Purification, characterization, and gene analysis of a chitosanase (ChoA) from Matsuebacter chitosanotabidus 3001

dThe extracellular chitosanase (34,000 M-r) produced by a novel gram-negati ve bacterium Matsuebacter chitosanotabidus 3001 was purified. The optimal p H of this chitosanase was 4.0, and the optimal temperature was between 30 a nd 40 degrees C. The purified chitosanase was most active on 90% deacetylat ed colloidal chitosan and glycol chitosan, both of which were hydrolyzed in an endosplitting manner, but this did not hydrolyze chitin, cellulose, or their derivatives. Among potential inhibitors, the purified chitosanase was only inhibited by Ag+ Internal amino acid sequences of the purified chitos anase were obtained. A PCR fragment corresponding to one of these amino aci d sequences was then used to screen a genomic library for the entire choA g ene encoding chitosanase. Sequencing of the choA gene revealed an open read ing frame encoding a 391-amino-acid protein. The N-terminal amino acid sequ ence had an excretion signal, but the sequence did not show any significant homology to other proteins, including known chitosanases. The 80-amino-aci d excretion signal of ChoA fused to green fluorescent protein was functiona l in Escherichia coli. Taken together, these results suggest that we have i dentified a novel, previously unreported chitosanase.