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 genu s Nocardioides on the basis of its chemotaxonomy, produced an extracel lular chitosanase and was highly active in chitosan degradation. A gen e library of Nocardioides sp. N106 was constructed in the shuttle vect or pFD666 and recombinant plasmids carrying the chitosanase gene (csnN 106) 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 chit osanase produced by 5. lividans(pCSN106-2) is a protein of 29.5 kDa, w ith a pl 8.1, and hydrolyses chitosan by an endo-mechanism giving a mi xture of dimers and trimers as endproducts. N-terminal sequencing reve aled that the mature chitosanase is a mixture of two enzyme forms diff ering by one N-terminal amino acid. the csnN106 gene is 79.5% homologo us to the csn gene from Streptomyces sp. N174. At the amino acid level , both chitosanases are homologous at 74.4% and hydrophobic cluster an alysis revealed a strict conservation of structural features. This chi tosanase is the third known member of family 46 of glycosyl hydrolases .