食用油生物制造高效特异性脂酶的创制

廖祥儒 喻晓蔚

摘 要：该研究利用酶的分子改造与高效制备技术，自主创制并筛选获得食用油制造用高效底物专一性脂酶总计11种，其中3种脂肪酶具有高1，3-位置选择性以及酯化能力，4种脂肪酶具有高转酯化活力及高热稳定性，1种磷脂酶具有专一性磷脂-2-酰基水解作用，适用于油脂酶法脱胶。代表性成果如下：（1）创制了适用于油脂改性的1，3-位置选择性脂肪酶，通过共表达伴侣蛋白、基因剂量调控以及氮源发酵调控将酶活提高到11719U/mL。构建了一系列解酯耶氏酵母Lip2基因工程菌，发酵活力达到30 800 U/mL，为目前国际报道最高。（2）创制了热稳定性酯交换特异性脂肪酶，该研究采用限定区域内的随机突变和结构热点位置的饱和突变的策略构建基因突变库，创造一些全新的酶反应，大大扩展酶催化的应用范围。（3）创制了油脂脱胶用磷脂酶，该酶对甘油三酯没有降解作用，与国外的酶制剂相比，专一性强，对水解条件和原料的要求宽松；适应性强，使用温度范围较宽，成本低；利用多拷贝质粒构建的链霉菌磷脂酶A2高产重组菌株的活力达到3 000 U/mL，对现有的酶法脱胶工艺进行了改进，使之更加简便和节能。

关键词：酯酶 脂肪酶 食用油

Abstract：In this study，by the technology of molecular evolution and preparation， totally 11 kinds of esterase and lipases for edible oil production were created，of which three kinds of lipase showed high 1，3-position selectivity，and esterification capabilities， four kinds of lipase exhibited high transesterification activity with high thermal stability， one kind of specific phospholipase for enzymatic degumming could hydrolysis phospholipids at sn-2 position. Representative results are as follows：（1）A 1，3-positions selective lipase used for oil modification was created. Through coexpression of chaperone， gene dosage optimization and fermentation the lipase activity reached 1 1719 U/mL. Construct a series of engineered yeast strains for production of Yarrowia Lip2 and the fermentation activity reached 3 0800 U/mL， which was the highest ever reported.（2）A series of thermal stable lipases and esterases were created with high transesterification specificity.This study constructed gene library by adopting random mutations within the defined protein region and hotspot of the structure，which created some new enzyme properties and greatly expanded the scope of application.（3）A phospholipase for oil degumming was created which showed no degradation activity towards triglyceride.This enzyme was also not stringent to reaction conditions and raw material. Taking advantage of multi-copy plasmid technology， Streptomyces phospholipase A2 activity reached 3 000 U/mL. The enzymatic degumming process has been improved to make it more convenient and efficient.

Key Words：Esterases；Lipases；Edible Oil

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