DESC:TECHNICAL FIELD OF THE INVENTION:
The present invention relates to a blended purified enzyme having origin of a mNPGA (mutant penicillin G acylase) from Achromobacter and Penicillin G acylase expressed in Escherichia coli.

The present invention further relates to an immobilized blended purified enzyme and an enzymatic process for synthesis of semisynthetic ß-lactam antibiotic in a single step reaction using Penicillin G as a starting material and corresponding activated phenylglycine derivative as acylating agent bypassing the need to isolate the intermediate nucleus and byproduct resulting in final semisynthetic antibiotic of desired purity.

BACKGROUND AND PRIOR ART OF THE INVENTION
ß-lactam antibiotics including penicillin, cephalopsorins and further semisynthetic ß-lactam antibiotics represent one of the world’s major biotechnology products constituting almost 65% of the total antibiotic market.

Due to rise in sustainable development and environmentally friendly practices, enzymatic process for ß-lactam antibiotic synthesis is preferred over conventional chemical process. Penicillin acylase enzyme available from various microbial sources have been widely exploited for their hydrolytic potential and suitably used on industrial scale for synthesis of ßlactam antibiotic intermediates nucleus like 6APA, 7ADCA, 7ACCA and 7AVCA. Further, evolution and development of enzymes have extended application of penicillin acylase enzymes for synthesis of antibiotic molecules, wherein the Penicillin acylase enzyme (PGA) catalyzes the acylation of intermediate nucleus with activated acyl donor under kinetically controlled reaction. Recombination and genetic engineering have enabled Penicillin acylase enzyme to be modified to catalyze acylation of range of ß-lactam antibiotic nucleus with activated side chain to generate the desired antibiotic. The application has been extended to synthesis of amoxicillin, ampicillin, cephalexin, cefadroxil, cefradine, to some of next generation cephalopsorins like cefaclor, cefprozil and cefazolin.

Due to obvious advantage of less environmental load, higher efficiency, aqueous reactions enzymatic reactions are industrially preferred and acceptable for ß lactam antibiotics.

Further, immobilization techniques evolved over the years have offered additional advantage for higher recyclability and stability of enzyme which in turn has reduced the overall cost of enzyme for the process, making it economically viable for commercial production.

Since, Penicillin acylase enzymes have both hydrolytic and synthetic catalytic potential, these have been immobilized on various natural and synthetic support materials to activate its desired property of catalysis. Selection of support material is based on the application, reaction kinetics and product isolation.

Reactions catalyzed by Penicillin acylase are generally in aqueous phase with minimal or no use of solvent or cosolvent in reactor vessel under stirring with precipitation of product in case of acylation which makes it imperative to use robust support material to withstand high shear and enable enzyme filtration and product isolation. Synthetic polymers as support for immobilization has emerged as most suitable platform for above conditions at industrial scale to use Penicillin acylase enzyme.

Presently, industrial process of ß-lactam antibiotic synthesis is a two-step process. The first step involves enzymatic hydrolysis of parent Penicillin or cephalosporin nucleus catalyzed by Penicillin acylase to form intermediate molecule -6APA, 7ADCA, 7ACCA, 7AVCA with phenyl acetic acid as by product.

The second step is acylation of intermediate with activated acyl donor generally a phenylglycine derivative catalyzed by penicillin acylase enzyme to form final antibiotic.

Penicillin acylase enzyme used in both the steps vary with respect to their ability to catalyze each reaction. Penicillin acylase enzyme (E.C.3.5.1.11) from different microbial sources - Escherichia coli, Arthrobacter, Achromobacter, Bacillus megaterium, Proteus rettgeri, Kluyera citrophilla etc. is a heterodimeric protein consisting of Small a-subunit and larger ß- subunit belonging to N-terminal nucleophile (Ntn) hydrolase superfamily that share common fold around the active site bearing serine, cysteine, threonine at the N terminal end wherein ßSer1 is the key role in catalytic mechanism.

Indian Patent Application No. 2020/21000782 discloses penicillin acylase from different source which vary with respect to their overall gene sequence which determines their catalytic potential and differentiating these for hydrolytic or acylating reaction. However, the inherent potential of various penicillin acylase enzymes have been modified by Genetic Engineering techniques to incorporate changes at specific site on the gene to develop a mutant Penicillin acylase with improved property for reaction.

Many such mutant Penicillin acylases have been reported which serve as ‘Booster Biocatalyst’ with desired enhanced activity. Further, the cloning and expressing the PGA gene in industrially suitable host like E coli expands the potential use of these enzymes on a commercial scale.

There is always need and scope to upgrade to improve industrial processes to make it faster, easier on operation scale and simpler than present processes with technological advances.

OBJECT OF THE INVENTION:
It is an object of the present invention to provide the bio-catalytic potential of penicillin acylases from two different bacterial sources integrated and designed to form a single biocatalyst to catalyze synthesis of semisynthetic ß lactam antibiotic in one pot from parent molecule without intermediate isolation step.

It is another object of the present invention to integrate the two enzymatic process of hydrolysis and acylation for the synthesis of semisynthetic ß-lactam antibiotic in a one pot - single reaction step with the blended purified penicillin acylase enzyme catalyzing the reaction.

SUMMARY OF INVENTION:
In an aspect, the present invention relates to a blended purified enzyme having origin of mNPGA (mutant penicillin G acylase) from Achromobacter spp. CCM4824 and Penicillin G acylase expressed in Escherichia coli.

In another aspect, the present invention describes a process for preparation of a beta lactam antibiotic, wherein the blended purified penicillin G acylase enzyme is used to catalyze synthesis of semisynthetic ß- lactam antibiotic in single step, in one pot from parent ß-lactam molecule Penicillin or cephalosporin without isolating the intermediate nucleus and phenylacetic acid by product generated in reaction.

In yet another aspect, the present blended purified Penicillin G acylase enzyme derived from Escherichia coli and Achromobacter spp. CCM4824 used in immobilized form exhibits both hydrolytic and synthetic activity with S/H ratio favoring the sequence of reactions from hydrolysis of Penicillin G to 6APA to acylation of 6APA with activated acyl sidechain to form the final antibiotic.

In further aspect, the invention relates to use of Penicillin G acylase enzyme from microbial sources other than Escherichia coli and Achromobacter CCM4824 which shows at least 80% homology with either or both said source.
In an advantageous aspect, the process being integration of a two-step reaction in single step with the present immobilized enzyme. The reaction is kinetically controlled to form the product as a precipitate which can be isolated at the end of reaction. Additionally, phenylacetic acid by-product can also be isolated for reuse.

Advantageously, the reaction proceeds towards acylation even in presence of phenylacetic acid by-product which is competitive inhibitor of Penicillin acylase enzyme.

The present process for synthesis of semisynthetic ß-lactam antibiotic using blended purified Penicillin G acylase enzyme derived from Escherichia coli and Achromobacter spp. CCM4824 and requires no isolation of antibiotic intermediate which reduces the time of downstream purification.

DETAILED DESCRIPTION OF DRAWINGS
Figure 1 depicts the Course of conversion for synthesis of amoxicillin from Penicillin and HPGMe (acyl donor) using Fermase PX biocatalyst from Example 1.

Figure 2 depicts the course of conversion for synthesis of amoxicillin from Penicillin and HPGMe (acyl donor) using Fermase PX biocatalyst from Example 2.

Figure 3 depicts the course of conversion for synthesis of amoxicillin from Penicillin and HPGMe (acyl donor) using Fermase PX biocatalyst from Example 1.

Figure 4 depicts the recyclability data of Fermase PX biocatalyst used for amoxicillin synthesis beginning from Penicillin G as the substrate.

Figure 5 depicts a one pot process for synthesis of amoxicillin using Fermase PX biocatalyst.

Source of biological material
Achromobacter spp. CCM4824 – mutant penicillin acylase gene in Escherichia coli (MTCC 25429) deposited in IMTECH Chandigarh.

Escherichia coli.: Escherichia coli wild type acylase recombinant culture is obtained from Institute of Microbiology, Czech Republic.

DETAILED DESCRIPTION OF INVENTION
The invention will now be described in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

In an embodiment, the present invention provides a blended purified enzyme having origin of mNPGA (mutant penicillin G acylase) from Achromobacter spp. CCM4824 and Penicillin G acylase expressed in Escherichia coli.

The enzyme Penicillin acylase can be derived from either or both Escherichia coli and Achromobacter CCM4824 of wild type and/or its functional equivalents such as mutants or other derivatives obtained by classical or recombinant DNA technology, as known to person skilled in art. The blended purified penicillin acylase of the invention shows tolerance to phenylacetic acid generated as hydrolytic by-product and display varying specificity and S/H ratio with respect to different acyl side chains.

In another embodiment, the present invention discloses a single step enzymatic synthesis of semisynthetic ß-lactam antibiotic wherein Penicillin G acylase enzyme catalyzes the in-situ synthesis of antibiotic intermediate from parent ß-lactam molecule which is simultaneously acylated to form the corresponding antibiotic.
Accordingly, the process comprises use of Penicillin acylase enzyme from both the sources Escherichia coli and Achromobacter CCM4824.

In an embodiment, the partially purified enzyme from combined sources has a specific activity of 40-60 U/mg more precisely between 45-55 U/mg which is a significant improvement over the individual enzyme upto 25-30% U/ mg.

In another embodiment, the present invention provides an immobilized blended purified enzyme having origin of mNPGA (mutant penicillin G acylase) from Achromobacter spp. CCM4824 and Penicillin G acylase expressed in Escherichia coli on polyacrylic polymer beads carrying epoxy or amine functional groups.

In accordance with above embodiment, the immobilized biocatalyst so generated includes functional derivative from chemical modification, crosslinking of one or both enzyme with the support material which impacts the properties like pH dependence, thermostability, specific activity towards enhancement of the said enzymatic reaction.

The polyacrylic polymer beads with functional epoxy groups offer an advantage for covalent binding of enzyme which imparts better operational stability and withstand physical shear in industrial reactor to achieve optimum recyclability and aid in filtration of enzyme during recycling. Additionally, immobilization on polymer with epoxy or amine spacer further comprises pre-activation with crosslinking agents such as glutaraldehyde or glyoxal at a concentration ranging from 1-3% which imparts improved mechanical and thermal stability to enzyme and enhanced activity expression with optimum protein load. The biocatalyst which is immobilized on the polymer support shows altered specificity to different substrates in different reaction conditions.

In accordance with above embodiment, the immobilization method of the invention includes obtaining purified penicillin acylase enzyme in soluble form from the fermentation process with activity ranging from 300-350 U/mL in terms of Penicillin hydrolysis with specific activity in range of 45-55 U/mg of protein. The penicillin acylase enzyme is suitably equilibrated with sodium and potassium phosphate buffer to desired molarity between 0.1-1M as per the polymer selected. The equilibrated enzyme is then mixed with polymer beads and incubated at desired temperature between 20-30°C for 24-48 hours. The enzyme immobilized polymer beads are obtained as Penicillin acylase Biocatalyst (Fermase PX Biocatalyst).

The activity of Biocatalyst is determined based on synthesis of product formed in kgs per hour per kg of enzyme used. In terms of amoxicillin, the value ranges between 0.15-0.25 units based on type immobilized biocatalyst used which is about 7-10% higher as compared to two-step process with isolation of intermediate.

Immobilization procedure of Penicillin acylase is suitably designed to express the optimum desired activity and S/H ratio in the reaction and display enhanced pH stability in the multiple reactions undergoing at particular time.

Accordingly, immobilization supports are selected from range of porous polyacrylate beads carrying epoxy or amine functional group with hydrophilic microenvironment suitable to retain the enzyme protein orientation throughout the multiple reaction in single pot.

Immobilization further favors the reaction with incorporation of both PGA so as to express its both hydrolytic and synthetic potential in the desired stage to drive the reaction towards synthesis of required ß-lactam antibiotic. The reaction conditions are optimized to drive the enzymatic synthesis. All the substrate beginning with ß-lactam penam/cepham nucleus and acyl donor are added in an aqueous reaction which is catalyzed with Penicillin acylase enzyme to form the corresponding ß-lactam antibiotic in precipitated form under controlled conditions of pH and temperature known to person skilled in art. On the completion of reaction, the product is separated from biocatalyst by filtration. Enzyme biocatalyst recycled by washing the biocatalyst for use in subsequent batches.

Activity of biocatalyst prepared by immobilization is expressed as amount of enzyme catalysing the formation of 1µmole of 6-Aminopenicillanic acid at 370c, pH 8.0 at 2 %(w/v) Penicillin G potassium salt initial concentration, in the milieu of 50mM Sodium Phosphate buffer, within the range of zero order kinetics.

The combined use of Penicillin acylase enzyme used has resulted in altered pH stability in the range from 6-8 enhancing its application suitability in the one pot reaction. Penicillin acylase from Escherichia coli and Achromobacter show pH tolerance in the range of 5 to 6 or 7 to 8 based on the individual source.

Due to the complex reactions undergoing at different rate in the enzymatic step for synthesis of amoxicillin /ampicillin from Penicillin viz, penicillin hydrolysis, Ester hydrolysis, acylation of 6APA, hydrolysis of product eventually leads to multiple byproducts. Enzyme of the present invention exhibits remarkable tolerance to the by-products, phenylacetic acid, hydrolyzed ester and methanol but also displays optimum performance towards synthesis resulting in product formed as precipitate.

In one of the embodiments, Phenyl acetic acid is separated as a side product in the process is extracted from the reaction mixture using either of solvents selected from n-butyl acetate, ethyl acetate, dichloromethane or toluene.

Phenyl acetic acid recovery from the process holds significance due to its application in other products and as component for drug intermediates.

Phenylacetic acid is used in some perfumes, as it possesses a honey-like odour even in low concentrations. It is also used in penicillin G production and diclofenac production. It is also employed to treat type II hyperammonaemia to help reduce the amounts of ammonia in a patient's bloodstream by forming phenylacetyl-CoA, which then reacts with nitrogen-rich glutamine to form phenylacetylglutamine. This compound is then excreted from the patient's body. It is also used in the illicit production of phenylacetone, which is used in the manufacture of methamphetamine.

The sodium salt of phenylacetic acid, sodium phenylacetate, is used as a pharmaceutical drug for the treatment of urea cycle disorders, including as the combination drug sodium phenylacetate/sodium benzoate.

One of the crucial kinetic parameters being S/H ratio with respect to each of the available substrate. S/H ratio is reaction driving relative parameter based on the substrates present and hence has specific significance in the reaction. S/H ratio is expressed as either SAmox/HPenG or SAmox/Hester.

The invention discloses trend of both S/H ratio of PGA enzyme in each stage of reaction which influence the reaction and display a unique behavior of combined enzyme in presence of two hydrolytic substrates Penicillin G and activated Phenylglycine derivative.

The rate of hydrolysis of Penicillin and acyl donor is equilibrated to drive reaction towards antibiotic synthesis.

The trend of S/H ratio is highlighted in following specific examples.

Since, Penicillin acylase activity and substrate specificity varies based on microbial source, use of combined enzyme in reaction has influenced to achieve kinetically controlled equilibrium favoring reaction completion within stipulated time ranging from 130-180 minutes which almost half than that required in two step process.

In accordance with one of the embodiments, enzymatic reaction includes ß-lactam substrate containing either of the below core nuclei, more specifically pertaining to Penicillin G or V or cephalosporin G enzymatically reacted with a side chain/acyl donor.

Accordingly, Penicillin or cephalosporin obtained by fermentation process, which may be as crude N-substituted ß-lactam in liquid form or further purified as salt wherein, either form may or may not contain traces of phenylacetate, used as inducer in fermentation, or solvent such as butyl acetate used for extraction of phenylacetate.

R1: hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl optionally containing one or more heteroatoms.

R and R2: phenylacetamido or phenoxyacetamido.

The side chain/acyl donor to prepare the corresponding ß-lactam antibiotic in accordance with the method of present invention may be compound recognized by Penicillin acylase enzyme as precursor for acylation onto the core nucleus forming amide linkage at the -NH2 position of core nucleus. Acyl donor is chosen from group of compounds containing D-(-) phenyl glycine, D-(-) hydroxyphenyl glycine, 2-thienylacetic acid, D-(-)2,5, dihydrophenylglycine and/or derivatives thereof. Preferably, activated derivatives of acyl donors, preferably methyl, ethyl, isopropyl esters, and amides are favorable for kinetics of the enzymatic reaction.

The enzymatic reaction of the invention comprises of adding the biocatalyst of the invention to an aqueous solution of acyl donor and ß-lactam core nucleus in suitable molar ratio, wherein the acyl donor is added in molar excess ranging from 1.1.to 5M for different forms used, more preferably between 1.3-2M.

Reaction conditions of pH and temperature vary between 5 to 8 more preferably between 7.0-7.8 and temperature between 20-30°C more preferably between 22-28°C based on different antibiotic molecule.

In this regard, it is most preferred to carry out the reaction in aqueous conditions wherein the addition of certain cosolvents such as glycols may or may not be favorable for reaction equilibrium.

According to one of the embodiments, the by-products of hydrolysis of core ß-lactam nucleus and excess acyl donor are separated by solvent extraction. In accordance with this embodiment, reaction mixture is filtered through 100-150 µ mesh which retains the biocatalyst. The filtrate is further used to separate the precipitated product from the enzyme. The filtrate obtained as mother liquor contains the by-products and desired product which are sequentially isolated beginning with acidification and extraction with nonpolar solvents selected from ethyl acetate, butyl acetate, dichloromethane to separate the phenylacetate by product. Product isolation includes dissolution of precipitated product with reaction mother liquor. Aqueous reaction mass pre acidified is crystallized under chilled conditions and raising the pH to precipitate the recrystallized product.

The biocatalyst is washed with water multiple times till free from reactants and products of reaction mixture. The final wash is analysed by HPLC to confirm the absence of any reaction mixture content. Biocatalyst is reused for subsequent cycles. Final product is dried and analysed for purity.

The invention discloses a process whereby the intermediate isolation stage is not required which reduces the cost of downstream to half and time for workup is reduced substantially with process also saving energy consumption due to combining of two process further reducing carbon footprint.

The present invention also encompasses recycling of the penicillin acylase biocatalyst in a subsequent reaction after filtration and separating the biocatalyst from the reaction mixture. The recyclability of biocatalyst varies for different antibiotics ranging from minimum 50-500 cycles.

The present embodiment encompasses one pot single step reaction of Penicillin acylase catalyzed synthesis of amoxicillin from Penicillin which further extends to antibiotics from range of ampicillin and cephalexin, cefadroxil and cefradine by methods disclosed hereinabove.

Examples: The invention is further described with reference examples which are illustration of invention but not limiting to the contents mentioned in the examples.

Example 1: Preparation of Penicillin acylase biocatalyst by immobilization on Epoxy acrylic polymer
The following determination method of immobilization on polymer beads is familiar per se to the person skilled in the art of covalent immobilization and is detailed only for the sake of completeness.

1 g of Dry Epoxy polymer bead are added to 25 U/mg of the said enzyme solution in 1.5 M potassium phosphate buffer pH 7.5, and incubated at 25° C for 48 h. The Epoxy 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 weight of the resulting Biocatalyst PGA is determined. Activity achieved corresponds to 2.2 g product /g of biocatalyst. The swell volume after immobilization is between 1.1 to 1.2 times of the initial volume of Epoxy polymer beads. Activity of biocatalyst for penicillin hydrolysis was achieved 320 U/ g dry.

Example 2: Preparation of Penicillin acylase biocatalyst by immobilization in Amide acrylic polymer
1 g of Dry Amide Acrylic polymer beads pre-treated with 2% glutaraldehyde are added to 35 U/mg of said enzyme in 0.25 M potassium phosphate buffer pH 7.5 and incubated at 25° C for 48 h. The Amide 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 weight of the resulting Biocatalyst PGA is determined. The activity obtained corresponds to 2.6 g product /g biocatalyst. The swell volume after immobilization is between 1.1 to 1.2 times of the initial volume of Amide acrylic polymer beads. Activity of biocatalyst for penicillin hydrolysis was achieved 250 U/ g dry.

Example 3: Preparation of Penicillin acylase biocatalyst by immobilization on Epoxy acrylic polymer
1 g of Dry Epoxy polymer bead are added to 40 U/mg of the said enzyme solution in potassium phosphate buffer pH 7.2, molarity 1.3 M and incubated at 25° C for 48 h. The Epoxy 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 weight of the resulting Biocatalyst PGA is determined. Activity achieved corresponds to 2.3 g product /g of biocatalyst. The swell volume after immobilization is between 1.1 to 1.2 times of the initial volume of Epoxy polymer beads. Activity of biocatalyst for penicillin hydrolysis was achieved 215 U/ g dry.

Example 4: Preparation of Penicillin acylase biocatalyst by immobilization in Amide acrylic polymer
1 g of Dry Amide Acrylic polymer beads pre-treated with 1% glutaraldehyde are added to 35 U/mg of said enzyme in 0.15 M potassium phosphate buffer pH 7.5 and incubated at 25° C for 48 h. The Amide 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 weight of the resulting Biocatalyst PGA is determined. The activity obtained corresponds to 2.5 g product /g biocatalyst. The swell volume after immobilization is between 1.1 to 1.2 times of the initial volume of Amide acrylic polymer beads. Activity of biocatalyst for penicillin hydrolysis was achieved 330 U/ g dry.

Example 5: Enzymatic synthesis of Amoxicillin.
The reaction is carried out at 22 °C, pH 7.2-7.5 for 3-4 hrs in a jacketed stirred tank reactor. Reaction mixture consists of 110 mM Penicillin G K salt and 300 mM HPGME HCl with 200 U of Penicillin acylase biocatalyst prepared as in Example 1 in 10 mL of water. pH was maintained with 11% ammonium hydroxide and 11% hydrochloric acid during the reaction. Course of the reaction monitored by withdrawing samples at regular interval of time and analysed by HPLC. Residual Penicillin was 6.07mg/mL and 6APA was 6.66mg/ mL, Ester 3.97mg/mL, PAA 30.11 mg/ mL

Table 1: Trend of S/H ratio in enzymatic amoxicillin from Penicillin G
Example 3 Time in hours SAmox/HPenG SAmox/Hester
Figure 1 2 2.13 0.44
4 7.55 1.66
6 13.06 2.03

Example 6: Enzymatic synthesis of Amoxicillin
The reaction is carried out at 25 °C, preferably pH 7.3 for 2-3 hrs in a jacketed stirred tank reactor vessel. Reaction mixture consists of liquid Penicillin G K solution containing about 110mM and 160 mM HPGME HCl with 200 U of Penicillin acylase biocatalyst as prepared in Example 2 in 10 mL of water. pH was maintained with 11% ammonium hydroxide and 11% hydrochloric acid during the reaction Course of the reaction monitored by withdrawing samples at regular interval of time and analysed by HPLC. Residual Penicillin was 0.89mg/mL, 6APA 0.51 mg/ mL, Ester 2.08 mg/mL and PAA 27.51mg/ mL

Table 2: Trend of S/H ratio in enzymatic amoxicillin from Penicillin G
Example 4 Time in minutes SAmox/HPenG SAmox/Hester
Figure 2 60 7.42 0.81
90 23.67 2.34
120 74.17 7.07
150 106.68 45.64

Example 7: Enzymatic synthesis of Amoxicillin
The reaction is carried out at 22 °C, pH 7.8 for 2 hours in a jacketed stirred tank reactor. Reaction mixture consists of liquid Penicillin G solution containing 60 mM and about 110 mM HPGME free ester with 300 U of Penicillin acylase biocatalyst as prepared in Example 2 in 10 mL of water. pH was maintained with 11% ammonium hydroxide and 11% hydrochloric acid during the reaction Course of the reaction monitored by withdrawing samples at regular interval of time and analysed by HPLC. Residual Penicillin G at 1.07 mg/ mL, 6APA 0.85mg/mL, Ester 2.8mg/ mL and PAA 32.1 mg/ mL

Example 8: Enzymatic synthesis of Amoxicillin
The reaction is carried out at 25 °C, pH 6.8 for 3-4 hrs in a stirred tank reactor. Reaction mixture consists of 60 mM Penicillin G K salt and 110mM HPGME free ester with 200 U of Penicillin acylase biocatalyst prepared as in Example 4 in 10 mL of water. pH was maintained with 11% ammonium hydroxide and 11% hydrochloric acid during the reaction Course of the reaction monitored by withdrawing samples at regular interval of time and analysed by HPLC. Residual Penicillin G was 3.72 mg/ mL, 6APA 3.25mg/ mL, Ester 5.09mg/ mL and PAA 28.05mg/ mL

Table 3: Trend of S/H ratio in Enzymatic amoxicillin from Penicillin G
Example 6 Time in hours SAmox/HPenG SAmox/Hester
Figure 3 2 0.98 0.47
4 4.27 1.56
6 10.08 4.5
6.5 15.25 6.09

Example 9: Enzymatic synthesis of Amoxicillin
The reaction is carried out at 23 °C for 2 hrs at pH 7.3 in a jacketed stirred tank reactor. Reaction mixture consists of liquid Penicillin G solution with 0.08%butyl acetate containing about 80 mM and 110mM HPGME HCl with 200 U of Penicillin acylase biocatalyst as prepared in Example 2 in 10 mL of water. pH was maintained with 11% ammonium hydroxide and 11% hydrochloric acid during the reaction Course of the reaction monitored by samples at regular interval of time and analysed by HPLC. Residual Penicillin G was 0.83 mg/ mL, 6APA mg/ mL, Ester 1.89 mg/ mL and PAA 29.45mg/ mL

Example 10: Enzymatic synthesis of Amoxicillin
The reaction is carried out at 25 °C, pH 7.3 for 3 hrs in a stirred tank reactor. Reaction mixture consists of liquid Penicillin G solution with 0.01% phenylacetic acid and 0.05% butyl acetate containing about 80 mM and 120 mM HPGME HCl with 300 U of Penicillin acylase biocatalyst as prepared in Example 3 in 25 mL of water. pH was maintained with 11% ammonium hydroxide and 11% hydrochloric acid during the reaction Course of the reaction monitored by samples at regular interval of time and analysed by HPLC. Residual Penicillin G was 1.65 mg/ mL, 6APA 1.74mg/ mL, Ester 1.3 mg/ mL and PAA 12.6 mg/ mL

Example 11: Enzymatic synthesis of Amoxicillin
The reaction is carried out at 24 °C, pH 7.0 for 3-4 hrs in a jacketed stirred tank reactor. Reaction mixture consists of 100 mM Penicillin G K salt and 200 mM HPGME HCl with 200 U of Penicillin acylase biocatalyst prepared as in Example 3 in 25 mL of water. pH was maintained with 10% sodium bicarbonate solution and 11% hydrochloric acid during the reaction. Course of the reaction monitored by withdrawing samples at regular interval of time and analysed by HPLC. Residual Penicillin was 7.0 mg/mL and 6APA was 6.7mg/ mL, Ester 3.5mg/mL, PAA 13.11 mg/ mL

Example 12: Enzymatic synthesis of Amoxicillin
The reaction is carried out at 24 °C, pH 7.0 for 3-4 hrs in a jacketed stirred tank reactor. Reaction mixture consists of 100 mM Penicillin G K salt and 200 mM hydroxy phenyl glycine amide with 300 U of Penicillin acylase biocatalyst prepared as in Example 3 in 25 mL of water. pH was maintained with 10% sodium bicarbonate solution and 11% hydrochloric acid during the reaction. Course of the reaction monitored by withdrawing samples at regular interval of time and analysed by HPLC. Residual Penicillin was 9.0 mg/mL and 6APA was 9.7mg/ mL, Ester 7.5mg/mL, PAA 10.11 mg/ mL

Example 13: Enzymatic synthesis of Ampicillin
The reaction is carried out at 25 °C, pH 7.5 for 4 hrs in a stirred tank reactor. Reaction mixture consists of liquid Penicillin G solution containing about 280mM and 420 mM PGME HCl with 200 U of Penicillin acylase biocatalyst as prepared in Example 1 in 25 mL of water. pH was maintained with 11% ammonium hydroxide and 11% hydrochloric acid during the reaction. Course of the reaction was monitored by withdrawing samples at regular interval of time and analysed by HPLC. Residual Penicillin was 4.4mg/ mL, 6APA 3.8mg/ mL, Ester 0.9 mg/ mL, PAA 48.mg/ mL. Ampicillin concentration was 76.3mg/ mL

Example 14: Enzymatic synthesis of Ampicillin
The reaction is carried out at 25 °C, pH 7.3 for 2.5 hrs in a stirred tank reactor. Reaction mixture consists of liquid Penicillin G solution containing about 100mM and 220 mM PGME HCl with 200 U of Penicillin acylase biocatalyst as prepared in Example 2 in 25 mL of water. pH was maintained with 11% ammonium hydroxide and 11% hydrochloric acid during the reaction. Course of the reaction was monitored by withdrawing samples at regular interval of time and analysed by HPLC. Residual Penicillin was 1.76 mg/ mL, 6APA 1.24 mg/ mL, Ester 1.5 mg/mL, PAA 13.2.mg/ mL. Ampicillin concentration was 83.5 mg/ mL

Example 15: Enzymatic synthesis of Ampicillin
The reaction is carried out at 25 °C, pH 7.3 for 2.5 hrs in a stirred tank reactor. Reaction mixture consists of liquid Penicillin G solution containing about 100mM and 220 mM PGME HCl with 200 U of Penicillin acylase biocatalyst as prepared in Example 3 in 25 mL of water. pH was maintained with 11% ammonium hydroxide and 11% hydrochloric acid during the reaction. Course of the reaction was monitored by withdrawing samples at regular interval of time and analysed by HPLC. Residual Penicillin was 4.76 mg/ mL, 6APA 3.24 mg/ mL, Ester 1.5 mg/ mL, PAA 9.2.mg/ mL. Ampicillin concentration was 72.5 mg/ mL

Example 16: Enzymatic synthesis of cefadroxil
The reaction is carried out at 22 °C, pH 7.8 for 3-4 hrs in a jacketed stirred tank reactor. Reaction mixture consists of 50 mM Cephalosporin G (Ceph G) and 220 mM HPGME HCl with 300 U of Penicillin acylase biocatalyst prepared as in Example 2 in 10 mL of water. pH was maintained with 11% ammonium hydroxide and 11% hydrochloric acid during the reaction Course of the reaction monitored by withdrawing samples at regular interval of time and analysed by HPLC. Residual Ceph G was 9.55mg/ mL, 7ADCA 8.1 mg/ mL, PAA 5.3mg/ mL.

Example 17: Enzymatic synthesis of cefalexin
The reaction is carried out at 24 °C, pH 7.8 for 4 hrs in a jacketed stirred tank reactor. Reaction mixture consists of 50 mM Cephalosporin G and 120 mM PGME HCl with 200 U of Penicillin acylase biocatalyst prepared as in Example 4 in 25 mL of water. pH was maintained with 11% ammonium hydroxide and 11% hydrochloric acid during the reaction Course of the reaction was monitored by withdrawing samples at regular interval of time and analysed by HPLC. Residual Ceph G was 9.78 mg/ mL, 7ADCA 8.3 mg/ mL, Ester 9.6 mg/ mL PAA 5.3 mg/ mL.

Example 18: Downstream: Product isolation and by product recovery for amoxicillin synthesis.
Reaction mixture is filtered through 100-150 µ mesh which retains the biocatalyst. The filtrate is further used to separate the precipitated product from the enzyme. The filtrate obtained as mother liquor contained the desired product, amoxicillin and by products, one being phenylacetic acid which are sequentially isolated under varying conditions. Amoxicillin as precipitated product and mother liquor containing dissolved amoxicillin and phenylacetic acid is acidified with concentrated hydrochloric acid to dissolve the precipitate and added with butyl acetate. Phenylacetic acid is extracted in butyl acetate. The acidified aqueous solution is cooled to freezing temperature and pH slowly raised to 5.2 to crystallize amoxicillin which is filtered and dried to obtain Amoxicillin trihydrate. The molar yield of final product is NLT 85% of theoretical amoxicillin yield based on Penicillin. The biocatalyst is washed with water multiple times till free from reactants and products of reaction mixture. The final wash is analysed by HPLC to confirm the absence of any reaction mixture content. Biocatalyst is reused for subsequent cycles. Final amoxicillin product is dried and purity achieved is NLT 98%.

Example 19: Recyclability of FERMASE PX biocatalyst.
FERMASE PX biocatalyst recycled in reactions carried out as in example 6 and Example 10 for 70 times with retention of Enzyme activity up to more than 95%. The graphical representation of recyclability data is represented in Fig 4.

Example 20: HPLC analysis for Amoxicillin (AMOX) synthesis:
Column: C18 ODS 3(250mm X 4.6mm X5u), Buffer: 8.0g potassium dihydrogen ortho phosphate buffer pH 5.8 with 1N sodium hydroxide acetonitrile, Flow rate: 1.2mL/ min., Wavelength: 225 nm, Injector Volume: 20 µl, Run Time: 30 minutes.

Gradient flow with following sequence of Mobile phase A: 96:4 Buffer: acetonitrile and Mobile phase B: 100%Acetonitrile

Table 4:
Time in minutes % A % B
0 100 0
10 100 0
12 70 30
17 100 0
20 100 0

Advantages of the invention:
1. The blended purified penicillin G acylase shows tolerance to phenylacetic acid generated as hydrolytic byproduct and display varying specificity and S/H ratio with respect to different acyl side chains.

2. The process being integration of a two-step reaction in single step with the present immobilized enzyme. The reaction is kinetically controlled to form the product as a precipitate which can be isolated at the end of reaction. Additionally, phenylacetic by product can also be isolated for reuse.

3. The reaction proceeds towards acylation even in presence of phenylacetic byproduct which is competitive inhibitor of Penicillin acylase enzyme.

4. The present process for synthesis of semisynthetic ß- lactam antibiotic using blended purified Penicillin G acylase enzyme derived from Escherichia coli and Achromobacter spp. CCM4824 requires no isolation of antibiotic intermediate which reduces the time of downstream purification.
,CLAIMS:1. A one-step process for enzymatic synthesis of ß-lactam antibiotics, wherein the process comprises of:
a. adding Enzyme Fermase PX biocatalyst in immobilized form consisting of blend of purified enzyme of the type Penicillin G acylase having origin of mNPGA (mutant penicillin G acylase) from Achromobacter spp. CCM4824 and Penicillin G acylase expressed in Escherichia coli to an aqueous solution of an activated acyl donor and a ß-lactam core nucleus of the general formula 1 or 2, and

wherein,
R1 is selected from hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl optionally containing one or more heteroatoms, and
R and R2 are selected from phenylacetamide or phenoxy acetamido.
b. isolating and recrystalizing the semisynthetic ß-lactam antibiotic from the aqueous reaction mass, and
c. sequentially recovering the by product by solvent extraction.
2. The process as claimed in Claim 1, wherein the blend of penicillin G acylase enzymes are selected from Escherichia coli and Achromobacter CCM4824 of wild type and/or its functional equivalents such as mutants or other derivatives obtained by classical or recombinant DNA technology.
3. The process as claimed in Claim 1, wherein the blend of Penicillin G acylase are selected from wild type or functional equivalents such as mutants or derivatives other than Achromobacter spp. CCM4824 or Escherichia coli which show 80% homology with either or both of the said organisms.
4. The process as claimed in claim 2 or 3, wherein the partially purified enzyme from combined sources has a specific activity is in range of 40U/mg – 60U/mg protein.
5. The process as claimed in claim 2 or 3, wherein the partially purified enzyme from combined sources is stable in pH range from 5-8.
6. The process as claimed in Claim 1, wherein the of blended purified enzyme having origin of mNPGA (mutant penicillin G acylase) from Achromobacter spp. CCM4824 and Penicillin G acylase expressed in Escherichia coli is immobilised on polyacrylic polymer beads carrying epoxy or amine functional groups.
7. The process as claimed in Claim 6, wherein the polyacrylic polymer beads carrying epoxy or amine functional groups are pre-activated with crosslinking agents selected from glyoxal and glutaraldehyde.
8. The process as claimed in Claim 7, wherein the concentration of crosslinking agent varies from 1-3% per gram of polymer beads.
9. The process as claimed in Claim 7, wherein the time of pre activation varies from 1 hour to 12 hours.
10. The process as claimed in Claim 6 wherein Fermase PX Biocatalyst express SProduct/ HNucleus ratio in the range of 0.6 to 0.9, more preferably 0.8-0.9 for Amoxicillin.
11. The process as claimed in Claim 6, wherein Fermase PX biocatalyst is stable at pH range of 5-8.
12. The process as claimed in Claim 6, wherein Fermase PX biocatalyst is stable at temperature range from 1°C- 40°C.
13. The process as claimed in Claim 6, wherein Fermase PX biocatalyst is tolerant to solvent selected from n-butyl acetate, Ethyl acetate, toluene, Methylene dichloride more preferably n-butyl acetate in concentration up to 0.5% in the reaction mixture.

14. The process as claimed in Claim 6, wherein the Fermase PX Biocatalyst activity loaded is at least 150 units per mM of ß-lactam nucleus.
15. The process as claimed in Claim 6, wherein the biocatalyst Fermase PX is tolerant to the by-products of the claimed process.
16. The process as claimed in Claim 1, wherein the ß-lactam nucleus is selected from Penicillin as crystalline Penicillin G salt or crude penicillin G obtained by downstream process of Penicillin G fermentation or cephalosporin derivative.
17. The process as claimed in Claim 1, wherein the acyl donor is activated form selected from D-(-)-phenyl glycine, D-(-)-hydroxy phenylglycine, 2-thienylacetic acid, D-(-)-2,5, dihydrophenylglycine or methyl, ethyl, isopropyl esters and amides derivatives thereof.
18. The process as claimed in Claim 1, wherein molar ratio of nucleus to acyl donor ranging from 1:1.05 to 1: 5 based on the type of final ß-lactam antibiotic being synthesized.
19. The process as claimed in Claim 1, wherein the reaction is carried out in controlled temperature in the range of 15°C to 30°C, preferably between 20°C -25°C.
20. The process as claimed in Claim 1, wherein the reaction is carried out in controlled pH in range of 5-8, preferably between 6.5-7.5
21. The process as claimed in Claim 1, wherein the ß-lactam antibiotic is isolated by recrystallization from the aqueous reaction mixture
22. The process as claimed in Claim 1, wherein the by-product is recovered sequentially with acidification, solvent extraction and raising to recrystallization pH under chilled conditions ranging from 0-5°C to obtain the product.
23. The process as claimed in Claim 21, wherein the by-product is isolated by using non-polar solvents, selected from butyl acetate, ethyl acetate, Toluene. Methylene Dichloride.

24. The process as claimed in Claim 1, wherein the process can be used for synthesis of products ranging from amoxicillin, ampicillin, cefalexin, cefadroxil and cefradine.
25. The process as claimed in Claim 1, wherein the molar product yield for amoxicillin is at least 85% of the theoretical yield with respect to Penicillin G.
26. The process as claimed in Claim 1, wherein the molar yield of final semisynthetic antibiotic product is at least 80% of the molar equivalent of intermediate formed in the reaction.