吡咯伯克霍尔德氏菌Burkholderia puraquae新型磷脂酶C催化特性及其应用研究  

Catalytic properties and application of a new phospholipase Cfrom Burkholderia puraquae

作　　者：李科娜 吴楠 杨旭 孙永威 段晨阳 张志平 宋丽丽 张靖楠 魏涛 LI Kena;WU Nan;YANG Xu;SUN Yongwei;DUAN Chenyang;ZHANG Zhiping;SONG Lili;ZHANG Jingnan;WEI Tao(School of Food and Biological Engineering,Zhengzhou University of Light Industry,Zhengzhou 450002,China)

机构地区：[1]郑州轻工业大学食品与生物工程学院,河南郑州450002

出　　处：《食品与发酵工业》2023年第19期38-43,共6页Food and Fermentation Industries

基　　金：中原科技创新领军人才项目(224200510017)。

摘　　要：酶法脱胶是一种植物油脂脱胶的绿色新工艺,具有反应条件温和、脱胶完全、副产物少、得油率更高、生产用水和废水排放少等特点,因此近年来受到油脂行业的广泛关注。磷脂酶C是油脂酶法脱胶技术重要的工业用酶之一,但目前已报道的磷脂酶C存在催化活性、稳定性不高、微生物来源酶不易分离纯化和重组酶多以包涵体表达等问题。该研究从吡咯伯克霍尔德氏菌(Burkholderia puraquae)克隆表达磷脂酶C基因plc_bp,构建重组大肠杆菌工程菌,研究重组酶酶学性质和油脂脱胶效果。通过镍柱亲和层析分子筛层析,得到纯化重组酶PLC_BP,SDS-PAGE分析表明该酶分子质量约为38 kDa。酶学性质研究表明,该酶最适温度和pH分别为60℃和8.5,具有较强的热稳定性,在60℃处理8 h保持50%左右活力。利用同源建模构建PLC_BP蛋白三维结构,发现该酶具有双核锰离子结合位点,通过定点突变鉴定D93和H169为锰离子结合关键位点。将该酶应用于不同来源毛油脱胶中,可以有效降低菜籽油、大豆油、花生油和葵花籽油的磷含量(5 mg/kg以下),满足后续油脂精炼要求,该研究成果为油脂酶法脱胶提供新的酶源和应用基础。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.

关 键 词：PLC_BP 酶学性质 酶法脱胶 双核锰离子结合位点 热稳定性 

分 类 号：TQ426.97[化学工程]