Cephalosporin C Deacetylase Research Articles

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

To maximize the expression of the cephalosporin-C deacetylase (CAH) gene isolated from Bacillus subtilis SHS 0133 in Escherichia coli, a series of expression plasmids was constructed with various spacings between the Shine-Dalgarno sequence and the ATG initiation codon. As the most efficient expression 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°C for 20 h in a 30- l jar fermentor. Since the amount of CAH reached about 70% of the total protein in the soluble fraction of the cells, and CAH was recovered from the cell extracts in an active form, the CAH was purified easily to homogeneity by only one column chromatography step. Twenty grams of 7-aminocephalosporanic acid was completely converted to deacetyl-7-aminocephalosporanic acid, a starting material for cefcapene pivoxil hydrochloride, by 12 mg of the purified enzyme without significant appearance of by-products. Thus, our expression and purification system has made the industrial production of CAH possible.

Purification and properties of cephalosporin-C deacetylase from the yeast, Rhodotorula glutinis 38B1, useful for bioconversion of 7-aminocephalosporanic acid derivatives

A 7-aminocephalosporanic acid (7-ACA) deacetylating enzyme was purified to homogeneity from Rhodotorula glutinis 38B1, whose resting cells have been previously reported as useful for the conversion of 7-ACA derivatives [Sakai et al., Appl. Environ. Microbiol., 62, 2667–2672, 1996]. The purified enzyme was a dimer comprised of identical subunits with a molecular mass of 82 kDa. The purified enzyme used cephalosporin C and several 7-ACA derivatives with low K m and high k cat values as substrates, as well as some acetyl esters with relatively long-chain alcohols. Based on this substrate specificity, the enzyme is classified as cephalosporin-C deacetylase (EC 3.1.1.41). The enzyme was most active at 35°C and pH 5.5, and was inhibited by several serine enzyme inhibitors. The purified enzyme was glycosylated on the addition of an asparagine-linked “hybrid type” oligosaccharide, and most of the enzyme activity was found in the purified cell wall fraction. The enzyme localization and kinetic properties explain the high efficiency of 7-ACA deacetylation in a resting-cell reaction.

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

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.

Purification, characterization and partial amino acid sequences of a novel cephalosporin-C deacetylase from Bacillus subtilis

Cephalosporin-C deacetylase [EC 3.1.1.41] was purified electrophoretically to homogeneity from the newly isolated Bacillus subtilis SHS 0133 (FERM BP-2755). The enzyme was purified about 27-fold with a yield of 9 % and a specific activity of 187.4 U/mg protein. The native enzyme (molecular weight, 280,000) was composed of eight identical subunits with apparent molecular weights of 35,000. The cephalosporin-C deacetylase was stable up to 60°C for 30 min at pH 7.0. The enzyme exhibited Michaelis-Menten kinetics with the substrates cephalosporin C, 7-aminocephalosporanic acid (7-ACA) and p-nitrophenyl acetate; the K m values were 24.0, 7.9 and 1.0 mM, respectively. One of the reaction products from 7-ACA, deacetyl-7-ACA, was a weak non-competitive inhibitor and other product, acetate, was a weak competitive inhibitor; the K i values were 171 and 290 mM, respectively. However, these weak product inhibitors did not prevent the completion of the deacylation of 7-ACA. The pI value of the enzyme was determined to be 5.3 using isoelectric focusing. The observed data indicate that the enzyme is different from known cephalosporin-C deacetylases. In addition, amino acid sequencing of the N-terminus and Achromobacter proteinase I-digested peptides yielded no sequences with similarities to other known proteins by a computer search.