低温脂肪酶产生菌的筛选、表达及酶学性质分析

Screening,Expression and Analysis of Enzymatic Properties of a Coldactive Lipase-producing Strain

为筛选高产低温脂肪酶的菌株,并为脂肪酶的工业化开发提供生产资料。利用罗丹明B培养法从漠河县兴安落叶松林为主的林下土壤中分离出1株低温脂肪酶产生菌,对其形态学、生理生化特性进行鉴定,并结合16S rDNA基因序列,确定该菌株为苏云金芽孢杆菌(Bacillus thuringiensis)。以苏云金芽孢杆菌基因组为模板克隆脂肪酶基因Lip240,对Lip240进行异源表达以及酶学性质的分析。结果表明:该重组脂肪酶Lip240的最适反应温度为30℃,在4~30℃条件下处理6 h能维持60%以上的活性,是一种低温脂肪酶,最适pH为8.0。Ca^(2+)、Mn^(2+)、Fe^(2+)、Fe^(3+)和Cr^(3+)对脂肪酶Lip240的酶活有一定的促进作用。该酶具有较好的有机溶剂耐受性,SDS能够显著(P<0.05)抑制脂肪酶Lip240的酶活。该酶更偏好于水解中短链底物。该结论可为微生物资源开发利用和低温脂肪酶的工业应用提供一定的理论依据。

High-yield strains of cold-active lipase are screened to provide production information for the industrial development of lipase.A cold-active lipase-producing strain was isolated from Xing'an larch forest soil in Mohe County using the Rhodamine B plate method.Its morphological,physiological,and biochemical characteristics were identified,and combined with the 16S rDNA gene sequence,the strain was determined to be Bacillus thuringiensis.The lipase gene Lip240 was cloned using the Bacillus thuringiensis genome as a template,and heterologous expression and enzymatic properties of Lip240 were analyzed.The results showed that the recombination lipase Lip240 had an optimal reaction temperature of 30℃,and could maintain more than 60%activity when treated at 4~30℃ for 6 hours,thus a cold-active lipase.Besides,its optimal pH was 8.0.Additionally,Ca^(2+),Mn^(2+),Fe^(2+),Fe^(3+),and Cr^(3+)had a certain enhancing effect on the activity of Lip240.Notably,Lip240 had a good organic solvent tolerance,while SDS could significantly(P<0.05)inhibit its activity.Lip240 exhibited good substrate specificity towards medium and short-chain substrates.The conclusions of this study would provide a theoretical basis for the development and utilization of microbial resources and the industrial application of cold-active lipase.