Cephalosporin C acylase

Details Cephalosporin C acylase Cephalosporin C acylase

1996-05-01

1998-09-08

Wax, Robert A.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

435 691, 435 49, 435 51, 4351723, 4352523, 4353201, 536 232, C12N 980, C12N 1500, C12P 2106, C07H 2104

Patent

active

058044299

DESCRIPTION:

BRIEF SUMMARY
The present application is a national stage application based on PCT/JP94/01799, filed Oct. 26, 1994.
The invention relates to a new cephalosporin C acylase (hereinafter referred to as "CC acylase"). More particularly, it relates to a new mutant CC acylase produced by protein engineering, a DNA coding therefor, an expression vector containing the said DNA, a microorganism transformed with the said expression vector, and the production of the CC acylase by culturing the said transformant.
The cephalosporin C acylase is a general term for an enzyme, which is, in common, capable of hydrolyzing cephalosporin C to 7-aminocephalosporanic acid (7-ACA).
Hitherto, there have been found three enzymes which should be classified as CC acylase, namely Cephalosporin C acylases SE83, N176 and V22, amino acid sequences of which are disclosed in Journal of Fermentation and Bioengineering Vol. 72, 232-243 (1991). In this literature, numbering of the amino acid sequence of CC acylase is begun at the methionine group of the N-terminal portion thereof. However, numbering of the amino acid sequence of CC acylase is begun at the threonine group adjacent to the methionine group of the N-terminal portion thereof in this Specification, because the N-terminal methionine of .alpha.-subunit of mature CC acylase obtained by expressing CC acylase gene in prokaryote is removed by an enzyme (e.g. aminopeptidase) to give a mature CC acylase having the threonine group as the N-terminal amino acid thereof. Production of native type CC acylase by recombinant DNA technology is also disclosed in the said literature and it has been found that the expressed CC acylase is intracellularly processed to give an active form composed of .alpha.-subunit and .beta.-subunit. However, efficiency of the processing is generally low in E. coli. From the results of extensive studies, the inventors of this invention have succeeded in producing mutant CC acylases which have more desirable properties which are characterized by higher enzymatic potency, alteration of pH profile, higher efficiency of processing and the like.
The new mutant CC acylase of this invention can be characterized by the following.
A mutant CC acylase wherein at least one amino acid at the Ala.sup.49, Met.sup.164, Ser.sup.166, Met.sup.174, Glu.sup.358, Met.sup.465, Met.sup.508 or Met.sup.750 position of the amino acid sequence of the native CC acylase is replaced by a different amino acid.
Preferred examples of the different amino acid to replace Met.sup.164 may include neutral amino acids such as glycine, alanine, leucine and the like.
Preferred examples of the different amino acid to replace Ser.sup.166, Met.sup.174, Met.sup.465, Met.sup.506 and/or Met.sup.750 may include neutral amino acids such as alanine and the like.
Preferred examples of the different amino acid to replace Glu.sup.358 may include neutral-amino acids (e.g. isoleucine, etc.), basic amino acids (e.g. lysine, etc.) and the like.
Most preferable example of the different amino acid to replace Ala.sup.49 is leucine.
The mutant CC acylase of this invention may also be a mutant CC acylase prepared by replacing at least one amino acid at another position of the amino acid sequence of native CC acylase with a different amino acid, for example, by replacing Met.sup.269 and/or Cys.sup.305 of the mutant CC acylase with (a) different amino acid(s).
A preferred example of the mutant CC acylase can be represented by the following formula in its precursor form before processing into .alpha.-subunit and .beta.-subunit thereof: A507-749-X8-A751-773 Glu.sup.48 of native CC acylase, Leu.sup.163 of native CC acylase, Arg.sup.173 of native CC acylase, Val.sup.357 of native CC acylase, Ala.sup.464 of native CC acylase, Ile.sup.505 of native CC acylase, Ala.sup.749 of native CC acylase, Ala.sup.773 of native CC acylase, different amino acid(s), and when X1 is Ala, X2, X4, X6, X7 and X8 are each Met, X3 is Ser and X5 is an amino acid other than Glu.
In this specification, a nomenclature for naming a specific mutant CC acylase is conveniently e

REFERENCES:
patent: 5336613 (1993-02-01), Niwa et al.
Journal of Fermentation and Bioengineering, vol. 72, No. 4, pp. 232-242, 1991, I. Aramori, et al., "Cloning and Nucleotide Sequencing of New Glutaryl 7-ACA and Cephalosporin C Acylase Genes from Pseudomonas Strains".
Biochimica Et Biophysica Acta, vol. 1132, No. 3, pp. 233-239, 1992, M. Ishiye, et al., "Nucleotide Sequence and Expression in Escherichia coli of the Cephalosporin Acylase Gene of a Pseudomonas Strain".

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