【摘要】：為研制具有自主知識(shí)產(chǎn)權(quán)的油脂脫膠用磷脂酶C（PLC）制劑，本文克隆了單核細(xì)胞增生性李斯特菌磷脂酶C基因（lm-plcB），構(gòu)建了重組大腸桿菌，通過(guò)優(yōu)化發(fā)酵條件，獲得了高效表達(dá)的重組PLC，對(duì)其酶學(xué)性質(zhì)進(jìn)行研究，最終應(yīng)用于植物毛油的酶法脫膠，取得了較好的效果。具體研究?jī)?nèi)容如下： 克隆了lm-plcB基因，構(gòu)建重組大腸桿菌E.coil BL21(DE3)/pET28a-lm-plcB，利用乳糖誘導(dǎo)劑對(duì)其進(jìn)行誘導(dǎo)表達(dá)，并對(duì)所獲重組PLC進(jìn)行鑒定分析。結(jié)果表明，lm-plcB基因在重組菌E.coil BL21(DE3)/pET28a-lm-plcB中實(shí)現(xiàn)高效表達(dá)，該重組PLC的分子質(zhì)量約為30.4kDa，對(duì)天然磷脂底物具有活性。 優(yōu)化了重組大腸桿菌E.coil BL21(DE3)/pET28a-lm-plcB的發(fā)酵條件，結(jié)果表明，當(dāng)接種量為4%時(shí)，37℃，200r/min培養(yǎng)2h后，利用2.5g/L乳糖進(jìn)行誘導(dǎo)，25℃搖瓶誘導(dǎo)發(fā)酵26h，可獲得重組PLC的最佳酶活力達(dá)到754.6U/mL。經(jīng)過(guò)離子交換層析對(duì)重組PLC進(jìn)行分離純化，最終得到的重組PLC純化度約為83%，比酶活為473.1U/mg。 研究了重組PLC的酶學(xué)性質(zhì)，結(jié)果表明，最適溫度為60℃；60℃條件下保溫30min，PLC酶活能維持在95%以上；最適pH為5.0；當(dāng)緩沖液pH在6.0~7.0左右時(shí)，殘留酶活力能保留80%以上，pH為6.0時(shí)具有最佳pH穩(wěn)定性。金屬離子對(duì)酶活力有一定影響，其中Zn2+、Ba2+、Mg2+、Mn2+對(duì)PLC具有激活作用；Ca2+、Fe3+對(duì)PLC酶活力幾乎沒(méi)有影響；Cd2+對(duì)PLC酶活力有抑制作用。利用不同的磷脂底物對(duì)其進(jìn)行底物特異性研究，結(jié)果表明，該P(yáng)LC對(duì)磷脂酰膽堿（PC）、磷脂酰乙醇胺（PE）水解作用較強(qiáng)，對(duì)磷脂酰肌醇（PI）也具一定水解作用，對(duì)磷脂酸（PA）無(wú)水解作用。 最后，利用該重組PLC對(duì)四種常見(jiàn)植物毛油進(jìn)行脫膠。結(jié)果表明：該P(yáng)LC能將高磷含量大豆毛油的磷含量從398.21mg/kg降至5.28mg/kg，，脫膠（磷）率達(dá)98.67%；低磷含量大豆毛油的磷含量從216.67mg/kg降至3.71mg/kg，脫膠（磷）率達(dá)98.40%；菜籽毛油的磷含量從183.70mg/kg降至4.50mg/kg，脫膠（磷）率達(dá)97.55%；米糠毛油的磷含量從306.82mg/kg降至50.19mg/kg，脫膠（磷）率達(dá)83.63%。該P(yáng)LC對(duì)高磷含量大豆毛油的優(yōu)化脫膠工藝條件為：pH5.5，55℃，添加量1000U/kg油，反應(yīng)1.5h，可將大豆毛油中磷含量從398.22mg/kg降至4.28mg/kg，滿(mǎn)足物理精煉的要求，同時(shí)將油脂得率提高約0.5%以上。 綜上，本文在大腸桿菌中成功克隆表達(dá)了單核細(xì)胞增生性李斯特菌PLC，最終應(yīng)用于四種不同植物毛油酶法脫膠，能夠降低毛油中的磷含量，滿(mǎn)足物理精煉的要求，并將油中磷脂水解成甘油二酯（DAG），提高油脂的精煉得率，應(yīng)用前景廣闊。
[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

本文編號(hào)：2256420