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 degrees 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.

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.

Site-directed mutagenesis of human pancreatic secretory trypsin inhibitor.

Arg-42 or Lys-43 or Arg-44 of human pancreatic secretory trypsin inhibitor (PSTI) was replaced by Thr or Ser by site-directed mutagenesis, and the inactivation rates of the mutants after mixing with human trypsin were compared with that of the natural form. The inactivation rate decreased for one mutant (Arg-44----Ser), whereas no change was observed for another (Arg-42----Thr) and an increase was observed for a third (Lys-43----Thr). Kinetic studies on the interactions between human trypsin and synthetic peptides, comprising the regions of Phe39-Ser47 of the respective PSTI species, showed that human trypsin cleaved the Arg42-Lys43 bond preferentially to the Arg44-Gln45 bond. However, it is cleavage of the latter bond that is thought to cause inactivation of human PSTI. These results suggest that the Arg44-Gln45 bond of human PSTI is responsible for its inhibitory activity, and inactivation of human PSTI is probably caused by deletion of the dipeptide Lys43-Arg44.

Production of 2-Keto-l-Gulonic Acid from d-Glucose by Two-Stage Fermentation.

A practical method for the production of calcium 2-keto-l-gulonate (an intermediate in the Reichstein synthesis of l-ascorbic acid) from d-glucose has been established by using a two-stage fermentation system. d-Glucose was first converted to calcium 2,5-diketo-d-gluconate by a mutant strain of Erwinia sp. in a medium containing d-glucose, corn steep liquor, (NH(4))(2)HPO(4), and CaCO(3). After a 26-h cultivation, 328.6 mg of calcium 2,5-diketo-d-gluconate per ml was obtained, with a 94.5% yield from d-glucose. This broth was used directly for the next conversion without removal of cells by treatment with sodium dodecyl sulfate. The stereospecific reduction of calcium 2,5-diketo-d-gluconate to calcium 2-keto-l-gulonate was performed with a mutant strain of Corynebacterium sp. When the cell growth reached a maximum (about 16 h) in a medium containing d-glucose, corn steep liquor, NaNO(3), KH(2)PO(4), and trace elements, NaNO(3) was added to the culture, and then the calcium 2,5-diketo-d-gluconate broth was fed over a period of about 50 h. Since the mutant strain requires a hydrogen donor for reduction, the calcium 2,5-diketo-d-gluconate broth was mixed with d-glucose before being fed. The results of four two-stage fermentations in 10-m conventional fermentors showed that an average of 106.3 mg of calcium 2-keto-l-gulonate per ml was obtained, with a 84.6% yield from d-glucose, the starting material of calcium 2,5-diketo-d-gluconate production. Calcium 2-keto-l-gulonate was stable in the broth. Neither 2-keto-d-gluconic acid nor 5-keto-d-gluconic acid was detected in the final broth.