High-level expression, purification, and some properties of a recombinant cephalosporin-C deacetylase

To maximize the expression of the cephalosporin-C deacetylase (CAH) gene is olated from Bacillus subtilis SHS 0133 in Escherichia coli, a series of exp ression plasmids was constructed with various spacings between the Shine-Da lgarno sequence and the ATG initiation codon. As the most efficient express ion plasmid, we selected pCAH431, which has the trp promoter, a replication origin derived from pAT153, and a spacing of 13 nucleotides. E. coli JM103 with pCAH431 produced 4.9 g of CAH per liter on cultivation at 37 degrees C for 20 h in a 30-l jar fermenter. Since the amount of CAH reached about 7 0% of the total protein h the soluble fraction of the cells, and CAB was re covered from the cell extracts in an active form, the CAH was purified easi ly to homogeneity by only one column chromatography step, Twenty grams of 7 -aminocephalosporanic acid was completely converted to deacetyl-7-aminoceph alosporanic acid, a starting material for cefcapene pivoxil hydrochloride, by 12 mg of the purified enzyme without significant appearance of by-produc ts. Thus, our expression and purification system has made the industrial pr oduction of CAH possible.