DESC:TECHNICAL FIELD OF THE INVENTION
The present invention relates to mutant penicillin G acylases (mNPGA) obtained by the amino acid substitution at a single or more than one point in nucleotide sequences in Achromobacter CCM4824, wherein the resultant mutants demonstrate improved S/H ratio, synthetic activity or both for different semi-synthetic ß-lactam antibiotics. The present invention further relates to immobilized mutant penicillin G acylase.

BACKGROUND AND PRIOR ART OF THE INVENTION
The process of industrial production of semi-synthetic ß-lactam antibiotics in undergoing transition from the conventional chemical route to the more eco-friendly efficient enzymatic route due to the obvious advantage of the enzymatic process, generating lesser by-products, not using organic solvents and harsh chemicals for protection and de-protection and avoiding extreme operating conditions of pressure, temperature and high energy costs. The synthesis of semi-synthetic ß-lactams by an enzymatic route involves a kinetically controlled acylation of ß-lactam nucleus with an amino acid derivative, catalyzed by enzymes belonging to Ntn-hydrolase classes mainly penicillin G acylase or a-amino acid ester hydrolase. However, the use of penicillin G acylase has been widely reported for application, possibly because of the presence of this enzyme in variety of micro-organisms and its characterization for application in hydrolysis and synthesis reaction. Penicillin G acylase from different microorganisms has been widely studied for its substrate specificity and selectivity.
Commercially, penicillin G acylase in immobilized form is known to catalyse hydrolysis reactions for antibiotic intermediate. Recently other variants of Penicillin acylases have been reported to be elucidated for application in synthesis reaction wherein the semi-synthetic ß-lactam antibiotic is synthesized by acylation of antibiotic intermediate. Following criteria are critical in the synthesis reaction.
Choice of enzyme: Kinetically controlled semi-synthetic ß-lactam antibiotic synthesis is governed by three simultaneous reactions catalysed by penicillin acylase namely hydrolysis of activated acyl donor (esterase activity-H), acylation of ß-lactam antibiotic intermediate (synthesis activity–S) and hydrolysis of ß-lactam antibiotic (amidase activity). Obviously enzyme with favourable S/H ratio is a preferred choice which would drive the reaction towards synthesis to yield higher product and generate lesser hydrolytic by-product.
Penicillin G acylases from E. coli, Providencia rettgerri, Klyuvera citrophilia and Bacillus megaterium which have been used for antibiotic intermediate synthesis like 6-APA and 7-ADCA generally show higher hydrolytic to synthetic activity. However, by site-directed mutagenesis, alteration in amino acid at specific site has resulted in mutant Penicillin acylase which shows enhanced synthesis activity and has made enzymatic synthesis of semi-synthetic antibiotic an industrially viable option.
The wild type penicillin acylases from various microbial sources with limited capacity of synthesis have been improved by altering amino acids at specific sites in nucleotide sequence. Various such mutant penicillin acylases have been reported to show altered S/H ratios or higher synthesis activity for different antibiotics based on amino acid changes made in either alpha or beta subunit of enzyme gene.
US Patent No. 8,541,199 discloses a mutant Penicillin G acylase from E. coli having amino acid substitution at different positions in alpha and beta subunit resulting in enhanced synthesis of penam and cepham semi-synthetic antibiotics. Also selective increase in S/H ratios with specific changes was reported.
Similar reports have been published of mutations in hydrophobic active sites such as ßF24A, ßF57W, ßF57Y, aF146L and aF146Y in E. coli penicillin acylases resulted in 5-10 fold decrease in Kcat values of synthesis of amoxicillin, ampicillin, cephalexin and cefadroxil with either hydroxy phenyglycine amide or phenylglycine amide and increased S/H ratio resulting in high yields compared to wild type.
Mutations at position B24 are known in WO96/05318 which particularly discloses the replacement of naturally occurring phenyl alanine amino acid residue at position B24 in the Penicillin G acylase produced by Alcaligenes faecalis by positively charged amino acid lysine or Arginine.
Immobilized enzyme: Commercially the use of enzyme is possible if the enzyme can be recycled and reused for repeated production cycles. This makes it imperative to develop an immobilized enzyme product on a suitable support which can be recycled. In addition synthetic reactions are suspension based and generate precipitate leading to physical attrition of immobilized enzyme. There is also a need to choose a support with good mechanical stability. Further, the support material chosen must be compatible with the enzyme protein so as to either maintain or enhance its desired S/H ratio.
Though PGA immobilization has witnessed extensive evolution to cater to industrial needs. This ranges from immobilization on agarose gel to monolithic silica supports, activated chitosan, magnetic nano-particles each with its distinct advantages and disadvantages for various substrates and products. On a commercial scale, enzyme carriers need to be more robust to withstand high attrition influencing operational stability. US Patent No. 6,218,138 reports EupergitRC 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 ß-lactam antibiotics.
The nature of supports hydrophobic or hydrophilic affects catalytic efficiency, kinetic parameters such as solvent, pH, temperature etc. and downstream process for product recovery. The extracellular penicillin acylase from Bacillus megaterium was immobilized by coupling to derivatives of polyacrylonitrile fibers was used for 50 cycles of cephalexin synthesis at 40°C and pH 6.5 with 83.9 % activity retention.
PGA from Arthrobacter viscosus immobilized on hydrophobic acrylic epoxy-supports (Eupergit C) during N –acylation of 7-aminocephalosporanic acid acted as a poor matrix and showed low turnover rate. It also reports cephalexin synthesis wherein the molar ratio of nucleus to acyl donor is 1:3 which may not prove to be an economically viable option. US 7,264,943 discloses polyvinyl alcohol-gelatin.
WO2010/055527 which uses the step of enzymatic acylation in the suspension reaction has a disadvantage of reduced operational stability, increased swell volume and filtration time thereby affecting process cost.
US2005/0084925 reports cephalexin use of hydrophobic copolymer for immobilization of penicillin G acylase for enzymatic cephalexin synthesis.
In addition, efforts to improve the immobilization efficiency were made by modifying the designed polymers by introducing additional reactive group by using aliphatic amines based on literature reports. Reports of immobilization on hetero-functional epoxy supports and novel amino epoxy polymers, wherein the polymer contain additional amino groups with epoxide making an additional site for enzyme binding are known in the prior art. However, considering the above factors, immobilization needs to be designed for this application and hence a critical factor.
Enzymatic process of antibiotic synthesis is influenced by various factors which determine the process viability in commercial context. Reported data on different process design and parameters indicate variety of influencing factors.
Input of substrate is one of the key factors which influence process economics. The kinetics of reaction ratio demands that nucleus to acyl donor ratio used in the process be optimum to drive the synthetic reaction, usually the acyl donor is in excess. However, for the process to be economically viable, ratio should be minimum which is influenced by the type of enzyme used. Enzyme input w.r.t. substrate is critical from the point of achieving maximum conversion in suitable time period.
PCT Publication No. WO 93/12250 discloses cephalexin synthesis wherein the product isolation is by complex formation with of beta-naphthol which requires further purification and hence may result in lower product yield.
The process parameters like pH, temperature and stirring are significant with respect to stability of substrate, enzyme and product. Further the precipitation of products results in suspended reaction which makes imperative that downstream process be designed so as to separate the product and enzyme to be recycled without incurring significant product loss and complete enzyme recovery.
In case of an in situ process wherein the substrate contains a byproduct such as liquid 6-APA or liquid 7-ADCA containing traces of PAA, the enzyme should tolerate PAA derived by catalysis of semi-synthetic ß-lactam. The process parameters also influence the enzyme kinetics for the reaction.
SUMMARY OF THE INVENTION
In an aspect, the present invention provides ß-lactam antibiotics enzymes obtained by site directed mutagenesis in genes encoding penicillin G acylase synthesized by Achromobacter CCM4824.
In another aspect, the present invention provides mutations in nucleotide sequence represented by SEQ ID No.1 encoding amino acid at one or more positions resulting in mutant penicillin acylase enzymes with high specificity and productivity are borne on plasmid pKRA9 carried on recombinant vector in host E. coli BL21 and the application of the enzyme for synthesis of semi-synthetic ß-lactam antibiotics like amoxicillin, cephalexin, cefaclor, cefadroxil, cefradine etc.
In yet another aspect, the present invention relates to penicillin G acylase mutants (mNPGA) obtained by amino acid substitution at single or more than one points or combination in gene sequence, wherein the resultant mutants show improved S/H ratio, synthetic activity or both for different semi-synthetic ß-lactam antibiotics.
Mutant Penicillin G acylase (mNPGA) variants so derived show tolerance to phenyl acetic acid (PAA) which is a competitive inhibitor generated as a by-product of hydrolysis reaction providing an added advantage for the feasibility of use of substrate containing traces of phenyl acetic acid.
The ter-polymer system each comprising of epoxy macroporous beads vary in composition with respect to epoxy content, hydrophilicity, porous structure & size all of which influence the immobilization and in turn recyclability and reusability of immobilized enzyme.
The present invention provides unique ter-polymers which are custom designed for binding of Penicillin acylase enzymes to form immobilized biocatalyst. Each of the disclosed polymers from the range of polymers used for immobilized biocatalyst are uniquely suitable for different antibiotic synthesis and recyclability by virtue of pore size, porosity, specific surface area, epoxy and cross-linking density. Amination of the said polymer and cross-linking thereafter creates additional spacer arm and further improves enzyme binding making the biocatalyst more stable for recyclability and reduces enzyme leaching from the polymer on use.
Since the immobilized enzyme undergoes change in the conformation due to binding on to supports, immobilization and immobilization supports also influence the catalytic efficiency, kinetic properties, thermal stability and tolerance to reaction conditions like pH, temperature, solvents of the enzyme.
The present invention provides a process for enzymatic synthesis of semi-synthetic ß-lactam antibiotic using the said mutant penicillin acylase enzyme and single pot enzymatic synthesis using dual enzyme system of parent and mutant penicillin acylase enzymes and its commercial viability for both the processes and distinct advantage over the native Penicillin G acylase (pNPGA) from Achromobacter sp CCM 4824 disclosed in US 8,039,604.
The series of semi-synthetic ß-lactam antibiotics which can be synthesized by enzymatic acylation of the said enzyme include amoxicillin, ampicillin, cephalexin, cefadroxil, cefaclor, cefprozil and cefradine.
The present invention provides stability of immobilized mutant PGA in repeated cycles of semi-synthetic ß-lactam antibiotic synthesized by enzymatic reaction under controlled conditions mentioned thereof. The invention also discloses the enzyme suitability for both reaction and product isolation from the reaction mixture.
DETAILED DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the substrate specificity comparison of mNPGA mutants,
Figure 2 depicts the antibiotic synthesis activity comparison of mNPGA mutants, and
Figure 3 depicts the synthesis to hydrolysis ratio comparison of mutants for different antibiotic substrate.

Source of biological material: E.coli BL-21 carrying the mutant penicillin G acylase is deposited as MTCC 25429. The DNA sequence encoding penicillin G acylase was isolated from Achromobacter sp. CCM4824 isolated by Institute of Microbiology, Czech Republic and mutated to be expressed in E.coli BL-21.

DETAILED DESCRIPTION OF THE INVENTION
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.
The present invention provides a series of mutant penicillin G acylases encoded by DNA sequence of penicillin acylase from Achromobacter sp. CCM4824 for producing semi-synthetic ß-lactam antibiotics, wherein the said mutation are generated by site directed mutations with substitution of one or more amino acid positions depicted in SEQ ID No.2.
In a preferred embodiment, the present invention provides a mutant penicillin G acylase encoded by DNA sequence of penicillin G acylase of Achromobacter sp. CCM4824 for producing semi-synthetic ß-lactam antibiotics, wherein the mutations by site directed mutagenesis with substitution of one or more amino acid position selected from the group consisting of amino acid positions A3, A27, A77, A144, A145, A146, A192, A195, A196, B23, B24, B31, B57, B69, B77, B313, B464 and B484 according to the amino acid numbering of the penicillin G acylase of Achromobacter sp. CCM4824 with the polypeptide having SEQ ID No: 2;
wherein the Achromobacter sp. CCM4824 penicillin G acylase consists of,
(a) a signal peptide consisting of 40 amino acids at amino acid positions 1-40,
(b) an a-subunit consisting of 209 amino acids at amino acid positions 41-249, represented by A1 to A209,
(c) a connecting peptide consisting of 57 amino acids at amino acid positions 250-306, and
(d) a ß-subunit consisting of 557 amino acid position at amino acid positions 307-863 represented by B1 to B557.

In an embodiment, the present invention provides for the site directed mutagenesis replacing specific amino acid positions of SEQ ID No. 2 selected from the group comprising of A3, A27, A77, A90, A144, A145, A146, A192, A195, A196, B23, B24, B31, B57, B69, B77, B148, B313, B464, B484 resulting in each mutant labelled in numerical sequence starting from 1 prefixed with mNPGA.
SEQ ID No: 2
MKQQWLSAALLAASSCLPAMAAQPVAPAAGQTSEAVAARPQTADGKVTIRRDAYGMPHVYADTVYGIFYGYGYAVAQDRLFQMEMARRSTQGRVAEVLGASMVGFDKSIRANFSPERIQRQLAALPAADRQVLDGYAAGMNAWLARVRAQPGQLMPKEFNDLGFAPADWTAYDVAMIFVGTMANRFSDANSEIDNLALLTALKDRHGAADAMRIFNQLRWLTDSRAPTTVPAEAGSYQPPVFQPDGADPLAYALPRYDGTPPMLERVVRDPATRGVVDGAPATLRAQLAAQYAQSGQPGI
AGFPTTSNMWIVGRDHAKDARSILLNGPQFGWWNPAYTYGIGLHGAGFDVVGNTPFAYPSILFGHNAHVTWGSTAGFGDDVDIFAEKLDPADRTRYFHDGQWKTLEKRTDLILVKDAAPVTLDVYRSVHGLIVKFDDAQHVAYAKARAWEGYELQSLMAWTRKTQSANWEQWKAQAARHALTINWYYADDRGNIGYAHTGFYPRRRPGHDPRLPVPGTGEMDWLGLLPFSTNPQVYNPRQGFIANWNNQPMRGYPSTDLFAIVWGQADRYAEIETRLKAMTANGGKVSAQQMWDLIRTTSYADVNRRHFLPFLQRAVQGLPADDPRVRLVAGLAAWDGMMTSERQPGYFDNAGPAVMDAWLRAMLRRTLADEMPADFFKWYSATGYPTPQAPATGSLNLTTGVKVLFNALAGPEAGVPQRYDFFNGARADDVILAALDDALAALRQAYGQDPAAWKIPAPPMVFAPKNFLGVPQADAKAVLCYRATQNRGTENNMTVFDGKSVRAVDVVAPGQSGFVAPDGTPSPHTRDQFDLYNTFGSKRVWFTADEVRRNATSEETLRYPR

The underlined region of the sequence is the alpha portion of the penicillin G acylase, the bold underline region is the beta potion of the penicillin G acylase. The signal peptide corresponds to 40 amino acids – in italics, the a-subunit (209 amino acids-single underlined), the linker peptide (57 amino acids) and the ß-subunit (557 amino acid-underlined in dark). The a-subunit amino acids are numbered A1-A209 and the ß-subunit amino acids are numbered B1 to B557. The signal peptide as well as the connecting peptide are removed during post-translational maturation thus yielding an enzymatically active penicillin acylase composed of a and ß subunit.

In another preferred embodiment, the present invention provides a mutant penicillin G acylase derived from Achromobacter CCM 4824 is having at least an amino acid substitution at position B24 and amino acid substitution at one or more positions selected from the group consisting of A3, A27, A77, A90, A144, A145, A146, A192, A195, A196, B23, B24, B31, B57, B69, B77, B313, B464, B484.
(i) The position A3, in penicillin G acylase derived from Achromobacter sp. CCM4824 harbours an alanine (A) amino acid residue. A preferred mutation at position A3 comprises the replacement of alanine by leucine (L).
(ii) Position A27, in the penicillin G acylase derived from Achromobacter sp. CCM4824, harbours an Isoleucine amino acid residue. A preferred mutation at position A27 comprises the replacement of isoleucine (I) by asparagine (N) or proline (P). A highly preferred mutation would be Asparagine (N).
(iii) Position A77, in penicillin G acylase derived from Achromobacter sp.CCM4824, harbours an arginine amino acid residue. A preferred mutation comprises the replacement of arginine (R) by threonine (T).
(iv) Position A144, in penicillin G acylase Achromobacter sp.CCM4824, harbours an Asparagine amino acid residue. A preferred mutation comprises the replacement of Asparagine (N) by threonine (T).
(v) Position A145, in penicillin G acylase Achromobacter sp.CCM4824, harbours an arginine amino acid residue. A preferred mutation comprises the replacement of Arginine (R) by leucine (L).
(vi) Position A146, in penicillin G acylase Achromobacter sp.CCM4824, harbours a phenyalanine amino acid residue. A preferred mutation comprises the replacement of phenylalanine (F) to alanine (A).
(vii) Position A192, in penicillin G acylase Achromobacter sp.CCM4824, harbours an alanine amino acid residue. A preferred mutation comprises the replacement of alanine (A) to glutamic acid (E).
(viii) Position A195, in penicillin G acylase Achromobacter sp.CCM4824, harbours a Glycine amino acid residue. A preferred mutation comprises the replacement of Glycine (G) by Leucine (L).
(ix) Position A196, in penicillin G acylase Achromobacter sp.CCM4824, harbours a Serine amino acid residue. A preferred mutation comprises the replacement of Serine by threonine (T) or Leucine (L).
(x) Position B23, in penicillin G acylase Achromobacter sp.CCM4824, harbours the Glutamine amino acid residue. A preferred mutation comprises the replacement of Glutamine (Q) by Glycine (G).
(xi) Position B24, in penicillin G acylase Achromobacter sp.CCM4824, harbours the phenylalanine amino acid residue. A preferred mutation comprises the replacement of phenylalanine (F) to alanine (A)
(xii) Position B31, in penicillin G acylase Achromobacter sp.CCM4824, harbours the Tyrosine amino acid residue. A preferred mutation comprises the replacement Tyrosine (Y) by Serine (S).
(xiii) Position B57, in penicillin G acylase Achromobacter sp.CCM4824, harbours the phenylalanine amino acid residue. A preferred mutation comprises the replacement of phenylalanine (F) to alanine (A).
(xiv) Position B69, in penicillin G acylase Achromobacter sp.CCM4824, harbours the Alanine amino acid residue. A preferred mutation comprises the replacement of Alanine (A) by Glycine (G).
(xv) Position B77, in penicillin acylase Achromobacter sp.CCM4824, harbours the Isoleucine amino acid residue. A preferred mutation comprises the replacement of Isoleucine (I) by Cysteine (C).
(xvi) Position B313, in penicillin acylase Achromobacter sp.CCM4824, harbours Glycine amino acid residue. A preferred mutation comprises the replacement of Glycine (G) by Valine (V).
(xvii) Position B464, in penicillin G acylase Achromobacter sp.CCM4824, harbours the Threonine amino acid residue. A preferred mutation comprises the replacement of Threonine (T) to glutamine (Q) or serine (S)
(xviii) Position B484, in penicillin G acylase Achromobacter sp.CCM4824, harbours the Asparagine amino acid residue. A preferred mutation comprises the replacement of Asparagine (N) to serine (S) or threonine (T).

In an embodiment, the present invention provides a nucleotide sequence encoding mutant penicillin acylase borne on plasmid pKARA9 is expressed in E. coli BL21. Both the plasmid and host E. coli BL21 were procured from Institute of Microbiology; Czech Republic. The mutant penicillin G acylase is encoded by a nucleotide sequence having at least 60% similarity with SEQ ID No.1 encoding mutant penicillin acylase.
The enzymes so obtained from recombinant E. coli BL21 showed improved penicillin acylase activity, S/H ratio, lower input ratio of acyl donor and improved yields compared to native penicillin acylase enzyme from Achromobacter sp CCM 4824. Enzymes so obtained are found suitable for enzymatic synthesis of semi-synthetic ß-lactam antibiotic like amoxicillin, ampicillin, cephalexin, cefadroxil, cefaclor etc. and enzymatic process using the said enzyme is commercially viable.
Further, the mutant penicillin acylase enzymes are immobilized on customized ter-polymer system of epoxy polyacrylate resins and evaluated for recyclability and suitability in process for production of semi-synthetic ß-lactam antibiotics. Mutant penicillin acylase enzymes in immobilized form and process parameters including raw material input, reaction conditions and product isolation are designed to enable the commercial viability.
The present invention provides mutants of penicillin G acylase produced by Achromobacter sp. CCM 4824 transformed in Escherichia coli BL21 borne on plasmid vector PKRA9 bearing the mutant penicillin G acylase enzyme gene.
The SEQ ID No. 2 of the present invention has 54% homology as compared to wild type E. coli Penicillin acylase. The SEQ ID No: 2 depicted herein above corresponds to the amino acid sequence of Penicillin G acylase produced by Achromobacter sp.CCM4824. A naturally occurring Penicillin G acylase is selected from microorganisms which have at homology in the range of 40-100% to SEQ ID No.2. More preferred penicillin acylases which are 50% to 100% homologous to SEQ ID No.2.
In an embodiment, the present invention provides mutant sequences of penicillin acylase obtained by replacement of amino acid mentioned above at more than one position in combinations in SEQ ID No. 2.
In another preferred embodiment, the present invention provides the substitution of an amino acid at B24 in combination with substitution at one or more positions selected from of A3, A27, A77, A90, A144, A145, A146, A192, A195, A196, B23, B31, B69, B77 and B313. Amongst the selected amino acid changes in combination with B24 include [A3+A192+196], [A3+ A145+A192+A27], [A145+B77], [A3+A145+B31], [A145 +A192+B69] and [A145 +A146+B313].
The mutant so obtained showed >95% homology to Penicillin acylase from Achromobacter sp.CCM4824 depicted in SEQ ID No: 2.
In one preferred embodiment, the present invention provides a mutant E.coli BL-21 carrying the mutant penicillin G acylase which is deposited as MTCC 25429.
Further, the mutants so obtained labelled starting numerically from mNPGA 1 and subsequently numbered further were expressed in host bacteria E. coli BL21 by recombination plasmid vector pKRA9 carrying the gene of mutant penicillin acylase.
In a preferred embodiment, the present invention provides cultivated mNPGA are compared with native penicillin G acylase (pNPGA) for the analytical parameters including its activity, substrate specificity, synthesis/Hydrolysis(S/H) ratio for various substrates, inhibitor tolerance, optimum substrate and enzyme concentration to achieve maximum conversion to final antibiotic under defined process conditions.
Penicillin G acylase enzyme obtained is quantified in terms of activity in Units (U) and protein in mg/mL
Proteins are determined by Folin-Lowry method and Penicillin G hydrolysis activity expressed as U/g 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 0.1N sodium hydroxide in the milieu of first order kinetics.
Activity in terms of synthesis of antibiotics in SU/g and S/H ratio with said enzymes and biocatalyst is determined by HPLC details listed below for respective antibiotics.
HPLC analysis for Amoxicillin (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 µl, Run Time: 20 minutes.
HPLC analysis for Ampicillin (AMP) synthesis:
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 2N Sodium hydroxide. Mobile phase: Buffer: Methanol: Acetonitrile::70: 23: 7, Column oven temperature 35 °C, Detection Wavelength: 215 nm, Flow Rate: 1.2 mL/min, Injection Volume: 20 µl.
HPLC analysis for Cephalexin (CPX) synthesis:
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 litre HPLC grade water, pH 6.4 with 2N 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 Cefprozil (CPZL) synthesis:
Column: Inertsil C8 250mm X 4.6 mm (5micron), temperature 30 °C, Buffer: 11.5 g ammonium hydrogen phosphate (pH 5.4) in 1 litre HPLC grade water, pH 5.4 with 2N 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 µl.
HPLC analysis for Cefaclor (CCL) synthesis:
Column: Inertsil C8 250mm X 4.6 mm (5micron), temperature 35 °C, Buffer: 0.1 M ortho-phosphoric acid + 0.4% Triethylamine in 1 litre HPLC grade water, pH 2.5 with 2N 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 Cefadroxil (CDL) synthesis:
Column: Inertsil C8 250mm X 4.6 mm (5micron), temperature 25 °C, Buffer: 13.6 g potassium dihydrogen phosphate dissolved in 1 litre 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 µl.
HPLC analysis for Cefradine (CFD) synthesis:
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 litre HPLC grade water, pH 6.4 with 2N 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.
Further, an embodiment describes epoxy ter-polymer combination obtained by suspension polymerization of acrylic monomers as beads of specific surface area and porosity and particle size, modification process to include an additional spacer arm in epoxy beads by amination with various aliphatic amines to enable maximum expression of enzyme bound on these support with improved mechanical stability, filtration ability and recyclability on use of immobilized enzyme in suspension reaction for antibiotic synthesis.
Ter-polymer are synthesized by free radical suspension polymerization wherein the monomer components added either or all used in different combination include glycidyl methacrylate (GMA), allyl glycidyl ether, Ethylene glycol dimethacrylate (EGDM), divinylbenzene (DVB), ethyl methacrylate (EMA), methylmethacrylate (MMA) and acrylonitrile (AN).
By virtue of nature of each monomers and the quantity used on polymerization with itself and other monomers impart specific characteristic to the beads resulting in variable epoxy content, cross-linking density, porosity, hydrophilicity and rigidity.
The molar ratios of monomers like Ethylene glycol dimethacrylate, divinyl benzene to other monomers result in variable cross-linking density in polymer beads. Epoxy content varies with quantity of glycidyl methacrylate or allyl glycidyl ether. The acrylonitrile content imparts rigid backbone to the polymer beads making them mechanical stronger than ones without the component.
The present invention discloses cross-linking density range from 25 to 200 for series of ter- polymers.
The macroporous nature imparted to the beads is by use of either aliphatic or aromatic alcohols or both chosen from dodecanol, octanol, hexanol and cyclohexanol which act as porogens added between 1.1 to 1.5 times of total monomer content.
Free radical polymerization is initiated by agents like benzoyl peroxide or azobisisobutyronitrile used between 2-4% and stabilized by either poly vinyl pyrollidone or poly vinyl alcohol used up to 8% of the total monomer weight.
Polymer beads were selectively prepared to obtain a degree of hydrophobicity by use of additives chosen from Span 60, Span 80, Span 85 and Sodium lauryl sulphate. Additives were added in concentration ranging from 0.01 % to 5% based on monomer concentration.
The additives imparted hydrophobic microenvironment in the pores which favoured the driving of synthesis reaction by the enzyme as compared to hydrolysis. This in turn favoured the suitability of biocatalyst in suspension reaction.
In the present embodiment, the designed and optimized process parameters using the said enzyme /enzymes makes the process viable for scale-up on industrial scale.
Further, an additional effort created a mechanically more stable polymer beads wherein the polymer were treated with aliphatic amines chosen from range of ethylamine, triethylamine, diethyl amine, butylamine and ethylene diamine in concentration of 25-50% of weight of polymer with solvents chosen from toluene or methanol at temperature between 40-80°C for 10h to 150h. The degree of amination was checked with color development of polymer with 0.1% w/v ninhydrin solution in water.
The aminated polymer beads were washed and further treated with 2%-8% v/w glutaraldehyde for 1h to 20h to crosslink the amino group to from activated amino epoxy polymer.
The polymer beads disclosed in the invention consist of poly (GMA-ter-EGDM-ter AN) and poly (GMA-EGDM-DVB-AN) are labelled as DILBEADS EG, DILBEADS EGX and activated amino epoxy polymers are referred as DILBEADS EGXAT.
The present embodiment discloses the analytical parameters pertaining to particle size, porosity, pore diameter and surface area of the above described polymer beads analyzed by laser diffraction analyzer (Particle size analyzer, HELOS H1004) and mercury intrusion porosimetry (Fisons Instruments Pascal 140/240 porosimeter) and BET (Brunaer-Emmett-Teller) respectively. Particle size between 200-500 microns, average pore diameter between 30-120 nm, pore volume between 0.4-1.5 cm3/g and surface area between 50 m2/g-160 m2/g.
In another embodiment, the invention discloses a process for synthesis of semi-synthetic ß-lactam antibiotics (SSA) using mNPGA enzymes more preferably biocatalyst immobilized on DILBEADS EG, DILBEADS EGX and DILBEADS EGXAT wherein the reaction conditions include the following:
(a) The substrates in synthesis reaction wherein in the said enzyme catalyses the acylation to synthesize semi-synthetic ß-lactam antibiotic are ß-lactam nucleus selected from 6-aminopenicillanic acid (6-APA), 7-desacetoxycephalosporanic acid (7-ADCA), 7-aminocephalosporanic acid (7-ACA), 7-Amino-3-Chloro-3-Cephem-4-Carboxylicacid (7-ACCA), and 7-Amino-3-[(Z)-propen-1-yl] -3-cephem-4-carboxylic acid (7-APCA) and activated acyl donor selected from ester or amide of hydroxyphenyl glycine (HPGME) or phenylglycine (PGME) or dihydroxyphenylglycine (DHPGME).
(b) The molar ratio of acyldonor to nucleus varies between 1:1 to1:1.2 more preferably between 1:1.02 to 1: 1.1 based on form of acyl donor as free acid or hydrochloride salt for different antibiotics.
(c) The concentration of nucleus in the reaction mixture varies in range of 6-12% w/v more preferably between 8-10%w/v.
(d) Enzyme quantity used more preferably in the ratio of 90-250 SU/g of activity per gram of ß-lactam nucleus.
(e) Conversion of ß-lactam nucleus achieved between 50-99 % in 90-240 minutes.
(f) Suitability of reusing immobilized biocatalyst for repeated cycles for different antibiotics and product isolation after enzymatic conversion in suspension reaction.
Representative enzymatic reaction cycles for amoxicillin synthesis and product isolation using immobilized biocatalyst of mNPGA have been disclosed in the examples.
The series of ter-polymer forming macroporous beads prepared by free radical polymerization consists of 3 or 4 monomers, diluent, initiator and stabilizer wherein;
(a) Monomer 1 comprising of 15-60% of total weight of the monomers is GMA capable of free radical polymerization and has epoxy groups.
(b) Monomer 2 comprising of 20-55% of total weight of the monomers is DVB or EGDM or both capable of free radical polymerization and is cross linking agent.
(c) Monomer 3 comprising of 20-55% of total weight of the monomers is AN capable of free radical polymerization and is a rigid backbone of the ter-polymer system.
(d) The porogen are from the group of hydrophilic or cyclic alcohols comprises of cyclohexanol, octanol, lauryl alcohol and combinations of these, are 1.2-1.5 times of total weight of the monomers.
(e) The initiator is selected from the group of benzoyl peroxide, azobisisobutyronitrile; methyl ethyl ketone peroxide is between 3-3.5% with respect to total weight of monomers.
(f) The suspension stabilizer is selected from the group of poly vinyl pyrollidone, poly vinyl alcohol; poly acrylic acid is between 7-7.5% with respect to total weight of monomers.
The macroporous beads formed in series of ter-polymer system are aminated with aliphatic amines selected from group from Diamino ethane, butylamine, triethylamine and diethylamine
Accordingly, the Immobilized biocatalyst is used in the ratio of 90-250 U/g of activity per gram of ß-lactam nucleus. The conversion of ß-lactam nucleus is achieved between 50-99 %with product yield achieved up to 90-95% in 120-240 minutes for 100 repeated cycles. The Immobilized biocatalyst is reused for 300 repeated cycles in suspension reaction containing substrates, immobilized enzyme catalyst and precipitated product. The precipitated product as semi-synthetic ß-lactam antibiotic isolated from the enzymatic reaction mixture.
In one preferred embodiment, the immobilized biocatalyst comprising the mutant penicillin G acylase produced by Achromobacter CCM 4824 shows up to 90% binding protein, up to 97.7% activity binding and up to 40-100% enzyme expression.
Further, the S/H ratio in immobilized biocatalyst in series of ter-polymer system varies between 0.3-1.3 for Amoxicillin synthesis and 0.1-1.49 for Cephalexin synthesis. The concentration of ß-lactam nucleus and activated acyl donor used in the molar ratio of 1:1.02 to 1:1.5.
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:
The isolation and purification of the nucleotide sequence as described in US 8,039,604 was done using Invitrogen’s Pure Link plasmid extraction kit. The isolated plasmid was run on Agarose gel electrophoresis and the plasmid size was confirmed. Using restriction enzyme XbaI and PstI the nucleotide sequence was isolated and then purified by using gel purification kit.

Example 2:
For mutant generation, Toyoba’s Mutagenesis kit was used. Initially suitable primers were designed and mutagenesis was carried out as per the protocol given in Toyoba’s mutagenesis kit. The vector carrying plasmid pKRA9 was grown at 37°C and plasmid isolated using Invitrogen’s Pure Link plasmid extraction kit. The vector was digested with XbaI and PstI, purified and made ready for ligation. Ligation of the vector with the nucleotide sequence obtained in example1 was done using standard protocol as known to the art. The ligated plasmid was then transformed into E.coli BL-21 cells. Earlier, competent cells of BL-21 were prepared using calcium chloride method as known to the art. Transformation was done using heat shock method and plated on LB agar plates and incubated at 37°C overnight. Colonies obtained were screened for activity.

Example 3:
Each of the mutant clones were obtained in the above examples were grown in 10 mL Luria Bertani (LB) medium with kanamycin concentration of 30-50 µg/ mL at 28°C for 18 h at 150 rpm. The culture growth was checked for pH, optical density at 600 nm and centrifuged to obtained cell pellet which was washed and resuspended to dry weight of 3-4%. Each of 100 µl cell suspension was incubated in 1%w/v Penicillin, 0.5%w/v amoxicillin, 0.5 %w/v cephalexin, in 100 mM phosphate buffer pH 7.5 for 15 min and resultant product was determined by color development reaction with 4-(Dimethylamino) benzaldehyde in methanolic acetic acid. The increasing color intensity indicated by +sign measured at 415nm correspond to specificity of each mutant clone to the respective substrate.
Mutants Corresponding mutations Penicillin Amoxicillin Cephalexin
1 A3+A192+A196 +B24 A145+A192+A27],[A145+B77],[A3 + A145+B31],[A145 +A192+B69], [A145 +A146+B313]. ++++ + ++
2 A3+A145+A192+A27 +B24 + + +++
3 A145+B77+B24 - ++ +
4 A3+A145+B24+B31 _ _ +
5 A145+A192+B24+B69 _ ++ ++++
6 A145+A146+B24+B313 +++ ++ -
7 A77+B224+B71+B313 _ _ +
8 A27+B24+B71+B77 + + -
9 A27+B24+B69 ++++ ++ +
10 A3+A144+B24+B313 - + ++
11 A77+A195+B24+B464 +++ _ +
12 A3+B24+B484 - + _
13 A196+B24+B484+B23 ++ ++ +++
14 A144+145+B24+B57 +++++ + -
15 A27+B23+B24+B69 + + +

Examples 4-8: poly(GMA-ter-EGDM -ter-AN) ter-polymers also referred to as DILBEADS-EG consist of composition and quantities as described in Table 1.
Polymer No. GMA-M1 EGDM-M2 AN-M3 % Cross Link Density
Wt. in g Wt. in g Wt. in g
Example 4 30 40 30 25
Example 5 38 50 12 50
Example 6 37 50 13 100
Example 7 32 63 5 150
Example 8 22 73 5 200

In the inert atmosphere of Nitrogen, the respective monomers in mentioned quantities with 110 g cyclohexanol are stirred with 300 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 9: ter-polymer system prepared by the procedure as per Example-4, replacing cyclohexanol with mixture of cyclohexanol and lauryl alcohol in ratio of 90:10 by weight

Example 10: ter-polymer system prepared by the procedure as per Example-4, replacing cyclohexanol with mixture of Cyclohexanol and Hexanol in ratio of 90:10 by weight
Example 11-15: ter-polymer system prepared by the procedure as per Example-4 with following additions
Example Additions Quantity
11 Span 60 1%w/w of total monomer weight
12 Span 60 3% w/w of total monomer weight
13 Sodium dodecyl sulphate 0.015% w/w of total monomer weight
14 Span 80 1% w/w of total monomer weight
15 Span 85 1 % w/w of total monomer weight

Example 16-17: Compositions of Poly(ter-GMA-EGDM-DVB-ACN)a referred as DILBEADS EGX
Polymer No. GMA-M1 EGDM-M2 AN-M3 DVB-M4 % Cross Link Density
Wt. in g Wt. in g Wt. in g Wt in g
Example 16 30 20 30 23.6 25
Example 17 20 30 25 35 65

In the inert atmosphere of Nitrogen, the respective monomers in mentioned quantities with 125-150 g cyclohexanol are stirred with 300 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 18: ter-polymer system prepared by the procedure as per Example-8, replacing cyclohexanol with mixture of isoamyl alcohol and cyclohexanol in ratio of 10:90 by weight.

Example 19: terpolymer system prepared by the procedure as per Example-16, replacing cyclohexanol with mixture of Dodecanol and cyclohexanol in ratio of 3: 5 by weight.
Example 20: DILBEADS EGXAT: 1 g dry DILBEADS EGX added with 5 mL toluene and 1 mL ethyelenediamine. Incubated for 150h at 40° C. Wash the beads with 10 volumes of water and soak in 10 mL methanol for 24 h. wash with 10 mL water twice and air dry. Add 5 mL water and 4 mL glutaraldehyde and stir for 1h at 200rpm. Wash with water till glutaraldehyde is removed completely and air dry.

Example 21:
Table 3: Physical appearance and porosity data of ter-polymers.
Macroporous beads Average Pore Diameter (nm) Total porosity (%) Pore volume (cm3/g) Specific surface area (m2/g) Bulk Density (g/cm3)

Example 4 0.0488 30.355 0.354 55.997 0.859
Example 5 0.0514 44.937 0.628 80.862 0.715
Example 6 0.0624 47.274 0.866 122.16 0.546
Example 7 0.0624 53.466 0.861 110.35 0.621
Example 8 0.123 35.395 0.586 47.329 0.6043
Example 9 0.106 54.908 1.213 93.926 0.452
Example 10 0.11 41.62 0.602 51.02 0.69
Example 16 0.096 32.96 1.376 155.88 0.239
Example 17 0.061 20.293 0.997 142.95 0.203
Example 19 0.11 41.62 0.602 51.02 0.69
Example 20 0.125 7.99 0.261 21.41 0.3055

Example 22-27 Hydrolysis of substrates
Example mNPGA Pen G-H Ceph G-H Nipab-H HPGME-H PGME-H
22 1 710.29 312.08 452.30 361.52 445.19
23 3 0.00 0.00 261.30 9.78 19.13
24 4 97.50 0.00 215.80 48.39 0.71
25 2 108.55 100.00 225.40 57.61 44.10
26 5 172.12 106.49 286.00 134.13 50.24
27 6 0.00 0.00 280.90 38.44 3.52

Substrate hydrolysis of mNPGA
Example mNPGA Amox-S Ampi-S Cpx-S Cefaclor-S Cefadroxil-S Cefprozil-S
22 1 1202.91 441.83 1480.14 972.08 1200.4 357.271
23 3 50.65 0 38.68 34.95 29.6 9.130
24 4 59.29 1.03 0 8.5 1.4 0.000
25 2 76.24 2.75 100.33 71.42 58.3 14.872
26 5 111.06 3.29 150.23 114.63 46.9 11.779
27 6 15.83 0 0 11.75 2.0 0.000

Antibiotic synthetic activity
Example mNPGA SAmox/Hester SAmpi/Hester Scpx/Hester SCefaclor/Hester SCDL/Hester S CFL/H ester
22 1 3.33 1.42 3.325 2.18 1.523 0.819
23 3 5.18 0.02 2.022 1.83 1.107 2.625
24 4 1.23 1.72 0.070 11.90 0.800 0.007
25 2 1.32 0.74 2.275 1.62 1.405 0.604
26 5 0.83 1.57 2.990 2.28 1.311 0.237
27 6 0.41 0.10 0.025 3.34 0.007 0

Example 28:
Immobilized biocatalysts prepared by immobilizing enzyme isolated by process described in Indian Patent No. 253959 from mNPGA mutants of the present invention cultures as described in Example 3. 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 macroporous bead are added to 100 Units of Penicillin G 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 weight of the resulting macroporous beads loaded with PGA is determined. The swell volume after immobilization is between 1.1 to 1.2 times of the initial volume of macroporous beads.

Example Immobilized Biocatalyst with polymer prepared in Protein binding % Activity binding % Activity expression
28 A Example 4 81.36 95.01 96.51
28B Example 5 80.18 90.73 95.44
28C Example 6 74.32 95.28 88.54
28D Example 7 66.00 81.98 44.83
28E Example 8 40.57 63.37 10.85
28F Example 9 70.24 93.64 27.63
28G Example 10 71.11 90.20 36.83
28H Example 11 92.20 72.20 47.12
28I Example 12 89.95 80.50 81.64
28J Example 13 90.10 84.30 38.00
28K Example 14 92.20 88.20 38.80
28L Example 15 56.60 46.40 12.10
28M Example 16 93.20 91.23 71.30
28N Example 17 89.90 87.50 73.20
28O Example 18 79.65 80.41 32.10
28P Example 19 89.35 91.23 74.50
28Q Example 20 85.45 89.66 72.30

Example Immobilized Biocatalyst with polymer prepared in Amoxicillin activity
SU/ g S amox / HHPGME
Cephalexin
Activity
SU/ g S CPX/ HPGME

28 A Example 4 190.00 2.42 160.0 2.45
28H Example 11 54.24 2.40 45.0 2.20
28 I Example 12 103.29 2.60 80.5 2.40
28 J Example 13 75.99 2.80 100.4 2.55
28K Example 14 77.66 2.65 88.6 2.30
28L Example 15 43.25 2.35 45.0 2.60
28M Example 16 175.00 2.45 165.0 2.55
28N Example 17 175.00 2.55 160.0 2.45
28O Example 18 150.00 1.68 140.0 2.20
28 P Example 19 160.00 1.80 150.0 2.15
28Q Example 20 150.00 2.10 160.0 1.88

Example 29: Enzymatic synthesis of Amoxicillin
The reaction is carried out at 20-25 °C, pH 6.3 for 2 hrs in a stirred tank reactor. Reaction mixture consists of 100 mM 6-APA and 102-110mM HPGME in free or hydrochloride form with 200 U of immobilized enzyme catalyst per gram of 6APA in 10 mL of water. Course of the reaction monitored by withdrawing samples at regular interval of time and analyzed by HPLC.

Example Enzyme
From Example HPGME form 6APA: HPGME ratio Reaction time in min % conversion of 6APA % conversion of ester
29 A 28A hydrochloride 1:1.1 75 99.50 96.53
29B 28A hydrochloride 1.04 80 98.70 97.53
29C 28A hydrochloride 1.02 85 98.53 97.88
29D 28A Free 1.04 100 98.40 97.90
29E 28A Free 1.02 105 98.30 98.10
29 F 28 M hydrochloride 1.02 100 98.40 97.78
29G 28 N Free 1.02 85 98.03 96.54
29 H 28N
(quantity of enzyme is half) Free 1.02 160 98.86 97.36
29 I 28Q Free 1.02 90 98.31 97.71
29J 28 Q hydrochloride 1.02 90 98.80 96.54

Example 30: Enzymatic synthesis of Ampicillin
The reaction is carried out at 20-25 °C, pH 6.3-6.8 for 2 hrs in a stirred tank reactor. Reaction mixture consists of 100 mM 6APA and 105-110 mM PGME hydrochloride form with 150-170 U of immobilized enzyme catalyst per gram of 6APA in 10 mL of water. Course of the reaction monitored by withdrawing samples at regular interval of time and analyzed by HPLC.
Example Enzyme
From Example
6APA: HPGME ratio
Reaction time in min
% conversion of 6APA % conversion of ester
30A 28A 1:1.10 90 99.00 96.39
30B 28 A 1:1.05 135 98.54 97.63
30C 28A 1:1.02 200 98.80 97.70
30D 28M 1:1.05 120 98.62 96.51
30E 28 N 1:1.05 110 98.53 96.23
30F 28 Q 1.1.05 110 98.56 96.41
30G 28 M 1:1.10 80 98.80 96.34
30H 28N 1:1.10 90 98.40 95.92

Example 31: Enzymatic synthesis of Cephalexin
The reaction is carried out at 20-25 °C, pH 6.3-6.8 for 2 hrs in a stirred tank reactor. Reaction mixture consists of 100 mM 7ADCA and 105-110 mM PGME hydrochloride form with 150-170 U of immobilized enzyme catalyst per gram of 7ADCA in 10 mL of water. Course of the reaction monitored by withdrawing samples at regular interval of time and analyzed by HPLC.
Example Enzyme
from Example 6APA: HPGME ratio Reaction time in min % conversion of 6APA % conversion of ester
31 A 28A 1:1.1 70 98.55 96.93
31 B 28 B 1:1.1 90 98.45 96.33
31C 28 C 1:1.1 95 98.55 97.45
31D 28 D 1:1.1 95 98.24 97.12
31E 28 E 1:1.1 100 98.76 97.97
31 F 28 M 1:1.1 80 98.56 97.4

Example 32: Enzymatic synthesis of cefadroxil
The reaction is carried out at 20-25 °C, pH 6.3-6.8 for 3 hrs in a stirred tank reactor. Reaction mixture consists of 100 mM 7ADCA and 110 mM HPGME hydrochloride form with 200 U of immobilized enzyme catalyst per gram of 7ADCA in 10 mL of water. Course of the reaction monitored by withdrawing samples at regular interval of time and analyzed by HPLC.
Example Enzyme
From Example
Reaction time in min
% conversion of 6APA % conversion of ester
32 A 28A 85 98.56 97.36
32 B 28 B 110 98.2 97.88
32C 28 C 125 98.01 97.23
32D 28 D 135 98.11 97.45
32E 28E 120 98.24 97.10
32 F 28 M 95 98.76 97.97

Example 33: Enzymatic synthesis of cefaclor
The reaction is carried out at 20-25 °C, pH 6.5-6.8 for 2 hrs in a stirred tank reactor. Reaction mixture consists of 100 mM 7ACCA and 120 mM PGME hydrochloride form with 225 U of immobilized enzyme catalyst per gram of 7ACCA in 10 mL of water. Course of the reaction monitored by withdrawing samples at regular interval of time and analyzed by HPLC.
Example Enzyme from Example 7-ACCA (%) PGME (%)
33 A 28A 98.66 98.99
33 B 28B 75.49 74.49
33 C 28C 77.25 76.39
33 D 28D 75.36 72.12
33 E 28E 76.49 72.3

Example 34: Enzymatic synthesis of cefprozil
The reaction is carried out at 20-25 °C, pH 6.5-6.8 for 2 hrs in a stirred tank reactor. Reaction mixture consists of 100 mM 7APCA and 120 mM PGME hydrochloride form with 225 U of immobilized enzyme catalyst per gram of 7APCA in 10 mL of water. Course of the reaction monitored by withdrawing samples at regular interval of time and analyzed by HPLC.
Example Enzyme from Example % 7-APCA conversion % HPGME hydrolysis
34A 28A 84.24 90.05
34B 28B 82.71 94.47
34C 28C 83.00 95.09
34D 28D 83.53 92.54
34E 28E 86.07 91.3

Example 35: Enzymatic synthesis of cefradine
The reaction is carried out at 20-25 °C, pH 6.3-6.8 for 2 hrs in a stirred tank reactor. Reaction mixture consists of 100 mM 7ADCA and 130 mM HPGME form with 250 U of immobilized enzyme catalyst per gram of 7ADCA in 10 mL of water. Course of the reaction monitored by withdrawing samples at regular interval of time and analyzed by HPLC.
Example Enzyme from Example % 7-APCA conversion % HPGME hydrolysis
35A 28A 54.92 51.68
35 B 28 C 50.5 48.33

Advantages of the invention:
1. Enzyme so obtained found suitable for enzymatic synthesis of semi-synthetic ß-lactam antibiotic like amoxicillin, ampicillin, cephalexin, cefadroxil, cefaclor etc and enzymatic process using the said enzyme is commercially viable.
2. Further mutant penicillin acylase enzymes are immobilized on customized ter-polymer system of epoxy polyacrylate resins and evaluated for recyclability and suitability in process for production of semi-synthetic ß-lactam antibiotics.
3. Mutant penicillin acylase enzymes in immobilized form and process parameters including raw material input, reaction conditions and product isolation are designed to enable the commercial viability.
4. The activity of immobilized biocatalyst retained to 90% after recycling in enzymatic process.

SEQUENCE LISTING
<110> Fermenta Biotech Ltd.
<120> Mutant Penicillin G acylases of Achromobacter CCM4824
<140>
<141>
<150> Indian Patent Application No.202021000782
<151> 08/01/2020
<160> 2

<210> 1
<211> 2592
<212> DNA
<213> Artificial sequence
<220>
<223> Recombinant DNA
<400> 1
atgaagcagc aatggttgtc ggccgccctg ttggcggcca gttcgtgcct gcccgcgatg 60
gcggcgcagc cggtggcgcc agccgccggc cagacgtccg aggcggttgc ggcacggccc 120
caaaccgccg atggcaaggt cacgatccgg cgcgatgcct acggcatgcc gcatgtctat 180
gccgacacgg tgtacggcat cttctacggc tacggctacg cggtggcgca ggaccggctg 240
ttccagatgg agatggcgcg gcgcagcacc cagggccggg tggccgaggt gctgggcgcc 300
tcgatggtgg gcttcgacaa gtcgatccgc gccaatttct cgcccgagcg catccagcgc 360
cagttggcgg cgctgccggc cgccgaccgc caggtgctgg acggctacgc ggctggcatg 420
aacgcctggc tggcgcgggt gcgggcccag ccgggccaac tgatgcccaa ggaattcaat 480
gacctgggtt tcgcgccggc cgactggacc gcctacgacg tggcgatgat cttcgtcggc 540
accatggcca accgcttttc ggacgccaac agcgagatcg acaacctggc gctgctgacg 600
gcgttgaagg accggcatgg cgccgccgat gccatgcgca tcttcaacca gttgcgctgg 660
ctgaccgaca gccgcgcgcc gaccacggtg ccggccgaag cgggcagcta ccagccgccg 720
gtgttccagc cggacggcgc ggacccgctg gcctacgcgc tgccgcgcta cgacggcacg 780
ccgccgatgc tcgagcgggt ggtgcgcgac ccggccacgc ggggcgtggt cgacggcgcg 840
ccggcgacgc tgcgggcgca actggccgcc caatacgcgc aatcgggcca gcccggcatc 900
gccggctttc cgaccaccag caatatgtgg atcgtgggcc gcgaccacgc caaggacgcg 960
cgctcgatcc tgctgaacgg cccgcagttc ggctggtgga atccggccta tacctacggc 1020
atcggcttgc acggcgccgg cttcgacgtg gtcggcaaca cgccgttcgc ctatcccagc 1080
attctgttcg gccacaatgc acacgtgacg tggggttcga ccgcgggctt cggcgatgac 1140
gtcgacatct ttgccgaaaa gctcgatccc gccgaccgca cgcgctattt ccacgacggc 1200
caatggaaga cgctggaaaa gcgcaccgac ctgatcctgg tgaaggacgc ggcgccagtg 1260
acgctggacg tgtaccgcag cgtgcatggc ctgatcgtca agttcgacga cgcgcagcac 1320
gtggcctacg ccaaggcgcg cgcctgggaa ggctatgaac tgcaatcgct gatggcctgg 1380
acccgcaaga cgcaatcggc caactgggaa cagtggaagg cgcaggcggc gcgccatgcg 1440
ctgaccatca actggtacta cgccgacgac cgcggcaaca ttggctacgc gcacacgggc 1500
ttctatccca ggcgccgtcc gggccacgat ccgcgcctgc cggtgcccgg caccggcgag 1560
atggactggc tgggcctgct gccgttctct accaatccgc aggtctacaa cccgcgccag 1620
ggcttcatcg ccaactggaa caaccagccg atgcgcggct acccgtccac cgacctgttc 1680
gccatcgtct ggggccaggc cgaccgctac gccgagatcg agacgcgcct gaaggccatg 1740
accgcgaacg gaggcaaggt cagcgcgcag cagatgtggg acctgatccg caccaccagc 1800
tacgccgacg tcaaccgccg tcatttcctg ccgttcctgc aacgcgcggt gcaagggctg 1860
ccggcggatg atccgcgcgt gcgcctggtg gccggcctgg cggcctggga cggcatgatg 1920
accagcgagc gccaaccggg ttacttcgac aacgccggcc cggcggtcat ggacgcgtgg 1980
ctgcgcgcca tgctgcggcg cacgctggcc gacgagatgc cggccgactt cttcaagtgg 2040
tacagcgcca ccggctaccc gacaccgcag gcgccggcca ccggttcgct caacctgacc 2100
accggcgtca aggtgctgtt caacgccctg gccgggcccg aggctggcgt gccgcagcgc 2160
tatgacttct tcaacggcgc gcgcgccgac gacgtcatcc tcgcggcgct ggacgatgcg 2220
ctggcggcgc tgcgccaggc ctatggccag gatccggcgg catggaagat cccggcgccg 2280
ccgatggtgt tcgcgcccaa gaacttcctg ggcgtgccgc aggccgacgc caaggcggtg 2340
ctgtgctatc gggccacgca gaaccgcggc accgagaaca acatgacggt gttcgacggt 2400
aaatcggtgc gcgcggtgga tgtggtggcg ccggggcaga gcggcttcgt cgccccggac 2460
ggcacgccgt cgccgcacac ccgcgaccag ttcgacctgt acaacacctt cggcagcaaa 2520
cgggtgtggt tcacggccga tgaggtgcgg cgcaacgcta cgtcggaaga gacgttgcgc 2580
tacccgcggt aa 2592

<210> 2
<211> 863
<212> PRT
<213> Penicillin G acylase
<220>
<223> Protein sequence of penicillin G acylase produced by Achromobacter CCM4824
5 10 15
Met Lys Gln Gln Trp Leu Ser Ala Ala Leu Leu Ala Ala Ser Ser Cys
20 25 30
Leu Pro Ala Met Ala Ala Gln Pro Val Ala Pro Ala Ala Gly Gln Thr
35 40 45
Ser Glu Ala Val Ala Ala Arg Pro Gln Thr Ala Asp Gly Lys Val Thr
50 55 60
Ile Arg Arg Asp Ala Tyr Gly Met Pro His Val Tyr Ala Asp Thr Val
65 70 75 80
Tyr Gly Ile Phe Tyr Gly Tyr Gly Tyr Ala Val Ala Gln Asp Arg Leu
85 90 95
Phe Gln Met Glu Met Ala Arg Arg Ser Thr Gln Gly Arg Val Ala Glu
100 105 110
Val Leu Gly Ala Ser Met Val Gly Phe Asp Lys Ser Ile Arg Ala Asn
115 120 125
Phe Ser Pro Glu Arg Ile Gln Arg Gln Leu Ala Ala Leu Pro Ala Ala
130 135 140
Asp Arg Gln Val Leu Asp Gly Tyr Ala Ala Gly Met Asn Ala Trp Leu
145 150 155 160
Ala Arg Val Arg Ala Gln Pro Gly Gln Leu Met Pro Lys Glu Phe Asn
165 170 175
Asp Leu Gly Phe Ala Pro Ala Asp Trp Thr Ala Tyr Asp Val Ala Met
180 185 190
Ile Phe Val Gly Thr Met Ala Asn Arg Phe Ser Asp Ala Asn Ser Glu
195 200 205
Ile Asp Asn Leu Ala Leu Leu Thr Ala Leu Lys Asp Arg His Gly Ala
210 215 220
Ala Asp Ala Met Arg Ile Phe Asn Gln Leu Arg Trp Leu Thr Asp Ser

225 230 235 240
Arg Ala Pro Thr Thr Val Pro Ala Glu Ala Gly Ser Tyr Gln Pro Pro
245 250 255
Val Phe Gln Pro Asp Gly Ala Asp Pro Leu Ala Tyr Ala Leu Pro Arg
260 265 270
Tyr Asp Gly Thr Pro Pro Met Leu Glu Arg Val Val Arg Asp Pro Ala
275 280 285
Thr Arg Gly Val Val Asp Gly Ala Pro Ala Thr Leu Arg Ala Gln Leu
290 295 300
Ala Ala Gln Tyr Ala Gln Ser Gly Gln Pro Gly Ile Ala Gly Phe Pro
305 310 315 320
Thr Thr Ser Asn Met Trp Ile Val Gly Arg Asp His Ala Lys Asp Ala
325 330 335
Arg Ser Ile Leu Leu Asn Gly Pro Gln Phe Gly Trp Trp Asn Pro Ala
340 345 350
Tyr Thr Tyr Gly Ile Gly Leu His Gly Ala Gly Phe Asp Val Val Gly
355 360 365
Asn Thr Pro Phe Ala Tyr Pro Ser Ile Leu Phe Gly His Asn Ala His
370 375 380
Val Thr Trp Gly Ser Thr Ala Gly Phe Gly Asp Asp Val Asp Ile Phe
385 390 395 400
Ala Glu Lys Leu Asp Pro Ala Asp Arg Thr Arg Tyr Phe His Asp Gly
405 410 415
Gln Trp Lys Thr Leu Glu Lys Arg Thr Asp Leu Ile Leu Val Lys Asp
420 425 430
Ala Ala Pro Val Thr Leu Asp Val Tyr Arg Ser Val His Gly Leu Ile
435 440 445
Val Lys Phe Asp Asp Ala Gln His Val Ala Tyr Ala Lys Ala Arg Ala
450 455 460
Trp Glu Gly Tyr Glu Leu Gln Ser Leu Met Ala Trp Thr Arg Lys Thr
465 470 475 480
Gln Ser Ala Asn Trp Glu Gln Trp Lys Ala Gln Ala Ala Arg His Ala
485 490 495
Leu Thr Ile Asn Trp Tyr Tyr Ala Asp Asp Arg Gly Asn Ile Gly Tyr
500 505 510
Ala His Thr Gly Phe Tyr Pro Arg Arg Arg Pro Gly His Asp Pro Arg
515 520 525
Leu Pro Val Pro Gly Thr Gly Glu Met Asp Trp Leu Gly Leu Leu Pro
530 535 540
Phe Ser Thr Asn Pro Gln Val Tyr Asn Pro Arg Gln Gly Phe Ile Ala
545 550 555 560
Asn Trp Asn Asn Gln Pro Met Arg Gly Tyr Pro Ser Thr Asp Leu Phe
565 570 575
Ala Ile Val Trp Gly Gln Ala Asp Arg Tyr Ala Glu Ile Glu Thr Arg
580 585 590
Leu Lys Ala Met Thr Ala Asn Gly Gly Lys Val Ser Ala Gln Gln Met
595 600 605
Trp Asp Leu Ile Arg Thr Thr Ser Tyr Ala Asp Val Asn Arg Arg His
610 615 620
Phe Leu Pro Phe Leu Gln Arg Ala Val Gln Gly Leu Pro Ala Asp Asp
625 630 635 640
Pro Arg Val Arg Leu Val Ala Gly Leu Ala Ala Trp Asp Gly Met Met
645 650 655
Thr Ser Glu Arg Gln Pro Gly Tyr Phe Asp Asn Ala Gly Pro Ala Val
660 665 670
Met Asp Ala Trp Leu Arg Ala Met Leu Arg Arg Thr Leu Ala Asp Glu
675 680 685
Met Pro Ala Asp Phe Phe Lys Trp Tyr Ser Ala Thr Gly Tyr Pro Thr
690 695 700
Pro Gln Ala Pro Ala Thr Gly Ser Leu Asn Leu Thr Thr Gly Val Lys
705 710 715 720
Val Leu Phe Asn Ala Leu Ala Gly Pro Glu Ala Gly Val Pro Gln Arg
725 730 735
Tyr Asp Phe Phe Asn Gly Ala Arg Ala Asp Asp Val Ile Leu Ala Ala
740 745 750
Leu Asp Asp Ala Leu Ala Ala Leu Arg Gln Ala Tyr Gly Gln Asp Pro
755 760 765
Ala Ala Trp Lys Ile Pro Ala Pro Pro Met Val Phe Ala Pro Lys Asn
770 775 780
Phe Leu Gly Val Pro Gln Ala Asp Ala Lys Ala Val Leu Cys Tyr Arg
785 790 795 800
Ala Thr Gln Asn Arg Gly Thr Glu Asn Asn Met Thr Val Phe Asp Gly
805 810 815
Lys Ser Val Arg Ala Val Asp Val Val Ala Pro Gly Gln Ser Gly Phe
820 825 830
Val Ala Pro Asp Gly Thr Pro Ser Pro His Thr Arg Asp Gln Phe Asp
835 840 845
Leu Tyr Asn Thr Phe Gly Ser Lys Arg Val Trp Phe Thr Ala Asp Glu
850 855 860
Val Arg Arg Asn Ala Thr Ser Glu Glu Thr Leu Arg Tyr Pro Arg
,CLAIMS:
1. A mutant penicillin G acylase encoded by DNA sequence of penicillin G acylase of Achromobacter sp. CCM4824 for producing semi-synthetic ß-lactam antibiotics, wherein the mutations by site directed mutagenesis with substitution of one or more amino acid position selected from the group consisting of amino acid positions A3, A27, A77, A144, A145, A146, A192, A195, A196, B23, B24, B31, B57, B69, B77, B313, B464 and B484 according to the amino acid numbering of the penicillin G acylase of Achromobacter sp. CCM4824 with the polypeptide having SEQ ID No: 2;
wherein the Achromobacter sp. CCM4824 penicillin G acylase consists of,
(a) a signal peptide consisting of 40 amino acids at amino acid positions 1-40,
(b) an a-subunit consisting of 209 amino acids at amino acid positions 41-249, represented by A1 to A209,
(c) a connecting peptide consisting of 57 amino acids at amino acid positions 250-306, and
(d) a ß-subunit consisting of 557 amino acid position at amino acid positions 307-863 represented by B1 to B557.

2. The mutant penicillin G acylase as claimed in claim 1, wherein the mutation are generated by site directed mutations with substitution of one or more amino acid position selected from the group consisting of;
(i) mutation at amino acid position A3 comprises the replacement of alanine (A) by leucine (L),
(ii) mutation at amino acid position A27 comprises the replacement of isoleucine by asparagine (N) or proline (P),
(iii) mutation at amino acid position A77 comprises the replacement of arginine (R) by threonine (T),
(iv) mutation at amino acid position A144 comprises the replacement of Asparagine (N) by threonine (T),
(v) mutation at amino acid position A145 comprises the replacement of Arginine (R) by leucine (L),
(vi) mutation at amino acid position A146 comprises the replacement of phenylalanine (F) to alanine (A),
(vii) mutation at amino acid position A192 comprises the replacement of alanine (A) to glutamic acid (E),
(viii) mutation at amino acid position A195 comprises the replacement of Glycine (G) by Leucine (L),
(ix) mutation at amino acid position A196 comprises the replacement of Serine by threonine (T) or Leucine (L),
(x) mutation at amino acid position B23, comprises the replacement of Glutamine (Q) by Glycine (G),
(xi) mutation at amino acid position B24 comprises the replacement of phenylalanine (F) to alanine (A),
(xii) mutation at amino acid position B31 comprises the replacement Tyrosine (Y) by Serine (S),
(xiii) mutation at amino acid position B57 comprises the replacement of phenylalanine (F) to alanine (A),
(xiv) mutation at amino acid position B69 comprises the replacement of Alanine (A) by Glycine (G),
(xv) mutation at amino acid position B77 comprises the replacement of Isoleucine (I) by Cysteine (C),
(xvi) mutation at amino acid position B313 comprises the replacement of Glycine (G) by Valine (V),
(xvii) mutation at amino acid position B464 comprises the replacement of Threonine to glutamine (Q) or serine (S), and
(xviii) mutation at amino acid position B484 comprises the replacement of Aspargine (N) to serine(S) or threonine (T).

3. The mutant penicillin G acylase as claimed in claim 2, wherein the amino acid substitution is at B24 position in combination with substitutions selected from the group consisting of [A3+A192+196], [A3+A145+A192+A27], [A145+B77], [A3+A145+B31], [A145+A192+B69] and [A145 +A146+B313].

4. The mutant penicillin G acylase as claimed in claim 1, wherein the mutant penicillin G acylase is encoded by a nucleotide sequence having at least 60% similarity with SEQ ID No.1 encoding mutant penicillin acylase

5. The mutant penicillin G acylase as claimed in claim 4, wherein the SEQ ID No.1 is borne on plasmid pKARA9 is expressed in E. coli BL21.

6. The mutant penicillin G acylase as claimed in claim 5, wherein the resultant mutant E. coli BL21 is deposited as MTCC 25429.

7. The mutant penicillin G acylase as claimed in claim 1, wherein the mutant penicillin acylase enzyme are immobilized on ter-polymer synthesized by free radical suspension polymerization wherein the monomer components is selected from the group comprising of glycidyl methacrylate (GMA), allyl glycidyl ether, Ethylene glycol dimethacrylate (EGDM), divinylbenzene (DVB), ethyl methacrylate (EMA), methylmethacrylate (MMA) and acrylonitrile (AN).

8. A process for the production of a semi-synthetic ß-lactam antibiotic comprising an mutant penicillin G acylase catalysing the acylation to synthesize semi-synthetic ß-lactam antibiotic are ß-lactam nucleus selected from 6-aminopenicillanic acid (6-APA), 7-desacetoxycephalosporanic acid (7-ADCA), 7-aminocephalosporanic acid (7-ACA), 7-Amino-3-Chloro-3-Cephem-4-Carboxylicacid (7-ACCA), and 7-Amino-3-[(Z)-propen-1-yl] -3-cephem-4-carboxylic acid (7-APCA) and activated acyl donor selected from ester or amide of hydroxyphenyl glycine (HPGME) or phenylglycine (PGME) or dihydroxyphenylglycine (DHPGME).

9. The process for the production of a semi-synthetic ß-lactam antibiotic as claimed in claim 8, wherein the semi-synthetic ß-lactam antibiotic is selected from ampicillin, amoxicillin, cefalexin, cefadroxil or cefradine.

10. A composition comprising mutant penicillin G acylase encoded by DNA sequence of penicillin G acylase of Achromobacter sp. CCM4824 for producing semi-synthetic ß-lactam antibiotics, wherein the mutations by site directed mutagenesis with substitution of one or more amino acid position selected from the group consisting of amino acid positions A3, A27, A77, A144, A145, A146, A192, A195, A196, B23, B24, B31, B57, B69, B77, B313, B464 and B484 according to the amino acid numbering of the penicillin G acylase of Achromobacter sp. CCM4824 with the polypeptide having SEQ ID No: 2;
wherein the Achromobacter sp. CCM4824 penicillin G acylase consists of,
(a) a signal peptide consisting of 40 amino acids at amino acid positions 1-40,
(b) an a-subunit consisting of 209 amino acids at amino acid positions 41-249, represented by A1 to A209,
(c) a connecting peptide consisting of 57 amino acids at amino acid positions 250-306, and
(d) a ß-subunit consisting of 557 amino acid position at amino acid positions 307-863 represented by B1 to B557.