Production optimization and expression of pectin releasing enzyme from Aspergillus oryzae PO

Protopectinase is an enzyme that solubilizes protopectin forming highly polymerized soluble pectin. Protopectinase activity was detected from Aspergillus oryzae PO isolated from soil of persimmon orchard. Response surface methodology of Box–Behnken Design with three fermentation variables (temperature, NaNO3 and apple pomace concentration) was used to optimize protopectinase production of A. oryzae PO, and protopectinase activity was improved to 270.0 U/ml. Endo-polygalacturonase belonged to A-type PPase from A. oryzae PO was cloned and expressed in Pichia pastoris GS115. The endo-polygalacturonase expression was 0.418 mg/ml and the specific activity of purified recombinant endo-polygalacturonase was 7520 U/mg toward polygalacturonic acid. The optimal temperature and pH of recombinant endo-polygalacturonase were 45 °C and 5.0, respectively. The recombinant endo-polygalacturonase activity was enhanced by the presence of Mg2+, while Ca2+, Ni2+ Mn2+, Cu2+ and SDS strongly inhibited the enzyme activity. The apparent Km value and Vmax value were 5.59 mg/ml and 1.01 μmol/(min ml), respectively.