单核细胞增生性李斯特菌磷脂酶C的克隆表达及其在油脂脱胶中的应用研究

发布时间：2018-10-08 10:22

【摘要】：为研制具有自主知识产权的油脂脱胶用磷脂酶C（PLC）制剂，本文克隆了单核细胞增生性李斯特菌磷脂酶C基因（lm-plcB），构建了重组大肠杆菌，通过优化发酵条件，获得了高效表达的重组PLC，对其酶学性质进行研究，最终应用于植物毛油的酶法脱胶，取得了较好的效果。具体研究内容如下： 克隆了lm-plcB基因，构建重组大肠杆菌E.coil BL21(DE3)/pET28a-lm-plcB，利用乳糖诱导剂对其进行诱导表达，并对所获重组PLC进行鉴定分析。结果表明，lm-plcB基因在重组菌E.coil BL21(DE3)/pET28a-lm-plcB中实现高效表达，该重组PLC的分子质量约为30.4kDa，对天然磷脂底物具有活性。 优化了重组大肠杆菌E.coil BL21(DE3)/pET28a-lm-plcB的发酵条件，结果表明，当接种量为4%时，37℃，200r/min培养2h后，利用2.5g/L乳糖进行诱导，25℃摇瓶诱导发酵26h，可获得重组PLC的最佳酶活力达到754.6U/mL。经过离子交换层析对重组PLC进行分离纯化，最终得到的重组PLC纯化度约为83%，比酶活为473.1U/mg。 研究了重组PLC的酶学性质，结果表明，最适温度为60℃；60℃条件下保温30min，PLC酶活能维持在95%以上；最适pH为5.0；当缓冲液pH在6.0~7.0左右时，残留酶活力能保留80%以上，pH为6.0时具有最佳pH稳定性。金属离子对酶活力有一定影响，其中Zn2+、Ba2+、Mg2+、Mn2+对PLC具有激活作用；Ca2+、Fe3+对PLC酶活力几乎没有影响；Cd2+对PLC酶活力有抑制作用。利用不同的磷脂底物对其进行底物特异性研究，结果表明，该PLC对磷脂酰胆碱（PC）、磷脂酰乙醇胺（PE）水解作用较强，对磷脂酰肌醇（PI）也具一定水解作用，对磷脂酸（PA）无水解作用。 最后，利用该重组PLC对四种常见植物毛油进行脱胶。结果表明：该PLC能将高磷含量大豆毛油的磷含量从398.21mg/kg降至5.28mg/kg，，脱胶（磷）率达98.67%；低磷含量大豆毛油的磷含量从216.67mg/kg降至3.71mg/kg，脱胶（磷）率达98.40%；菜籽毛油的磷含量从183.70mg/kg降至4.50mg/kg，脱胶（磷）率达97.55%；米糠毛油的磷含量从306.82mg/kg降至50.19mg/kg，脱胶（磷）率达83.63%。该PLC对高磷含量大豆毛油的优化脱胶工艺条件为：pH5.5，55℃，添加量1000U/kg油，反应1.5h，可将大豆毛油中磷含量从398.22mg/kg降至4.28mg/kg，满足物理精炼的要求，同时将油脂得率提高约0.5%以上。 综上，本文在大肠杆菌中成功克隆表达了单核细胞增生性李斯特菌PLC，最终应用于四种不同植物毛油酶法脱胶，能够降低毛油中的磷含量，满足物理精炼的要求，并将油中磷脂水解成甘油二酯（DAG），提高油脂的精炼得率，应用前景广阔。
[Abstract]:The phospholipase C gene (lm-plcB) of Listeria monocytogenes (Listeria monocytogenes) was cloned and recombinant Escherichia coli was constructed in order to produce phospholipase C (PLC) for oil degumming with intellectual property rights. The high expression recombinant PLC, was obtained to study its enzymatic properties. Finally, the recombinant PLC, was applied to the enzymatic degumming of plant hair oil, and good results were obtained. The specific research contents are as follows: lm-plcB gene was cloned, recombinant Escherichia coli E.coil BL21 (DE3) / pET28a-lm-plcBwas constructed, and its expression was induced by lactose inducer, and the recombinant PLC was identified and analyzed. The results showed that lm-plcB gene was highly expressed in E.coil BL21 (DE3) / pET28a-lm-plcB, and the molecular weight of the recombinant PLC was about 30.4 kDa, which was active to natural phospholipid substrate. The fermentation conditions of recombinant Escherichia coli E.coil BL21 (DE3) / pET28a-lm-plcB were optimized. The results showed that the best enzyme activity of recombinant PLC was 754.6 UmL after 2 h culture at 37 鈩

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