黑曲霉脂肪酶盖子结构域突变对其活性的影响

Effect of Mutation at the Lid Domain of Aspergillus niger Lipase on Activity

比较黑曲霉脂肪酶与黑曲霉酯酶的3-D结构发现二者在盖子结构域存在显著差异。根据已解析的酯酶的3-D结构信息,运用重叠延伸PCR技术,对黑曲霉脂肪酶的4个位点进行突变,以期获得开盖型黑曲霉脂肪酶。4个突变位点分别为形成黑曲霉脂肪酶盖子结构的α-螺旋与酯酶对应区域的α-螺旋相互置换;Ser84突变为Gly;Asp99突变为Pro;Lys108突变为Glu。4个重组质粒导入毕赤酵母GS115菌株进行异源表达后,仅pPCI9K-anl-D99P和pPCI9K-anl-K108E实现了活性表达。

There existed obvious difference at the lid domain between A. niger lipase and A. niger feruloyl esterase by superimposing the predicted 3-D molecular structure of A. niger lipase on the X-ray three-dimensional structure of A. niger feruloyl esterase. Four sites of A. niger lipase were mutated to design the lipase molecular with a permanently open lid conformation. The four sites were as follows, a mutual exchange of the amino acid residues fragment between the lid domain of A. niger lipase and the corresponding a-helix domain of A. niger feruloyl esterase ;Ser-84 was mutated to Gly;Asp-99 was mutated to Pro;Lys108 was mutated to Glu. The mutated lipase genes were transformed into Pichia pastoris GS115 and only two mutated lipase genes,pPCI9K-anl-D99P and pPCI9K-anl-K108E, were functionally expressed.