黑曲霉脂肪酶盖子结构域突变对其活性的影响

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。

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

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.