Process for the enzymatic preparation of 7-aminocephalosporanic

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound having a 1-thia-5-aza-bicyclo

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43525233, 4353201, 435228, 4351723, C12P 3502, C12P 119, C12N 121, C12N 1563, C12N 980

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active

053546679

ABSTRACT:
A process for the production of an E. coli strain producing high yields of 7.beta.-(4-carboxybutanamido) cephalosporin acylase, comprises: (a) digesting the DNA of a microorganism whose DNA includes the sequence encoding 7.beta.-(4-carboxybutanamido) cephalosporin acylase, and forming a plasmidic library; (b) transforming, with the sequences in the plasmidic library, an auxotrophic E. coli host; (c) selecting for transformed E. coli hosts containing the acylase sequence by growth on a suitable medium; (d) isolating the vector containing the acylase sequence, digesting the vector, and ligating the DNA sequences obtained into an E. coli vector under control of an E. coli promoter; (e) repeating selection procedure of step (c) ; (f) using the vectors from the selected E. coli hosts to transform an E. coli host lacking substantial .beta.-lactamase activity. 7-Aminocephalosporanic acid and its derivatives can be prepared by reaction of substrates like 7.beta.-(4-carboxybutanamido) cephalosporanic acid with 7.beta.-(4-carboxybutanamido) cephalosporin acylase produced by the E. coli strain produced by the above process.

REFERENCES:
patent: 4774179 (1988-09-01), Ichikawa et al.
Garcia et al. "An improved method to clone penicilin acylace genes . . . ," J. of Biotech. 3:187-195 (1986).
Forney et al. "Selection of Amidases with Novel Substrate Specificities . . . " Applied and Environ. Microbiology 55(10):2550-2555 (Oct. 1989).
Shibuya et al. "Isolation and Properties of 7.beta.-(4-carboxybutanamido) cephalosporanic Acid Acylase-producing Bacteria," Agric. Biol. Chem. 45(7):1561-7 (1981).
Matsuda et al., "Molecular Cloning and Structure of the Gene for 7.beta.-(4-carboxybutanamido) cephalosporic Acid Acylase from a Pseudomonas strain," J. of Bacteriology, 163(3):1222-1228 (Oct. 1985).

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