枯草芽孢杆菌脂肪酶基因lipaseA突变文库构建及其生物柴油转酯研究

Construction of the mutant library of Bacillus subtilis lipase A gene and study on its transesterification efficiency of biodiesel

采用易错PCR方法对枯草芽孢杆菌脂肪酶基因lipase A进行定向进化,并首次采用对硝基苯棕榈酸酯(p NPP)法进行96孔板高通量筛选。结果表明:第一轮易错PCR没有产生随机突变,第二轮易错PCR反应在Mn^(2+)浓度为0.2 mmol·L^(-1)时,产生了突变菌株。对该条件下构建的文库中124株突变菌株进行p NPP高通量筛选,突变株4B_2的吸光度值A_(405)为1.395,与未突变株PET32a-lipase A(A_(405)=0.448)差异显著。测序结果表明,突变株4B_2脂肪酶基因lipase A有5个核苷酸位点发生了突变,其中3个是同义突变,2个是错义突变,分别是82位的天冬酰胺(AAU)突变为酪氨酸(UAU),143位的赖氨酸(AAG)突变为苏氨酸(ACG)。突变株4B_2发酵上清液转化生物柴油的转酯效率较对照菌PET 32a-lipase A有明显提高,前者为79.5%,后者为49.72%。本研究为枯草芽孢杆菌脂肪酶lipase A转酯活性位点的探索奠定了基础。

The two rounds of error-prone PCR was used to directly evolve Bacillus subtilis lipase A gene with highthroughput screening of nitrobenzene palmitate（ p NPP） in 96-pore plate. The results showed that the first round of error-prone PCR had no random mutation. After the second round of error-prone PCR reaction with Mn^（2 ＋）concentration of 0. 2 mmol·L^（-1）,124 mutant strains were screened. The absorbance value A_（405） of mutant strain 4B_2was1. 395,which was significantly different from non-mutant strain PET32a-lipase A（ A_（405）= 0. 448）. Mmutant strain4B_2 lipase A gene was found to have five mutant nucleotide sites,three of which were synonymous mutations,two were missense mutations with 82 site of asparagine（ AAU） changed to tyrosine（ UAU） and 143 site of lysine（ AAG）changed to threonine（ ACG）. The transesterification efficiency of biodiesel of the mutant strain 4B_2 was significantly improved contrast to that of non-mutant strain. The former was 79. 5% and the latter was 49. 72%. These results laidthe foundation for the study of the key sites related to lipase A transesterification activity of Bacillus subtilis.