This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mitsushima, K. Articles by Yagi, S. Search for Related Content PubMed PubMed Citation Articles by Mitsushima, K. Articles by Yagi, S. Agricola Articles by Mitsushima, K. Articles by Yagi, S.

 Previous Article  |  Next Article 

Appl. Environ. Microbiol., Jun 1995, 2224-2229, Vol 61, No. 6
Copyright © 1995, American Society for Microbiology

Gene cloning, nucleotide sequence, and expression of a cephalosporin-C deacetylase from Bacillus subtilis

K Mitsushima, A Takimoto, T Sonoyama and S Yagi
Bioprocess Development Department, Shionogi & Co., Ltd., Osaka, Japan.

The gene encoding a cephalosporin-C deacetylase (CAH) from Bacillus subtilis SHS 0133 was cloned and sequenced. The nucleotide sequence contained an open reading frame encoding a polypeptide consisting of 318 amino acids, the molecular weight of which was in good agreement with the value obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence contained the common sequence Gly-X-Ser-X-Gly found in many esterases, lipases, and serine proteases. This indicates that CAH is a serine enzyme. A possible promoter sequence which is very similar to the consensus sequences of - 35 and -10 regions recognized by B. subtilis RNA polymerase utilizing sigma factor H was found in the 5'-flanking region of the CAH structural gene. Two repeated A+T-rich blocks consisting of 24 bp were also found in the upstream region of the initiation codon. We constructed a series of expression plasmids by inserting the CAH gene into Escherichia coli ATG vectors. The degree of CAH gene expression depended on promoters and vector plasmids, which have different replication origins. The expressed CAH protein was an active form in the soluble fraction obtained after cell disruption. The highest expression level was accomplished with an expression plasmid, pCAH400, which has the trp promoter and the replication origin derived from pAT153. In the fermentation using a 30-liter jar fermentor, the transformant E. coli JM103(pCAH400) produced 440 U of CAH per ml of culture during a 24-h incubation. This value corresponded to 2.1 g of CAH protein in 1 liter of culture broth.

This article has been cited by other articles:

• Degrassi, G., Kojic, M., Ljubijankic, G., Venturi, V. (2000). The acetyl xylan esterase of Bacillus pumilus belongs to a family of esterases with broad substrate specificity. Microbiology 146: 1585-1591 [Abstract] [Full Text]