Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound having a 1-thia-5-aza-bicyclo
Reexamination Certificate
2000-05-26
2002-05-28
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound having a 1-thia-5-aza-bicyclo
C435S188000, C435S228000, C435S195000
Reexamination Certificate
active
06395507
ABSTRACT:
The present invention relates to a process for delaying the deactivation of glutaryl amidase during enzyme catalysis.
7-Aminocephalosporic acid is of great commercial interest for the production of semisynthetic cephalosporin antibiotics.
The enzymatic synthesis of the antibiotic precursor 7-aminocephalosporic acid (7-ACS) is—as shown in FIG.
1
—carried out in two reaction steps. Cephalosporin C is initially oxidized by action of D-amino acid oxidase (DAO) to &agr;-ketoadipyl-7-ACS. In the next step, this compound is hydrolyzed to 7-ACS by glutaryl amidase (GAE).
The synthesis is carried out with the enzymes DAO and GAE, which are generally immobilized on supports and which are, after the reaction has ended, separated off from the solution of the product and can be reused for the next batch. However, if the catalyst is used repeatedly, the enzymes are deactivated, which is equivalent to enzyme consumption.
From the literature, it is known that structure-changing oxidations of proteins can be suppressed by using thiol reagents, such as, for example, 2-mercaptoethanol (P. Golini et al.,
Enzyme and Microbial Technology
17 (1995) 324-329;
Int J. Peptide Protein Res.
48 (1996) 532-538).
DAO, for example, can be regenerated using thiols. The flavoprotein DAO catalyzes the stereospecific deamination of D-amino acids to the corresponding &agr;-keto acids and ammonium, for example, as shown in
FIG. 1
, the conversion of cephalosporin C to &agr;-ketoadipyl-7-ACS. &agr;-Ketoadipyl-7-ACS decarboxylates in situ to glutaryl-7-ACS (G-7-ACS) (P. Golini et al.,
Enzyme and Microbial Technology
17 (1995) 324-329). In industry, the enzyme is frequently not employed in soluble form but immobilized by binding to polymers, such as, for example, amino-alkylated polymers or oxirane-activated polymers. Thus, after the reaction, the enzyme catalyst can be separated off by filtration, thus being available for reuse. The immobilized DAO catalyst suffers partial inactivation. When the enzyme catalyst, which has already been used once, is reemployed in a further reaction under otherwise identical conditions, the reaction time required for maximum conversion of the substrate is longer. This prolongation of the reaction time, which occurs on each reuse, is a measure of the stability of the catalyst in the preparation process of G-7-ACS. The extent of the change of the reaction time to maximum substrate conversion, determined over a plurality of production cycles, is referred to as operational stability. The operational stability of immobilized DAO can be improved by separating off the enzyme catalyst from the reaction mixture after the reaction by filtration, and treating it with a thiol, for example 2-mercaptoethanol. DAO contains some easily oxidizable sulfhydryl groups, predominantly as functional groups of the cysteine amino acids of the protein. The action of 2-mercaptoethanol is based on the reducing action of the thiol on the oxidation-sensitive sulfhydryl groups of the cysteines. The regeneration of these oxidized sulfhydryl groups results in a considerable improvement in operational stability. In the particular case of DAO, regeneration has to be carried out after separation of the enzyme from the reaction mixture, because H
2
O
2
is formed during the enzyme catalysis. As a strong oxidizing agent, this would inactivate any added thiol.
Addition of thiol may also result in a reduction in the activity of proteins. This effect, too, can be explained by the presence of cysteine radicals. One example for this is aminoacylase. Aminoacylase is a dimeric enzyme having one Zn
2+
atom per subunit. Each subunit of the enzyme contains two cysteine SH groups and two disulfide bonds. The chemical modification of the SH groups, such as the breaking of the disulfide bonds, can result in an inactivation of the enzyme. It has been demonstrated that the activity of aminoacylase is reduced by addition of 2-mercaptoethanol, whereas, when the 2-mercaptoethanol is removed by dialysis or gel filtration, the original enzyme activity can be reestablished almost completely (W. Kördel and F. Schneider,
Biochem. Biophys. Acta
445 (1976) 446-457).
Since the action of the thiol is apparently mediated by the oxidation or reduction of sulfhydryl groups of cysteine radicals, the addition of thiols to enzymes which are known not to contain any cysteine radicals should consequently not result in any change of the enzyme activity.
During repeated use of GAE in catalytical conversions of the catalyst, the enzyme is, as described at the outset for the synthesis of 7-ACS (cf. FIG.
1
), deactivated, which corresponds to a consumption. The stability of the catalyst correlates with important production costs of the process, such as the time required, the waste produced, and the costs of the catalyst. A process that is comparable to the process described above for DAO and which is suitable for stabilizing GAE or increasing its operational stability has hitherto not been disclosed. The enzyme GAE comprises two peptide chains (protein A and B). The interaction of the chains is via hydrogen bonds and hydrophilic and hydrophobic interactions of protein domains.
It is an object of the present invention to provide a process for delaying the deactivation of glutaryl amidase during enzyme catalysis.
As can be seen from the amino acid analysis (Table 1), GAE lacks the amino acid cysteine. Thus, the use of thiol reagents for stabilization should not result in any enhanced operational stability of this biocatalyst. Contrary to expectation, it was possible to experimentally prove the opposite. In the case of GAE, the addition of various thiol-containing reagents, for example 2-mercaptoethanol or cysteine, resulted in a drastic increase in operational stability, depending on the concentration.
Accordingly, the object of the present invention is achieved by a process for delaying the inactivation during enzyme catalysis of GAE, comprising bringing the enzyme into contact with at least one thiol. The enzyme can optionally be present in free or supported form. A preferred support is, for example, an oxirane-activated polyacrylate.
Thiol or mercaptan is understood as meaning a chemical compound such as 2-mercaptoethanol, glutathione, or the amino acid cysteine, the common feature of which is that they contain a thiol group (—SH) in the molecule.
Supported GAE consists of the enzyme catalyst GAE which is attached, for example, to oxirane-activated or else amino-alkylated polyacrylates (=supports). A process for preparing supported GAE is described in D. Bianchi et al.,
Enzyme and Microbiological Technology
20 (1997) 368-372. Examples of supports are EUPERGIT® (oxirane containing polymer), AMBERLITE® XAD7 (nonionic polymeric adsorbent), and DUOLITE® A365 (ion-exchange resin)(all from Röhm and Haas).
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Fernandez-Lafuente et al., Tetrahedron Letters, vol. 38, No. 26, pp. 4693-96, 1997.*
P. Golini et al., “Immobilization of D-amino acid oxidase from different yeasts: Characterization and application . . . ,”Enzyme Microb. Technol., 17:324-329 (1995).
Y. Yang et al., “Kinetics of inhibition of aminoacylase activity by dithiothreitol or 2-mercaptoethanol,”Int. J. Peptide Protein Res., 48:532-538 (1996).
D. Bianchi et al., “Immobilization of glutaryl-7-ACA acylase on aminoalkylated polyacrylic supports,”Enzyme Microb. Technol., 20:368-372 (1997).
W. Kördel et al., “Chemical Investigations on Pig Kidney Aminoacylase,”Biochimica et Biophysica Acta, 445:446-457 (1976).
E. Battistel et al., “Purification and Stability of Glutaryl-7-ACA Acylase fromPesudomonas sp.,”Applied Biochemistry and Biotechnology, 69:53-67 (1998).
Bongs Jürgen
Koller Klaus-Peter
Kruse Wolfgang
Meiwes Johannes
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Meller Mike
Naff David M.
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