Studied On Screening Of A Thermostable Phospholipase C Producing Bacterium And Its Enzyme Characterization

Phospholipase is phospholipid metabolism related enzymes in organisms that catalyze the hydrolysis of glycerol phospholipid reaction, hydrolysis products include a variety of phosphatidic acid and amino alcohol, such as choline, serine, ethanolamine, etc. According to the position of the hydrolyze phospholipids, phospholipases can be divided into phospholipase A1, A2, B, C, D. They play an important physiological function in the organism.The commercial application prospect of phospholipase is widespreaded. PLA1, PLA2and PLC, PLD can be widely used in oil refining, phospholipids modification, food and agricultural pesticides. PLC was applied to antitumor drugs.Phospholipase especially have broad application prospects in vegetable oil degumming, etc. Phospholipase widely exists in animals, plants and microorganisms.The microbial sources of phospholipase are varied and easy to mass production, which become the main source of industrial enzymes.Eighty phospholipase producing strains were isolated from the soil samples collected in Gansu province by the method of egg yolk plate. One strain JYSY1was selected for the high growth and production of phospholipases. The strain JYSY1was preliminarily identified as Bacillus cereus by morphological characteristics, physiological and biochemical, and sequence analysis of16S rDNA. The optimum temperature for cell growth and phospholipase production were25℃and30℃.The optimum pH value for the cell growth and phospholipase production was pH7.0. Optimum culture time is30h. A phospholipase was purified from the culture supernatant of the strain JYSY1by means of ammonium sulfate precipitation, column chromatography on DEAE-Sepharose fast flow and Sephadex-G100. The enzyme appeared to be a single peptide of48kDa on SDS-PAGE. The optimal temperature and pH of for the enzyme reactivity were50℃and8.0respectively. Addition of0.lmmol/L Co2+and Ca2+increased the activity to111.59%and110.11%respectively, but Zn2+and Mg2+inhibited the enzymatic activity respectively. Addition of1mM Zn2+and Mg2+inhibited the activity to58.13%and62.65%.Addition of1mM of PMSF, SDS, EGTA and EDTA also inhibited the enzymatic activity. At 65℃, there still remained about55%enzyme activity. The purified enzyme was identified to be a thermal stability phospholipase C. The enzyme was also suggested to be a metallophospholipase.