A NEW CHITOSANASE GENE FROM A NOCARDIOIDES SP IS A 3RD MEMBER OF GLYCOSYL HYDROLASE FAMILY-46

Strain N106, a newly isolated soil actinomycete classified in the genus Nocardioides on the basis of its chemotaxonomy, produced an extracellular chitosanase and was highly active in chitosan degradation. A gene library of Nocardioides sp. N106 was constructed in the shuttle vector pFD666 and recombinant plasmids carrying the chitosanase gene (csnN106) were identified using the 5'-terminal portion of the chitosanase gene from Streptomyces sp. N174 as a hybridization probe. One plasmid, pCSN106-2, was used to transform Streptomyces lividans TK24. The chitosanase produced by 5. lividans(pCSN106-2) is a protein of 29.5 kDa, with a pl 8.1, and hydrolyses chitosan by an endo-mechanism giving a mixture of dimers and trimers as endproducts. N-terminal sequencing revealed that the mature chitosanase is a mixture of two enzyme forms differing by one N-terminal amino acid. the csnN106 gene is 79.5% homologous to the csn gene from Streptomyces sp. N174. At the amino acid level, both chitosanases are homologous at 74.4% and hydrophobic cluster analysis revealed a strict conservation of structural features. This chitosanase is the third known member of family 46 of glycosyl hydrolases.