| As a new technology in oil refining, enzyme degumming has several advantages overtraditional oil degumming, such as mild reaction condition, wide range of application, lesspollution to environment and so on. However, PLC has drew wide attention of oil companynot only for its short reaction time, but the efficiency of converting phospholipid into1,2-Diacylglycerol(1,2-DAG), which could increase the oil yield obviousely. In this study,PLC from Clostridium perfringens (Cp-PLC) was cloned and expressed in E. coli BL21(DE3),and the recombinantional enzyme was applied to plant oil degumming.First of all, gene plc (gene encoding PLC of full length) and gene nplc (gene encodingthe N domain of PLC) of Clostridium perfringens were cloned respectively, and therecombinants E. coli BL21(pET-28a/Cp-plc) and E. coli BL21(pET-28a/Cp-nplc) aresuccessfully constructed. After identification, only the recombinational protein of E. coliBL21(pET-28a/Cp-plc) showed PLC activity.Secondly, after single factor optimization, the optimal fermentation condition of E. coliBL21(pET-28a/Cp-plc) was decided: OD600=1.0, final concentration of lactose8g/L, with theinduction temperature of30℃for32h. Under this condition, the ultimate PLC activityreached396.40±9.7U/mL.In order to study the enzymology properties of Cp-PLC, affinity chromatography wasused, and the purity of the recombinational Cp-PLC reached85.0%after purification. Gelfiltration chromatography was performed for further purification. Finally,93.3%purity ofCp-PLC was obtained and the enzyme activity reached248.3±5.6U/mg.The hydrolysis effect of purified Cp-PLC was analyzed with different soybeanphospholipids (PE、PC、PAand PI) via high performance liquid chromatography (HPLC). Theresults indicated that Cp-PLC could hydrolyze PE and PC, whereas, there were no obvioushydrolysis effect of Cp-PLC to PA or PI. Further analysis of hydrolysate of PE and PCshowed that1,2-DAG is the main production. According to the new enzyme activitydefinition defined by PE, the activity of recombinational Cp-PLC reached357.5U/ml.In addtion, recombinational Cp-PLC was applied to degumming process of cruderapeseed oil and soybean oil. The optimal degumming condition of crude rapeseed oil byCp-PLC was determined as follows:57℃, pH5.0, enzyme dosage of600U/kg, reaction timeof1.5h. Under this condition, the phosphorus content of rapeseed oil decrease from183.71mg/kg to5.66±0.61mg/kg. To obtain accurate results, Crude soybean oil was chosed for yieldmeasurement, the initial phosphorus content of which was398.22mg/kg. The degummingprocess indicated that the yield of oil reached95.74%and increased by0.48%with Cp-PLC,which provided theoretical reference for the application of Cp-PLC in plant oil enzymedegumming.In conclusion, recombinational Cp-PLC could effectively reduce phosphorus content ofplant oil, and improve oil yield during refining process. Therefore, Cp-PLC has broadapplication prospect in plant oil degumming. |
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