Studies On Screening, Identification And Optimization Of Cultivation Conditions Of High Phospholipase C Strains

The studies were done to obtain new microorganisms by isolation and screening from plenty of collected soil samples,which have high phospholipase C production and phospholipase C of which has vigorous anti-platelets function,and to study purification and characteristics of their phospholipase C at the same time.These studies are hoped to lay strong foundations for developing phospholipase C into a new drug of anti-platelets in the future.The main methods used were as follows:1.LB plate agar with egg yolk was used to isolate individual colony.2.Qualitative determination of phospholipase C activity by borate plate with egg yolk, biomass,quantitative determination of phospholipase C activity by NPPC method and rate of anti-platelet aggregation were applied to screen strains for many times.3.Selected strains were identified according to their morphological,physiological and biochemical characteristics and 16s rRNA sequences.4.The effects of inoculum size,culture temperature,culture time and rotate speed on phospholipase C production were considered,and the optimal fermentation conditions were determined by orthogonal experiments.5.Molecular weight of phospholipase C was estimated by SDS-PAGE.6.The enzyme activity of Phospholipase C was assayed by NPPC method.7.Hemolytic activity of phospholipase C was investigated by blood agar.The main results obtained were as follows:1.Four new strains,which have high phospholipase C production,were obtained by isolation and screening.They were identified as B.cereus Shenzhen 754-1,B.mycoides Shenzhen 779,B.mycoides Shenzhen 970 and B.mycoides Shenzhen 1107 respectively.2.Optimal conditions of B.cereus Shenzhen 754-1 phospholipase C production by shaker were as follows:medium of 1%peptone and 0.3%beef extract,culture temperature 28℃,culture time 12h,inoculum size 0.5%and rotate speed 200 r/min. Phospholipase C activity of B.cereus Shenzhen 754-1 was up to 28.8 U/ml on these optimal conditions.3.Molecular weight of purified phospholipase C was estimated to be 75.1 KDa.It was heat-labile and no activity of phospholipase C was remained aider treatment in 70℃water bath for 5 minutes.4.The purified phospholipase C indicated no hemolytic activity on blood agar.The optimal temperature and pH of this enzyme was 60"C and pH7.0-7.2 in NPPC system respectively.5.In a word,four new bacteria of phospholipase C were obtained by the study.One of the four new Phospholipase C strains,B.cereus Shenzhen 754-1,had exceeded that of other microorga production of nisms reported.Phospholipase C from B.cereus Shenzhen 754-1 had different characteristics from that of other microorganisms.