Screening Of Protease Producing Marine Yeast, The Optimization Of Fermentation Conditions And Enzyme Properties

A strain HN311with high protease activity was isolated from sediments of saltern near Qingdao, China. It was identified belong to the Aureobasidium pullulans. The optimal compositions of the medium for HN311 strain were studied as follows(%,w/v): soluble starch 2.5, NaNO3 1.0,the initial pH6.0. The optimal producing protease conditions for HN311 were illustrated below: amount of inoculation10%(v/v) , incubating temperature 24℃, shaking cultivation 150 rpm and aeration 8L/min. The protease activity of fermented broth could reach 623.1U/mL when HN311 was incubated under the optimal cultivition conditions. The protease of the strain was purified by gel filtration (Sephadex G-100 colum) and ion-exchange chromatography (CM-Sepharose Fast Flow colum), SDS-PAGE showed a single band for the purified protease and the molecular weight was estimated to be 33kD, Km=0.654mg/mL and Vmax =25.64μmol/min/mL。The optimal temperature and pH for hydrolysis of the protease were 45℃and 10, respectively. The changes of pH and temperature had the significant influences on the protease activity. Mn2+ ion enhanced the enzyme activity significantly. 10mmol/l or 0.01mmol/l Mg2+,Ca2+,and Na+ and 0.01mmol/l Fe3+,Fe2+,Zn2+ and Cu2+ cannot effect the protease activity ,whereas10mmol/l Fe3+,Fe2+,Zn2+ and Cu2+ inhibited the enzyme activity. The enzyme showed activity for hydrolysis of protein. PMSF,SDS can inhibit the enzyme activity obviously.The eazyme was the serine protease which can seen from above.