Studies On Screening Producing Phospholipase C And Its Enzymatic Properties

Ceramide is a lipid second messager, which is involved in diverse cell activities, such as cell proliferation, differentiation, cell cycle arrest, senescence and apoptosis. It also has great value in market and adds in pharmaceutical health care products, food and cosmetic. Binding phosphorylcholine by the Sphingomyelin (SM) in konjac fly power, Ceramide is difficult to isolate and the yield is too low, so it seriously affects the industrialization and application of ceramide. Camparetly, biosynthesis of ceramide invirto is an effective method to produce ceramide in large quantities. Whereas the lack of phospholipase C (PLC) restricts biosynthesis of ceramide in vitro. In order to solve this key problem, researches in thesis are based on the enzyme resources of microorganism due to microorganism being cultured easily and excreted PLC in large amount from the culture medium. The aim of this study, firstly was to screen a stain producing PLC in high quantity, secondly was to purify PLC for further determination of its characteristics, and finally was to lay strong foundations for PLC developing konjac ceramide in the future. The main results obtained are as follows:1. Screening a strain producing phospholipase C and its identificationStrain Z-13 is screened primally from borate plate with egg yolk, and then followed by determination of biomass, activity of PLC by method of p-nitrophenylphosphorylchoine. It is classfied to Bacillus cereus according to morphological, physiological and biochermical characteristics2. Optimization of fermenting conditions for PLC by B. cereus Z-13The optimal conditions for fermenting PLC of B. cereus Z-13 are conformed by single-factor experiments and uniform design experiments. The results show optimal conditions are as follows:Konjac powder 2.5g/L, Soybean flour 30g/L, K2HPO4·3H2O 2.8g/L, Zn2SO4·7H2O 1.3 g/L, with inatial pH 9.0, inoculating ratio 1.0%, rotating speed 150 r/min when cultured in shaking flask at 32℃for 15 hrs. The living cells reach 4.5×109 CFU/mL and the diameter of sedimentary circle (milky white spot) reach 30 mm by egg yolk cylinder plate method under the optimal conditions. The activity of phospholipase C reaches 23.31 U/mL 3. Purification of PLC by B.cereus. Z-13The PLC was successfully purified by ammonium sulfate precipitation followed by application of ion-exchange chromatography (DEAE-52), sepharose 6B and gel filtration (Sephadex G-75 chromatography). Results show purification fold of PLC is 39.98, with Specific activity 135.15 U/mg protein and activity of PLC recovery 20.93%. The molecular mass and purity of the PLC is determined as one subuint with molecular mass 3.4 ku by methods of Native-PAGE and SDS-PAGE.4. Partial charaeterization of PLC by B.cereus. Z-13The optimum temperature and pH of the PLC is 55℃and pH7.5, respectively. The activity of PLC decreases sharply when temperature is over 60℃, suggesting that the PLC was heat-labile. The activity of PLC is stable at pH 6.0-8.0, but decreases sharply when pH is lower 6.0. Ions of Zn+2, Ca+2, Mg+2 are able to active activity of PLC, while inhibited by Mn+2,Cu+2.5. Preparation of ceramide from sphingomyelin degraded by PLC of B.cereus Z-13Free ceramide is released from sphingomyelin in konjac fly power by hydrolyzation of PLC in two-liquid-phase system. The content of ceramide reaches 0.725 mg/mL determinated by method of HPLC-ELSD, indcating 2.9% ceramide increaded camparetively to method of direct extraction.