【摘要】：采用易錯(cuò)PCR方法對(duì)枯草芽孢桿菌脂肪酶基因lipase A進(jìn)行定向進(jìn)化,并首次采用對(duì)硝基苯棕櫚酸酯(p NPP)法進(jìn)行96孔板高通量篩選。結(jié)果表明:第一輪易錯(cuò)PCR沒有產(chǎn)生隨機(jī)突變,第二輪易錯(cuò)PCR反應(yīng)在Mn~(2+)濃度為0.2 mmol·L~(-1)時(shí),產(chǎn)生了突變菌株。對(duì)該條件下構(gòu)建的文庫中124株突變菌株進(jìn)行p NPP高通量篩選,突變株4B_2的吸光度值A(chǔ)_(405)為1.395,與未突變株P(guān)ET32a-lipase A(A_(405)=0.448)差異顯著。測(cè)序結(jié)果表明,突變株4B_2脂肪酶基因lipase A有5個(gè)核苷酸位點(diǎn)發(fā)生了突變,其中3個(gè)是同義突變,2個(gè)是錯(cuò)義突變,分別是82位的天冬酰胺(AAU)突變?yōu)槔野彼?UAU),143位的賴氨酸(AAG)突變?yōu)樘K氨酸(ACG)。突變株4B_2發(fā)酵上清液轉(zhuǎn)化生物柴油的轉(zhuǎn)酯效率較對(duì)照菌PET 32a-lipase A有明顯提高,前者為79.5%,后者為49.72%。本研究為枯草芽孢桿菌脂肪酶lipase A轉(zhuǎn)酯活性位點(diǎn)的探索奠定了基礎(chǔ)。
[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.
【作者單位】： 成都理工大學(xué)材料與化學(xué)化工學(xué)院;成都理工大學(xué)礦產(chǎn)資源化學(xué)四川省高校重點(diǎn)實(shí)驗(yàn)室;
【基金】：四川省教育廳科研項(xiàng)目(SZS013)
【分類號(hào)】：Q78;TE667

【參考文獻(xiàn)】

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