Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1997-04-23
1999-04-06
Jacobson, Dian C.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435 44, 435 45, 435 691, 4351723, 4352523, 43525233, 4353201, 536 232, 935 10, 935 14, 935 29, 935 73, C12N 984, C12N 1511, C12N 1555, C12N 1570
Patent
active
058917039
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to mutations of prokaryotic Penicillin G acylase or its preenzyme or preproenzyme, resulting in altered properties of the mutant penicillin G acylase.
BACKGROUND OF THE INVENTION
The basic antibiotics of the .beta.-lactam type are principally obtained by fermentation. Fungi of the genus Penicillium and Cephalosporium (Acremonium) are used for the production of raw material for .beta.-lactam antibiotics as penicillin G, penicillin V and cephalosporin C. These fermentation products, also referred to as PenG, PenV and CefC, respectively, are the starting materials for nearly all currently marketed penicillins and cephalosporins. In general the acyl group at the 6-amino of the penicillin nucleus or at the 7-amino position of the cephalosporin nucleus is referred to as `side chain`, the corresponding acid as `side chain acid`. The side chains of PenG, PenV and CefC are phenylacetyl, phenoxyacetyl and aminoadipyl, respectively. The side chains are removed by cleavage of an amide linkage (deacylation), resulting in 6-aminopenicillanic acid (6-APA) in case of the penicillin molecules and 7-aminocephalosporanic acid (7-ACA) in case of the cephalosporin molecule. In this respect also phenylacetyl-7-aminodesacetoxycephalosporanic acid (CefG) should be mentioned as a precursor of 7-ADCA, although CefG is not a fermentation product. CefG is usually produced chemically from Penicillin G.
In order to obtain .beta.-lactam compounds with an altered activity spectrum, an increased resistance against .beta.-lactamases or improved clinical performance of .beta.-lactam compounds, 6-APA, 7-ACA and 7-ADCA are used as starting points for synthetic manipulation to produce the various penicillins and cephalosporins of choice. At present these semisynthetic penicillins and cephalosporins form by far the most important market of .beta.-lactam antibiotics.
The production of semisynthetic .beta.-lactam products requires the deacylation of the penicillins and cephalosporins produced from fermentation. Although rather efficient chemical routes are available for the deacylation (J. Verweij & E. de Vroom, Recl. Trav. Chim. Pays-Bas 112 (1993) 66-81), nowadays the enzymatic route is preferred in view of the high energy and solvents cost together with some environmental problems associated with the chemical route (Dunnill, P., Immobilised Cell and Enzyme Technology. Philos, Trans. R. Soc. London B290 (1980) 409-420). The enzymes which may accomplish the deacylation of .beta.-lactam compounds are classified as hydrolases based on the chemical reaction they catalyse. However, those hydrolases which are particularly useful in the deacylation of .beta.-lactam compounds are usually referred to in the art as `acylases` or `amidases`. These denominations as used in this specification have the same meaning. In connection with .beta.-lactam antibiotics these acylases usually are further specified as `.beta.-lactam acylases` as not all amidases accept a .beta.-lactam nucleus as an acceptor/donor moiety for the acyl group. According to the literature several types of .beta.-lactam acylases may be envisaged, based on their substrate specificity and molecular structure (B. S. Deshpande et al., World J. Microbiology & Biotechnology 10 (1994) 129-138).
Acylase, Nomenclature & Classification
The substrate specificity of the acylase is determined by a side chain binding pocket at the enzyme which recognizes the side chain moiety of .beta.-lactam molecules. In general, the acylases are not very specific for the moiety adjacent to the nitrogen atom of the amide group (this might be a cephem group, a penem group, an amino acid, sugars, etc. (J. G. Shewale et al., Process Biochemistry International, June 1990, 97-103). In case of the Penicillin G acylases (Benzylpenicillin amidohydrolase, also named Penicillin amidase, EC 3.5.1.11) this acyl moiety must be very hydrophobic and is preferably phenylacetyl or (short) alkyl. Penicillin G acylase is used commercially to hydrolyse PenG or CefG to phenylacetic
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Quax Wilhelmus J.
Riemens Adriana M.
Van Der Laan Jan M.
Gist-Hrocades
Jacobson Dian C.
Moore William W.
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