between 6.3-6.5 with 12% ammonium hydroxide solution and temperature at 23-25. °C. The substrate and product conversion is determined by HPLC analysis, samples taken at regular interval.

The synthesis of antibiotic to hydrolysis of the respective acyl donor determined by HPLC analysis under the said reaction conditions mentioned in the embodiment and S/H ratio calculated thereof.
The said reaction is described for AMOX, CEX, CPZL, CCL, CDL as representative antibiotics and likely to function for other antibiotics mentioned in the embodiment. The reaction functions with said PGA but likely to function with alpha-amino ester hydrolase.
HPLC analysis for AMOX synthesis:
Column: C18 Inertsil, Mobile phase: 97:3 0.1 M potassium phosphate buffer pH 5.0: acetonitrile, Flow rate: 1.2mL/min., Wavelength: 215 nm., Injector Volume: 20 ul, Run Time: 20 minutes.
HPLC analysis for CEX:
Column: Inertsil C8 250mm X 4.6 mm (5micron), temperature 35 °C, Buffer: 16.95 g Tetra-n-butyl ammonium hydrogen sulphate + 6.8 g potassium dihydrogen orthophosphate + 2.0 ml Triethylamine dissolved in 1 liter HPLC grade water, pH 6.4 with 2 N Sodium hydroxide. Mobile phase: Buffer: Methanol: Acetonitrile::70: 23: 7, Column oven temperature 35 °C, Detection Wavelength: 225 nm, Flow Rate: 1.2 mL/min, Injection Volume: 20 μL
HPLC analysis for CPZL:
Column: Inertsil C8 250mm X 4.6 mm (5micron), temperature 30 °C, Buffer: 11.5 g ammonium hydrogen phosphate (pH 5.4) in 1 liter HPLC grade water, pH 5.4 with 2 N Sodium hydroxide. Mobile phase: Acetonitrile: Methanol::70:30, Column oven temperature 30 °C, Detection Wavelength: 240 nm, Flow Rate: 1.00 mL/min, Injection volume: 20 ul.
HPLC analysis for CCL:
Column: Inertsil C8 250mm X 4.6 mm (5micron), temperature 35 °C, Buffer: 0.1 M ortho-phosphoric acid + 0.4% Triethylamine in 1 liter HPLC grade water, pH 2.5 with 2 N potassium hydroxide. Mobile phase: Buffer: Methanol::80:20, Column oven

temperature 35 °C, Detection Wavelength: 240 nm, Flow Rate: 1.2 mL/min, Injection volume: 20 μl.
HPLC analysis for CDL:
Column: Inertsii C8 250mm X 4.6 mm (5micron), temperature 25 °C, Buffer: 13.6 g potassium dihydrogen phosphate dissolved in 1 liter HPLC grade water pH 5.0 with 2 N Sodium hydroxide. Mobile phase: Buffer: Acetonitrile:: 97:3, Column oven temperature 25 °C, Detection Wavelength: 240 nm, Flow Rate: 1.2 mL/min, Injection volume: 20 u.1.
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 1-5: poly(AGE-ter-EGDM-ter-AN) terpolymers consist of composition and quantities as described in Table 1.
Table 1: Compositions of poly(AGE-ter-EGDM-ter-AN) terpolymers with varying cross-link density

Polymer No. AGE
Ml EGDM M2 AN M3 % Cross
Link Density
Wt. in g Wt. in g Wt. in g

Example 1 14.6 6.4 9 25
Example 2 11.2 9.8 9 50
Example 3 7.7 13.4 9 100 Figure 1(A) and Figure 1(B)
Example 4 5.8 15.2 9 150
Example 5 4.7 16.4 9 200

In the inert atmosphere of Nitrogen, the respective monomers in mentioned quantities with 38g cyclohexanol are stirred with 135 ml distilled water at 300 rpm and polymerized using polyvinyl pyrollidone and azobisisobutyronitrile for 4 hrs at 70 °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 oven drying at 40°C.
Example 3A: terpolymer system prepared by the procedure as per Example-3, replacing cyclohexanol with lauryl alcohol. [Figure 1(C)]
Example 4A: terpolymer system prepared by the procedure as per Example-4, replacing cyclohexanol with lauryl alcohol.
Example 3B: terpolymer system prepared by the procedure as per Example-3, replacing cyclohexanol with octanol.
Example 4B: terpolymer system prepared by the procedure as per Example-4, replacing cyclohexanol with octanol.
Example 6-10: poly(GMA-ter-DVB-ter-AN) terpolymers composition consist of composition and quantities as described in Table 2.
Table 2: Compositions of poly (GMA- ter-DVB-ter-AN) terpolymers with varying cross-link density

Polymer No. GMA
Ml DVB
M2 AN M3 % Cross
Link Density
Wt. in g Wt. in g Wt. in g

Example 6 17.10 3.95 9.00 25
Example 7 14.40 6.60 9.00 50
Example 8 11.00 10.10 9.00 100 Figure 2(A) and Figure 2(B)
Example 9 8.85 12.18 9.00 150
Example 10 7.40 13.60 9.00 200

In the inert atmosphere of nitrogen, the respective monomers in mentioned quantities with 38g cyclohexanol are stirred with 135 mL distilled water at 300 rpm and polymerized using polyvinyl pyrollidone and azobisisobutyronitrile for 4 hrs at 70 °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 oven drying at 40°C.
Example 8A: terpolymer system prepared by the procedure as per Example-8, replacing cyclohexanol with lauryl alcohol.
Example 9A: terpolymer system prepared by the procedure as per Example-9, replacing cyclohexanol with lauryl alcohol.
Example 8B: terpolymer system prepared by the procedure as per Example-8, replacing cyclohexanol with octanol.
Example 9B: terpolymer system prepared by the procedure as per Example-9, replacing cyclohexanol with octanol.
Example 10: Physical Data of macroporous beads from Example 1-10, 3A, 8A, and 8B listed in Table 3.
Table 3: Physical and textural data of terpolymers.

Macroporous beads Average Pore
Diameter
(nm) Total porosity (%) Pore volume (cm3/g) Specific surface area Bulk Density (g/cm3)
Example 1 90.7 37.779 1.173 98.530 0.322
Example 2 93.7 51.059 1.009 81.737 0.506
Example 3 75.8 40.020 1.085 104.390 0.369
Example 4 80.5 45.907 1.125 106.680 0.408
Example 5 69.4 38.030 1.020 106.500 0.373
Example 3A 121.0 35.990 0.901 54.740 0.399

Example 6
47.5 40.199 0.625 83.760 0.64
Example 7 52.3 42.540 0.776 110.570 0.547
Example 8 52.0 48.660 0.754 104.830 0.644
Example 9 47.0 45.160 0.710 105.720 0.635
Example 10 33.9 28.270 0.611 109.930 0.462
Example 8A 64.0 48.355 0.909 60.750 0.531
Example 8B 63.8 36.211 0.776 55.140 0.466
Example 11: Protein binding and Substrate specificity of enzyme in terpolymer series
The resultant immobilized enzyme catalysts mentioned in the present embodiment are found to have higher protein binding and broad substrate specificity. The following determination method is familiar per se to the person skilled in the art of covalent immobilization and is detailed only for the sake of completeness.
Determination of the binding capacity for said PGA.
1 g of macroporous bead are added to 100 Units of said PGA in 1 M potassium phosphate buffer pH 7.5 and incubated at 25° C for 48 h. The macroporous polymer beads are then vacuum filtered and washed thrice with deionized water and then twice with 0.1 M potassium phosphate buffer pH 7.5. The moist weight of the resulting macroporous beads loaded with PGA is determined. The swell volume after immobilization is between 1.2-1.3 times of the initial volume of macroporous beads.
Table 4: The protein binding, activity binding and activity expression of immobilized enzyme catalyst.

Immobilized enzyme catalyst % Protein Binding % Activity binding % Activity Expression
Example 1 60.42 93.1 20.15
Example 2 74.39 93.64 26.82

Example 3
73.05 95.28 26.25
Example 4 66.94 94.8 18.84
Example 5 90.14 97.91 40.92
Example 3A 48.00 57.00 15.40
Example 3B 38.300 83.00 19.80
Example 4A 39.00 54.00 20.00
Example 4B 53.00 58.00 16.70
Example 6 73.81 72.68 32.88
Example 7 83.93 87.8 35.83
Example 8 86.46 82.67 33.34
Example 9 80.38 86.28 31.95
Example 10 85.31 60.21 33.57
Example 8A 55.12 68.00 11.40
Example 8B 72.40 80.01 12.72
Example 9A 69.00 69.34 23.11
Example 9B 60.17 97.93 10.43
Table 5: Immobilized enzyme catalyst activity, S/H ratio of AMOX and CEX.

Immobilized enzyme catalyst Activity (U/g Wet) (S/H) AMOX (S/H) CEX
Example 1 63.65 1.26 0.33
Example 2 85.95 1.07 0.76
Example 3 80.84 1.04 0.76
Example 4 56.69 1.18 1.49
Example 5 126.98 1.05 0.45
Example 3A 11.30 0.45 0.22
Example 3B 88.68 1.19 0.73
Example 4A 9.11 0.47 0.21
Example 4B 22.73 0.34 0.09
Example 6 110.70 0.6 0.39

Example 7
142.56 0.55 0.41
Example 8 143.57 0.53 0.44
Example 9 124.18 0.73 0.31
Example 10 91.37 1.23 0.34
Example 8A 29.19 0.28 0.07
Example 8B 58.27 1.34 0.13
Example 9 A 13.39 0.35 0.17
Example 9B 49.01 1.33 0.16
Example 12: Enzymatic synthesis of Cephalexin
The reaction is carried out at 25 °C, pH 6.3 for 2 hrs in a stirred tank reactor. Reaction mixture consists of 233 mM 7-ADCA and 326 mM PGME with 225 U of immobilized enzyme catalyst per gram of 7-ADCA in 10 mL of water. Course of the reaction monitored by withdrawing samples at regular interval of time and analyzed by HPLC.
The respective immobilized enzyme catalyst from Example 1-10, 8B, and 9B were used for synthesis of cephalexin as in process described above and results of each of the reaction listed in Table 6. The course of reaction with use of immobilized enzyme catalyst from Example 1 to Example 10 in terms of % conversion of 7ADCA and PGME is represented in Figure 4(A), 5(A) and Figure 4(B), 5(B) respectively.
Table 6: % Conversion of 7ADCA and PGME in synthesis of CEX with immobilized enzyme catalyst

Immobilized enzyme catalyst % 7-ADCA Conversion % PGME Conversion
Example 1 86.85 84.18
Example 2 86.03 83.74
Example 3 84.17 78.63
Example 4 74.39 70.04
Example 5 77.65 70.59
Example 6 85.33 83.62

Example 7
82.90 73.72
Example 8 84.05 77.89
Example 9 82.69 75.52
Example 10 78.30 73.9
Example 8B 81.61 75.71
Example 9B 81.03 74.24
Example 13: Enzymatic synthesis of Cefprozil (CPZL)
The reaction is carried out at 25 °C, pH 6.5 for 2 hrs in a stirred tank reactor. Reaction mixture consists of 208 mM 7-APCA and 312 mM HPGME with 225 U of immobilized enzyme catalyst per gram of 7-APCA in 10 mL of water. Course of the reaction monitored by withdrawing samples at regular interval of time and analyzed by HPLC.
The respective immobilized enzyme catalysts from Example 6-10 were used for synthesis of cefprozil as in process described above and results of each of the reaction listed in Table 7. The course of reaction with use of immobilized enzyme catalyst from Example 6 to Example 10 in terms of % conversion of 7APCA and HPGME is represented in Figure 6(A) and Figure 6(B) respectively.
Table 7: % Conversion of 7APCA and HPGME in synthesis of CPZL with immobilized enzyme catalyst

Immobilized enzyme catalyst % 7-APCA
Conversion % HPGME Conversion
Example 6 83.67 85.13
Example 7 86.99 80.47
Example 8 82.43 83.76
Example 9 87.98 84.33
Example 10 87.30 84.33
Example 14: Enzymatic synthesis of Cefaclor (CCL)

The reaction is carried out at 25 °C, pH 6.5 for 2 hrs in a stirred tank reactor. Reaction mixture consists of 213 mM 7-ACCA and 319 mM PGME with 225 U of immobilized enzyme catalyst per gram of 7-ACCA in 10 mL of water. Course of the reaction monitored by withdrawing samples at regular interval of time and analyzed by HPLC.
The respective immobilized enzyme catalyst from Example 1-5 were used for synthesis of cefaclor as in process described above and results of each of the reaction listed in Table 8. The course of reaction with use of immobilized enzyme catalyst from Example 1 to Example 5 in terms of % conversion of 7ACCA and PGME is represented in Figure 7(A) and Figure 7(B) respectively.
Table 8: % Conversion of 7ACCA and PGME in synthesis of CCL with immobilized enzyme catalyst

Immobilized enzyme catalyst % 7-ACCA Conversion % PGME Conversion
Example 1 61.4 59.8
Example 2 60.4 58.4
Example 3 54.0 52.2
Example 4 58.4 55.3
Example 5 52.6 50.4
Example 15: Enzymatic synthesis of Cefadroxil (CDL)
The reaction is carried out at 25 °C, pH 6.5 for 2 hrs in a stirred tank reactor. Reaction mixture consists of 233 mM 7-ADCA and 350 mM HPGME with 225 U of immobilized enzyme catalyst per gram of 7-ADCA in 10 mL of water. Course of the reaction monitored by withdrawing samples at regular interval of time and analyzed by HPLC.
The respective immobilized enzyme catalysts from Example 3(A), 3(B), 4(A), 4(B), 8(B) and 9(B) were used for synthesis of cefprozil as in process described above and results of each of the reaction listed in Table 9. The course of reaction with use of immobilized enzyme catalyst from Example 3(A), 3(B), 4(A), 4(B), 8(B) and 9(B) in terms of %

conversion of 7ADCA and HPGME is represented in Figure 8(A) and Figure 8(B) respectively.
Table 9: % Conversion of 7ADCA and HPGME in synthesis of CDL with immobilized enzyme catalyst

Immobilized enzyme catalyst % 7-ADCA Conversion % HPGME Conversion
Example 3(A) 83.4 82.6
Example 3(B) 89.8 88.4
Example 4(A) 90.1 89.4
Example 4(B) 91.3 89.9
Example 8(B) 90.6 79.8
Example 9(B) 81.4 80.6
Example-16: Stability of macroporous beads in immobilized enzyme catalyst
The immobilized enzyme catalysts were recycled for 10 batches in the said reaction mentioned in Example 13. The immobilized enzyme catalyst retained the activity and particle size after 10 batches. The % conversion of 7ADCA after 10 cycles also remained consistent.
Advantages:
1. The macroporous beads serve as efficient support to improve the S/H ratio of PGA enzyme and show minimal swelling after enzyme binding.
2. The enzyme biocatalyst so formed can be suitably used in heterogenous system.
3. It catalyses the acylation of p-lactam nucleus with phenylated side chain with efficient conversion of substrate to product in time frame and limiting undesirable product hydrolysis which influence process economics.
4. The immobilized enzyme retains the enzyme activity, resists particle attrition.
5. Limits adherence of substrate or the product to the enzyme on subsequent use, can be separated from reaction and reused.
6. This process improves viability of process and cost gain ratio on industrial scale.

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We Claim,
1. Aerylonitrile based macroporous terpolymer beads, for immobilization of enzymes to form immobilized enzyme catalyst useful in enzymatic synthesis of semi-synthetic β-lactam antibiotics.
2; Acrylonitrile based macroporous terpolymer beads according to claim 1, wherein said terpolymer beads comprises monomer with epoxy functional group, cross linker monomer, acrylonitrile with a porogenic diluent, polymerization initiator and stabilizer.
3. Acrylonitrile based macroporous terpolymer beads according to claim 2, wherein said terpolymer beads comprise;
a) 20-55% of total weight of the monomer acrylonitrile (AN);
b) 15-60% of total weight of monomers with epoxy functional group selected from AGE or GMA;
c) 20-55% of total weight of cross linker monomer selected from DVB or EGDM;
d) porogenic diluent selected from the group of hydrophilic or cyclic alcohols comprises of cyclohexanol, octanol, lauryl alcohol and/or combinations thereof, in an amount of 1.2-1.5 times of total weight of the monomers;
e) initiator selected from the group of benzoyl peroxide, azobi si sobutyron itrile, methyl ethyl ketone peroxide, in an amount of 3-3.5% with respect to total weight of monomers; and
f) suspension stabilizer selected from the group of poly vinyl pyrollidone, poly vinyl alcohol, poly acrylic acid, in an amount of 7-7.5% with respect to total weight of monomers.
4.. Acrylonitrile based macroporous terpolymer beads according to claim 1 to 3, wherein said terpolymer beads is selected from poly(AGE-ter-EGDM-ter-AN) and poly(GMA-ter-DVB-ter-AN).

5. Acrylonitrile based macroporous terpolymer beads according to claim 1, wherein particle size distribution of the said macroporous beads ranging from 200 microns to 500 microns, average pore diameter between 30 nm-120 nm, pore volume between 0.6 cm3/g and 1.2 cm3/g and BET (Brunaer-Emmett-Teller) surface area between 50 m2/g. and 110 m2/g.
6. Acrylonitrile based macroporous terpolymer beads according to claim 1, wherein semi-synthetic p-lactam antibiotics are selected from Amoxicillin, Cephalexin, Ampicillm, Cefaclor, Cefadroxil, and Cefprozil.
7. Immobilized enzyme catalyst on terpolymer macroporous beads according to claim 1.
8. Immobilized enzyme catalyst on terpolymer macroporous beads according to claim 7, wherein the enzyme is selected from PGA from Achromobacter sp. 7394 cloned in E.coli RE3 bearing the plasm id pKX 1P1 and a-amino ester hydrolase.
9. Immobilized enzyme catalyst on terpolymer macroporous beads according to claim 7, wherein the swell volume of macroporous beads after immobilization is in the range of 1.2-1.3 times of the initial volume before immobilization.
10. Immobilized enzyme catalyst on terpolymer macroporous beads according to claim 7, wherein the Immobilized enzyme catalyst shows upto 60- 90% binding protein, upto 97.7% activity binding and upto 15-40 % % enzyme expression.
11. Immobilized enzyme catalyst on terpolymer macroporous beads according to claim 7, wherein the S/H ratio in immobilized enzyme catalyst varies between 0.3-1.3 for Amoxicillin synthesis and 0.1-1.49 for Cephalexin synthesis.
12. The process for synthesis of semi-synthetic p-lactam antibiotics using immobilized enzyme catalyst according to claim 7 comprising, reacting (J-Iactam nucleus with activated acyl donor in the molar ratio of 1:1.2 to 1:1.5, in suspension reaction to precipitate semi-synthetic p-lactam antibiotic product.

13. The process according to claim 12, wherein p-lactam nucleus is selected from 6-aminopenicillanic acid (6-APA), 7-desacetoxycephalosporanic acid (7ADCA), 7-aminocephalosporanic acid (7-ACA), 7-Amino-3-Chloro-3-Cephem-4-Carboxylicacid (7ACCA), and 7-Amino-3-propylene-3-cephem-4-carboxylic acid (7APCA) and activated acyl donor selected from ester or amide of hydroxyphenyl glycine (HPGME) or phenylglycine (PGME).
14. The process according to claim 12, wherein the concentration of immobilized enzyme catalyst in the ratio of 250U/g of activity per gram of β-lactam nucleus.
15. The process according to claim 12, wherein the conversion of β-lactam nucleus is achieved between 74-86 % in 120 minutes for 10 repeated cycles.
16. The process according to claim 12, wherein the activity of immobilized enzyme catalyst used in process of semi-synthetic β-lactam antibiotics remain unchanged after 10 repeated cycles.