Over-expression of a bacterial chitosanase gene in Yarrowia lipolytica and chitosan hydrolysis by the recombinant chitosanase

摘要

The bacterial strain OUC1 was found to be closely related to Pseudomonas sp. A-01. A chitosanase gene (chi) cloned from the marine bacterium Pseudomonas sp. 0UC1 was over-expressed in Yarrowia lipolytica which is generally regarded as safe. After the expression vector carrying two copies of the chi gene was transformed into V. lipolytica, the transformants obtained could secrete much more chitosanase (9.0U/ml) than those (4.3 U/ml) transformed with the expression vector carrying one copy of the chi gene. The molecular mass of the recombinant chitosanase was 31.5 kDa. The optimal pH and temperature of the recombinant chitosanase were 6.0 and 50 °C, respectively. Mn~(2+) was found to greatly enhance its activity. K_m and V_(max) for chitosan were 6.77 mg/ml and 0.224 mg/ml min, respectively. Chitosan was actively hydrolyzed by the recombinant chitosanase into GluN2, GluN3, GluN4, GluN5 and GluN6.