WO2004062558A2

WO2004062558A2 - Process for enzymatic synthesis of βετα-lactam antibiotics

Info

Publication number: WO2004062558A2
Application number: PCT/EP2004/000182
Authority: WO
Inventors: Paolo Linda; Lucia Gardossi; Micaela Toniutti
Original assignee: Universita' Degli Studi Di Trieste
Priority date: 2003-01-14
Filing date: 2004-01-14
Publication date: 2004-07-29
Also published as: WO2004062558A3; ITMI20030042A1

Abstract

The present invention describes a process for the preparation of β-lactam antibiotics, cephalexin and ampicillin, wherein the β-lactam nucleus is acylated with an acylating agent, D-phenylglycine, by means of a reaction biocatalyzed by a catalytic enzyme. The acylation reaction is carried out in solid phase in solvent free systems (water or organic solvents) for the dispersion of reagents and at controlled temperatures.

Description

PROCESS FOR ENZYMATIC SYNTHESIS OF β-LACTAM ANTIBIOTICS

FIELD OF THE INVENTION

The present invention relates to a process for the preparation of β-lactam antibiotics, cephalexin and ampicillin, obtained with enzymatic synthesis of acylation of a β-lactam nucleus with an ester or a mixture of esters or a mixture of an ester and an amide of D-phenylglycine .

STATE OF THE ART β-lactam antibiotics, and in particular cephalexin and ampicillin, are among the most widely used in therapy as they are suitable for a wide spectrum of bacterial infections and have a good level of activity and tolerability. For example, cephalexin has, at worldwide level, a consumption that reaches 3000 tonnes per year.

Synthesis of these antibiotics is obtained by a condensation reaction between an acylating agent composed of activated D-phenylglycine and a β-lactam nucleus and two different processes can be applied at industrial level: chemical synthesis or biocatalyzed synthesis.

Briefly, chemical condensation is obtained by the formation of an intermediate -

Dane anhydride- obtained starting out from D-phenylglycine and ethylacetoacetate subsequently converted into an mixed anhydride with pivaloyl chloride or with methyl chlorofomiate. As the mixed anhydride produced in situ at low temperatures cannot be isolated, a salt, for example a salt of triethylamine, of the β-lactam nucleus 7-ADCA, is added. At the end of condensation it is acidified to bring the pH to 1.5 to release the protected amine group of the D-phenylglycine from the side chain. The chemical process is therefore somewhat disadvantageous at industrial level as to obtain the final product various reaction stages are required; the reagents, β- lactam nucleus and acylating agent D-phenylglycine, respectively, require protection or activation on reactive groups with groups, which must then be removed, after condensation has been carried out, in drastic reaction conditions. For example, to obtain Dane anhydride a process temperature of - 60°C is required. Moreover, the activation and protection processes mentioned produce large quantities of waste that cannot be recycled and are not biodegradable. For example, for cephalexin this waste is between 30 and 40 kg for each 1 kg of finished product.

From the industrial viewpoint, chemical synthesis is also limited by its reduced possibility of optimization and high use of organic solvents that consequently must be disposed of.

Chemical synthesis is currently still the most widely utilized, although some industries have developed production processes, for example for cephalexin, utilizing biocatalytic processes. In fact, biocatalyzed synthesis has the unquestionable advantage of reducing the condensation reaction to a single stage that can be carried out in an aqueous environment and in the presence of organic solvents. However, the process of enzymatic synthesis at the state of the art also has limits.

The condensation reaction is in fact considerably long due to the poor solubility of the reagents; moreover, in the conditions of synthesis utilized they are unstable, to an extent that they influence the final yields of the product. The synthesis reaction in an aqueous environment in fact determines hydrolysis of the acylating agent, utilized as a donor of the side chain, and of the finished product, as the water acts as a competitive nucleophile. As well as reducing yields, this makes the recovery process complex.

In order to solve these problems, the patent WO 99/20786 describes a process for enzymatic synthesis that proposes reducing the condensation times between a β- lactam nucleus and an acylating agent represented by D-phenylglycine activated with substituents on the carboxyl group (D-phenylglycine amide or esters of lower alcohols) and obtaining higher yields of antibiotic per quantity of β-lactam nucleus and/or acylating agent utilized. For this purpose, the acylating agent and/or β- lactam nucleus are utilized in the reaction mixture in conditions of supersaturation, of one or both reagents in an aqueous environment and in the presence of organic solvents, obtained by varying (increasing or decreasing) the pH and decreasing the temperature with respect to the usual conditions required to obtain complete dissolution of the two reagents, or their dissolution by at least 5% in weight. A supersaturated solution of the β-lactam nucleus can be obtained starting out from a solution thereof increasing or decreasing the pH for example in a range between 3.0 and 9.0, preferably between 4.0 and 8.5 and in particular between 4.5 and 8.0. Likewise for the acylating agent, a supersaturated mixture can be obtained starting out from a mixture, or preferably from a solution, thereof, increasing the pH for example above a value of 4.5, preferably above 5.5 and in particular above 6.0. Dissolution of the β-lactam nucleus and/or of the acylating agent can usually be obtained at temperatures above 15°C, preferably above 20°C and in particular above 25°C and a solution or supersaturated suspension can be obtained by decreasing the temperature to below 15°C and preferably below 10°C. To obtain a solution or supersaturated suspension of the two reagents the variations in pH and in temperature are in practice applied simultaneously. Although producing significant yields of final product, the process described does not solve the problems indicated hereinbefore, as it is considerably complex due to the necessity to maintain conditions of supersaturation during the biocatalyzed acylation reaction. Moreover, it does not seem to have solved the problem of hydrolysis of the acylating agent and/or of the final product, as synthesis is carried out in an aqueous environment, nor does it seem to have solved the problems of recovery of the unreacted substrates and isolation of the final product due to the fact that the species present in the reaction mixture have similar chemico-physical properties. Moreover, although preferably contained below 30% in volume, the process described does not eliminate the use of organic solvents during the synthesis reaction wjth the consequent problem of their disposal. OBJECTS AND SUMMARY OF THE INVENTION

In order to obviate the disadvantages of the biocatalyzed syntheses highlighted hereinbefore, the Inventors have established an enzymatic synthesis in solid phase in solvent free systems, wherein the reaction mixture has no aqueous phase for dispersion of the reagents and no organic solvents are used for the same purpose and wherein the enzyme catalyzing the acylation reaction contains the minimum quantity of water required for catalytic activity. The absence of an aqueous phase in the reaction mixture reduces the incidence of the hydrolytic reaction which is the main disadvantage of enzymatic syntheses currently utilized or described, all carried out in an aqueous environment. A further object of the invention is to establish a simple method that can be applied at industrial level for recovery from the reaction mixture of the reagents - D- phenylglycine and the β-lactam nucleus - and of the final product of the reaction by means of selective precipitations with organic solvents and variations in pH. Therefore, the object of the invention is a process for enzymatic synthesis of β- lactam antibiotics characterized in that the acylation reaction between the β-lactam nucleus and the acylating agent activated D-phenylglycine is carried out in solid phase in a solvent free system, at temperatures up to 5°C and is catalyzed by an enzyme from the hydrolase family containing the minimum quantity of water required for catalytic activity.

A further object of the invention is a process for recovery of the unreacted reagents and of the final product of the enzymatic synthesis reaction of β-lactam antibiotics characterized by the following phases:

- recovery by solubilization of the unreacted activated D-phenylglycine with aprotic organic solvents not miscible with water and in which it is soluble;

- solubilization of the residue in water with an acid pH and acidification or alkalinization of the solution to the isoelectric point of the β-lactam antibiotic obtained from the acylation reaction and selective precipitation thereof with organic solvents;

- solubilization of the remaining residue containing the unreacted β-lactam nucleus with an alkaline or acid aqueous solution and acidification of the solution obtained and selective precipitation of the β-lactam nucleus with organic solvents.

The process described hereinbefore also allows recovery of the enzyme utilized for catalysis, which may be separated at the beginning or at the end thereof.

The process for enzymatic synthesis of β-lactam antibiotics in solvent free systems and for recovery of reagents and of the final reaction product forming the object of the invention can be better understood in the detailed description hereunder of the phases of which it is composed.

DETAILED DESCRIPTION OF THE INVENTION

To solve the principal problem of hydrolysis of the biocatalyzed reactions of β- lactam antibiotics, concurrently producing a synthesis process simple to apply at industrial level, the aqueous phase in reaction systems would have to be reduced or eliminated. Nonetheless, this solution is difficult to obtain in practice both due to the need to solubilize the reagents, i.e. β-lactam nucleus and acylating agent, and due to the fact that the catalytic enzyme requires water to carry out its activity, as these enzymes are not active in anhydrous environments. This makes it necessary to operate in systems with low water activity that allow the degree of hydrolysis in reaction products and reagents to be limited. The problem to be solved is therefore how to modulate hydration of the system in order to balance these two opposed needs.

With the process forming the object of the invention, the technical problem mentioned hereinbefore is solved by operating in solid phase in solvent free systems, i.e. in a system wherein the reaction mixture has no aqueous phase for dispersion of the reagents and there are no organic solvents for this purpose.

Moreover, to limit the presence of water in the reaction mixture the enzyme catalyzing the acylation reaction is dehydrated in a controlled manner.

The enzymatic synthesis reaction of β-lactam antibiotics is carried out in a solvent free system between a β-lactam nucleus, if necessary protected at the carboxyl group, and D-phenylglycine in the activated form as acylating agent. The β-lactam nuclei useful for the purpose of synthesizing the antibiotics involved are 6- aminόpenicillanic acid (6-APA) or 7-aminodeacetooxycephalosporanic acid (7- ADCA), while the activated D-phenylglycine is in the form of an ester with a short alkyl chain with G between 1 and 5, and preferably methyl and ethyl ester, or these esters mixed with each other or with an amide thereof wherein the amide may be present up to 60%.

For the biocatalyst acylation reaction, in general enzymes from the hydrolase family (for example proteases or amide hydrolases) can be utilized as catalysts of the acylation reaction (De Martin L., Ebert C, Gardossi L, Linda P., "High isolated yields in thermolysin-catalysed synthesis of Z-aspartyl-L-phenylalanine methyl ester in toluene at controlled water activity." Tetrahedron Lett., 2001, 42, 3395- 3397; Ebert C, Gardossi L, Linda P., "Control of enzyme hydration in penicillin amidase catalysed of amide bond" Tetrahedron Lett., 1996, 37, 9377-9380). In the case of the process forming the object of the invention it is preferably to use amide hydrolases, such as Penicillin G Acylases (PGA) from different sources or even produced using biotechnology or glutaryl acylases. Preferably, for enzymatic synthesis in solvent free systems acylating enzymes, immobilized covalently on inert solid supports usually constituted by variably functionalized poiyacrylamide polymers, such as PGA 450 (Roche), PGA immobilized on Eupergit C (Fluka) and PGA immobilized on poiyacrylamide (Fluka), are used.

The reaction catalyzed by the enzyme chosen is carried out in a solvent free system at a temperature of up to 5°C and preferably between 0°C and 5°C. The reaction may also be carried out preferably, although not necessarily, in an inert gas atmosphere, i.e. nitrogen.

At the temperatures considered, the acylating agent, activated D-phenylglycine, is liquid. This allows dispersion of the β-lactam nucleus, chosen according to the antibiotic to be synthesized, in the same acylating agent and in the same reactor without any solution or previous dispersion in water and/or organic solvents. In order to eliminate relevant quantities of water which may trigger hydrolysis processes, the enzyme required for synthesis is previously dehydrated, as published in (Basso A. et al. J. Mol. Catalysis B. Enzymatic 2001, 11, 851-855) and described briefly hereunder, to a minimum water content between 40 and 10% w/w.

The reaction is carried out using the acylating agent and the β-lactam nucleus in molar ratios of the acylating agent with the β-lactam nucleus between around 5:1 and 2:1. The enzymatic catalyst is added in the quantity of at least 35 enzymatic units per 100 g of β-lactam nucleus and preferably between around 176 and 35 enzymatic units per 100 g of β-lactam nucleus. Both the reagents and the catalyst are placed in the same reactor maintaining a controlled temperature of up to 5°C and the reaction is carried out preferably in an inert gas atmosphere, i.e. nitrogen, for a time of at least 3 hours, and preferably between 3 and 24 hours, under continuous stirring.

At the end of the reaction the unreacted reagents, the enzyme and the final reaction product are recovered with a process composed of the following phases, already mentioned briefly. In particular, recovery of the unreacted substituted D- phenylglycine is obtained by its selective solubilization with organic aprotic solvents that are not miscible with water and wherein the D-phenylglycine is soluble with a logP between 0.7 and 2.5, and preferably 1.2, such as dichloromethane, chloroform, ethyl acetate and toluene. The organic solution is then evaporated at reduced pressure with the formation of an oily residue composed of the unreacted substituted D-phenylglycine. After addition of the organic solvent such as for example dichloromethane required for solubilization of the D-phenylglycine, the immobilized enzyme can be separated using sieves or filters. In fact, the crude reaction residue insoluble in organic solvent passes through the sieves or filters and may therefore be recovered. After separation of the D-phenylglycine derivative, the reaction product is recovered by solubilization of the residue in water with an acid pH between 1 and 2, and preferably between 1.5 and 1.6, of the solution subsequently taken to the isoelectric point of the β-lactam antibiotic obtained by the acylation reaction and by its selective precipitation with organic solvents such as acetonitrile. Solubilization of the remaining residue, containing the β-lactam nucleus is obtained with an aqueous solution with a pH between 6.5 and 9 and preferably 8 and acidification of the solution obtained up to a pH between 4 and 6 and preferably 5 and selective precipitation of the β-lactam nucleus with organic solvents such as acetonitrile.

If the enzyme has not been recovered in the first phase it will be separated as remaining residue and if necessary regenerated with buffer solutions with an alkaline pH (for example 8).

The enzyme, just as the recovered reagents, can be re-utilized for subsequent synthesis cycles. Hereunder are some examples of synthesis processes according to the invention, which must not be intended as a limitation of the invention and must therefore be considered for the purposes of its illustration.

Experimental Procedures

Example 1 : procedure for releasing the phenylglycine ester from hydrochloride

The methyl (D-PGM) or ethyl (D-PGE) ester of D-phenylglycine available commercially in the form of hydrochloride is obtained, as a free base, by the following treatment.

1 g of hydrochloride phenylglycine ester (equal to 5x10^"3 moles) is suspended in dichloromethane and kept under magnetic stirring in the presence of 1x10^"2 moles of sodium carbonate decahydrate. The suspension is filtered and the organic phase eliminated by evaporation at reduced pressure, to obtain a yellow oily residue corresponding to phenylglycine methyl or ethyl ester (3.5x10^"3 moles corresponding to a yield of 70%).

Example 2: dehydration and storage of the Penicillin G Acylase (PGA).

The enzyme of the aminohydrolase family employed is PGA-450 by Roche which is supplied with an initial water content of around 60%. This enzyme is partially dehydrated before use with the following procedure: 2 g of enzyme are suspended in 100 mL of petroleum ether together with 4g of anhydrous Celite R-6450. The preparation is conserved for 15 days at 4°C. Subsequently the Celite is removed and the PGA conserved in the same solvent at 4°C. The final water content of the preparation is between 10% and 30% w/w (determined with Mettler Karl Fischer titrator) and its activity, measured as hydrolysis of the benzylpenicillin, is equal to 453 U per dry gram of enzymatic preparation. This value remains constant even after 4 months of storage in petroleum ether at 4°C. When necessary, samples of enzymatic preparation' are taken and the organic solvent evaporated at ambient temperature and atmospheric pressure without any detriment of catalysis. Example 3: synthesis of the β-lactam antibiotic Cephalexin

Solvent free enzymatic synthesis of Cephalexin is carried out varying the quantity of enzyme utilized (from 35 to 176U), and of the substrates D-PGM and 7-ADCA in a ratio between 2.3:1 and 4.6:1 respectively according to reaction process 1. The catalyst utilized is a preparation of Penicillin G. Acylase from E-Coli immobilized covalently on a solid polymer functionalized with commercial epoxy groups (PGA-450, Roche) with a water content of around 60% and dehydrated as described in example 2 with a water content of around 30%.

Reaction process 1 : acylation reaction of 7-aminodeacetooxycephalosporanic acid

(7-ADCA) with D-phenylglycine methyl ester (PGM, in presence of PGA-450 at

4°C.

The composition of the various synthesis reactions and the yields of Cephalexin

(CEX), to form phenylglycine acid (PGOH) and the dipeptide D-phenylglycyl- phenylglycine methyl ester (D-PG-D-PGM) are indicated in table 1.

Table 1: composition of the syntheses carried out with D-PGM, 7-ADCA and PGA- 450 as catalyst and yields in percentages of the syntheses in the reactions to form cephalexin, phenylglycine acid and dipeptide.

The conversion is calculated as millimoles of cephalexin over initial millimoles of β- lactam nucleus and millimoles of phenylglycine acid or D-pheηylglycyl- phenylglycine methyl ester over initial millimoles of phenylglycine methyl ester.

The reactions of enzymatic synthesis indicated in Tab.1 as synthesis 4-B (example 3 A) and 4-C (example 3 B) are described hereunder by way of an example. The synthesis 4 A is essentially identical to the synthesis 4-B.

Example 3 A: in a 20 mL glass test tube with cap provided with a Teflon septum the following are weighed: 4.6x10^"3 moles of 7-ADCA (equal to 0.99 g), 1.05x10^"2 moles of D-PGM (equal to 1.75 g) equivalent to 2.3 times the moles of 7-ADCA. The reaction is started by adding 349 enzymatic units of PGA-450 (equal to 0.158 g). Samples from the reaction are diluted in 2 mL of water acidified with trifluoroacetic acid and ethanol (3:2).

Analyses are carried out with RP-HPLC. The concentration in time of 7-ADCA, cephalexin and D-PGM and hydrolyzed D-phenylglycine D-PGOH is measured. In the specific reaction, carried out as described, at a maximum (3 hours) the conversion of 7-ADCA into the product (cephalexin) is observed in a percentage of

30%. Hydrolysis of the methyl ester of the D-phenylglycine is between 4% and

10%.

Example 3 B: in a 20 mL glass test tube with cap provided with a Teflon septum the following are weighed: 1.4x10^"2 moles of 7-ADCA (equal to 2.97 g), 0.032 moles of D-PGM (equal to 5.3 g) equivalent to 3.3 times the moles of 7-ADCA.

The reaction is started by adding 1050 enzymatic units (4.79 g) of PGA-450,

Roche. After 3 hours of reaction a sample is taken and analyzed with RP-HPLC.

Conversion into cephalexin observed is of 16% and the formation of phenylglycine acid is below 10%.

Example 4: procedure to recover the Cephalexin

At the end of the synthesis reaction as described in example 3 A, the reaction mixture is washed on a bϋchner filter, with 100 mL of dichloromethane to recover the unreacted D-PGM. The filtered organic phase is subjected to evaporation of the solvent at reduced pressure obtaining a yellow oily residue that is analyzed by

RP-HPLC. 0.9014 g of D-PGM equal to 5.4x10^"3 moles is recovered.

After washing with dichloromethane the solid residue is suspended in 20 mL of ultrapure water, subsequently acidified to pH 1.5 with concentrated hydrochloric acid 36 N (a few drops). The suspension is filtered at reduced pressure.

The filter product, slightly yellow in colour, is taken to pH 5, the isoelectric point of Cephalexin, with ammonium hydroxide at 28% (a few drops). Acetonitrile (around

40 mL) is added to the solution presenting the formation of the first crystals until an evident white precipitate is formed. 0.7461 g of a solid composed of cephalexin and phenylglycine acid in equimolar quantities are recovered.

In order to recover the unreacted 7-ADCA, the residue obtained after filtering, containing 7-ADCA and the enzyme, is suspended in 50 mL of ultrapure water alkalinized to pH 8 with concentrated ammonium. The solution obtained is acidified to pH 5 with concentrated hydrochloric acid. Subsequently around 100 mL of acetonitrile is added until an evident white precipitate is formed; 0.2797g of

7-ADCA equal to 1.3x10^"3 moles, equivalent to the recovery of 52% of 7-ADCA are recovered.

Analyses with RP-HPLC are carried out by diluting small portions of the samples with 2.5 mL of ultrapure water acidified to pH 1.5 with concentrated hydrochloric acid and 2.5 mL of ethanol (0.01% of trifluoroacetic acid). A mobile phase composed of acetonitrile and water (0.1% trifluoroacetic acid) is used, in variable proportions according to a gradient wherein the organic phase increases from 15% to 50% in 10 minutes.

Cephalexin:

¹H-NMR (D₂O) δ : 2.94(s, 3H, CH₃), 3.14(d, 1 H, S-CHH), 3.24(d, 1H, S-CHH),

4.79(d, 1 H, NH-CH-CH-S), 4.94(s, 1H,CH- NH₂), 5.38(d, 1 H, NH-CH-CH-S), 7.24( s, 5H, C₆H₅).

7-ADCA:

¹H-NMR (D₂O) δ : 1.99(s, 1H, CH₃), 3.32 (m, 2H, CH₂), 4.87 (d, 1 H, CH), 5.04(d,

1 H, CH-NH₂).

Characterization of Cephalexin RP-HPLC: the reaction product is analyzed with RP-HPLC: the retention time of the standard is equal to 7.0 minutes, the product has a retention time equal to 7.2.

ES-MS: the product is analyzed with electrospray mass spectrometry ES-MS, the mass spectrum of the standard has a signal equal to 348 m/z (PM cephalexin

347+1 H⁺). The product analyzed with ES-MS has a signal corresponding to the molecular ion equal to 348 m/z.

¹H-NMR: characteristic signals of Cephalexin in the isolated mixture

¹H-NMR (D₂O) δ : 2.94(s, 3H, CH₃), 3.14(d, 1 H, S-CHH ), 3.24 (d, 1 H, S-CHH),

4.79(d, 1H, NH-CH-CH-S), 4.94(s, 1 H, CH-NH₂ ), 5.38(s, 1 H, NH-CH-CH-S), 7.24( s, 5H, C₆H₅).

Example 5: synthesis of the β-lactam antibiotic ampicillin

The reaction is carried out utilizing D-PGM and 6-APA in a ratio between 4.6:1 and

1 :1 , The catalyst utilized is PGA-450 (Roche).

The synthesis reactions are carried out varying the ratios of the substrates and of the catalyst PGA-450 with 30% in water content obtained by dehydration starting out from commercial PGA containing around 60% in water.

Table 2: composition of the synthesis reactions carried out with D-phenylglycine methyl ester (D-PGM), 6-aminopenicillanic acid (6-APA) and Penicillin G Acylase immobilized covalently (PGA-450) as catalyst and percentage yields of the synthesis in the reactions to form ampicillin, phenylglycine acid and dipeptide.

* conversion is calculated as millimoles of ampicillin over initial millimoles of β- lactam nucleus and millimoles of phenylglycine acid or D-phenylglycyl- phenylglycine methyl ester over initial millimoles of phenylglycine methyl ester.

Characterization of Ampicillin

RP-HPLC: the reaction product is analyzed with RP-HPLC: the retention time of the standard is 13.0 minutes, the product has a retention time equal to 13.2 minutes.

ES-MS: the product is analyzed with electrospray mass spectrometry ES-MS, the mass spectrum of the standard has a signal equal to 349.4 m/z (PM cephalexin

349+1 H⁺). The product analyzed with ES-MS has a signal corresponding to the molecular ion equal to 349.4 m/z.

From the experimental results obtained it may be concluded that the process forming the object of the invention fulfils the pre-established objects: a) the process is very simple and easy to apply at industrial level and is completed in a single phase carried out in only one reactor; b) with the process described hereinbefore the presence of water is limited to the water necessary for catalytic activity of the enzyme, thus drastically reducing the hydrolysis reactions that take place in other processes carried out in aqueous environments. Dehydration of the enzyme at hydration concentrations controlled with a hygroscopic agent in fact make it possible to operate without an aqueous phase and to minimize hydrolysis of the acylating agent.

Moreover, the absence of an aqueous phase allows quantitative recovery of the substrates, activated D-phenylglycine and β-lactam nucleus, while hydrolysis of the activated phenylglycine is greatly reduced (below 15%) with a considerable benefit from the aspect of reducing the costs of synthesis.

Moreover, as the process is extremely simple and allows several reaction cycles, it is extremely advantageous from the aspect of application at industrial level.

Claims

1. Process for enzymatic synthesis of β-lactam antibiotics, characterized in that the acylation reaction between a β-lactam nucleus and the acylating agent, activated D-phenylglycine, is carried out in solid phase in a solvent free system at temperatures of up to 5°C and is catalyzed by a hydrolase containing the minimum quantity of water required for catalytic activity.

2. Process for enzymatic synthesis of β-lactam antibiotics as claimed in claim 1 , wherein the molar ratios of the acylating agent with the β-lactam nucleus are between around 5:1 and 2:1.

3. Process for enzymatic synthesis as claimed in claim 1 , wherein the catalyzing enzyme is an amide hydrolase with a water content between 40 and 10 % w/w and is present in the reaction mixture in the quantity of at least 35 enzymatic units per 100 g of β-lactam nucleus.

4. Process for enzymatic synthesis as claimed in claim 1 , wherein the reaction process is carried out at temperatures comprised from 0°C to 5°C.

5. Process for enzymatic synthesis as claimed in claim 1 , wherein the acylation reaction is carried out for at least 3 hours under continuous stirring.

6. Process for enzymatic synthesis as claimed in claim 1 , wherein the acylation reaction is carried out in an inert gas atmosphere.

7. Process for enzymatic synthesis as claimed in claims 1 to 6, wherein the β- lactam nucleus can be 7-aminodeacetooxycephalosporanic acid (7-ADGA) or 6-aminopenicillanic acid (6-APA).

8. Process for enzymatic synthesis as claimed in claims 1 to 6, wherein the acylating agent activated D-phenylglycine is in the form of an ester with a short alkyl chain with C between 1 and 5 or mixtures of said esters or mixtures of said esters with an amide thereof.

9. Process for recovery of unreacted reagents and of the final product of the enzymatic synthesis reaction of β-lactam antibiotics characterized by the following phases:

10. Process as claimed in claim 9, wherein the catalyzing enzyme is recovered by separation on sieves or filters during the first phase of selective solubilization of the unreacted activated D-phenylglycine or after selective precipitation of the β-lactam nucleus with organic solvents.

11. Process as claimed in claim 9, wherein selective solubilization of the activated D-phenylglycine is obtained with aprotic organic solvents with a logP between 0.7 and 2.5.

12. Process as claimed in claim 9, wherein the β-lactam antibiotic obtained from the acylation reaction present in the residue is solubilized in water with an acid pH between 1 and 2.

13. Process as claimed in claim 9, wherein the unreacted β-lactam nucleus present in the residue is recovered with solubilization in an aqueous solution with a pH between 6.5 and 9 and acidification of the solution obtained up to a pH between 4 and 6.