RU2537845C2 - Method of synthesising peptides, including beta-lactam antibiotics with application of penicillin acylase version - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to engineering enzymology, molecular biology and biotechnology, in particular to increase of efficiency of biocatalytic conversions by means of applying mutant forms of enzymes with improved catalytic properties. In claimed invention increase of efficiency of fermentative synthesis of peptide bond is achieved by application of version of penicillin acylase from E.coli, containing substitution of amino acid residue of enzyme beta-chain in position 484.

EFFECT: application of said penicillin acylase mutant makes it possible to increase output and rate of accumulation of target product in reactions of peptide synthesis, including beta-lactam antibiotics, containing peptide bond in side chain with nucleus.

3 cl, 2 tbl, 2 ex

Description

Penicillin acylase (Penicillin G acylase) from E. coli belongs to amidohydrolase class enzymes. The ability of penicillin acylase to catalyze the synthesis of a peptide bond in an aqueous medium has been used for many years in the pharmaceutical industry for the production of semi-synthetic β-lactam antibiotics [1], however, interest in increasing the efficiency of this process does not decrease. The search for new and improvement of existing catalysts based on penicillin acylase does not stop, since their use can give a significant economic effect. One of the main directions of improving the efficiency of the process is to obtain mutant forms of penicillin acylase by genetic engineering by replacing one or more amino acids, leading to an improvement in the catalytic properties of the enzyme.

The full-length amino acid sequence of E. coli penicillin acylase includes a signal peptide (amino acids 1-26), an alpha subunit (amino acids 27-235), a spacer peptide (amino acids 236-289), and a beta subunit (amino acids 290-846):

In works related to the production of penicillin acylase variants, the enzyme subunits are usually numbered separately, and this numbering will be used in the further presentation (it is shown as α1-209 and β1-557 on the full-sized amino acid sequence).

Several scientific works are known on the preparation of mutant forms of an enzyme from E. coli at the positions αR145 and αF146 with improved ability to catalyze peptide bond formation reactions in the synthesis of ampicillin and amoxicillin by the acyl transfer method [2, 3], which leads to an increase in the yield in the reaction of synthesis of ampicillin from 15% to 24% for low concentration systems and from 51% to 77% for highly concentrated systems. The greatest effect is characteristic of αR145G / S / L mutants. For mutants at the αF146 position, a significant decrease in catalytic activity is observed.

In [4], the authors obtained recombinant hybrids in the PA family. Among random mutations, substitutions of αD148G and βG385S were found, including which in the composition of E.Coli PA, it is possible to improve the yield in the reaction of synthesis of ampicillin by 20%

By using the βF24A mutation, it is possible to increase yields in the synthesis of ampicillin, amoxicillin, cephalexin and cefadroxil using esters as an acyl donor. [5] The use of amides does not improve the yield. This effect is associated with a general weakening of the catalytic abilities of the enzyme.

The patent literature on improving the efficiency of peptide synthesis reactions by improving the properties of penicillin acylase is limited to several patents related mainly to the use of mutant penicillin acylases for the synthesis of β-lactam antibiotics.

In the patents [WO9605318, 22-02-1996 and US6033823, 07-03-2000], belonging to one group of authors, a change in the substrate specificity and activity of penicillin acylase from prokaryotes by mutation of amino acid residues by a significant number of alpha chain sites (α139-152) and beta chains (β20-27, β31, β32, β49-52, β56, β57, β65-72, β154-157, β173-179, β239-241, β250-263, β379-387, β390, β455, β474- 480), although patents are based on only a few examples of the influence of mutations in certain positions of αM143, αF147, βL56, βA67, βI177 in penicillin acylase from Alcaligenes faecalis on the enzyme's ability to catalyze the hydrolysis of natural penises Illins G and V, as well as ampicillin synthesis.

In the patents [WO9820120, 14-05-1998; US6403356, 11-06-2002] the range of E. coli penicillin acylase variants is substantially narrowed and limited by mutations at the αM142, αF146, βF24, βV56, βI177 sites. The priority rights of these patents are limited by the method of producing 6-aminopenicillanic and 7-aminodetacetoxycephalosporanic acids by enzymatic hydrolysis of their acylated forms, as well as by the method of producing β-lactam antibiotics by enzymatic acylation of 6-aminopenicillinic and 7-aminodetacetoxycephalosporanic acids. The greatest effect was achieved at the β24 site mutation by replacing L-phenylalanine with L-alanine, which allowed increasing the yield in the synthesis of penicillins and cephalosporins when using esters as an acyl donor.

In the patent [US2005124029, 09-06-2005], the authors replaced the amino acid residue αR145 in L, K, or C in penicillin acylase from E. coli. For the αR145L mutant, the yield of the antibiotic in the synthesis of cephalexin increased from 70 to 81% under slightly alkaline conditions, however, a significant (more than 10 times) loss of catalytic activity was observed. An object of the present invention is to develop a method for producing peptides, including semi-synthetic β-lactam antibiotics containing a peptide bond in the side chain, by using a penicillin acylase preparation with improved properties.

The technical result of the invention is the creation of an improved method for producing peptides, including semi-synthetic β-lactam antibiotics containing a peptide bond in the side chain, characterized by a higher yield and the rate of formation of the target product.

The specified technical result is achieved by using a penicillin acylase preparation from E. coli with a modified structure, namely, containing the replacement of the aspartic acid residue of the beta chain of the enzyme at position β484, with another amino acid residue, in particular with an asparagine residue. The closest analogues of the present invention are the above patents [WO9820120, 14-05-1998; US6403356, 11-06-2002, US2005124029, 09-06-2005].

In the scientific and patent literature, examples of improving the catalytic properties of penicillin acylase in the synthesis of peptides not related to β-lactam antibiotics are not known, and the obtained increase in the efficiency of the synthesis of peptides, including those related to β-lactam antibiotics, using the penicillin acylase βD484N mutant does not follow with evidence from the known structure of penicillin acylase and its functions.

The present invention relates to the field of engineering enzymology and biotechnology, in particular to the use of penicillin acylase from E. coli obtained from an enzyme precursor by replacing the amino acid residue at position 484 of the enzyme beta chain with any available genetic engineering methods aimed at evolution or selection.

As the acyl donors in enzymatic reactions of the synthesis of peptides, including β-lactam antibiotics with a peptide bond in the side chain with the nucleus, esters and amides of amino acids, their substituted derivatives in the α-position and in the aromatic ring, preferably amides and esters of R, S-phenylglycine.

The penicillin acylase according to the invention can be obtained and used in the form of a homogeneous preparation, a solution containing other chemical compounds or proteins, micelles, aggregates, or in the solid state in the form of crystals, aggregates or immobilized in a gel, on various substrate and carrier preparations, or used in in the form of cell culture, immobilized cells, combined enzyme preparations with cells, immobilized cells or enzymes. The claimed method is implemented by contacting the drug penicillin acylase and the reaction mixture. As the reaction medium, water, single-phase and multiphase aqueous-organic mixtures, solutions containing organic and inorganic compounds, in particular salts, acids, bases, can be used. As a reaction medium, it is more preferable to use water or aqueous solutions with a content of not more than 40% (by volume) of organic solvents. Some examples of the implementation of the claimed method are given below. Variants of penicillin acylase from E. coli were obtained by constructing the mutant gene by site-specific mutagenesis according to the methods described in the literature [6]. After cloning, culturing, isolation and purification procedures, penicillin acylase mutant preparations were obtained.

Example 1. An increase in the yield in the reaction of the enzymatic synthesis of cephalexin.

The enzymatic synthesis of cephalexin by transferring the acyl group to the core of the antibiotic 7-aminodecetoxycephalosporanic acid was carried out as described in the experimental part. As the acyl donor, D-phenylglycine amide was used. When using the βD484N penicillin acylase variant, an increase in the yield of the reaction product is observed from 1.5 to 1.9 times as compared with wild-type penicillin acylase.

Table 1
The yield of the reaction product of the enzymatic synthesis of cephalexin [7-ADCA] / [D-PGA], mM The yield of cephalexin,% Wild type βD484N 200/200 40 60 200/400 39 73

Example 2. An increase in the yield in the enzymatic synthesis reaction of D-phenylglycyl-glycine.

The enzymatic synthesis of D-phenylglycyl-glycine by transferring the acyl group to glycine was carried out in an aqueous medium, as described in the experimental part. As the acyl donor, D-phenylglycine amide was used. It turned out that in the case of the βD484N variant of penicillin acylase, the yield of the reaction product increased by approximately 4.7 times compared to wild-type penicillin acylase (Table 2).

table 2
The product yield in the enzymatic synthesis of D-phenylglycyl-glycine Penicillin Acylase Option The yield of D-phenylglycyl-glycine,% Wild type 17 βD484N 80

Description of the experimental part

Determination of enzymatic activity. The activity of penicillin acylase variants was determined spectrophotometrically from the accumulation of the chromophore during the hydrolysis of a 1 mM solution of a colored substrate of 6-nitro-3- (phenylacetamido) benzoic acid at 400 nm on a Shimadzu UV-1601 spectrophotometer (Japan). The reaction was carried out at 25 ° C in 0.01 M phosphate buffer, pH 7.5, 0.1 M KCl.

Determination of the concentration of active centers. The absolute concentration of active centers of each of the mutant forms of penicillin acylase was determined by titration of the active centers of the enzyme with an irreversible phenylmethylsulfonyl fluoride inhibitor according to the procedure [7]. Residual enzymatic activity was determined spectrophotometrically by hydrolysis of a colored substrate, as described above.

HPLC analysis. The quantitative determination of the components of the reaction mixture was performed by reverse phase high performance liquid chromatography on a chromatographic system Perkin Elmer 200 Series (USA); Kromasil Eternity C18 column (AkzoNobel, USA), 250 × 4.6 mm, particle size 5 μm; mobile phase CH ₃ CN / water 30:70, 0.68 g / l KH ₂ PO4, 0.1 g / l sodium dodecyl sulfate, pH 3.0; flow rate 0.7 ml / min; injection volume 10 μl; UV detection at 210 nm.

Carrying out the enzymatic synthesis of D-phenylglycyl-glycine. The enzymatic synthesis of D-phenylglycyl-glycine was carried out in an aqueous medium in a thermostatic cell of a pH stat Titrino 719 (Metrohm, Switzerland) at 25 ° C, pH 9.5 with constant stirring. Initial reagent concentrations: 200 mM D-phenylglycine, 200 mM glycine, 1 μM active centers of penicillin acylase, reaction volume 3 ml. The time of the experiment did not exceed 5 hours. Along the course of the reaction, aliquots were taken from the reaction mixture, diluted with their mobile phase and analyzed by HPLC as described above.

Carrying out the enzymatic synthesis of cephalexin. The enzymatic synthesis of cephalexin was carried out in an aqueous medium in a thermostatic cell of a pH stat Titrino 719 (Metrohm, Switzerland) at 25 ° C, pH 8.5 with constant stirring. Initial concentrations of reagents: 200-400 mM D-phenylglycine and 200 mM 7-aminodedetoxycephalosporanic acid, 1 μM active centers of penicillin acylase, reaction volume 3 ml. The time of the experiment did not exceed 5 hours. Along the course of the reaction, aliquots were taken from the reaction mixture, diluted with their mobile phase and analyzed by HPLC as described above.

Bibliography

1. Bruggink, A., Roos, E.C., de Vroom, E. Penicillin acylase in the industrial production of b-lactam antibiotics. Org. Proc. Res. Dev. (1998) 2, 128-33.

2. Alkema, W.B.L., Prins, A.K., de Vries, E. & Janssen, D.B. Role of a Arg 145 and b Arg 263 in the active site of penicillin acylase of Escherichia coli. Biochem. J. (2002) 365, 303-309.

3. Jager, S.A.W., Shapovalova, I.V., Jekel, P.A., Alkema, W.B.L., Svedas, V.K., Janssen, D.B. Saturation mutagenesis reveals the importance of residues alphaR145 and alphaF146 of penicillin acylase in the synthesis of beta-lactam antibiotics. J. Biotechnol. (2008) 133, 18-26.

4. Jager, S.A.W., Jekel, P.A. and Janssen, D.B. Hybrid penicillin acylases with improved properties for synthesis of β-lactam antibiotics. Enzyme and Microb.l Technol (2007) 40, 1335-1344.

5. Alkema, W.B.L., Dijkhuis, A.-J., de Vries, E. & Janssen, D.B. The role of hydrophobic active-site residues in substrate specificity and acyl transfer activity of penicillin acylase. Eur. J. Biochem. (2002) 269, 2093-2100.

6. Dominy, C.N., Andrews, D.W. in Methods in Molecular Biology. Vol. 235 (E. coli Plasmid Vectors) (Eds .: N. Casali, A. Preston), Humana, Totowa, NJ, 209-223 (2003).

7. Shvyadas, V.K., Margolin, A.L., Sherstyuk. S.F., Berezin. I.V. Inactivation of soluble and immobilized penicillin amidase from E. coli by phenylmethylsulfonyl fluoride: kinetic analysis and titration of active sites. Bioorg. Chemistry (1977) 3, 546-554.

The full-length amino acid sequence of penicillin acylase from E. coli with two types of numbering: through numbering and numbering of subunits individually, used to describe the invention.

Claims (3)

1. A method for producing peptides, including β-lactam antibiotics containing a peptide bond in the side chain with the nucleus, by enzymatic transfer of the acyl moiety to a nucleophile, characterized in that a variant of penicillin acylase (Penicillin G acylase) from E. coli is used as a catalyst, comprising replacing the amino acid residue of aspartic acid at position 484 of the beta chain (numbering begins with the first amino acid residue of the beta chain containing 557 amino acid residues) with an asparagine residue. 2. The method according to claim 1, where the peptide is cephalexin. 3. The method according to claim 1, where the peptide is D-phenylglycyl-glycine.