Properties And Gene Cloning Of Chitosanase From Penicillium Sp. D-1

A strain producing chitosanase was isolated from soil samples and identified as Penicillium sp. D-1. It is the only one Penicillium strain that can produce two chitosanases reported so far. The optimal fermentation conditions for Penicillium sp. D-l producing chitosanase were incubated at 32, in 500ml Erlenmeyer flask containing 100ml medium (pH 5.0) with 2.5% chitosan as substrate. The chitosanase activity of fermented broth was 4.696U/ml when Penicillium sp. D-l was cultured under the optimal conditions at 108h. Two inducible and extracellular chitosanases (ChoA and ChoB) secreted by Penicillium sp. D-l were purified by ammonium sulfate precipitation as well as gel filtration. The purified enzymes of ChoA and ChoB had molecular masses of 93 and 21 kDa by SDS-PAGE. Both ChoA and ChoB have an acidic pH optimum, which were 4.0. The enzymes were stable over the pH range 3 to 5 for 1 h. The optimum temperatures for hydrolysis of the chitosanases were determined to be 52 for ChoA and 48 for ChoB, respectively. 80% activity of ChoA was maintained below 52 for 1 h, while 60% activity of ChoB was maintained below 40 . Metal ions of Mn2+ stimulate the ChoA activity and Ca2+ significantly stimulate the ChoB activity, while Cu2+, Fe3+, and Ag+ inhibited them. The Km and Vmax were 7.35 mg/ml and 0.206 nmol/min-mg for ChoA, and 30.42 mg/ml and 0.376(xmol/min-mg for ChoB, respectively. A 270bp DNA fragment, the partial chitosanase gene from Penicillium sp. D-1, was cloned and determined. The deduced amino sequence showed significant homology with the fungal chitosnases in NCBI.