Improved purification and some properties of a novel phospholipase C from Bacillus mycoides strain 970

A novel microbial phospholipase C from Bacillus mycoides strain 970 was purified to apparent homogeneity by an improved purification process, in which DEAE-Cellulose adsorption was first used to remove bulk of dark brown impurities and then a combination of DEAE-Cellulose ion-exchange, Phenyl Sepharose hydrophobic interaction and Sephacryl S-100 gel filtration chromatography was used to separate the enzyme from remained contaminants. The enzyme appeared to be a single peptide of 75.1 kDa on SDS-PAGE. The optimum temperature and pH of the enzyme were 60 degrees C and 7.0 - 7.5, respectively. The enzyme was stable at temperatures lower than 50 degrees C and pH 5 - 9.5. This purified phospholipase C was characterized as a metallophospholipase C and Ca2+, Mg2+, Zn2+, Ba2+ were essential for its PLC activity. The enzyme showed no lecithinase activity on egg yolk agar and also no hemolytic activity on blood agar.