KR20220125219A - Mutant penicillin G acylase of Acromobacter CCM4824 - Google Patents

Abstract

The present invention relates to a mutant semi-synthetic β-ramtam antibiotic-synthetase, which is a penicillin G acylase from Achromobacter sp. CCM4824 produced by site-directed mutagenesis with substitution of one or more amino acid positions. to start The gene sequence encoding the mutant penicillin acylase on plasmid pKARA9 is this . It is expressed in E. coli BL21. Recombinant E. The enzyme obtained from E. coli BL21 has improved penicillin acylase activity, S/H ratio, acyl donor compared to the parent penicillin acylase enzyme of Achromobacter sp. CCM4824. showed a lower input rate and improved yield of

Description

Mutant penicillin G acylase of Acromobacter CCM4824

The present invention relates to a mutant penicillin G acylase (mNPGA) obtained by amino acid substitution at a single or more than one site in the nucleotide sequence of Achromobacter CCM4824, wherein the obtained mutant is a different semisynthetic β-lactam antibiotic. for improved S/H ratio, synthetic activity, or both. The present invention also relates to an immobilized mutant penicillin G acylase.

The industrial production method of semi-synthetic β-lactam antibiotics is in the process of converting from the conventional chemical route to a more environmentally friendly and efficient enzymatic route, which generates less by-products, and uses organic solvents and harsh chemicals for protection and deprotection. This is due to the obvious advantage of the enzymatic method of avoiding the extreme process conditions of pressure, temperature and high energy costs. The synthesis of semisynthetic β-lactams by the enzymatic pathway involves the kinetically controlled acylation of β-lactam nuclei with amino acid derivatives, which are enzymes belonging to the Ntn-hydrolase classes, mainly It is catalyzed by penicillin G acylase or α-amino acid ester hydrolase. However, the use of penicillin G acylase has been extensively reported for its application, due to the presence of this enzyme in various microorganisms and its characterization for application to hydrolysis and synthesis reactions. Penicillin G acylases from different microorganisms have been extensively studied for their substrate specificity and selectivity.

Commercially, an immobilized form of penicillin G acylase is known to catalyze hydrolysis reactions to antibiotic intermediates. Recently, it has been reported that other variants of penicillin acylase have been found for application in synthetic reactions, in which semisynthetic β-lactam antibiotics are synthesized by acylation of an antibiotic intermediate. The following criteria are important in synthetic reactions.

Selection of Enzymes: Kinetics-controlled semisynthetic β-lactam antibiotic synthesis is catalyzed by penicillin acylase, three simultaneous reactions: hydrolysis of activated acyl donors (esterase activity-H), β-lactam antibiotics It is governed by acylation of the intermediate (synthetic activity-S) and hydrolysis of the β-lactam antibiotic (amidase activity). Obviously, enzymes with favorable S/H ratios are preferred choices to drive the reaction towards synthesis to provide products in higher yields and also produce fewer hydrolysis by-products.

E. used in the synthesis of antibiotic intermediates such as 6-APA and 7-ADCA. Penicillin G acylases from E. coli , Providencia rettgerri , Klyuvera citrophilia and Bacillus megaterium are generally more sensitive to synthetic activity. It exhibits high hydrolysis properties. However, alteration of amino acids at specific sites by site-directed mutagenesis results in mutant penicillin acylases with enhanced synthetic activity, making the enzymatic synthesis of semisynthetic antibiotics an industrially viable option. do.

Wild-type penicillin acylases from various microbial sources with limited synthetic capacity have been improved by altering amino acids at specific positions in the nucleotide sequence. It has been reported that these various mutant penicillin acylases exhibit altered S/H ratios or higher synthetic activities for different antibiotics, which are based on amino acid changes made in the alpha or beta subunit of the enzyme gene.

U.S. Pat. No. 8,541,199 describes E. with amino acid substitutions at different positions in the alpha and beta subunits that enhance the synthesis of phenam and cepam semisynthetic antibiotics. A mutant penicillin G acylase derived from E. coli is disclosed. A selective increase in the S/H ratio with certain modifications was also reported.

this. Mutations of hydrophobic active sites, such as βF24A, βF57W, βF57Y, αF146L and αF146Y in E. coli penicillin acylase, amoxicillin, ampicillin, cephalexin and cefadroxyl synthesis using hydroxyphenylglycinamide or phenylglycinamide A similar report has been published that causes a 5-10 fold decrease in the K _cat value of

Mutations at the B24 position are known from WO96/05318, which is, in particular, of the naturally occurring phenylalanine amino acid residue of the B24 site of penicillin G acylase produced by Alcaligenes faecalis , the positive charge It discloses substitution with amino acids lysine or arginine with

Immobilized enzymes: Enzymes are commercially available if they can be recycled and reused in repeated production cycles. Therefore, it is essential to develop an enzyme product immobilized on a suitable support that can be recycled. In addition, the synthesis reaction is suspension based and produces a precipitate that leads to physical consumption of the immobilized enzyme. There is also a need to select a support having good mechanical stability. In addition, the selected support material should be compatible with the enzyme protein to maintain or enhance the desired S/H ratio.

PGA immobilization has witnessed extensive evolution to meet industrial needs. These range from immobilization on agarose gels to monolithic silica supports, activated chitosan, and magnetic nanoparticles, each with unique advantages and disadvantages for a variety of substrates and products. On a commercial scale, enzyme carriers must be more robust to withstand high attrition that affects operational stability. U.S. Pat. No. 6,218,138 describes the covalent immobilization of PGA from E. coli and the suitability of the immobilized enzyme for reuse for 5 cycles in the synthesis of β-lactam antibiotics, methacrylamide, N,N, methylene ^{Eupergit R C} copolymers of bis-acrylamide, glycidyl methacrylate and allyl glycidyl ether have been reported.

The nature of the hydrophobic or hydrophilic support affects catalytic efficiency, kinetic parameters such as solvent, pH, temperature, etc., and downstream processes for product recovery. Extracellular penicillin acylase derived from Bacillus megaterium was immobilized by coupling to a derivative of polyacrylonitrile fiber, maintaining 83.9% activity at 40°C and pH 6.5 and during 50 cycles of cephalexin synthesis. was used During N-acylation of 7-aminocephalosporanoic acid, PGA from Arthrobacter viscosus immobilized on a hydrophobic acrylic epoxy-support (Upergit C) acted as a poor substrate, resulting in low The turnover rate is shown. It also reports cephalexin synthesis with a molar ratio of nuclei to acyl donor of 1:3, which may not be an economically viable option. US 7,264,943 discloses polyvinyl alcohol-gelatin.

WO2010/055527, which uses an enzymatic acylation step in a suspension reaction, has the disadvantages of reduced operational stability and increased swelling volume and filtration time, thereby affecting the manufacturing cost.

US2005/0084925 reports the use of cephalexin in hydrophobic copolymers for the immobilization of penicillin G acylase for enzymatic cephalexin synthesis.

In addition, based on literature reports, efforts were made to improve the immobilization efficiency by modifying the designed polymer by introducing an additional reactive group using an aliphatic amine. Reports of immobilization on hetero-functional epoxy supports and novel amino epoxy polymers are known in the prior art, wherein the polymers have an additional amino group with an epoxide that makes an additional site for enzymatic binding. contains However, considering the above factors, immobilization must be designed for this application and is therefore an important factor. The enzymatic method of antibiotic synthesis is influenced by various factors that determine process viability in commercial terms. Data reported for different process designs and parameters indicate various influencing factors.

The input of the substrate is one of the key factors influencing the economics of the manufacturing method. The kinetics of the reaction ratio should be such that the ratio of nuclei to acyl donor used in the preparation method is optimal to drive the synthesis reaction, and generally the acyl donor is in excess. However, in order for the manufacturing method to be economically feasible, the ratio affected by the type of enzyme used should be minimal. Enzyme input w.r.t. The substrate is important in that it achieves maximum conversion in an appropriate time period.

PCT Publication No. WO 93/12250 discloses cephalexin synthesis, wherein product separation is by complex formation with beta-naphthol, which requires further purification and thus may lead to lower product yields. .

Process parameters such as pH, temperature and agitation are important with regard to the stability of substrates, enzymes and products. In addition, precipitation of the product results in a suspended reaction, which makes it essential to design the downstream process to separate the product and the enzyme to be recycled without causing significant product loss and complete enzyme recovery.

For in situ preparations where the substrate contains by-products such as liquid 6-APA or liquid 7-ADCA containing trace amounts of PAA, the enzyme must withstand the PAA induced by the catalysis of semisynthetic β-lactam . Process parameters also affect enzyme kinetics for the reaction.

Summary of the invention

In one aspect, the present invention provides a β-lactam antibiotic enzyme obtained by site-directed mutagenesis in a gene encoding penicillin G acylase synthesized by Achromobacter CCM4824.

In another aspect, the present invention provides a host E. Nucleotide represented by SEQ ID NO: 1 encoding an amino acid at one or more positions, resulting in a mutant penicillin acylase enzyme with high specificity and productivity retained in plasmid pKRA9 carried on recombinant vector in E. coli BL21 mutation in sequence; and for the synthesis of semisynthetic β-lactam antibiotics such as amoxicillin, cephalexin, cefaclor, cefadroxil, cepradin, and the like.

In another aspect, the present invention relates to a penicillin G acylase mutant (mNPGA) obtained by amino acid substitutions at single or more than one site or by combination of gene sequences, wherein the resulting mutants are different semisynthetic β-lactams. It exhibits improved S/H ratio, synthetic activity, or both for antibiotics. The mutant penicillin G acylase (mNPGA) variant thus derived exhibits resistance to phenylacetic acid (PAA), a competitive inhibitor produced as a by-product of the hydrolysis reaction, adding to the potential for use of substrates containing trace amounts of phenylacetic acid. provides the advantages of

Terpolymer systems, each containing epoxy macroporous beads, vary in composition with respect to epoxy content, hydrophilicity, porous structure and size, all of which affect immobilization and thus recyclability and reusability of the immobilized enzyme.

The present invention provides unique terpolymers custom designed for the binding of penicillin acylase enzymes to form immobilized biocatalysts. From a series of polymers used in immobilized biocatalysts, each disclosed polymer is uniquely suited for different antibiotic synthesis and recyclability due to its pore size, porosity, specific surface area, epoxy and crosslink density.

Amination of the polymer and subsequent cross-linking creates additional spacer arms, further improving enzyme binding, making the biocatalyst more stable to recyclability and reducing enzyme leaching from the polymer in use.

Because immobilized enzymes undergo conformational changes due to binding to the support, immobilized and immobilized supports also affect catalytic efficiency, kinetic properties, thermal stability, and resistance to reaction conditions such as pH, temperature, and solvents of the enzyme. crazy

The present invention relates to a method for enzymatic synthesis of a semisynthetic β-lactam antibiotic using the mutant penicillin acylase enzyme and a single pot using a dual enzyme system of parent and mutant penicillin acylase enzymes. It provides a method for enzymatic synthesis and also provides commercial applicability and distinct advantages of the preparation method for the native penicillin G acylase (pNPGA) from Achromobacter sp CCM 4824 disclosed in US Pat. No. 8,039,604 .

A series of semisynthetic β-lactam antibiotics that can be synthesized by enzymatic acylation of the enzyme include amoxicillin, ampicillin, cephalexin, cefadroxil, cefaclor, cefprozil and cepradin.

The present invention provides the stability of immobilized mutant PGA in repeated cycles of a semisynthetic β-lactam antibiotic synthesized by enzymatic reaction under the above-mentioned controlled conditions. The present invention also discloses enzyme suitability for both reaction and product separation from reaction mixtures.

1 depicts a comparison of substrate specificity of mNPGA mutants.
Figure 2 depicts a comparison of the antibiotic synthesis activity of mNPGA mutants.
3 depicts a comparison of synthesis versus hydrolysis ratios of mutants for different antibiotic substrates.
Source of Biological Material: E. lice carrying a mutant penicillin G acylase. E. coli BL-21 has been deposited as MTCC 25429. The DNA sequence encoding penicillin G acylase was isolated from Achromobacter sp. CCM4824 isolated at the Institute of Microbiology, Czech Republic, and E. It was mutated to be expressed in E. coli BL-21.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in detail with reference to certain preferred and optional embodiments, so that various aspects of the invention may be more fully understood and appreciated.

The present invention provides a series of mutant penicillin G acylases encoded by the DNA sequence of penicillin acylase from Achromobacter sp. CCM4824 for producing semisynthetic β-lactam antibiotics, wherein the mutation comprises It is generated by site-directed mutagenesis with substitution of one or more amino acid positions shown in SEQ ID NO:2.

In a preferred embodiment, the present invention provides a mutant penicillin G acylase encoded by the DNA sequence of a penicillin G acylase of Achromobacter sp. CCM4824 for producing a semisynthetic β-lactam antibiotic, The mutation is amino acid positions A3, A27, A77, A144, A145, A146, A192, A195, according to amino acid numbering of penicillin G acylase of Achromobacter sp. CCM4824 having the polypeptide of SEQ ID NO: 2 produced by site-directed mutagenesis having a substitution of one or more amino acid positions selected from the group consisting of A196, B23, B24, B31, B57, B69, B77, B313, B464 and B484;

The penicillin G acylase of Achromobacter sp. CCM4824 is

(a) a signal peptide consisting of 40 amino acids at amino acid positions 1-40;

(b) the α-subunit consisting of 209 amino acids at amino acid positions 41-249, represented by A1 to A209;

(c) a linking peptide consisting of 57 amino acids at amino acid positions 250-306, and

(d) is composed of a β-subunit consisting of 557 amino acids at amino acid positions 307-863, denoted by B1 to B557.

In one embodiment, the present invention relates to the group consisting of A3, A27, A77, A90, A144, A145, A146, A192, A195, A196, B23, B24, B31, B57, B69, B77, B148, B313, B464, B484 It provides a site-directed mutation substituted at a specific amino acid position of SEQ ID NO: 2 selected from, which generates each mutant indicated in numerical order starting with the prefix 1 in mNPGA.

SEQ ID NO: 2

The underlined region of the sequence is the alpha site of penicillin G acylase, and the bold underlined region is the beta site of penicillin G acylase. The signal peptide corresponds to 40 amino acids - italicized α-subunit (209 amino acids - single underlined), linker peptide (57 amino acids) and β-subunit (557 amino acids - dark underlined). The α-subunit amino acids are numbered A1-A209 and the β-subunit amino acids are numbered B1 through B557. The signal peptide and the linking peptide are removed during post-translational maturation to produce an enzymatically active penicillin acylase composed of α and β subunits.

In another preferred embodiment, the present invention provides amino acid substitutions at position B24 and A3, A27, A77, A90, A144, A145, A146, A192, A195, A196, B23, B24, B31, B57, B69, B77, B313, Provided is a mutant penicillin G acylase derived from Achromobacter CCM4824 having an amino acid substitution at one or more positions selected from the group consisting of B464, B484.

(i) Position A3 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains an alanine (A) amino acid residue. A preferred preferred mutation at position A3 involves the substitution of alanine for leucine (L).

(ii) position A27 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains an isoleucine amino acid residue. Preferred mutations at position A27 include the substitution of isoleucine (I) with asparagine (N) or proline (P). A more preferred mutation is asparagine (N).

(iii) position A77 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains an arginine amino acid residue. Preferred mutations include the substitution of arginine (R) for threonine (T).

(iv) position A144 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains an asparagine amino acid residue. Preferred mutations include the substitution of asparagine (N) for threonine (T).

(v) position A145 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains an arginine amino acid residue. Preferred mutations include the substitution of arginine (R) for leucine (L).

(vi) position A146 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains a phenylalanine amino acid residue. A preferred mutation involves the substitution of alanine (A) for phenylalanine (F).

(vii) position A192 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains an alanine amino acid residue. A preferred mutation involves substituting alanine (A) for glutamic acid (E).

(viii) position A195 of penicillin G acylase derived from Achromobacter sp. CCM4824 comprises a glycine amino acid residue. Preferred mutations include the substitution of glycine (G) for leucine (L).

(ix) position A196 of penicillin G acylase derived from Achromobacter sp. CCM4824 comprises a serine amino acid residue. Preferred mutations include substituting serine for threonine (T) or leucine (L).

(x) Position B23 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains a glutamine amino acid residue. Preferred mutations include the substitution of glutamine (Q) for glycine (G).

(xi) Position B24 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains a phenylalanine amino acid residue. A preferred mutation involves the substitution of alanine (A) for phenylalanine (F).

(xii) position B31 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains a tyrosine amino acid residue. Preferred mutations include the substitution of tyrosine (Y) for serine (S).

(xiii) position B57 of a penicillin G acylase derived from Achromobacter sp. CCM4824 contains a phenylalanine amino acid residue. A preferred mutation involves the substitution of alanine (A) for phenylalanine (F).

(xiv) position B69 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains an alanine amino acid residue. Preferred mutations include the substitution of alanine (A) for glycine (G).

(xv) position B77 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains an isoleucine amino acid residue. Preferred mutations include the substitution of isoleucine (I) for cysteine (C).

(xvi) Position B313 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains a glycine amino acid residue. Preferred mutations include the substitution of glycine (G) for valine (V).

(xvii) Position B464 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains a threonine amino acid residue. Preferred mutations include the substitution of threonine (T) with glutamine (Q) or serine (S).

(xviii) position B484 of penicillin G acylase derived from Achromobacter sp. CCM4824 contains an asparagine amino acid residue. Preferred mutations include substituting asparagine (N) for serine (S) or threonine (T).

In one embodiment, the present invention provides a nucleotide sequence encoding a mutant penicillin acylase, which comprises E. It is present on plasmid pKARA9 expressed in E. coli BL21. The plasmid and host E. E. coli BL21 were all obtained from the Institute of Microbiology, Czech Republic. The mutant penicillin G acylase is encoded by a nucleotide sequence having at least 60% similarity to SEQ ID NO: 1, which encodes the mutant penicillin acylase.

Recombinant E. Enzyme obtained from E. coli BL21, compared with the native penicillin acylase enzyme of Achromobacter sp CCM 4824, improved penicillin acylase activity, S/H ratio, lower acyl donor input ratio and improved yield. The enzyme thus obtained was found to be suitable for the enzymatic synthesis of semisynthetic β-lactam antibiotics such as amoxicillin, ampicillin, cephalexin, cefadroxil, cefaclor, and the like, and the enzymatic method using the enzyme is commercially viable. In addition, a mutant penicillin acylase enzyme was immobilized in a custom terpolymer system of epoxy polyacrylate resin to evaluate recyclability and suitability in a semisynthetic β-lactam antibiotic production method. The immobilized form of the mutant penicillin acylase enzyme and process parameters including raw material input, reaction conditions and product isolation are designed to enable commercial applications.

The present invention is produced by Achromobacter sp CCM 4824 transformed in Escherichia coli BL21, present in a plasmid vector PKRA9 carrying a mutant penicillin G acylase enzyme gene, Mutants of penicillin G acylase are provided.

SEQ ID NO: 2 of the present invention is wild-type E. It has 54% homology compared to E. coli penicillin acylase. SEQ ID NO: 2 shown above corresponds to the amino acid sequence of penicillin G acylase produced by Achromobacter sp CCM 4824. The naturally occurring penicillin G acylase is selected from microorganisms having homology in the range of 40-100% to SEQ ID NO:2. More preferred is a penicillin acylase having 50% to 100% homology with SEQ ID NO:2.

In one embodiment, the present invention provides a mutant sequence of penicillin acylase obtained by substitution of the above-mentioned amino acids in one or more position combinations in SEQ ID NO:2.

In another preferred embodiment, the present invention provides B24 in combination with substitution at one or more positions selected from A3, A27, A77, A90, A144, A145, A146, A192, A195, A196, B23, B31, B69, B77 and B313. amino acid substitutions in Of these, selected amino acid alterations in combination with B24 are [A3+A192+196], [A3+A145+A192+A27], [A145+B77], [A3+A145+B31], [A145+A192+B69] and [A145+A146+B313].

The mutant thus obtained showed >95% homology to penicillin acylase derived from Achromobacter sp. CCM 4824 shown in SEQ ID NO: 2.

In a preferred embodiment, the present invention relates to a mutant E. mutant carrying a mutant penicillin G acylase, deposited as MTCC 25429. E. coli BL-21 is provided. In addition, the mutations thus obtained were numerically labeled, starting at mNPGA 1, and subsequently further numbered, by recombinant plasmid vector pKRA9 carrying the gene of the mutant penicillin acylase to the host bacterium E. It was expressed in E. coli BL21.

In a preferred embodiment, the present invention provides cultured mNPGA, which has activity, substrate specificity, synthesis/hydrolysis (S/H) ratio for various substrates, inhibitor resistance, optimal substrate, and final antibiotic under defined process conditions. compared to native penicillin G acylase (pNPGA) for assay parameters including enzyme concentration to achieve maximal conversion to

The obtained penicillin G acylase enzyme was quantified as unit (U) activity and protein in mg/mL.

Proteins were determined by the Folin-Lowry method, and penicillin G hydrolytic activity catalyzes the hydrolysis of 25 mM Pen G K in 50 mM sodium phosphate buffer pH 8.0 at 37 °C to release phenylacetic acid, which has a first-order kinetic ( It is expressed in U/g of biocatalyst, neutralized with 0.1N sodium hydroxide as first order kinetics).

The activity for the synthesis of antibiotics at SU/g and S/H ratios using these enzymes and biocatalysts is determined by the HPLC details described below for each antibiotic.

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: 1.2 mL/min, wavelength: 215 nm, injector volume: 20 μl, run time: 20 min.

HPLC analysis for ampicillin (AMP) synthesis:

Column: Inertsil C8 250 mm X 4.6 mm (5 micron), temperature 35° C., buffer: 16.95 g tetra-n-butyl ammonium hydrogen sulfate + 6.8 g potassium dihydrogen orthophosphate + dissolved in 1 liter HPLC grade water 2.0 ml triethylamine, 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 250 mm X 4.6 mm (5 micron), temperature 35° C., buffer: 16.95 g tetra-n-butyl ammonium hydrogen sulfate + 6.8 g potassium dihydrogen orthophosphate + dissolved in 1 liter HPLC grade water 2.0 ml triethylamine, 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 250 mm X 4.6 mm (5 micron), temperature 30° C., buffer: 11.5 g ammonium hydrogen phosphate in 1 liter HPLC grade water, pH 5.4, 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 250 mm X 4.6 mm (5 micron), temperature 35° C., buffer: pH 2.5 with 0.1M ortho-phosphoric acid + 0.4% triethylamine, 2N sodium hydroxide in 1 liter HPLC grade water. 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 250 mm X 4.6 mm (5 micron), temperature 25° C., buffer: 13.6 g potassium dihydrogen phosphate in 1 liter HPLC grade water, pH 5.0 using 2N 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 250 mm X 4.6 mm (5 micron), temperature 35° C., buffer: 16.95 g tetra-n-butyl ammonium hydrogen sulfate + 6.8 g potassium dihydrogen orthophosphate + dissolved in 1 liter HPLC grade water 2.0 ml triethylamine, 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.

In addition, one embodiment describes an epoxy terpolymer combination obtained by suspension polymerization of acrylic monomers as beads of specific surface area and porosity and particle size, wherein the modification process has improved mechanical stability, filtration capacity, and for antibiotic synthesis. Additional spacer arms in the epoxy beads by amination with various fatty amines are included to allow for maximal expression of enzymes bound to these supports, with recyclability for use of immobilized enzymes in suspension reactions.

Terpolymers are synthesized by free radical suspension polymerization, wherein one or both of the added monomer components used in different combinations are glycidyl methacrylate (GMA), allyl glycidyl ether, ethylene glycol dimethacrylic rate (EGDM), divinylbenzene (DVB), ethyl methacrylate (EMA), methyl methacrylate (MMA) and acrylonitrile (AN).

The properties of each monomer and the amount used to polymerize itself and with other monomers impart specific properties to the beads, resulting in varying epoxy content, crosslinking density, porosity, hydrophilicity and stiffness. The molar ratio of monomers such as ethylene glycol dimethacrylate, divinyl benzene to other monomers results in variable crosslink densities in the polymer beads. The epoxy content varies depending on the amount of glycidyl methacrylate or allyl glycidyl ether. The acrylonitrile content gives the polymer bead a hard backbone, which makes it mechanically stronger than without its components.

The present invention discloses crosslink densities ranging from 25 to 200 for a series of terpolymers.

The macroporosity imparted to the beads acts as porogens which are added at 1.1 to 1.5 times the total monomer content using an aliphatic or aromatic alcohol selected from dodecanol, octanol, hexanol and cyclohexanol or both. do.

Free radical polymerization is initiated by agents such as benzoyl peroxide or azobisisobutyronitrile used between 2-4% polyvinylpyrrolidone or polyvinyl used up to 8% of the total monomer weight Stabilized by alcohol. Polymer beads were optionally prepared using additives selected from Span 60, Span 80, Span 85 and Sodium Lauryl Sulfate to achieve some degree of hydrophobicity. Additives were added in concentrations ranging from 0.01% to 5% based on the monomer concentration.

The additive imparted a hydrophobic microenvironment to the pores, which supported the driving of the enzymatic synthesis reaction compared to hydrolysis. This in turn supported the suitability of the biocatalyst in suspension reactions.

In this embodiment, the process parameters designed and optimized using the enzyme/enzymes allow the manufacturing method to be scaled up to an industrial scale. Additionally, additional efforts have resulted in more mechanically stable polymer beads, wherein the polymer is an aliphatic amine selected from ethylamine, triethylamine, diethylamine, butylamine and ethylene diamine toluene or at a concentration of 25-50% by weight of the polymer. It was treated with a solvent selected from methanol at a temperature of 40-80° C. for 10 to 150 hours. The degree of amination was confirmed by color development of the polymer using a 0.1% w/v ninhydrin solution in water.

The aminated polymer beads were washed and further treated with 2%-8% v/w glutaraldehyde for 1 to 20 hours to crosslink the amino groups to the activated amino epoxy polymer.

The polymer beads disclosed herein are composed of poly(GMA-ter-EGDM-ter-AN) and poly(GMA-EGDM-DVB-AN), denoted DILBEADS EG, DILBEADS EGX, and the activated amino epoxy polymer is DILBEADS It is referred to as EGXAT.

This embodiment discloses analytical parameters for particle size, porosity, pore diameter, surface area of the polymer beads described above, which are laser diffraction analyzer (particle size analyzer, HELOS H1004) and mercury intrusion porosimeter (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 cm ³ /g and surface area between 50 m ² /g-160 m ² /g.

In another embodiment, the present invention discloses a method for synthesizing a semisynthetic β-lactam antibiotic (SSA) using a biocatalyst immobilized on an mNPGA enzyme, more preferably DILBEADS EG, DILBEADS EGX and DILBEADS EGXAT, wherein the reaction conditions are includes:

(a) The substrate of the synthesis reaction catalyzing the acylation for the synthesis of semisynthetic β-lactam antibiotics in the enzyme is 6-aminopenicillanic acid (6-APA), 7-desacetoxycephalosporanoic acid (7- ADCA), 7-aminocephalosporanoic acid (7-ACA), 7-amino-3-chloro-3-cephem-4-carboxylic acid (7-ACCA), and 7-amino-3-[(Z)- β-lactam nucleus selected from propen-1-yl]-3-cephem-4-carboxylic acid (7-APCA) and hydroxyphenylglycine (HPGME) or phenylglycine (PGME) or dihydroxyphenylglycine (DHPGME) ) is an active acyl donor selected from esters or amides of

(b) the molar ratio of acyl donor to nucleus varies from 1:1 to 1:1.2, more preferably from 1:1.02 to 1:1.1, based on the form of the acyl donor as free acid or hydrochloride salt for different antibiotics do.

(c) the concentration of nuclei in the reaction mixture varies in the range of 6-12 %w/v, more preferably 8-10 %w/v.

(d) The amount of enzyme is more preferably used at an activity ratio of 90-250 SU/g per gram of β-lactam nucleus.

(e) Conversion of β-lactam nuclei is achieved between 50-99% within 90-240 min.

(f) Suitability to reuse immobilized biocatalysts for product isolation after repeated cycles of different antibiotics and enzymatic conversion in suspension reactions.

A representative enzymatic reaction cycle for amoxicillin synthesis and product isolation using an immobilized biocatalyst of mNPGA is disclosed as an example.

A series of terpolymer forming macroporous beads prepared by free radical polymerization consists of three or four monomers, a diluent, an initiator and a stabilizer, wherein:

(a) Monomer 1 contained in 15-60% of the total weight of the monomer is GMA capable of free radical polymerization and has an epoxy group.

(b) Monomer 2 contained in 20-55% of the total weight of the monomer is DVB or EGDM capable of free radical polymerization or both, and is a crosslinking agent.

(c) Monomer 3 contained in 20-55% of the total weight of the monomer is AN capable of free radical polymerization, and is a rigid backbone of the terpolymer system.

(d) porogens are derived from the group of hydrophilic or cyclic alcohols, including cyclohexanol, octanol, lauryl alcohol, and combinations thereof, at 1.2-1.5 times the total weight of the monomers.

(e) the initiator is selected from the group of benzoyl peroxide, azobisisobutyronitrile, methyl ethyl ketone peroxide and is 3-3.5% based on the total weight of the monomers.

(f) The suspension stabilizer is selected from the group of polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, and is 7-7.5% based on the total weight of the monomer.

The macroporous beads formed in a series of terpolymer systems are aminated using an aliphatic amine selected from the group consisting of diamino ethane, butylamine, triethylamine and diethylamine.

Therefore, the immobilized biocatalyst is used at a rate of 90-250 U/g activity per gram of β-lactam nucleus. Conversion of β-lactam nuclei is achieved between 50-99% and product yields of up to 90-95% are achieved within 120-240 minutes for 100 repeated cycles. The immobilized biocatalyst is reused for 300 repeated cycles in a suspension reaction containing substrate, immobilized enzyme catalyst and precipitated product. As a semi-synthetic β-lactam antibiotic, the precipitated product is isolated from the enzymatic reaction mixture.

In a preferred embodiment, the immobilized biocatalyst comprising a mutant penicillin G acylase produced by Achromobacter CCM 4824 contains up to 90% binding protein, up to 97.7% active binding and up to 40-100% enzyme expression. indicates

In addition, the S/H ratio of the immobilized biocatalyst in a series of terpolymer systems varies from 0.3-1.3 for amoxicillin synthesis and 0.1-1.49 for cephalexin synthesis. The concentration of β-lactam nucleus and activated acyl donor is used in a molar ratio of 1:1.02 to 1:1.5.

The following examples, including preferred embodiments, serve to illustrate the practice of the present invention, and specific details are to be understood by way of example and for purposes of illustrative description of preferred embodiments of the present invention.

Example

Example 1:

Isolation and purification of the nucleotide sequences described in US 8,039,604 was performed using Invitrogen's Pure Link plasmid extraction kit. Agarose gel electrophoresis was performed on the isolated plasmid, and the size of the plasmid was confirmed. After the nucleotide sequence was isolated using restriction enzymes XbaI and PstI, it was purified using a gel purification kit.

Example 2:

For mutagenesis, a mutagenesis kit from Toyoba was used. First, appropriate primers were designed and mutagenesis was performed according to the protocol provided in Toyoba's mutagenesis kit. The vector carrying the plasmid pKRA9 was grown at 37°C, and the plasmid was isolated using Invitrogen's Pure Link plasmid extraction kit. The vector was digested with XbaI and PstI, purified and prepared for ligation. Conjugation of the vector with the nucleotide sequence obtained in Example 1 was performed using a standard protocol known in the art. The conjugated plasmid was . E. coli BL21 cells were transfected. Early competent cells of BL-21 were prepared using calcium chloride methods known in the art. Transfection was performed using the heat shock method, plated on LB agar plates, and incubated overnight at 37°C. The resulting colonies were screened for activity.

Example 3:

Each of the mutant clones obtained in the above example was grown in 10 mL Luria Bertani (LB) medium having a kanamycin concentration of 30-50 μg/mL at 28° C. for 18 hours at 150 rpm. Culture growth was checked for optical density at pH, 600 nm, and centrifuged to obtain a cell pellet, which was washed and resuspended to 3-4% dry weight. Each 100 μl cell suspension was incubated for 15 minutes in 1% w/v penicillin, 0.5% w/v amoxicillin, 0.5% w/v cephalexin in 100 mM phosphate buffer pH 7.5 and 4-in methanolic acetic acid. The resulting product was determined by a color reaction using (dimethylamino)benzaldehyde. The increasing color intensity indicated by the + sign measured at 415 nm corresponds to the specificity of each mutant clone for each substrate.

Examples 4-8: Poly(GMA-ter-EGDM-ter-AN) terpolymer, also referred to as DILBEADS-EG, consists of the composition and content as described in Table 1.

In a nitrogen inert atmosphere, the mentioned amounts of each monomer comprising 110 g of cyclohexanol are stirred with 300 ml of distilled water at 300 rpm, and polyvinylpyrrolidone and azobisisobutyronitrile at 70° C. for 4 hours. was used for polymerization. The macroporous beads formed at the end of the reaction were vacuum filtered, washed with water, soaked in methanol overnight, vacuum filtered and dried in a vacuum oven at 40°C.

Example 9: A ter-polymer system was prepared by the procedure according to Example 4, replacing cyclohexanol with a mixture of cyclohexanol and lauryl alcohol in a ratio of 90:10 by weight.

Example 10: A terpolymer system was prepared by the procedure according to Example 4, replacing cyclohexanol with a mixture of cyclohexanol and hexanol in a ratio of 90:10 by weight.

Examples 11-15: A terpolymer system was prepared by the procedure according to Example 4 using the following additives.

Examples 16-17: Composition of poly(ter-GMA-EGDM-DVB-ACN) termed DILBEADS EGX

In an inert nitrogen atmosphere, the mentioned amounts of each monomer comprising 125-150 g of cyclohexanol are stirred with 300 ml of distilled water at 300 rpm, and polyvinylpyrrolidone and azobisisobuty are stirred at 70° C. for 4 hours. Polymerization was carried out using ronitrile. The macroporous beads formed at the end of the reaction were vacuum filtered, washed with water, soaked in methanol overnight, vacuum filtered and dried in a vacuum oven at 40°C.

Example 18: A terpolymer system was prepared by the procedure according to Example 8, replacing cyclohexanol with a mixture of isoamyl alcohol and cyclohexanol in a ratio of 10:90 by weight.

Example 19: A terpolymer system was prepared by the procedure according to Example 16, replacing cyclohexanol with a mixture of dodecanol and cyclohexanol in a 3:5 weight ratio.

Example 20: DILBEADS EGX ^AT : 1 g of dry DILBEADS EGX was added to 5 mL of toluene and 1 mL of ethylenediamine. Incubated at 40° C. for 150 hours. The beads were washed with 10 volumes of water and immersed in 10 mL of methanol for 24 hours. Washed twice with 10 mL of water and dried in air. 5 mL of water and 4 mL of glutaraldehyde were added, and the mixture was stirred at 200 rpm for 1 hour. It was washed with water until glutaraldehyde was completely removed and dried in air.

Example 21:

Table 3: Physical appearance and porosity data of terpolymers

Examples 22-27: Hydrolysis of Substrates

Substrate hydrolysis of mNPGA

antibiotic synthesis activity

Example 28:

From the mNPGA mutant of the present invention as described in Example 3, the enzyme isolated by the method described in Indian Patent No. 253959 was immobilized to prepare an immobilized biocatalyst. The following methods of determination of immobilization to polymer beads are familiar per se to those skilled in the art of covalent immobilization and are described in detail only for completeness.

1 g of macroporous beads were added to Penicillin G 100 Units of the PGA in 1M potassium phosphate buffer (pH 7.5) and incubated at 25° C. for 48 hours. The macroporous polymer beads were vacuum filtered, washed three times with deionized water, and then washed twice with 0.1M calcium phosphate buffer (pH 7.5). The resulting macroporous beads loaded with PGA were weighed. The swelling volume after immobilization is 1.1 to 1.2 times the initial volume of the macroporous beads.

Example 29: Enzymatic synthesis of amoxicillin

The reaction was carried out in a stirred tank reactor at 20-25° C., pH 6.3 for 2 hours. The reaction mixture consists of 100 mM 6-APA and 102-110 mM HPGME in free or hydrochloride form, with 200 U of immobilized enzyme catalyst per gram of 6-APA in 10 mL of water. Samples were taken at regular time intervals to monitor the course of the reaction and analyzed by HPLC.

Example 30: Enzymatic Synthesis of Ampicillin

The reaction was carried out in a stirred tank reactor at 20-25° C., pH 6.3-6.8 for 2 hours. The reaction mixture consists of 100 mM 6-APA and 105-110 mM PGME hydrochloride form, with 150-170 U of immobilized enzyme catalyst per gram of 6-APA in 10 mL of water. Samples were taken at regular time intervals to monitor the course of the reaction and analyzed by HPLC.

Example 31: Enzymatic Synthesis of Cephalexin

The reaction was carried out in a stirred tank reactor at 20-25° C., pH 6.3-6.8 for 2 hours. The reaction mixture consists of 100 mM 7-ADCA and 105-110 mM PGME hydrochloride form, with 150-170 U of immobilized enzyme catalyst per gram of 7-ADCA in 10 mL of water. Samples were taken at regular time intervals to monitor the course of the reaction and analyzed by HPLC.

Example 32: Enzymatic Synthesis of Cefadroxil

The reaction was carried out in a stirred tank reactor at 20-25° C., pH 6.3-6.8 for 3 hours. The reaction mixture consists of 100 mM 7-ADCA and 110 mM HPGME hydrochloride form, with 200 U of immobilized enzyme catalyst per gram of 7-ADCA in 10 mL of water. Samples were taken at regular time intervals to monitor the course of the reaction and analyzed by HPLC.

Example 33: Enzymatic Synthesis of Cefaclor

The reaction was carried out at 20-25° C., pH 6.5-6.8 in a stirred tank reactor for 2 hours. The reaction mixture consists of 100 mM 7-ACCA and 120 mM PGME hydrochloride form, with 225 U of immobilized enzyme catalyst per gram of 7-ACCA in 10 mL of water. Samples were taken at regular time intervals to monitor the course of the reaction and analyzed by HPLC.

Example 34: Enzymatic Synthesis of Cefprozil

The reaction was carried out at 20-25° C., pH 6.5-6.8 in a stirred tank reactor for 2 hours. The reaction mixture consists of 100 mM 7-APCA and 120 mM PGME hydrochloride form, with 225 U of immobilized enzyme catalyst per gram of 7-APCA in 10 mL of water. Samples were taken at regular time intervals to monitor the course of the reaction and analyzed by HPLC.

Example 35: Enzymatic Synthesis of Cepradine

The reaction was carried out in a stirred tank reactor at 20-25° C., pH 6.3-6.8 for 2 hours. The reaction mixture consists of 100 mM 7-ADCA and 130 mM HPGME form, with 250 U of immobilized enzyme catalyst per gram of 7-ADCA in 10 mL of water. Samples were taken at regular time intervals to monitor the course of the reaction and analyzed by HPLC.

Advantages of the present invention:

1. The enzyme thus obtained was found to be suitable for the enzymatic synthesis of semisynthetic β-lactam antibiotics such as amoxicillin, ampicillin, cephalexin, cefadroxil, cefaclor, etc., and the enzymatic method using the enzyme is commercially viable .

2. Additional mutant penicillin acylase enzymes are immobilized in a custom terpolymer system of epoxy polyacrylate resins and evaluated for recyclability and suitability in the production method of semisynthetic β-lactam antibiotics.

3. The immobilized form of the mutant penicillin acylase enzyme and the process parameters including raw material input, reaction conditions and product isolation are designed to be commercially viable.

4. The activity of the immobilized biocatalyst is maintained at 90% after recycling in the enzymatic process.

<110> Fermenta Biotech Ltd. <120> Mutant Penicillin G acylases of Achromobacter CCM4824 <130> PN0978IM <150> IN 202021000782 <151> 2020-01-08 <160> 2 <170> KoPatentIn 3.0 <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> Artificial Sequence <220> <223> Protein sequence of penicillin G acylase produced by Achromobacter CCM4824 <400> 2 Met Lys Gln Gln Trp Leu Ser Ala Ala Leu Leu Ala Ala Ser Ser Cys 1 5 10 15 Leu Pro Ala Met Ala Ala Gln Pro Val Ala Pro Ala Ala Gly Gln Thr 20 25 30 Ser Glu Ala Val Ala Ala Arg Pro Gln Thr Ala Asp Gly Lys Val Thr 35 40 45 Ile Arg Arg Asp Ala Tyr Gly Met Pro His Val Tyr Ala Asp Thr Val 50 55 60 Tyr Gly Ile Phe Tyr Gly Tyr Gly Tyr Ala Val Ala Gln Asp Arg Leu 65 70 75 80 Phe Gln Met Glu Met Ala Arg Arg Ser Thr Gln Gly Arg Val Ala Glu 85 90 95 Val Leu Gly Ala Ser Met Val Gly Phe Asp Lys Ser Ile Arg Ala Asn 100 105 110 Phe Ser Pro Glu Arg Ile Gln Arg Gln Leu Ala Ala Leu Pro Ala Ala 115 120 125 Asp Arg Gln Val Leu Asp Gly Tyr Ala Ala Gly Met Asn Ala Trp Leu 130 135 140 Ala Arg Val Arg Ala Gln Pro Gly Gln Leu Met Pro Lys Glu Phe Asn 145 150 155 160 Asp Leu Gly Phe Ala Pro Ala Asp Trp Thr Ala Tyr Asp Val Ala Met 165 170 175 Ile Phe Val Gly Thr Met Ala Asn Arg Phe Ser Asp Ala Asn Ser Glu 180 185 190 Ile Asp Asn Leu Ala Leu Leu Thr Ala Leu Lys Asp Arg His Gly Ala 195 200 205 Ala Asp Ala Met Arg Ile Phe Asn Gln Leu Arg Trp Leu Thr Asp Ser 210 215 220 Arg Ala Pro Thr Thr Val Pro Ala Glu Ala Gly Ser Tyr Gln Pro Pro 225 230 235 240 Val Phe Gln Pro Asp Gly Ala Asp Pro Leu Ala Tyr Ala Leu Pro Arg 245 250 255 Tyr Asp Gly Thr Pro Pro Met Leu Glu Arg Val Val Arg Asp Pro Ala 260 265 270 Thr Arg Gly Val Val Asp Gly Ala Pro Ala Thr Leu Arg Ala Gln Leu 275 280 285 Ala Ala Gln Tyr Ala Gln Ser Gly Gln Pro Gly Ile Ala Gly Phe Pro 290 295 300 Thr Thr Ser Asn Met Trp Ile Val Gly Arg Asp His Ala Lys Asp Ala 305 310 315 320 Arg Ser Ile Leu Leu Asn Gly Pro Gln Phe Gly Trp Trp Asn Pro Ala 325 330 335 Tyr Thr Tyr Gly Ile Gly Leu His Gly Ala Gly Phe Asp Val Val Gly 340 345 350 Asn Thr Pro Phe Ala Tyr Pro Ser Ile Leu Phe Gly His Asn Ala His 355 360 365 Val Thr Trp Gly Ser Thr Ala Gly Phe Gly Asp Asp Val Asp Ile Phe 370 375 380 Ala Glu Lys Leu Asp Pro Ala Asp Arg Thr Arg Tyr Phe His Asp Gly 385 390 395 400 Gln Trp Lys Thr Leu Glu Lys Arg Thr Asp Leu Ile Leu Val Lys Asp 405 410 415 Ala Ala Pro Val Thr Leu Asp Val Tyr Arg Ser Val His Gly Leu Ile 420 425 430 Val Lys Phe Asp Asp Ala Gln His Val Ala Tyr Ala Lys Ala Arg Ala 435 440 445 Trp Glu Gly Tyr Glu Leu Gln Ser Leu Met Ala Trp Thr Arg Lys Thr 450 455 460 Gln Ser Ala Asn Trp Glu Gln Trp Lys Ala Gln Ala Ala Arg His Ala 465 470 475 480 Leu Thr Ile Asn Trp Tyr Tyr Ala Asp Asp Arg Gly Asn Ile Gly Tyr 485 490 495 Ala His Thr Gly Phe Tyr Pro Arg Arg Arg Pro Gly His Asp Pro Arg 500 505 510 Leu Pro Val Pro Gly Thr Gly Glu Met Asp Trp Leu Gly Leu Leu Pro 515 520 525 Phe Ser Thr Asn Pro Gln Val Tyr Asn Pro Arg Gln Gly Phe Ile Ala 530 535 540 Asn Trp Asn Asn Gln Pro Met Arg Gly Tyr Pro Ser Thr Asp Leu Phe 545 550 555 560 Ala Ile Val Trp Gly Gln Ala Asp Arg Tyr Ala Glu Ile Glu Thr Arg 565 570 575 Leu Lys Ala Met Thr Ala Asn Gly Gly Lys Val Ser Ala Gln Gln Met 580 585 590 Trp Asp Leu Ile Arg Thr Thr Ser Tyr Ala Asp Val Asn Arg Arg His 595 600 605 Phe Leu Pro Phe Leu Gln Arg Ala Val Gln Gly Leu Pro Ala Asp Asp 610 615 620 Pro Arg Val Arg Leu Val Ala Gly Leu Ala Ala Trp Asp Gly Met Met 625 630 635 640 Thr Ser Glu Arg Gln Pro Gly Tyr Phe Asp Asn Ala Gly Pro Ala Val 645 650 655 Met Asp Ala Trp Leu Arg Ala Met Leu Arg Arg Thr Leu Ala Asp Glu 660 665 670 Met Pro Ala Asp Phe Phe Lys Trp Tyr Ser Ala Thr Gly Tyr Pro Thr 675 680 685 Pro Gln Ala Pro Ala Thr Gly Ser Leu Asn Leu Thr Thr Gly Val Lys 690 695 700 Val Leu Phe Asn Ala Leu Ala Gly Pro Glu Ala Gly Val Pro Gln Arg 705 710 715 720 Tyr Asp Phe Phe Asn Gly Ala Arg Ala Asp Asp Val Ile Leu Ala Ala 725 730 735 Leu Asp Asp Ala Leu Ala Ala Leu Arg Gln Ala Tyr Gly Gln Asp Pro 740 745 750 Ala Ala Trp Lys Ile Pro Ala Pro Pro Met Val Phe Ala Pro Lys Asn 755 760 765 Phe Leu Gly Val Pro Gln Ala Asp Ala Lys Ala Val Leu Cys Tyr Arg 770 775 780 Ala Thr Gln Asn Arg Gly Thr Glu Asn Asn Met Thr Val Phe Asp Gly 785 790 795 800 Lys Ser Val Arg Ala Val Asp Val Val Ala Pro Gly Gln Ser Gly Phe 805 810 815 Val Ala Pro Asp Gly Thr Pro Ser Pro His Thr Arg Asp Gln Phe Asp 820 825 830 Leu Tyr Asn Thr Phe Gly Ser Lys Arg Val Trp Phe Thr Ala Asp Glu 835 840 845 Val Arg Arg Asn Ala Thr Ser Glu Glu Thr Leu Arg Tyr Pro Arg 850 855 860

Claims (10)

A mutant penicillin G acylase encoded by the DNA sequence of penicillin G acylase of Achromobacter sp. CCM4824 for the preparation of semi-synthetic β-ramtam antibiotics,
Amino acid positions A3, A27, A77, A144, A145, A146, A192, A195, according to amino acid numbering of penicillin G acylase of Achromobacter sp. CCM4824, wherein the mutation has the polypeptide of SEQ ID NO: 2; produced by site-directed mutagenesis having substitution of one or more amino acid positions selected from the group consisting of A196, B23, B24, B31, B57, B69, B77, B313, B464 and B484;
The penicillin G acylase of Achromobacter sp. CCM4824
(a) a signal peptide consisting of 40 amino acids at amino acid positions 1-40;
(b) the α-subunit consisting of 209 amino acids at amino acid positions 41-249, represented by A1 to A209;
(c) a linking peptide consisting of 57 amino acids at amino acid positions 250-306, and
(d) a β-subunit consisting of 557 amino acids at amino acid positions 307-863, denoted by B1 to B557
A mutant penicillin G acylase composed of The mutant penicillin G acylase of claim 1 , wherein said mutation is generated by site-directed mutation with substitution of one or more amino acid positions selected from the group consisting of:
(i) the mutation at amino acid position A3 comprises substituting alanine (A) for leucine (L);
(ii) the mutation at amino acid position A27 comprises substituting isoleucine for asparagine (N) or proline (P);
(iii) the mutation at amino acid position A77 comprises substituting arginine (R) for threonine (T);
(iv) the mutation at amino acid position A144 comprises substituting asparagine (N) for threonine (T);
(v) the mutation at amino acid position A145 comprises replacing arginine (R) with leucine (L);
(vi) the mutation at amino acid position A146 comprises substituting alanine (A) for phenylalanine (F);
(vii) the mutation at amino acid position A192 comprises substituting alanine (A) for glutamic acid (E);
(viii) the mutation at amino acid position A195 comprises replacing glycine (G) with leucine (L);
(ix) the mutation at amino acid position A196 comprises substituting serine for threonine (T) or leucine (L);
(x) the mutation at amino acid position B23 comprises replacing glutamine (Q) with glycine (G);
(xi) the mutation at amino acid position B24 comprises substituting alanine (A) for phenylalanine (F);
(xii) the mutation at amino acid position B31 comprises replacing tyrosine (Y) with serine (S);
(xiii) the mutation at amino acid position B57 comprises substituting alanine (A) for phenylalanine (F);
(xiv) the mutation at amino acid position B69 comprises substituting alanine (A) for glycine (G);
(xv) the mutation at amino acid position B77 comprises replacing isoleucine (I) with cysteine (C);
(xvi) the mutation at amino acid position B313 comprises replacing glycine (G) with valine (V);
(xvii) the mutation at amino acid position B464 comprises substituting threonine for glutamine (Q) or serine (S);
(xviii) the mutation at amino acid position B484 comprises the substitution of asparagine (N) with serine (S) or threonine (T). 3. The method of claim 2, wherein said amino acid substitution is [A3+A192+196], [A3+A145+A192+A27], [A145+B77], [A3+A145+B31], [A145+A192+B69] and A mutant penicillin G acylase having a substitution at position B24 in combination with a substitution selected from the group consisting of [A145+A146+B313]. The mutant penicillin G acylase of claim 1, wherein the mutant penicillin G acylase is encoded by a nucleotide sequence having at least 60% similarity to SEQ ID NO: 1, which encodes the mutant penicillin acylase. . 5. The method of claim 4, wherein SEQ ID NO: 1 is E. A mutant penicillin G acylase, characterized in that it is present on the plasmid pKARA9 expressed in E. coli BL21. 6. The method of claim 5, wherein the resulting mutant E. A mutant penicillin G acylase, characterized in that E. coli BL21 is deposited as MTCC 25429. The method of claim 1, wherein the mutant penicillin acylase enzyme is immobilized on a terpolymer synthesized by free radical suspension polymerization, and the monomer component is glycidyl methacrylate (GMA), allyl glycidyl ether, ethylene Mutant penicillin, characterized in that it is selected from the group consisting of glycol dimethacrylate (EGDM), divinylbenzene (DVB), ethyl methacrylate (EMA), methyl methacrylate (MMA) and acrylonitrile (AN) G acylase. 6-aminopenicillanic acid (6-APA), 7-desacetoxycephalosporanoic acid (7-ADCA), 7-aminocephalosporanoic acid (7-ACA), 7-amino-3-chloro- β selected from 3-cephem-4-carboxylic acid (7-ACCA), and 7-amino-3-[(Z)-propen-1-yl]-3-cephem-4-carboxylic acid (7-APCA) -acylation of lactam nuclei and active acyl donors selected from esters or amides of hydroxyphenylglycine (HPGME) or phenylglycine (PGME) or dihydroxyphenylglycine (DHPGME) to synthesize semi-synthetic β-lactam antibiotics A method for producing a semi-synthetic β-lactam antibiotic comprising a mutant penicillin G acylase that catalyzes The method according to claim 8, wherein the semi-synthetic β-lactam antibiotic is selected from ampicillin, amoxicillin, cephalexin, cefadroxil, or cepradin. A composition comprising a mutant penicillin G acylase encoded by the DNA sequence of a penicillin G acylase of Achromobacter sp. CCM4824 for the preparation of a semi-synthetic β-ramtam antibiotic,
Amino acid positions A3, A27, A77, A144, A145, A146, A192, A195, according to amino acid numbering of penicillin G acylase of Achromobacter sp. CCM4824, wherein the mutation has the polypeptide of SEQ ID NO: 2; produced by site-directed mutagenesis having a substitution of one or more amino acid positions selected from the group consisting of A196, B23, B24, B31, B57, B69, B77, B313, B464 and B484;
The penicillin G acylase of Achromobacter sp. CCM4824
(a) a signal peptide consisting of 40 amino acids at amino acid positions 1-40;
(b) the α-subunit consisting of 209 amino acids at amino acid positions 41-249, represented by A1 to A209;
(c) a linking peptide consisting of 57 amino acids at amino acid positions 250-306, and
(d) a β-subunit consisting of 557 amino acids at amino acid positions 307-863, denoted by B1 to B557
A composition comprising a mutant penicillin G acylase consisting of.

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