Enzymatic degumming is a new green process for vegetable oil degumming. It has the characteristics of mild reaction conditions, complete degumming, less by-products, higher oil yield, less production water and wastewater discharge, and so it has been widely concerned by the oil industry in recent years. Phospholipase C is one of the most important industrial enzymes in the enzymatic degumming technology. However, the reported phospholipase C has some disadvantages, such as low catalytic activity and stability, difficult separation and purification of microbial enzymes, and recombinant enzymes are mostly expressed by inclusion bodies. In this study, the recombinant PLC_BP protein was cloned and expressed from Burkholderia puraquae, and the engineering bacteria of Escherichia coli was constructed. Enzymatic properties of the recombinant enzyme and the effect of oil degumming were studied. The purified PLC_BP was obtained by nickel column affinity chromatography and gel filtration. SDS-PAGE analysis showed that the molecular weight of the enzyme was about 38 kDa. The study of enzymatic properties showed that the optimum temperature and pH were 60℃ and 8.5, respectively. PLC_BP displayed strong thermal stability, and maintained about 50% activity after being treated at 60℃ for 8h.The three-dimensional structure of PLC_BP was constructed by homologous modeling. It was found that the enzyme had a binuclear manganese binding site. D93 and H169 were found to be the key binding sites for manganese ion through site directed mutagenesis. The enzyme was applied to the degumming of crude oil from different sources, and the phosphorus content of rapeseed oil, soybean oil, peanut oil, and sunflower seed oil were effectively reduced to below 5 mg/kg, which meeting the requirements of subsequent oil refining. The research results provide a new enzyme source and application basis for enzymatic degumming of oils.
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