DeSantis Grace, Wong Kelvin, Farwell Bob, Chatman Kelly, Zhu Zoulin, Tomlinson Geoff, Huang Hongjun, Tan Xuqiu, Bibbs Lisa, Chen Pei, Kretz Keith, Burk Mark J
Diversa Corporation, 4955 Directors Place, San Diego, California 92121, USA.
J Am Chem Soc. 2003 Sep 24;125(38):11476-7. doi: 10.1021/ja035742h.
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-1 d-1.
基因位点饱和诱变（GSSM）技术被应用于腈水解酶的定向进化。该腈水解酶可有效催化前手性底物3-羟基戊二腈的去对称化反应，生成（R）-4-氰基-3-羟基丁酸，这是一种用于生产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底物浓度下能有效进行反应，但产物对映体过量仅为87.6% ee。通过基因位点饱和诱变（一种使蛋白质中的每个氨基酸与其他19种氨基酸进行组合饱和的诱变技术），结合一种新型的高通量质谱分析方法，鉴定出了许多改进变体，其中最佳变体是Ala190His突变体，它在3 M底物负载量下产物对映体过量为98.5%，体积产率为619 g L-1 d-1。
