Cloning and overexpression of a new chitosanase gene from Penicillium sp D-1

A chitosanase gene, csn, was cloned from Penicillium sp. D-1 by inverse PCR. The cDNA sequence analysis revealed that csn had no intron. The deduced CSN protein consists of 250 amino acids including a 20-amino acid signal peptide, and shared 83.6% identity with the family 75 chitosanase from Talaromyces stipitatus (B8M2R4). The mature protein was overexpressed in Escherichia coli and purified with the affinity chromatography of Ni2+-NTA. The novel recombinant chitosanase showed maximal catalytic activity at pH 4.0 and 48 degrees C. Moreover, the activity of CSN was stable over a broad pH range of 3.0-8.0, and the enzymatic activity was significantly enhanced by Mg2+ and Mn2+. The CSN could effectively hydrolyze colloidal chitosan and chitosan, while could not hydrolyze chitin and carboxymethylcellulose (CMC). Due to the particular acidophily, CSN has the potential application in the recycling of chitosan wastes. The GenBank/EMBL/DDBJ accession numbers for the 18S rRNA gene and chitosanase gene of strain D-1 are JF950269 and JF950270, respectively.