Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM)
Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM)
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DOI：
10.1021/ja035742h
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发表时间：
2003-09-24
影响因子：
15
通讯作者：
Burk, MJ
中科院分区：
文献类型：
--
作者：
DeSantis, G;Wong, K;Burk, MJ
应用基因位点饱和诱变（GSSM）技术对腈水解酶进行定向进化。腈水解酶有效地催化前手性底物3-羟基戊二腈的去对称化，得到（R）-4-氰基-3-羟基丁酸，其是有价值的降胆固醇药物立普妥的前体。所发现的野生型酶在工业相关的3 M底物浓度下有效地进行反应，但提供仅87.6%ee的产物对映体过量。通过GSSM，一种诱变技术，其影响蛋白质中的每个氨基酸对其他19个氨基酸中的每个氨基酸的组合饱和，结合新的高通量质谱分析，鉴定了许多改进的变体，其中最好的是Ala 190 His突变体，其在3 M底物负载下产生98.5%的产物对映体过量和619 g L-1d-1的体积生产率。
Gene site saturation mutagenesis (GSSM) technology is applied for the directed evolution of a nitrilase. The nitrilase effectively catalyzes the desymmetrization of the prochiral substrate 3-hydroxyglutaronitrile to afford (R)-4-cyano-3-hydroxybutyric acid, a precursor to the valuable cholesterol-lowering drug Lipitor. The discovered wild-type enzyme effectively performs the reaction at the industrially relevant 3 M substrate concentration but affords a product enantiomeric excess of only 87.6% ee. Through GSSM, a mutagenesis technique that effects the combinatorial saturation of each amino acid in the protein to each of the other 19 amino acids, combined with a novel high-throughput mass spectroscopy assay, a number of improved variants were identified, the best of which is the Ala190His mutant that yields product enantiomeric excess of 98.5% at 3 M substrate loading and a volumetric productivity of 619 g L-1d-1.