Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound having a 1-thia-4-aza-bicyclo
Patent
1998-01-15
2000-05-09
Naff, David M.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound having a 1-thia-4-aza-bicyclo
435 50, 435177, 435178, 435180, C12P 3704, C12P 3504, C12N 1102, C12N 1110
Patent
active
060602686
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to an improved immobilized Penicillin G acylase. Furthermore, the invention relates to the preparation of .beta.-lactam-antibiotics by enzymatic acylation of the parent amino .beta.-lactam nucleus with the corresponding acylating agent using said immobilized enzyme.
BACKGROUND AND FIELD OF THE INVENTION
Enzymatic production of semisynthetic .beta.-lactam antibiotics by acylation of the parent amino .beta.-lactam moiety with an activated side chain acid derivative, such as an amide or an ester, is known from Dutch patent 158847, European patent applications 339751 and 473008, international patent applications WO 92/01061 and WO 93/12250, U.S. Pat. No. 3,816,253, and West German patent documents 2163792 and 2621618. The enzymes used in the art are in most cases penicillin acylases obtained from Escherichia coli and are immobilized on various types of water-insoluble materials.
A drawback of the known enzymatic methods for the production of amoxycillin, ampicillin, cephadroxil, cephalexin, and cephradine is the high cost due to the selectivity of the immobilized enzyme. Said immobilized enzymes are capable of condensing activated side chain derivatives such as D(-)-phenylglycine amide (PGA), D(-)-phenylglycine methyl ester (PGM), D(-)-4-hydroxyphenylglycine amide (HPGA), D(-)-4-hydroxyphenylglycine methyl ester (HPGM), D(-)-2,5-dihydro-phenylglycine amide (DPGA), and D(-)-2,5-dihydrophenylglycine methyl ester (DPGM) with amino .beta.-lactams such as 6-amino-penicillanic acid (6-APA), 7-aminocephalosporanic acid (7-ACA), 7-amino-3-chloro-3-cephem-4-carboxylic acid (7-ACCA), 7-aminodesacetoxycephalosporanic acid (7-ADCA) and 7-amino-3-[(Z)-1-propenyl]-3-cephem-4-carboxylic acid. On the other hand, said immobilized enzymes will also hydrolyse the activated side chain derivatives to worthless side chain acids. Also, the desired product hydrolyses to form side chain acid and the parent amino .beta.-lactam. A high ratio between synthesis and hydrolysis will lower the cost of activated side chain derivative.
From international patent application Wo 93/12250 it is known that the ratio synthesis/hydrolysis for cephadroxil and cephalexin synthesis by Escherichia coli penicillin G acylase immobilized on Eupergit PCA is strongly dependent on the reaction conditions such as pH, concentration of reactants and temperature. The influence of the nature of the carrier material on the ratio synthesis/hydrolysis however, has not been taught.
From European Patent No. 222,462 and corresponding U.S. Pat. No. 5,137,818. It is known that amino groups can be introduced onto the carrier material by adding aminopolymers such as alginate amine, chitosan, pectin, or poly-ethylene imine to the base gelling constituent of the carrier.
Surprisingly, it has been found that immobilization of Escherichia coli penicillin G acylase on a carrier consisting of a gelling agent and a polymer containing free amino groups gives an enzymatic catalyst with superior characteristics regarding the ratio synthesis/hydrolysis in the condensation reaction of activated side chain derivatives with amino .beta.-lactams as compared to penicillin G acylases immobilized on other carriers.
SUMMARY OF THE INVENTION
The present invention provides Penicillin G acylase immobilized on a carrier comprising a gelling agent and a polymer containing free amino groups. Preferably the polymer is selected from the group consisting of alginate amine, chitosan, pectin, or polyethylene imine, and more preferably, the gelling agent is gelatin. Furthermore, by applying such an immobilized enzyme, an improved process for the preparation of a .beta.-lactam derivative by an enzymatic reaction of the parent amino .beta.-lactam with the corresponding acylating agent has been provided for.
SPECIFIC EMBODIMENTS
Examples of .beta.-lactam derivatives that may be produced by the process of this invention are amoxycillin, ampicillin, cephaclor, cephadroxil, cephprozil, cephalexin, and cephradine. The acylase activity is independent of the
Gist-Brocades B.V.
Naff David M.
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