HK1021205A - A METHOD FOR PREPARING A β-LACTAM ANTIBIOTIC - Google Patents

Description

Method for preparing beta-lactam antibiotic

Field and background of the invention

The invention relates to a method for preparing beta-lactam antibiotics.

Beta-lactam antibiotics, such as penicillins and cephalosporins, contain many compounds and have their respective activity characteristics. In general, β -lactam antibiotics comprise a core, the so-called β -lactam core, which is linked with its primary amino group to the so-called side chain via a linear amide bond.

The beta-lactam nucleus is a very important intermediate in the preparation of semi-synthetic penicillin and cephalosporin antibiotics. The preparation routes for these semisynthetic penicillins and cephalosporins mostly start from fermentation products such as penicillin G, penicillin V and cephalosporin C, which are converted into the corresponding β -lactam nucleus, for example: matsumoto in Bioprocess.technology (1993,16:67-88), J.G.Shewale and H.Sivaraman in Biochemical methods (Process Biochemistry,1989,8 months: 146-: 97-103).

Examples of β -lactam parent nuclei used as several antibiotic precursor species are: 6-aminopenicillanic acid (penicilanic acid) (6-APA), 7-aminocephalosporanic acid (cephalosporanic acid) (7-ACA), 3-chloro-7-aminodesacetoxycetamidomethylcephalosporanic acid (7-ACCA), 7-aminodesacetylcephalosporanic acid (7-ADAC) and 7-aminodesacetoxycephalosporanic acid (7-ADCA).

The β -lactam nucleus is converted to the desired antibiotic by coupling an appropriate side chain, as described, inter alia, in EP 0339751, JP53005185 and CH 640240. By different combinations of side chains and beta-lactam nucleus, a number of different penicillin and cephalosporin antibiotics can be obtained, all of which have their own respective activity profile.

For example, D- (-) -phenylglycine or a suitable derivative thereof (e.g., an amide or ester) may be conjugated to any of 7-ACA, 7-ACCA, 7-ADCA and 6-APA to form cephalosporin III, cefaclor, cephalexin or ampicillin, respectively. Other examples of side chains that are often useful are: d- (-) -4-hydroxyphenylglycine, 2-cyanoacetic acid and 2- (2-amino-4-thiazolyl) -2-methoxyiminoacetic acid.

The known enzymatic preparation of beta-lactam antibiotics involves the preparation of the beta-lactam nucleus and its subsequent coupling to a suitable side chain. Reference may be made to enzymatic synthesis methods such as: t.a. savidge, industrial antibiotic biotechnology (e.j.vandame eds.) Marcel Dekker, new york 1984; shewale et al, international methods of biochemistry, month 19906: 97 to 103; J.Vandamm, applied microbiology development (Advances in applied microbiology),21, (1977),89-123 and E.J.Vandamm, enzymemeicrob.Techniol, 5, (1983): 403-. In addition, novel routes which report direct fermentative production of 7-ADCA and 7-ACA are also disclosed in EP 0532341, EP 0540210, WO93/08287, WO95/04148 and WO 95/04149.

One disadvantage of these methods is that the side chain coupling reaction starts with the β -lactam nucleus, which must be isolated prior to the coupling reaction. The separation of the β -lactam nucleus is usually carried out by crystallization, which can lead to losses of up to 10% of the theoretical yield. Because of the amphiprotic character of the beta-lactam parent nucleus, the beta-lactam parent nucleus is easy to dissolve in a water-soluble system with any pH value, so that most of the produced beta-lactam parent nucleus is lost in the crystallization mother liquor.

The present invention overcomes the above-mentioned disadvantage that the side chain introduction reaction starts from a different material than the beta-lactam nucleus.

Summary of The Invention

It is an object of the present invention to provide a process for the preparation of beta-lactam antibiotics, wherein the side chain introduction reaction is initiated with a different substance than the beta-lactam nucleus.

It is a further object of the present invention to provide a process for the preparation of beta-lactam antibiotics which can be conveniently combined with known enzymatic processes starting from some fermentation products, such as penicillin G or cephalosporin C.

It is another object of the present invention to provide a process for the preparation of a beta-lactam antibiotic which is a clean, efficient, economical and easy to implement process, in other words which does not cause waste water problems or involve expensive chemicals.

The above-mentioned object is met in a process for the preparation of a beta-lactam antibiotic, wherein an N-substituted beta-lactam has the general structure shown in formula (i):wherein R is₀Is H or C_1-3An alkoxy group; y is CH₂O, S or an oxidized form of S; z is

Wherein R is₁Is H, hydroxy, halogen, C_1-3Alkoxy, optionally substituted, saturated or unsaturated, branched or straight chain C optionally containing one or more heteroatoms_1-5Alkyl, preferably methyl, optionally substituted, optionally containing one or more hetero atomsC of an atom_5-8Cycloalkyl, optionally substituted aryl or heteroaromatic ring, or optionally substituted benzyl; and is

X is (CH)₂)_m-A-(CH₂)_nWherein m and n are the same or different and should be one of an integer of 0, 1, 2, 3 or 4, and A is CH = CH, C ≡ C, CHB, C = O, optionally substituted N, O, S or an optionally oxidized form of S, while B is H, halogen, hydroxy, C_1-3An alkoxy group or an optionally substituted methyl group,

or a salt form of the N-substituted beta-lactam, which is contacted with at least one dicarboxylic acid acyltransferase or isoenzyme thereof, and reacted with a precursor substance in the presence of at least one penicillin acylase or isoenzyme thereof to obtain a side chain of the beta-lactam antibiotic.

Surprisingly, beta-lactam antibiotics can be efficiently prepared by the introduction of the side chain of the beta-lactam antibiotic. The side chain introduction reaction starts with an N-substituted beta-lactam and two enzymes with different substrates are used. In the process of the invention there is no need to recover the intermediate product, i.e.the product of the first enzymatic reaction, before the second enzyme is applied.

Since N-substituted beta-lactams are also obtainable from fermentation products, such as: penicillin G, penicillin V, cephalosporin C, adipoyl-7-ADCA, 3-carboxyethylthiopropionyl-7-ADCA, 2-carboxyethylthioacetyl-7-ADCA, 3-carboxyethylthiopropionyl-7-ADCA, adipoyl-7-ACA, 3-carboxyethylthiopropionyl-7-ACA, 2-carboxyethylthioacetyl-7-ACA and 3-carboxyethylthiopropionyl-7-ACA, and a major advantage of the present invention is that beta-lactam antibiotics can be produced starting from these fermentation products by enzymatic reactions without the necessity of isolating the beta-lactam nucleus intermediate, which would result in the loss of large amounts of the fermentation product.

The method according to the invention is a clean and highly specific method. This means that no or almost no by-products are produced and thus no waste water and/or purification problems result. Furthermore, the method according to the invention does not require the use of complex and expensive reagents, which are generally sensitive and difficult to handle.

Surprisingly, no significant enzyme inhibitory effect occurs in the process according to the invention. Until now, it was thought that it was not possible to use one or both enzymes to transfer acyl groups in the preparation of β -lactam antibiotics due to an enzyme inhibitory effect. It is generally believed that phenylacetic acid or phenoxyacetic acid may be formed during the transacylation reaction, and these acids are inhibitors of particular enzymes, such as: schomer et al in application and environmental microbiology (Applied and environmental microbiology), (2,1984: 307-) (312) and A.L.Margolin et al in Biochim.Biophys.acta, (1980),616: 283-) (289).

The starting material in the process according to the invention is an N-substituted beta-lactam having the general structure according to formula (I) or in the form of a salt thereof. In the above definitions of the various symbols of formula (I), an oxidized form of S refers to groups such as: sulfur oxides and sulfones. By optionally substituting alkyl, cycloalkyl, aryl, heteroaromatic, and benzyl groups, such that groups containing, for example, alkyl groups of 1-3 carbon atoms are prepared. Optionally substituted nitrogen atoms include primary, secondary and tertiary amino groups, which may be substituted, for example, with alkyl groups of from 1 to 3 carbon atoms. Optionally substituted methyl includes one methyl group and various substituted methyl groups such as: -CHpDq, wherein D is a halogen and p and q are integers adding up to 3.

It is contemplated that formula (I) comprises N-substituted β -lactams based on any of the β -lactam cores disclosed in cephalosporin and penicillin, chemistry and biology, E.H. Flynn, Academic Press, 1972,151-166 and "β -lactam organic chemistry", G.I. Georg, ed, VCH,1992,89-96, which are incorporated herein by reference. Preferred starting materials are those in which R is₁Is CH₂-E or CH = CH-E group, wherein E is H, hydroxy, halogen, C_1-3Alkoxy, optionally substituted, optionallySaturated or unsaturated, branched or straight-chain C containing one or more hetero atoms_1-5Alkyl, optionally substituted C optionally containing one or more hetero atoms_5-8Cycloalkyl, optionally substituted aryl or heteroaromatic ring, or optionally substituted benzyl.

Suitable salts as N-substituted β -lactam starting materials include any non-toxic salt such as: an alkali metal salt (e.g., sodium or potassium), an alkaline earth metal salt (e.g., calcium or magnesium), an ammonium salt, or an organic base salt (e.g., trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine, N, N' -dibenzyldivinyldiamine).

Starting materials for N-substituted beta-lactams of formula (I) can be prepared by enzymatic reaction, as described in one of the processes disclosed in EP 0532341, WO95/04148 or WO 95/04149. Preferred starting materials are N-glutaryl, N-succinyl, N-adipyl, N-3- (carboxymethylthio) propionyl, N-trans-beta-hydrohexadienedioyl, N-pimeloyl or N-3, 3' -thiodipropionyl beta-lactam or in the form of its salts. Such dicarboxylic acid-based starting materials can be efficiently converted by those enzymes used in the present invention.

More preferred starting materials are N-substituted 6-aminopenicillanic acid (6-APA), N-substituted 7-aminocephalosporanic acid (7-ACA), N-substituted 3-chloro-7-aminodesacetoxycetamidomethylcephalosporanic acid (7-ACCA), N-substituted 7-aminodesacetylcephalosporanic acid (7-ADAC) or N-substituted 7-aminodesacetoxycephalosporanic acid (7-ADCA), these N-substituted β -lactams yielding the most active β -lactam antibiotics.

A suitable dicarboxylic acid acyltransferase to be contacted with the N-substituted beta-lactam in the process according to the present invention is an enzyme isolated from a number of naturally occurring microorganisms, such as fungi and bacteria. Such microorganisms having enzymes of the desired dicarboxylic acid specificity can be screened by monitoring the hydrolysis of some suitable substrate. Such suitable substrates may be, for example, chromophores such as succinyl-, glutaryl-or adipyl-p-nitroanilides. The hydrolysis of the corresponding N-substituted beta-lactams can also be used as the enzyme required for the identification. It has been found that suitable pH ranges relied upon by these enzymes are between about 6 (preferably about 7) and about 9 (preferably about 8).

Organisms which have been found to produce dicarboxylic acid acyltransferases are: alcaligenes (Alcaligenes), Arthrobacter (Arthrobacter), Achromobacter (Achromobacter), Aspergillus (Aspergillus), Acinetobacter (Acinetobacter), Bacillus (Bacillus) and Pseudomonas (Pseudomonas). In particular the following microbial species can produce very suitable dicarboxylic acid acyltransferases: alcaligenes xylosoxidans (Achromobacter xylosoxins), Arthrobacter (Arthrobacter viscosus), Arthrobacter CA128, Bacillus CA78, Bacillus megaterium (Bacillus megaterium) ATCC53667, Bacillus cereus (Bacillus cereus), Bacillus laterosporus (Bacillus laterosporus) J1, Paecilomyces (Paecilomyces) C2106, Pseudomonas dimorpha (Pseudomonas dimomina) N176, Pseudomonas defectiveness V22, Pseudomonas oligovata (Pseudomonas papulobacter), Pseudomonas defectiveness BL072, Pseudomonas strain C, Pseudomonas SE83, Pseudomonas 495, Pseudomonas ovorans (Pseudomonas dimyrialis) ATCC950, Pseudomonas Comamonas SY77, Pseudomonas GK16, Pseudomonas pseudomonads-SY-1, Pseudomonas aeruginosa A14, Pseudomonas aeruginosa ATCC 10790, Pseudomonas aeruginosa B (Pseudomonas aeruginosa) 3, Pseudomonas putida bacterial vesicle PS 3, Pseudomonas putida bacterial strain P2, Pseudomonas putida (Pseudomonas putida) P2, Pseudomonas putida bacterial vesicle PS 3, Pseudomonas putida bacterial strain P3, Pseudomonas putida bacterial strain P2, Pseudomonas putida strain P3, Pseudomonas putida strain P2, Pseudomonas putida strain P3, Pseudomonas put.

The dicarboxylic acid acyltransferase may be obtained from these microorganisms in any suitable manner, for example: a method for obtaining the enzyme from Pseudomonas SE83 strain as described in US4,774,179. The genes for some enzymes, such as SE83 or SY77 dicarboxylic acid acyltransferase, can be expressed in different suitable hosts, such as e. The SE83 strain SY77 strain was described in Matsuda et al, J.Bacteriology, 1987,169:5818-5820 and US5,457,032, respectively.

The enzyme isolated from the above sources is commonly referred to as glutaryl acyltransferase. However, the side chain specificity of the enzyme is not limited to glutaryl side chains but can also be applied to smaller and larger dicarboxylic acid side chains. Some dicarboxylic acid acyltransferases also express gamma-glutaryl transpeptidase activity and are therefore sometimes also assigned to the gamma-glutaryl transpeptidase class.

In the process according to the invention, a suitable penicillin acylase to be contacted with the N-substituted beta-lactam is an enzyme isolated from a number of naturally occurring microorganisms, such as fungi and bacteria. Microorganisms producing enzymes of the desired specificity can be screened in a monitoring test similar to the one desired for a dicarboxylic acid acyltransferase. The enzymes are preferably present at a pH of about 4 (preferably about 5) to about 7 (preferably about 6).

Microorganisms which have been found to produce penicillin acylases are: acetobacter (Acetobacter), Aeromonas (Aeromonas), Alcaligenes (Alcaligenes), Bacillus, Cephalosporium (Cepalosporium), Escherichia (Escherichia), Aphanocandium, Flavobacterium (Flavobacterium), Kluyvera (Kluyvera), Acinetobacter (Mycoplana), Protaminobacter (Protaminobacter), providencia (Providentia), Pseudomonas or Xanthomonas (Xanthomonas). Enzymes obtained from Acetobacter pasteurianum, Alcaligenes faecalis (Escherichia coli), Escherichia coli, providencia rettgeri and Xanthomonas citri (Xanthomonas citrii) have proven suitable for use in the method of the present invention. Penicillin acylases are also known in the literature as penicillin amidases.

Dicarboxylic acid acylases and penicillin acylases can be used not only in the free enzyme form but also in any immobilized enzyme form, for example: has been described in EP 0222462 and WO 97/04086. When carrying out the process according to the invention, the two enzymes may be immobilized on one support or may be immobilized on different supports. In addition, isozymes of one or both enzymes may be used, wherein the properties of these enzymes are such as: the pH-dependent, thermal stability or specific activity can be influenced by chemical modification or crosslinking, but in the enzymes involved in the process of the invention their activity, although quantitatively altered, is not substantially altered per se. Also, isoenzymes such as: some mutants or other derivatives, enzyme biologically active parts or hybrids obtainable by classical methods or by recombinant DNA methods. As part of the ordinary knowledge of those skilled in the art, the methods of the invention may in some cases be beneficially chemically or otherwise modified.

The precursor material for the preparation of the side chains of the beta-lactam antibiotic in the process according to the invention may be any compound which is recognized by the penicillin acylase mentioned above and which leads to the production of this class of beta-lactam antibiotics. Preferably, the substrate used is selected from the following groups: d- (-) -phenylglycine, D- (-) -4-hydroxyphenylglycine, D- (-) -2, 5-dihydrophenylglycine, 2-thienylacetic acid, 2- (2-amino-4-thiazolyl) -2-methoxyiminoacetic acid, α - (4-pyridylthio) acetic acid, 3-thienylmalonic acid or 2-cyanoacetic acid and their derivatives, since highly active β -lactam antibiotics are produced from these substrates. Suitable derivatives of these substrates are esters or amides, wherein the side chain molecule is linked to a C via an ester or amide bond₁-C₃On the alkyl group of (a).

In the process according to the invention, the dicarboxylic acid acyltransferase, the β -lactam antibiotic side chain precursor substance and the penicillin acyltransferase may be added together or separately to the N-substituted β -lactam starting material. Preferably, these enzymes are added together to the N-substituted β -lactam and its side chain precursor species.

In a preferred embodiment of the invention, a process is employed which does not require isolation and/or purification of any intermediate which is present in the reaction mixture once or twice. Thus, no product is lost during the isolation or purification process.

In a highly preferred embodiment of the invention, a so-called "one-pot process" is used. By "one-pot process" is meant a process in which the entire process is carried out in one reaction pot. In other words, according to the process of the invention, substantially no majority of the reactants are removed from the reaction tank during any reaction carried out according to the process of the invention. The advantages of such an embodiment will be apparent to those skilled in the art.

The conditions as applied in the process of the invention depend on a number of parameters, in particular: reactant type, reactant concentration, reaction time, titrant, temperature, pH, enzyme concentration and enzyme morphology. If a particular N-substituted beta-lactam is to be converted to a particular beta-lactam antibiotic using a particular dicarboxylic acid acyltransferase and penicillin acyltransferase, one of ordinary skill in the art can appropriately select the optimal reaction conditions.

However, in the process of the present invention it has been found that the optimum reaction temperature is between 0 and 80 ℃ and preferably between 10 and 50 ℃. The optimum pH value for preparing the beta-lactam antibiotic according to the invention is between 4.5 and 9.0. In this regard, it is noted that it is highly preferred that the process according to the invention is carried out in an aqueous system, thus overcoming the use of organic solvents which cause waste problems. Moreover, dicarboxylic acid acyltransferases and penicillin acyltransferases have been shown to be most effective in catalyzing this conversion reaction in aqueous systems.

In general, the amount of reactants in each step ranges between 0.01 (preferably 0.5) and 3 (preferably 2) moles per kg of reaction mixture.

The enzyme concentration is chosen such that the total reaction time does not exceed 4 hours. To convert 10 mmoles of substrate to product in 1 hour, it is necessary to use approximately 500-3000 enzyme reaction units, one of which is defined as the amount of enzyme required to convert 1. mu. mole of substrate to product in 1 minute under the conditions of actual operation. In general, for the conversion of a specific amount of substrate in 1 hour, the enzyme concentration is preferably between 50 and 300 KU/mol. However, to compensate for the activity lost during the reaction, a very large dose of enzyme is usually applied.

Suitable titrants are inorganic acids and inorganic bases, such as: hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, ammonium hydroxide, and the like, or an organic acid such as: formic acid, acetic acid, succinic acid, adipic acid, glutaric acid, and the like. The titrant concentration varies between 0.01 and 8M depending on the scale of the reaction and the solubility of the titrant.

Of course, the present invention also includes a beta-lactam antibiotic obtained by the process disclosed above.

The invention will now be illustrated by the following non-limiting examples.

EXAMPLES Definitions and methods enzyme Activity

Penicillin G acyltransferase activity is defined as follows: one unit (U) corresponds to the amount of enzyme required to hydrolyze 1 micromole of penicillin G per minute under standard conditions: 100G/l of penicillin G potassium salt, 0.05M potassium phosphate buffer, pH8.0,28 ℃.

The dicarboxylic acid acyltransferase activity is defined as follows: one unit (U) corresponds to the amount of enzyme required to hydrolyze 1 millimole of N-adipoyl-7-ADCA per minute under standard conditions: 10mM N-adipoyl-7-ADCA, 100mM Tris buffer, pH8.0,37 ℃. Determination of pH

A Mettler DL21 titrator equipped with an automatic burette and a Brother M1509 printing device was used. HPLC analysis for amoxicillin: a chromatographic column: chromosphere C18,5Mm (100 × 3.0nm) solvent: 25% acetonitrile in 12mM potassium phosphate buffer (containing 0.2% sodium lauryl sulfate) flow rate: 1ml/min detection wavelength: 214nm for cephalexin: a chromatographic column: chromsphere C18,3Mm (100X 4.6Mm) solvent: flow rate of 29% acetonitrile in 14mM potassium dihydrogen phosphate buffer (containing phosphoric acid, pH 3.0): 1ml/min detection wavelength: 254nm

Example 1 Amoxicillin from N-adipoyl-6 β -aminopenicillanic acid and D- (-) -4-hydroxyphenylglycine methyl ester

Dicarboxylic acid acyltransferase from Pseudomonas SE83 (1.044g,96U/g) and penicillin acylase from Escherichia coli (0.80g,125U/g) were added to an aqueous solution (10ml) of dipotassium N-adipoyl-6-beta-aminopenicillanate (0.71g, 59% purity, 1.0mmol) and methyl D- (-) -4-hydroxyphenylglycine (0.45g, more than 97% purity, 2.4 mmol). The mixture was stirred at room temperature and the pH was maintained at 6.9 by the application of 1M aqueous sodium hydroxide solution. HPLC analysis can be used to monitor the formation of the product. The results are shown in table 1: TABLE 1

Time (h)	adipoyl-6-APA (mM)	6-APA(mM)	Amoxicillin (mM)
				00.51.0	1218180	01312	047

Example 2 cephalexin from N-adipoyl-7-amino-3-methyl-ceph-3-em-4-carboxylate and D- (-) -phenylglycine amide

The dicarboxylic acid acyltransferase (4.00g,369U/g) from Pseudomonas SE83 andpenicillin acylase from E.coli (1.6g,250U/g) was added to an aqueous solution (20ml) of N-adipoyl-7-amino-3-methyl-ceph-3-em-4-carboxylate (0.68g, purity 97.1%, 2.0mmol) and D- (-) -phenylglycine amide (0.75g, purity 96%, 4.8 mmol). The reaction mixture had an initial pH of 6.3 and was stirred at 35 ℃ after 30 minutes (pH =6.8) HPLC analysis showed the formation of cephalexin. For HPLC analysis, 0.5ml of the reaction mixture was removed from the reaction tank, centrifuged, filtered and 0.2ml of buffer pH 7 was added to 50 ml. The results are shown in table 2: TABLE 2

Time (h)	adipoyl-7-ADCA (mM)	7-ADCA(mM)	Cefalexin (mM)
				00.5	9534	035	09.9

Example 3 cephalexin from N-adipoyl-7-amino-3-methylcephalo-3-em-4-carboxylate and D- (-) -phenylglycine amide

The dicarboxylic acid acyltransferase from Pseudomonas SE83 (4.00g,369U/g) was added to an aqueous solution (20ml) of N-adipoyl-7-amino-3-methylcephalosporin-3-em-4-carboxylate (0.68g, 97.1% purity, 2.0 mmol). The reaction mixture was stirred at 35 ℃ and the pH was maintained at 8.0 by the application of 2M aqueous potassium hydroxide. After about 1 hour the reaction was filtered. D- (-) -phenyl groupGlycine amide (0.75g, 96% purity, 4.8mmol) and penicillin acylase from E.coli (1.6g,250U/g) were added to the filtrate. The reaction was maintained at 13 ℃ and the pH was maintained at 7.5 by using 1M hydrochloric acid. The formation of cephalexin was shown by HPLC analysis. For HPLC analysis, 0.5ml of the reaction mixture was removed from the reaction tank, centrifuged, filtered and 0.2ml of buffer pH 7 was added to 50 ml. The results are shown in Table 3: TABLE 3

Time (h)	adipoyl-7-ADCA (mM)	7-ADCA(mM)	Cefalexin (mM)
				01.02.0	952.23.7	03214	0037

Claims (11)

1. A process for the preparation of a beta-lactam antibiotic, wherein an N-substituted beta-lactam has the general structure shown in formula (I)Wherein R is₀Is H or C_1-3An alkoxy group; y is CH₂O, S, or an oxidized form of S; z is:wherein R is₁Is H, hydroxy, halogen, C_1-3Alkoxy, optionally substituted, optionally containing oneOr more hetero atoms, saturated or unsaturated, branched or straight-chain C_1-5Alkyl, preferably methyl, optionally substituted C optionally containing one or more hetero atoms_5-8Cycloalkyl, optionally substituted aryl or heteroaromatic ring, or optionally substituted benzyl; and X is (CH)₂)_m-A-(CH₂)_nWherein m and n are the same or different and should be one of an integer of 0, 1, 2, 3 or 4, and A is CH = CH, C ≡ C, CHB, C = O, optionally substituted N, O, S or an optionally oxidized form of S, while B is H, halogen, hydroxy, C_1-3An alkoxy group or an optionally substituted methyl group,

or a salt form of the N-substituted β -lactam, which is contacted with at least one dicarboxylic acid acyltransferase or an isozyme thereof, and reacted with a precursor substance in the presence of at least one penicillin acylase or an isozyme thereof to obtain a side chain of the β -lactam antibiotic.

2. A process as claimed in claim 1, wherein no intermediate product is isolated and/or purified.

3. A process as claimed in claim 2, which is carried out as a "one-pot" process.

4. A process according to any one of the preceding claims wherein the N-substituted β -lactam is N-glutaryl, N-succinyl, N-adipyl, N-3- (carboxymethylthio) propionyl, N-trans- β -hydrohexadienoyl, N-pimeloyl or N-3, 3' -thiodipropionyl β -lactam, or a salt form thereof.

5. A process according to any preceding claim, wherein the N-substituted β -lactam is N-substituted 6-aminopenicillanic acid (6-APA), 7-aminocephalosporanic acid (7-ACA), 3-chloro-7-aminodesacetoxycetamidomethylcephalosporanic acid (7-ACCA), 7-aminodesacetylcephalosporanic acid (7-ADAC), 7-aminodesacetoxycephalosporanic acid (7-ADCA), or a salt thereof.

6. A process according to any preceding claim, wherein the side chain precursor of the β -lactam antibiotic is D- (-) -phenylglycine, D- (-) -4-hydroxyphenylglycine, D- (-) -2, 5-dihydrophenylglycine, 2-thienylacetic acid, 2- (2-amino-4-thiazolyl) -2-methoxyiminoacetic acid, α - (4-pyridylthio) acetic acid, 3-thienylmalonic acid or 2-cyanoacetic acid, or an amide or ester form thereof.

7. A process according to any preceding claim, wherein the dicarboxylic acid acyltransferase is obtained from a strain of Alcaligenes, Arthrobacter, Achromobacter, Aspergillus niger, Acinetobacter, Bacillus or Pseudomonas.

8. A method according to any preceding claim, wherein the penicillin acylase is obtained from a strain of Acetobacter, Aeromonas, Alcaligenes, Aphanocladium, Bacillus, Cephalosporium, Escherichia, Flavobacterium, Kluyveromyces, Acinetobacter, Protaminobacter, providencia, Pseudomonas or Xanthomonas.

9. A process according to any one of the preceding claims wherein the N-substituted β -lactam is obtained by an enzymatic process starting from a fermentation product.

10. A process as claimed in claim 9, wherein the fermentation product is selected from the group consisting of penicillin G, penicillin V, cephalosporin C, adipyl-7-ADCA, 3-carboxyethylthiopropionyl-7-ADCA, 2-carboxyethylthioacetyl-7-ADCA, 3-carboxyethylthiopropionyl-7-ADCA, adipyl-7-ACA, 3-carboxyethylthiopropionyl-7 ACA, 2-carboxyethylthioacetyl-7-ACA and 3-carboxyethylthiopropionyl-7-ACA.

11. Use of dicarboxylic acid acyltransferase and penicillin acyltransferase to convert an N-substituted β -lactam to a β -lactam antibiotic.