CLONING, SEQUENCING, AND EXPRESSION OF THE GENE ENCODING CLOSTRIDIUM-PARAPUTRIFICUM CHITINASE CHIB AND ANALYSIS OF THE FUNCTIONS OF NOVEL CADHERIN-LIKE DOMAINS AND A CHITIN-BINDING DOMAIN

The Clostridium paraputrificum chiB gene, encoding chitinase B (ChiB), consists of an open reading frame of 2,493 nucleotides and encodes 83 1 amino acids with a deduced molecular weight of 90,020. The deduced C hiB is a modular enzyme composed of a family 18 catalytic domain respo nsible for chitinase activity, two reiterated domains of unknown funct ion, and a chitin-binding domain (CBD). The reiterated domains are sim ilar to the repeating units of cadherin proteins but not to fibronecti n type III domains, and therefore they are referred to as cadherin-lik e domains. ChiB was purified from the periplasm fraction of Escherichi a coli harboring the chiB gene. The molecular weight of the purified C hiB (87,000) by sodium dodecyl sulfate-polyacrylamide gel electrophore sis (SDS-PAGE) analysis, was in good agreement with the value (86,578) calculated from the deduced amino acid sequence excluding the signal peptide. ChiB was active toward chitin from crab shells, colloidal chi tin, glycol chitin, and 4-methylumbelliferyl beta-D-N,N'-diacetylchito bioside [4-MU-(GlcNAc)(2)]. The pH and temperature optima of the enzym e was 6.0 and 45 degrees C, respectively. The K-m and V-max values for 4-MU-(GlcNAc)(2) were estimated to be 6.3 mu M and 46 mu mol/min/mg, respectively. SDS-PAGE, zymogram, acid Western blot analyses using ant iserum raised against purified ChiB suggested that ChiB was one of the major chitinase species in the culture supernatant of C. paraputrific um. Deletion analysis showed clearly that the CBD of ChiB plays an imp ortant role in hydrolysis of native chitin but not processed chitin su ch as colloidal chitin.