Sandström Anders G, Engström Karin, Nyhlén Jonas, Kasrayan Alex, Bäckvall J-E
Department of Organic Chemistry, Stockholm University, Stockholm, Sweden.
Protein Eng Des Sel. 2009 Jul;22(7):413-20. doi: 10.1093/protein/gzp019. Epub 2009 Jun 9.
We herein report the first directed evolution of Candida antarctica lipase A (CalA), employing a combinatorial active-site saturation test (CAST). Wild-type CalA has a modest E-value of 5.1 in kinetic resolution of 4-nitrophenyl 2-methylheptanoate. Enzyme variants were expressed in Pichia pastoris by using the episomal vector pBGP1 which allowed efficient secretory expression of the lipase. Iterative rounds of CASTing yielded variants with good selectivity toward both the (S)- and the (R)-enantiomer. The best obtained enzyme variants had E-values of 52 (S) and 27 (R).
我们在此报告首次利用组合活性位点饱和测试（CAST）对南极假丝酵母脂肪酶A（CalA）进行定向进化。野生型CalA在4-硝基苯基2-甲基庚酸酯的动力学拆分中具有适度的E值5.1。通过使用附加型载体pBGP1在毕赤酵母中表达酶变体，该载体允许脂肪酶高效分泌表达。经过多轮CAST迭代产生了对（S）-和（R）-对映体均具有良好选择性的变体。获得的最佳酶变体的E值分别为52（S）和27（R）。
