GENE CLONING, NUCLEOTIDE-SEQUENCE, AND EXPRESSION OF A CEPHALOSPORIN-C DEACETYLASE FROM BACILLUS-SUBTILIS

The gene encoding a cephalosporin-C deacetylase (CAH) from Bacillus su btilis SHS 0133 was cloned and sequenced. The nucleotide sequence cont ained an open reading frame encoding a polypeptide consisting of 318 a mino acids, the molecular weight of which was in good agreement with t he value obtained by sodium dodecyl sulfate-polyacrylamide gel electro phoresis. The deduced amino acid sequence contained the common sequenc e Gly-X-Ser-X-Gly found in many esterases, lipases, and serine proteas es, This indicates that CAH is a serine enzyme. A possible promoter se quence which is very similar to the consensus sequences of -35 and -10 regions recognized by B. subtilis RNA polymerase utilizing sigma fact or H was found in the 5'-flanking region of the CAH structural gene. T wo 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 e xpression 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 express ed CAH protein was an active form in the soluble fraction obtained aft er cell disruption. The highest expression level was accomplished with an expression plasmid, pCAH400, which has the trp promoter and the re plication origin derived from pBT153. In the fermentation using a 30-l iter jar fermenter, the transformant E. coli JM103(pCAH400) produced 4 40 U of CAH per ml of culture during a 24-h incubation. This value cor responded to 2.1 g of CAH protein in 1 liter of culture broth.