WO2006008296A1

WO2006008296A1 - A process for the preparation of enzymatic catalysts, catalysts obtainable by this process and use thereof

Info

Publication number: WO2006008296A1
Application number: PCT/EP2005/053474
Authority: WO
Inventors: Marco Terreni; Susanna Vaccaro; Ilona Estruch; Massimo Pregnolato; Antonio Giannelli; Salvatore Caruso
Original assignee: Fidia Farmaceutici S.P.A.; Innovate Biotechnology S.R.L.
Priority date: 2004-07-19
Filing date: 2005-07-19
Publication date: 2006-01-26
Also published as: EP1802751A1; ITPD20040191A1

Abstract

Herein described is a process for the preparation of enzymatic catalysts immobilised on epoxidic support, particularly suitable for the synthesis of β-lactamic antibiotics; the enzymatic catalysts obtainable by this process and their use.

Description

A process for the preparation of enzymatic catalysts, catalysts obtainable by this process and use thereof

FIELD OF THE INVENTION

The present invention relates to the field of enzymatic catalysts, and in particular to a process for preparing enzymatic catalysts wherein the enzyme is immobilised on an epoxidic support, having improved catalytic features and particularly useful in the synthesis of β-lactamic antibiotics and in other kinetically-controlled reactions, such as the resolution of racemic compounds.

STATE OF THE ART Penicillin-G-Acylase (PGA), in particular that isolated from Escherichia coli, is an enzyme that has been well known for some time and is used in industry as a catalyst in hydrolysis and acylation processes, and more precisely:

• in the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid (6-APA) and in the hydrolysis of cephalosporin G, obtained by chemical means from Penicillin G, to obtain 7-aminodesacetoxycephalosporanic acid (7-ADCA) [Bruggink, A., et al. Org. Process Research and Development, 1998, 2: 128-133];

• in the enzymatic acylation of 6-APA, 7-ADCA, 7-aminocephalosporanic acid (7-ACA) or the derivatives thereof with analogues of phenylacetic acid (acylating agents) to obtain β-lactamic antibiotics. Indeed, PGA is capable of catalysing said acylation reaction far more simply than the chemical methods commonly used in industrial processes [Bruggink A. et al., Org. Process Research and Development, 1998, 2: 128-133].

Enzymatic synthesis normally requires bland reaction conditions that are compatible with the products stability and suitable to obtain compounds that are highly pure, often even purer that those obtained by the classic processes of chemical synthesis. Moreover, by avoiding the need to use toxic reagents and solvents, the impact on the environment normally linked with chemical synthesis processes is greatly reduced. Besides this, the possibility of linking the catalysing enzyme with a solid support so as to be able to recover and reuse it effectively renders the study and development of enzymatic processes for the synthesis of penicillin and cephalosporin increasingly interesting in many areas of industry. Patent literature reports numerous examples in which PGA is used as a catalyst in enzymatic acylation reactions of β-lactamic nuclei (see for example WO 98/56945). The enzyme most commonly used at an industrial level is acylase isolated from strains of Escherichia coli. However, the yields obtained from such processes are rarely competitive with those from classic chemical synthesis, mainly because of the notable hydrolytic activity of the enzyme towards most of β- lactamic antibiotics (and therefore the final product) as well as towards the side chains used as acylating agents (and therefore the intermediate products). In such situations it becomes necessary to use high molar excesses to obtain large yields, with negative consequences on the economics of the industrial process.

One of the most classic ways of obtaining catalysts with properties that are better suited to this type of process is to use acylases other than that obtained from Escherichia coli, i.e. acylases isolated from other micro-organisms, as described in the International Patent Application No. WO 02/86111 (α-amino ester hydrolase from Acetobacter turbidans, Acetobacter pasteuήanum, Xanthomonas citri, Zymomonas mobilis, XyIeIIa fastidiosa) and in the European Patent No. 775 210 B1 (PGA from mutant Alcaligenes faecalis, obtained by site-specific and random mutagenesis). A second way of improving the performance of acylases in synthesis processes concerns the engineering aspect. In European Patent No. 771 357 B1 , for example, a process is disclosed in which the concentration of the nucleus and of the acylating agent is kept constantly high, for example by the continuous addition of reagents. In this way, the active site of the enzyme is kept in a condition of continuous saturation, giving high values in the ratio between synthesis and hydrolysis of the acylating agent and, consequently, better yields. This method is applied, for example, to the synthesis of ampicillin, but it is hampered by the complexity of the process. The European Patent No. 771 357 B1 reports the use of acylase from Xantomonas citri and strains of Mycoplana, Protaminobacter, Aeromonas, Pseudomonas, Flavobacterium, Aphanocladium, Cephalosporium preferably embedded in gelatine. Similarly, the European Patent No. 869 961 B1 describes the synthesis of cephalexin with phenylglycin amide, carried out with high concentrations of reagents by using acylase from E. coli. The International Patent Application No. WO 99/20786 describes synthesis carried out with an excess of acylating agent. Besides PGA from E. coli it also describes the use, in these conditions, of acylase isolated from strains of Fusarium oxisporum, Fusarium solani and Aphanocladium sp. Lastly, the International Patent Application No. WO 95/34675 describes enzymatic synthesis using modulators at lower concentrations of reagents, to inhibit the hydrolysis of the final product and of intermediates, thus increasing the synthesis/hydrolysis ratio. In general this approach results in better yields, but the complexity of the process and of the mixtures obtained, the formation of precipitates during reaction and the consequent need for complex procedures to separate the precipitates from the immobilised enzymes, constitute a serious hurdle to the industrial application of such technology so that it is no longer cost-effective. In order for a process to be cost-effective, it is advisable to use enzymes that are sufficiently well supported, to facilitate recovery and reuse of the catalyst. Acylase from E. coli is commonly sold and used in immobilised form for covalent interaction on epoxide resins of a hydrophobic character (such as Eupergit^® C and Sepabeads^® EC-EP) which give it mechanical properties suitable for its use in industrial processes. Immobilisation occurs by interaction between the epoxide groups present on the surface of the supports and the amino residues present on the surface of the protein, leading to the formation of covalent bonds. The supports are characterised by a strongly lipophilic surface that makes it necessary to perform immobilisation in the presence of aqueous solutions at high saline concentrations. [Colemann P.L. et al. J Chromatogr 1990; 512: 345-363; Wheatley J. B. et al. J Chromatogr, 1993; 644: 11-16; Kircher V. et al. J Chromatogr B ; 1996; 667: 245-255; Wheatley J. B. et al. J Chromatogr A ; 1999; 849: 1-12]. In this way, an initial adsorption of the enzyme is obtained on the hydrophobic surface of the support. Only after such adsorption, formation of covalent bonds between protein and support may be induced. In these conditions, because of the high geometric congruence, the enzyme is preferentially arranged with its single planar surface containing the active site facing the surface of the support. Saline concentrations must be high during the immobilisation process so as to obtain a sufficiently strong and stable initial hydrophobic interaction and enable the subsequent formation of covalent bonds. Despite their good mechanical properties, the catalytic properties of these enzymatic preparations are not particularly suitable for their use in the synthesis of penicillin and cephalosporin. Indeed, the yields they give are far removed from those obtained using the free enzyme.

In an attempt to overcome these difficulties, procedures have been developed for immobilisation of the enzyme on various hydrophilic matrices with better catalytic features. The European patent application No. 839 192 describes a process for immobilising PGA from E. coli in a composite gelling agent and a polymer containing free amino groups (gelatine with alginates, chitosan, pectin, polyethyleneimine) and gives examples of its application in the synthesis of cephachlor, cephalexin, amoxicillin, cephradine and ampicillin, using also PGA from micro-organisms other than E. coli. In such cases, however, the increased yield is accompanied by difficulties in recovering and reusing the catalyst.

Better results have been obtained by immobilising PGA on agarose-CL according to the procedure published in the literature [Guisan J. M. Enzyme Microb. Technol. 1988; 10: 375-382]: after activating the agarose support by esterification with glycidol and subsequent oxidation with periodate, the surface is enriched with aldehyde functions (agarose-aldehyde support) capable of reacting with the amino groups of the lysin residues present in the protein molecule to form imine bonds. The subsequent reduction with sodium borohydride leads to the formation of covalent C-N bonds. These derivatives are stable, characterised by a high number of covalent bonds between enzyme and support, and they are an improvement on the immobilisation preparations on epoxide resins, both in terms of stability [Alvaro G. et al., Appl. Biochem. Biotechnol. 1990; 26: 181-95] and yield from synthesis processes [Terreni M. et al., Biorg. Med. Chem. Letter. 2001 ; 11 : 2429-32], but their performance is not good enough for use in industrial processes. There is still, therefore, a need for enzymatic catalysts having mechanical and physical properties that are suitable for industrial usage but without jeopardising their catalytic properties and the yields of synthesis processes. SUMMARY OF THE INVENTION Now the Applicant goes beyond the limits of existing technology, thanks to innovative process for the immobilisation of enzymes on solid supports. The immobilised enzymes obtainable according to this process act as efficient catalysts giving pure products and high yields that are comparable to those obtained using the free enzyme, and are therefore competitive in industrial terms. Moreover, their physical and mechanical characteristics guarantee easy recovery and reuse and make them particularly suitable in industrial processes. It is therefore subject of the present invention a process for the preparation of an enzymatic catalyst wherein an enzyme is immobilised on a solid epoxidic support, comprising the following steps: i) possible partial oxidation of the epoxide groups on the surface of an epoxidic support into aldehyde groups; ii) immobilisation of an enzyme onto the surface of an epoxide support, possibly derivatised by partial oxidation as coming from step i); iii) hydrophilisation of the surface of the support onto which the enzyme is immobilised as coming from step ii).

Further subjects of the present invention are the immobilised enzymes obtainable by the above said process, and their use as catalysts. Features and advantages of the present invention will be illustrated in detail in the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : shows schematically the formation of aldehyde groups starting from the epoxide groups on the surface of the epoxidic support by bland-condition hydrolysis of the epoxides with H₂SO₄ and subsequent oxidation with periodate of the so obtained diolic groups. The rectangle represents the surface of epoxidic support.

Figure 2: shows schematically the formation of covalent bonds between the epoxy groups on the surface of the epoxidic support and the amino groups on the surface of the enzyme, followed by hydrolisation with amino acids of the surface of the support on which the enzyme is immobilised. The rectangle represents the surface of epoxidic support, and the ovoidal forms represent the enzyme. Figure 3: shows schematically the formation of imine bonds between the amino groups on the surface of the enzyme and the aldehyde groups on the surface of the epoxidic support previously partially modified with aldehyde groups as showed in Figure 1. The interaction between enzyme and support is then followed by the hydrolisation step with amino acids, and finally by the reduction of the imine bonds with NaBH₄. The rectangle represents the surface of epoxidic support, and the ovoid-shaped structure represent the enzyme. DETAILED DESCRIPTION OF THE INVENTION The highly efficient catalysts that are the subject of the present invention are obtained by immobilising the enzyme on solid hydrophobic epoxide supports, in particular on hydrophobic epoxidic resins, and preferably the resins known with the trade names Eupergit^® C and Sepabeads^® EC-EP.

The present process for preparing the enzymatic catalysts according to the invention may comprise an optional step i) of derivatisation of the starting hydrophobic epoxide support by the partial transformation of the epoxide groups present on the surface into aldehyde groups, in order to obtain an epoxyaldehyde support. In this situation, the first interaction between the enzyme and the support occurs when imine bonds form between the primary amino groups of the enzyme lysins and the aldehyde groups introduced on the surface of the support. The above said partial oxidation of epoxide groups into aldehyde groups in step i) may be carried out by bland-condition hydrolysis of the epoxidic groups with H₂SO₄ and subsequent oxidation of the so obtained diolic groups with periodate, as schematically illustrated in Figure 1. In some cases, such as that of Sepabeads^® EC-EP₁ a certain amount of diolic groups are already present on the surface of the epoxide resin, and these may be directly oxidated to aldehydes.

Subsequently, in step ii) of the present process there is a reaction between the functional groups of the enzyme and the epoxide groups of the support. In this way, orientation of the enzyme in respect of the support depends, not on any geometric congruence, but on the reactivity of the various functional groups (e.g. lysin amino groups) of the enzyme. The immobilisation of the enzyme in step ii) may be preferably carried out by adding said epoxidic support to a suspension of said enzyme in a buffer at pH ranging from 8 to 10, such as a phosphate or a bicarbonate buffer, and maintaining the suspension under stirring for a period of time ranging from 12 to 48 hours.

The immobilisation of the enzyme, as said above, is achieved by formation of covalent bonds between amino groups of the enzyme and the epoxide groups - or the epoxide and aldehyde groups - of the support. In case aldehyde groups are present on the surface of support, they form with the amino groups of the enzyme imino bonds that are then preferably reduced to amino bonds by addition of a suitable reducing agent such as NaBH₄ (see Figure 3).

According to the process of the invention, in step iii) hydrophilisation of the surface of the support onto which the enzyme is immobilised as coming from step ii) occurs, that is a further derivatisation of the surface of the support with highly hydrophilic molecules of a polar and/or ionic character by reaction between the epoxide groups still free after the previous derivatisation and the reactive groups of the hydrophilic molecules described above, such as amino groups, with the formation of covalent bonds. This step iii), called herein below post-immobilisation hydrophilisation, is fundamental to the creation of a hydrophilic environment around the enzyme and at its active site that strongly favours contact between the reagent in the processes catalysed by the present enzymatic catalysts (for example the β-lactamic nucleus) and the active site of the enzyme, both of which are hydrophilic. According to a particular embodiment of the present process, hydrophilisation in step iii) is carried out at pH ranging from 7 and 11 , preferably ranging from 9 to 10, in the presence of the above said hydrophilic products in concentrations of between 0.1 and 3 M, and optionally in the presence of concentrations ranging from 50 to 200 mM of enzymatic inhibitors, preferably selected from between acetic phenyl acid and Penicillin G sulphoxide. According to the invention the above said highly hydrophilic molecules may be selected for example from the group consisting of sugars, ethylene diamines, bis- amines and amino acids, preferably selected in their turn from the group consisting of lysine, cysteine and aspartic acid.

Post-immobilisation hydrophilisation, described here, may be carried out also on known epoxide resins that have not previously been derivatised in step i). The presence of free epoxide groups on the surface of a resin after immobilisation of the enzyme according to the conventional techniques described above makes their reaction with the reactive groups of the above said hydrophilic molecules possible. By the present process, immobilised enzymes, such as acylase, esterase, protease and hydrolase, in particular acylase, and more particularly Penicillin-G- Acylase, are obtained with optimal catalytic, mechanical and physical features that are better than those of the same enzymes immobilised by the known methods, and are particularly suitable for industrial use, especially in reactions to synthesise β-lactamic antibiotics. The present process have been successfully applied also to acylases other than that isolated from E. coll, such as those isolated from Kluyvera citrophila, Bacillus megaterium (Staphylococcus) and Arthrobacter viscosus (Micrococcineae), and it may be used on other enzymes such as proteases, esterases and hydrolases, revealing in any case considerable advantage when used in kinetically controlled processes, such as the resolution of racemic mixtures coming from peptide synthesis.

The enzymatic catalysts obtained by the present process were also tested in reactions for the acylation of β-lactamic nuclei with activated acylating agents in the form of esters or amides. In such cases, condensation occurs between an acylating agent and the amino group in non-ionised form of a β-lactamic nucleus. According to the synthesis reaction, better results can be obtained using as acylating agent an acid activated as an ester or an amide.

In these processes the enzyme may also act, however, as a transferase and hydrolase, thus catalysing other parallel reactions that are in competition with one another, such as:

• hydrolysis of the acylating agent activated in the form of an ester (hi ) and

• hydrolysis of the same final antibiotic (h2). The acylating agent in activated form reacts with serine in the active centre of the enzyme, to form a tetrameric complex that can be attacked by the amino group of the β-lactamic nucleus (synthesis reaction) or by water (reaction hi, hydrolysis of the acylating agent). The same product of synthesis can form an activated tetrameric complex with serine from the active centre of the enzyme and undergo hydrolysis (reaction h2). The final yields of antibiotic are transitory (the concentration of the product reaches a peak and then drops because of hydrolysis of the product itself) and therefore depend on the relative rates of the three different reactions. In order to obtain good yields of antibiotic, the rate of the acylation reaction, that is, synthesis of the antibiotic (Vs), must be greater than the hydrolysis rate of the ester of the starting acylating agent (Vh i) and the hydrolysis rate of the final antibiotic (Vh2).

The yields therefore depend on various factors: • affinity of the active centre of the enzyme for the nucleophile (nucleo β- lactamic nucleus); this parameter depends on the ratio between the synthesis rate of the antibiotic and hydrolysis rate of the acylating agent (V₅ A/hi).

• ratio between the rate of acylation, i.e. synthesis, and the rate of hydrolysis of the antibiotic (V_s /Vh2). The V_sA/hi ratio parameter is indicative of the enzyme site that is active for the β- lactamic nucleus and establishes the maximum theoretical yield obtainable in the absence of hydrolysis of the final product. This parameter is linked with a capacity for adsorbing the β-lactamic nucleus in the active site of the enzyme and for excluding water from the active site that competes with the β-lactamic nucleus in the reaction with the acyl-enzyme complex. As the concentration of β-lactamic nucleus increases, the active site of the enzyme becomes saturated. In these conditions, it is difficult for water to enter and the acyl-enzyme complex reacts preferentially with the amino group of the β-lactamic nucleus. Consequently, as the concentration of the latter increases, there is an increase in the V₃ /Vh 1 ratio. When all the active sites are saturated by the β-lactamic nucleus, the hydrolysis reaction of the acylating ester will be nullified and the theoretical yield of the acylating process will be 100% (V_s/Vhi= ∞). The greater the affinity of the enzyme for a given nucleus, the lower the concentrations required to obtain complete saturation of the active site. This parameter is of fundamental importance in industrial processes because it determines the excess of acylating agent needed to obtain complete transformation of the nucleus into antibiotic, so it influences the cost-effectiveness of the process. Indeed, the higher the V_s/Vhi ratio, the smaller the excess of acylating agent required, at the same concentration of β-lactamic nucleus, to obtain quantitative yields. It can therefore be deduced that the catalytic characteristics of the enzyme are strongly influenced by both the nature of the support and the mechanism of immobilisation used.

According to state of the art, immobilisation of an enzyme such as PGA on a hydrophobic epoxide support guarantees good mechanical and physical properties but low yields in terms of antibiotic synthesis. Conversely, using hydrophilic supports improves the catalytic properties but does not guarantee that the mechanical and physical properties will be sufficient for industrial use. The limitations of the techniques known to the state of the art have been overcome by the present invention, which enables the enzyme to be immobilised by the interaction of its lysin residues with aldehyde groups suitably introduced on the surface of a starting hydrophobic epoxide support, by partial chemical modification of the epoxides, conducted in such a way as to obtain, a mixture of epoxide and aldehyde groups on the surface of the resin. Another particularly advantageous aspect of the present process is the applicability of the post-immobilisation hydrophilisation step starting also from known hydrophobic epoxide resins that have not previously been derivatised. Once the enzyme has been immobilised, the surface of the matrix is hydrophilised by reacting the epoxy groups that remain with amino acids, for example lysin, cysteine or aspartic acid, or with amines or other highly hydrophilic molecules such as sugars or heterocycles with suitable functional groups.

For the above reasons, the post-immobilisation hydrophilisation step enables easy contact between the active site of the enzyme and the reagents, thereby ensuring quantitative yields from the process for antibiotic synthesis, as can be confirmed by analysing the V_s /Vh 1 ratio.

Moreover, the use of commercial matrices such as Eupergit^® and Sepabeads^®, as starting supports, is advantageous because of their excellent physical and mechanical properties, which allow easy use and recovery of the bioreactor catalyst. This translates into lower industrial costs and lessens the impact on the environment linked with the disposal of chemical reagents and solvents. The validity of the present process of immobilisation was successfully confirmed by comparing the results obtained with the techniques already known in the art. 1. Effect of the interaction between enzyme and support

For this study, acylases from E. coli and other microbial sources were immobilised on epoxy resins of hydrophobic nature. We assessed various parameters indicative of the type of interaction between enzyme and solid support and in particular of the type of immobilisation mechanism and therefore also the orientation adopted by the enzyme during the immobilisation process. The derivatives obtained were compared with the free enzyme. lmmobilisationon on hydrophobic epoxide resins

As specified above, immobilisation on hydrophobic-type epoxide resins requires a medium with high ionic strength that favours a hydrophobic interaction between enzyme and support and then the formation of covalent bonds, by the reaction of the nucleophilic groups of the enzyme with the epoxides of the support. Therefore it follows that the orientation of the enzyme towards the surface of the support, for this type of immobilisation, is determined, ideally, by geometric congruence and the presence of particularly hydrophobic areas on the surface of the enzyme.

Acyclases from E. CoIi and other microbial sources were immobilised on epoxide resins such as Eupergit^® C and Sepabeads^® EC-EP according to the standard procedures known to any expert in the field and disclosed in the prior art above discussed [Colemann P.L. et al. J Chromatogr 1990; 512: 345-363; Wheatley J. B. et al. J Chromatogr, 1993; 644: 11-16; Kircher V. et al. J Chromatogr B ; 1996; 667: 245-255; Wheatley J. B. et al. J Chromatogr A ; 1999; 849: 1-12]. After immobilisation, the epoxide groups of the supports that had not reacted with the enzyme were disactivated by derivatisation with amine ethanol at pH 9 for 24-48 hours.

To assess the affinity of the active site of the PGA thus immobilised for the β- lactamic nuclei, we used acylation of 7-ACA with the methyl ester of R-mandelic acid (RMM) as a reaction test. In the following Scheme 1 , this reaction is schematically illustrated:

Scheme 1

RMM 7-ACA acylation product

mandelic acid

We calculated the V_s/Vhi ratio which, in its turn, establishes the theoretical maximum yield that can be obtained from the synthesis reaction in the absence of hydrolysis of the product being acylated, that is, the final antibiotic.

The so obtained results are summarised in the following Table 1 : it can be seen that the V_sΛ/hi ratio presents significant variations according to the type of support and the immobilisation technique used.

Table 1

Derivative Activation of the VS/Vh1 Theoretical support yield

Free PGA 12.7 92.7% Eupergit C Epoxide 3.3 76.7%

Sepabeads Epoxide 3.2 76.2%

EC-EP

The free enzyme presents a value of around 13 (corresponding to a theoretical yield of 93%), far greater than that showed by biocatalysts prepared by immobilisation on hydrophobic-type epoxide supports. For example, after immobilisation on the above said hydrophobic epoxide resins, a Vs /Vhi ratio of between 3 and 3.5, and a theoretical yield of less than 80%, are obtained. The catalysts thus obtained are therefore less efficient than free PGA. This can be explained by the fact that the necessary initial hydrophobic reaction causes the enzyme to orientate preferentially with its only planar face (on which the active site is present) turned towards the surface of the support. Only when this adsorption has occurred is the formation of covalent bonds possible, by interaction between the lysin amino groups and the epoxide groups of the support. It follows that the nearness of the hydrophobic surface of the support to the catalytic site of the enzyme creates a hydrophobic neighbourhood that is highly unfavourable for the entrance of hydrophilic substrates of considerable size, such as cephalosporanic nuclei.

When immobilisation on activated supports is brought about by the introduction of aldehyde groups, the preferential orientation of the protein is that determined by the area in which the greatest number of lysins is present, that is, those lysins with the greatest degree of reactivity. Because of the need to perform this type of immobilisation at high pH, all the primary amino groups of the lysin residues of the protein are completely undissociated, and therefore highly reactive as nucleophiles for attachment to the aldehyde groups of the support. Studying the surfaces of PGA from E. coli has revealed that both the distribution of the lysins and their reactivity are uniform over the surface of this enzyme. It therefore follows that the orientation of the protein will be casual and that many of the molecules of the enzyme will have a catalytic site far away from the surface of the support. 2. Preparation of enzymatic catalysts of the invention In order to overcome the limitations of the techniques that are already know in the art, the innovative enzymatic catalysts of the invention have been prepared. They take into account the orientation adopted by the enzyme during immobilisation as well as the characteristics of the support and, therefore, of the microenvironment that surrounds the immobilised enzyme.

In particular, by combining the effect obtained by an immobilisation technique that ensures proper orientation of the enzyme on the surface of the support, with hydrophilisation of the surface by post-immobilisation derivatisation, it is possible to obtain biocatalysts that are efficient in terms of catalytic activity and therefore yield, that are stable and easy to recover and recycle.

2.1 Post-immobilisation hydrophilisation on hydrophobic epoxide resins The derivatives were obtained by immobilisation on hydrophobic epoxide resins, subsequently performing derivatisation of the epoxide groups that had not reacted, with highly hydrophilic molecules such as amines, amino acids (in particular cysteine), heterocycles, sugars, or other hydrophilic molecules with functional groups able to react with the epoxide groups, as shown schematically in Figure 2. This derivatisation was performed at pH within the range of 7 to 11 , preferably between 9 and 10, in the presence of derivatising reagents at concentrations of between 0.1 and 3 Molar. When necessary, derivatisation of the surface of the support after immobilisation was performed in the presence of concentrations ranging from 50 to 200 mM of enzymatic inhibitors such as acetic phenyl acid and Penicillin G sulphoxide. The presence of these substances enables enzymatic activity to be maintained during particularly drastic derivatisation conditions (such as high pH), thus avoiding denaturation of the enzyme itself. 2.2 Post-immobilisation hydrophilisation on epoxide-aldehyde resins

Enzymatic derivatives according to the invention were obtained by immobilising the enzyme on an epoxide support, previously derivatised to epoxyaldehyde, to favour an initial interaction of its lysin residues with the aldehyde groups introduced on the surface of the support by the formation of double imine bonds. Subsequently, the enzyme was left to react again with the support (between 24 and 72 hours) in order to allow the reaction of the functional groups of the enzyme with the epoxide groups present on the surface of the support, as schematichally shown in Figure 3. In this way, it is possible to obtain the formation of covalent C-N bonds and the bond between the protein and the support becomes irreversible. Mixed epoxide-aldehyde functionalisation was obtained, for example, by partial hydrolysis of the epoxides in bland conditions and subsequent oxidation with periodate of the diolic groups obtained.

In some cases, for example when Sepabeads^® EC-EP is used as support, a certain quantity of diolic groups that can be directly oxidated to aldehydes are present on the surface of the epoxide resin. Subsequent to immobilisation, hydrophilisation of the matrix surface can be obtained by reaction of the remaining free epoxide groups of the support with amino acids (such as lysin, cysteine or aspartic acid), with amines or with other highly hydrophilic molecules such as sugars or heterocycles with suitable functional groups (see Figure 3). This derivatisation was performed at a pH of between 7 and 11 , preferably between 9 and 10, in the presence of derivatising reagents at concentrations of between 0.1 and 3 M. When necessary, post-immobilisation derivatisation was performed in the presence of concentrations of between 50 and 500 mM of molecules such as acetic phenyl and Penicillin G sulphoxide, in order to avoid denaturisation of the enzyme in the drastic conditions sometimes used in derivatisation. In the case of an immobilisation on activated supports by introducing aldehyde groups, the protein is orientated preferentially according to the zone in which the greatest number of lysins is present, or in any case, the lysins with the highest degree of reactivity. Because of the need to perform this kind of immobilisation at high pH values, all the primary amino groups of the lysin residues of the protein are completely undissociated, and therefore highly reactive as nucleophils for attachment to the aldehyde groups of the support. By this procedure, a large part of the enzyme molecules will be immobilised with the catalytic site facing the other way or in any case away from the surface of the support. Moreover, the presence of free epoxide groups on the surface of the support enables the formation of a hydrophilic microenvironment around the immobilised enzyme. Thus, even the fraction of enzymatic molecules with unfavourable orientation, that is, with their active site facing the surface of the support, will be in contact with a hydrophilic or even polar environment, depending on the derivatisation that has been performed. 2.3 Study of the catalytic properties of the enzymatic derivatives

We studied the above reported acylation of 7-ACA with the methyl ester of R- mandelic acid as reaction model (RMM), to assess the affinity of the various PGA derivatives for β-lactamic nuclei. The V_sΛ/hi ratio was calculated, which also gave the maximum theoretical yield that can be obtained from the synthesis reaction in the absence of hydrolysis of the acylation product. Analysis of the results reported in the following Table 2 clearly shows that by immobilising PGA from Escherichia coli on epoxide hydrophobic supports and performing post-immobilisation hydrophilisation, decidedly higher V₃ A/hi values and corresponding theoretical yields than those observed after immobilisation on epoxide resins alone can be obtained.

Table 2

Derivative Activation of Derivatisation VsWh₁ Theoretical the support yield

Free PGA 12.7 92.7%

Sepabeads^® EC-EP Epoxide 3.2 76.2 Eupergit^® C Epoxide 3.3 76.7%

Eupergit^® C Epoxide Ethanol amine 1.5 M 6.1 85.9%

Eupergit^® C Epoxide Glycine 3 M 5.4 84.3%

Eupergit^® C Epoxide Cysteine 1 M 8.6 89.6%

Sepabeads^® EC-EP Epoxide Cysteine 1 M 9.3 90.3%

Sepabeads^® EC-EP Epoxide- Cysteine 1 M 10.8 91.5% aldehyde

Even more notable are the results obtained when the enzyme is immobilised on an epoxide support, previously derivatised by introducing aldehyde groups (epoxyaldehyde support). Post-immobilisation hydrophilisation with cysteine on epoxyaldehyde Sepabeads^® gives V₅ /Vhi ratio and theoretical yield values that are similar in all respects to those of the free enzyme.

Completely analogous results were obtained immobilising acylases from sources other than E. coli in the same way. For example, by immobilising acylase isolated from A. viscosus, very high Vs /Vh1 values were obtained, according to the type of support and the immobilisation mechanism used.

3. Use of acylase derivatives according to the invention in the synthesis of β- lactamic antibiotics The enzymatic derivatives prepared by the present process were tested in the synthesis of different cephalosporins, in particular in kinetically-controlled synthesis of various cephalosporanic nuclei with derivatives of tetrazol-1-yl-acetic, thiophen-2-yl-acetic and 2-hydroxyphenylacetic acids, phenyl glycine and parahydroxy phenyl glycine. Acylases immobilised according to the present process have led to an increase in the yields obtained, compared to the enzymatic derivatives obtained by immobilisation on epoxide, hydrophobic resins obtained by known methods.

In particular, the acylase derivative isolated from E. coli obtained on epoxyaldehyde resins hydrophilised with cysteine, has proved to be particularly suitable as a catalyst for the synthesis of cephalosporins such as cephazoline and cephalotine.

The acylase derivative prepared with acylase isolated from A. viscosus has proved to be particularly suitable as a catalyst in the synthesis of cephalosporines such as cephonicid, cephaclor and cephprozyl. The following examples are reported to provide a non limiting illustration of the present invention.

EXAMPLE 1

Immobilisation of acyclase from E. coli on Euperαit C and post-immobilisation h ydrophilisation Immobilisation of the enzyme

Eupergit^® C (10 g) is suspended in 138.6 ml of phosphate buffer, 1 M at pH 8 containing 2000 U of enzyme. It is stirred at room temperature for 24h. Lastly, the derivative is filtered and washed abundantly with water. 99% immobilised activity

68% expressed activity value and measurement of the activity: 135.3 U/g

Post-immobilisation hvdroDhilisation.

After adding the enzymatic derivative on Eupergit^® C obtained as previously described to a solution of phenylacetic acid 100 mM in bicarbonate buffer 50 mM at pH 9.6 (56 ml), 8.6 g of cysteine is added (final concentration 1 M). The suspension is stirred at room temperature for 24h. Lastly, the derivative is filtered and washed with distilled water.

64% residue expressed activity (86.3 U/g) EXAMPLE 2

Immobilisation of acylase from E. coli on epoxyaldehvde Sepabeads^® and post- immobilisation hvdrophilisation.

Derivatisation of the resin

The Sepabeads^® EC-EP resin (10 g) is added to 135 ml of a solution of NaIO₄ 15 mM and stirred at room temperature for 2h. Subsequently, the support is filtered and washed thoroughly with water.

Immobilisation of the enzyme

The epoxyaldehyde Sepabeads^® EC-EP (10 g) obtained as described above, is suspended in 56 ml of a solution of phenylacetic acid, 100 mM, in bicarbonate buffer, 50 mM at pH 10.05, containing 2000 U of enzyme. After stirring at room temperature for 3h, 70 ml of a phenylacetic acid solution in 50 mM bicarbonate buffer at pH 10.05 is added. Subsequently, 140 mg of NaBH₄ is added and stirred at room temperature for 30 min. Lastly, the derivative is filtered and washed abundantly with water. 99% immobilised activity

23% expressed activity value and measurement of the activity: 46 U/g Post-immobilisation hydrophilisation.

After adding the enzymatic derivative on epoxyaldehyde Sepabeads^® EC-EP, obtained as previously described, to a solution of phenylacetic acid 100 mM in bicarbonate buffer 50 mM at pH 9.6 (56 ml), 8.6 g of cysteine is added (final concentration 1 M). It is stirred at room temperature for 24h. Lastly, the derivative is filtered and washed with distilled water.

75% residue activity initial expressed activity: 46 U/g final expressed activity: 35 U/g EXAMPLE 3

Assessment of the Vs/Vh^ ratio of the various enzymatic derivatives.

To assess the Vs/VM ratio, 20 ml of a solution of the 7-ACA nucleus (50 mM) and

R-methyl mandelate ester (10 mM) is prepared in phosphate buffer, 10 mM, at pH

6.5. The solution is then cooled to a temperature of 4⁰C. The enzymatic derivative (10 U) is added, having previously been washed with water and conditioned with the reaction buffer. During the reaction, the pH is kept constant with a diluted solution Of NaOH.

The synthesis rate/hydrolysis rate ratio of the ester (Vs/Vh1) is analysed and assessed by HPLC. HPLC conditions

Column: RP-select B

Eluent: phosphate buffer, 10 mM 70% : Acetonitrile 30% : pH 2.8

Flow rate: 0.8 ml/min

T = 30⁰C λ = 220

Dilution: 125 μl sample + 375 μl eluent (1:4)

Calculation of the Vs/Vh± value

The concentrations of R-mandelic acid and acylation product are calculated from the chromatograms obtained by HPLC analysis at various reaction times. The ratio between the derivative before the curve relative to the acylation product and the derivative before the line relative to the acid represents the Vs/Vhi value. As an example, in the following Table 3 some of the values obtained with acylase from E. co// are reported. Table 3

Similarly, in the following Table 4 the results obtained with acylase from A. viscosus are reported.

Table 4

EXAMPLE 4

Synthesis of cephazoline with a commercial derivative of acylase from E. coli immobilised on Euoergit C

The cephazoline nucleus is dissolved (50 mM, 0.344 g) in 20 ml of phosphate buffer 25 mM pH 7.5; once the nucleus has dissolved, the methyl ester of tetrazolyl-acetic acid is added (100 mM, 0.284 g). The solution is adjusted to pH 7 and cooled to a temperature of 4°C. Subsequently, the enzymatic derivative (1 g), previously washed with water and then with the reaction buffer is added. During the reaction, the pH is kept constant with a solution of NH₄OH 1 :10 by automatic titration. After about 3 hours, a maximum conversion of 57% is reached. Conversion of the nucleus into acylation product (cephazoline) is assessed by

HPLC:

Column: RP-select B

Eluent: phosphate buffer 10 mM 87.5% : Acetonitrile 12.5% ; pH 3.2 Flow rate: 1 ml/min

T = 25°C λ = 272 nm.

Dilution: 20 μl sample + 840 μl eluent

EXAMPLE 5 Synthesis of cephazoline with a derivative of acylase from E. coli immobilised on epoxide Sepabeads EC-EP

Cephazoline was synthesised according to the procedure reported above in

Example 4 using 1 g of an enzymatic derivative of from E. coli immobilised on commercial Sepabeads^® EC-EP resin (Resindion^®). Conversion of the 7-ZACA nucleus to cephazoline was performed by HPLC analysis as reported above in

Example 4 and after about 4 hours a maximum conversion of 67% was reached.

EXAMPLE 6

Synthesis of cephazoline with a derivative of acylase from E. coli immobilised on epoxide-aldehvde Sepabeads^® EC-EP, hvdrophilised after immobilisation with cysteine

Cephazoline was synthesised by the procedure reported above in Example 4 using 1.5 g of an enzymatic derivative of PGA from E. coli immobilised on commercial Sepabeads^® EC-EP resin (Resindion^®) with mixed epoxide-aldehyde activation, and hydrophilised after immobilisation with cysteine 1 M. Conversion of the 7-ZACA nucleus to cephazoline was performed by HPLC analysis as reported above in Example 4 and after about 4 hours a maximum conversion of 75% was reached.

Claims

1. A process for the preparation of an enzymatic catalyst wherein an enzyme is immobilised on a solid epoxidic support, comprising the following steps: i) possible partial oxidation of the epoxide groups on the surface of an epoxidic support into aldehyde groups; ii) immobilisation of an enzyme onto the surface of an epoxide support, possibly derivatised by partial oxidation as coming from step i); iii) hydrophilisation of the surface of the support onto which the enzyme is immobilised as coming from step ii).

2. The process according to claim 1, wherein said epoxidic support is a hydrophobic epoxidic resin.

3. The process according to claim 1, wherein said partial oxidation in step i) is carried out by bland-condition hydrolysis of the epoxidic groups with H₂SO₄ and subsequent oxidation of the so obtained diolic groups with periodate.

4. The process according to claim 1, wherein said immobilisation of the enzyme in step ii) is carried out by adding said epoxidic support to a suspension of said enzyme in a buffer at pH ranging from 8 to 10, and maintaining the suspension under stirring for a period of time ranging from 12 to 48 hours.

5. The process according to claim 1 , wherein said hydrophilisation in step iii) is carried out by reaction of the free epoxide groups of the support and the amino groups of highly hydrophilic products.

6. The process according to claim 5, wherein said highly hydrophilic molecules are selected from the group consisting of sugars, amino acids, ethylene diamines and bis-amines.

7. The process according to claim 6, wherein said amino acids are selected from the group consisting of lysin, cysteine and aspartic acid.

8. The process according to claim 5, wherein said hydrophilisation in step iii) is carried out at pH ranging from 7 and 11, in the presence of said hydrophilic products in concentrations of between 0.1 and 3 M.

9. The process according to claim 8, wherein said hydrophilisation in step iii) is carried out at pH ranging from 9 and 10.

10. The process according to claim 5, wherein said hydrophilisation in step iii) is carried out in the presence of concentrations ranging from 50 to 200 mM of enzymatic inhibitors.

11. The process according to claim 10, wherein said enzymatic inhibitors are selected from between acetic phenyl acid and Penicillin G sulphoxide.

12. The process according to claim 1, wherein said enzyme is selected from the group consisting of acylase, esterase, protease and hydrolase.

13. The process according to claim 12, wherein said enzyme is an acylase.

14. The process according to claim 13, wherein said enzyme is an acylase isolated from a microorganism selected from the group consisting of E. coli, Kluyvera citrophila, Bacillus megaterium and Arthrobacter viscosus.

15. The process according to claim 14, wherein said enzyme is an acylase isolated from E. coli.

16. The process according to any of claims 13-15, wherein said enzyme is Penicillin-G-Acylase.

17. An enzymatic catalyst wherein an enzyme is immobilised on a solid support, obtainable by the process as defined in claims 1-16.

18. The enzymatic catalyst according to claim 17, wherein said enzyme is selected from the group consisting of acylase, esterase, protease and hydrolase.

19. The enzymatic catalyst according to claim 18, wherein said enzyme is an acylase.

20. The enzymatic catalyst according to claim 19, wherein said enzyme is an acylase isolated from a microorganism selected from the group consisting of E. coli, Kluyvera citrophila, Bacillus megaterium and Arthrobacter viscosus.

21. The enzymatic catalyst according to claim 20, wherein said enzyme is an acylase isolated from E. coli.

22. The enzymatic catalyst according to any of claims 19-21, wherein said enzyme is Penicillin-G-Acylase.

23. The enzymatic catalyst according to claim 17, wherein said epoxidic support is an epoxidic resin.

24. The enzymatic catalyst according to claim 17, wherein said enzyme is an acylase isolated from E. coli and said epoxidic support is an epoxyaldehyde resin hydrophilised in step iii) of the process as defined in claim 5 wherein the hydrophilic product is cysteine.

25. The enzymatic catalyst according to claim 17, wherein said enzyme is an acylase isolated from A. viscosus and said epoxidic support is an epoxyaldehyde resin hydrophilised in step iii) of the process as defined in claim 5 wherein the hydrophilic product is cysteine.

26. Use of the enzymatic catalyst as defined in claims 18-25, as catalyst.

27. The use according to claim 26, as catalyst in a kinetically-controlled reaction.

28. The use according to claim 27, wherein said reaction is the resolution of racemic compounds coming from peptide synthesis.

29. Use of the enzymatic catalyst as defined in claim 19, as catalyst in the synthesis of β-lactamic antibiotics.

30. The use according to claim 29, wherein said β-lactamic antibiotics are selected from the group consisting of cephazoline, cephalotine, cephaclor and cephprozyl.

31. Use of the enzymatic catalyst as defined in claim 24, as catalyst in the synthesis of cephalosporins selected from cephazoline and cephalotine.

32. Use of the enzymatic catalyst as defined in claim 25, as catalyst in the synthesis of cephalosporines such as cephonicid, cephaclor and cephprozyl.