Characterization of a novel fungal chitosanase Csn2 from Gongronella sp. JG.

A 28kDa chitosanase designated as Csn2 was purified from the culture broth of the fungus Gongronella sp. JG through three chromatography steps: CM-Sepharose FF, Superdex 200 and SP-Sepharose FF. Its optimal reaction pH and temperature were pH 5.6 and between 55 degrees C and 60 degrees C. The half-lives of Csn2 at 50 degrees C and 55 degrees C were estimated to be 30min and 11min, respectively. The K(m) value of Csn2 in sodium acetate buffer (pH 5.6) at 55 degrees C was 8.86mg/mL. Mn(2+), Ca(2+) and Sr(2+) were activators of Csn2; ETDA was an inhibitor. Cu(2+) stimulated Csn2 at 1mM, but inhibited Csn2 activity at 10mM. Csn2 displayed strong activity on colloidal chitosan, but did not hydrolyze colloidal chitin and carboxylmethyl cellulose. Thin layer chromatography analysis showed the end products of colloidal chitosan hydrolyzed by Csn2 were chitobiose, chitotriose and chitotetraose with chitotriose as the major product. The N terminus of Csn2 was determined to be YQLPANLKKIYDSHKSGTC. Part of the genomic DNA sequence corresponding to Csn2 was cloned. Sequence alignment showed DNA sequence of Csn2 was partly identical to chitosanase genes from Metarhizium anisopliae var. acridum, Hypocrea lixii and Aspergillus fumigatus. Based on sequence similarity, Csn2 was classified as a GH-75 chitosanase.