CAJ | 학술논문

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a-螺旋与酯酶对应区域的a-螺旋相互置换;Ser84突变为Gly;Asp99突变为Pro;Lys108突变为Gh.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.
비교흑곡매지방매여흑곡매지매적3-D결구발현이자재개자결구역존재현저차이.근거이해석적지매적3-D결구신식,운용중첩연신PCR기술,대흑곡매지방매적4개위점진행돌변,이기획득개개형흑곡매지방매.4개돌변위점분별위형성흑곡매지방매개자결구적a-라선여지매대응구역적a-라선상호치환;Ser84돌변위Gly;Asp99돌변위Pro;Lys108돌변위Gh.4개중조질립도입필적효모GS115균주진행이원표체후,부pPCI9K-anl-D99P화pPCI9K-anl-K108E실현료활성표체.
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.