偏甘油酯脂肪酶PrLip的重组表达及酶学性质表征

Recombination Expression and Enzymatic Characteristics of Mono-and Diacylglycerol Lipase PrLip

偏甘油酯脂肪酶因其独特的底物偏好性具有广阔的应用前景。研究从食品安全菌娄地青霉的基因组中发现了一个假定的偏甘油酯脂肪酶prlip基因,通过全基因合成技术获得该酶基因序列,并构建了PrLip组成型表达的毕赤酵母基因工程菌。工程菌在30℃发酵培养60 h,发酵活力达到22.26 U/mL。利用阴离子交换层析法纯化,获得纯度大于90%的脂肪酶PrLip。酶学性质研究发现该酶的最适反应温度为45℃,最适反应pH值为7.0,40℃的半衰期为6 h,水解及酯化反应证实PrLip是一种偏甘油酯脂肪酶。脂肪酶PrLip具有良好的温度耐受性及对大多数表面活性剂的耐受性,使其具有广阔的工业应用前景。

Mono-and diacylglycerol lipase is broadly applied due to its unique substrate specificity.In this study,a putative mono-and diacylglycerol lipase gene prlip was identified from the genome of a food safety strain Penicillium roqueforti.The lipase PrLip was obtained by artificial synthesis strategy and constitutively expressed in Pichia pastoris X33.The lipolytic activity in the broth supernatant reached 22.26 U/mL after fermentation for 60 h at 30℃.The lipase PrLip was purified by anion exchange chromatography with the purity greater than 90%.The optimal reaction temperature and pH of PrLip were 45℃and 7.0,respectively,and its thermostable half-life at 40℃was about 6 h.The lipase PrLip was characterized as a mono-and diacylglycerol lipase through hydrolysis and esterification.PrLip had a good tolerance against temperature and resistance to most surfactants,which is promising for potential application in industrial scale.