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Chitinases Al and D were purified from the periplasmic proteins produced by Escherichia coli HBlOl harbouring recombinant plasmids carrying respectively the chiA and chiD genes of Bacillus circulans WL-12. HPLC analysis indicated that during the hydrolysis of chitotriose, both chitinases initially produce N-acetylglucosamine and only one anomer of chitobiose. 'H NMR spectroscopy of the hydrolysis of chitotetraitol showed that this anomer corresponds to p-chitobiose, demonstrating that chitinases Al and D act by a molecular mechanism that retains the anomeric configuration. This mechanism is similar to that of lysozymes although both chitinases belong to a family of proteins sharing no demonstrable amino acid sequence similarity with lysozymes.

The gene (bdb) for protein thiol-disulfide oxidoreductase cloned from Bacillus brevis was found to encode a polypeptide consisting of 117 amino acid residues with a signal peptide of 27 residues. Bdb contains a well-conserved motif, Cys-X-X-Cys, which functions as the active center of disulfide oxidoreductases such as DsbA, protein disulfide isomerase, and thioredoxin. The deduced amino acid sequence showed significant homology with those of several bacterial thioredoxins. The bdb gene complemented the Escherichia coli dsbA mutation, restoring motility by means of flagellar and alkaline phosphatase activity. The Bdb protein overproduced in B. brevis was enzymatically active in both reduction and oxidization of disulfide bonds in vitro. Immunoblotting indicated that Bdb could function at the periphery of the cell.Disulfide bonds play an important role in producing the three-dimensional structures responsible for the specific properties of proteins. Although disulfide bonds in a protein can form spontaneously in vitro, the process is much slower and less effective than that in vivo (20), suggesting the existence of proteins which catalyze native disulfide bond formation in vivo. Disulfide oxidoreductases such as thioredoxin and protein disulfide isomerase (PDI) contain the Cys-X-X-Cys motif as the catalytic active site (16) and facilitate disulfide exchange in vitro (10).The dsbA gene in Escherichia coli has been discovered independently by two groups (3, 12). DsbA is a periplasmic protein with the sequence motif Cys-Pro-His-Cys, which resembles the active site of thioredoxin and PDI. The dsbA mutation causes a defect in the disulfide bond formation of several periplasmic proteins such as alkaline phosphatase, the OmpA protein, ␤-lactamase (3), and the P-ring protein of the flagellar basal body (7). DsbA appears to be required for the formation of disulfide bonds. DsbA homologs are also found in bacteria such as Vibrio cholerae (19) and Haemophilus influenzae (26). They are thought to form a new family of bacterial disulfide oxidoreductase.All bacterial disulfide oxidoreductases have been isolated only from gram-negative bacteria, while no data have been obtained on those of gram-positive bacteria. In gram-positive bacteria, secretory nascent polypeptides are transported to the external medium and must fold into precise shapes to produce functional proteins outside or on the surface of the cell. It is of interest to examine how extracellular proteins acquire disulfide bonds in the protein secretory pathway. We describe here the cloning and characterization of a gene termed bdb (for Bacillus disulfide bond formation), which encodes the protein thioldisulfide oxidoreductase of Bacillus brevis, a gram-positive bacterium used as a host for heterologous protein production (31). MATERIALS AND METHODSBacterial strains, media, plasmids, and transformation. The bacterial strains used were B. brevis HPD31 (24) and E. coli XL1-Blue (Stratagene), JCB502 ( D69 lacZ::Tn10 tetS by fusaric acid) (3), JCB572 (JCB502 dsbA::kan-1) (...

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