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
发布时间：2019-07-28 18:05
【摘要】：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。
[Abstract]:The lipase gene lipase A of Bacillus subtilis was evolved by error-prone PCR, and the 96-well plate was screened by p-nitrophenylpalmitate (p NPP) method for the first time. The results showed that the first round of error-prone PCR did not produce random mutation, and the second round of error-prone PCR reaction produced mutant strains when the concentration of Mn~ (2) was 0.2 mmol 路L ~ (- 1). The absorbance value of the mutant 4B_2 was 1.395, which was significantly different from that of the unmutant PET32a-lipase A (A405A). The results showed that the absorbance value of the mutant 4B_2 was 1.395, which was significantly different from that of the unmutated strain PET32a-lipase A (A405A), which was significantly different from that of the unmutated strain PET32a-lipase A (A405A). The results of sequencing showed that there were five nucleotides mutations in the mutant 4B_2 lipase gene lipase A, of which 3 were synonymous mutations and 2 were missense mutages. asparagine (AAU) at position 82 was mutated to lysine (AAG) at tyrosine (UAU), 143. lysine (AAG) was mutated to threonine (ACG). The conversion efficiency of biodiesel into biodiesel was significantly higher than that of the control strain PET 32a-lipase A (79.5%) and 49.72% (49.72%). This study laid a foundation for the exploration of lipase A transester activity site of Bacillus subtilis.
【作者单位】： 成都理工大学材料与化学化工学院;成都理工大学矿产资源化学四川省高校重点实验室;
【基金】：四川省教育厅科研项目(SZS013)
【分类号】：Q78;TE667
[Abstract]:The lipase gene lipase A of Bacillus subtilis was evolved by error-prone PCR, and the 96-well plate was screened by p-nitrophenylpalmitate (p NPP) method for the first time. The results showed that the first round of error-prone PCR did not produce random mutation, and the second round of error-prone PCR reaction produced mutant strains when the concentration of Mn~ (2) was 0.2 mmol 路L ~ (- 1). The absorbance value of the mutant 4B_2 was 1.395, which was significantly different from that of the unmutant PET32a-lipase A (A405A). The results showed that the absorbance value of the mutant 4B_2 was 1.395, which was significantly different from that of the unmutated strain PET32a-lipase A (A405A), which was significantly different from that of the unmutated strain PET32a-lipase A (A405A). The results of sequencing showed that there were five nucleotides mutations in the mutant 4B_2 lipase gene lipase A, of which 3 were synonymous mutations and 2 were missense mutages. asparagine (AAU) at position 82 was mutated to lysine (AAG) at tyrosine (UAU), 143. lysine (AAG) was mutated to threonine (ACG). The conversion efficiency of biodiesel into biodiesel was significantly higher than that of the control strain PET 32a-lipase A (79.5%) and 49.72% (49.72%). This study laid a foundation for the exploration of lipase A transester activity site of Bacillus subtilis.
【作者单位】： 成都理工大学材料与化学化工学院;成都理工大学矿产资源化学四川省高校重点实验室;
【基金】：四川省教育厅科研项目(SZS013)
【分类号】：Q78;TE667
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