Institute of Bioengineering, Zhejiang University of Technology , Hangzhou, Zhejiang 310014, People's Republic of China.
J Agric Food Chem. 2014 May 21;62(20):4685-94. doi: 10.1021/jf405683f. Epub 2014 May 13.
Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(-)-mandelic acid after 7.5 h of conversion reached 693 mM with an enantiomeric excess of 99%, and the space-time productivity of Mut3 was 21.50-fold higher than that of wild-type nitrilase. The Km, Vmax, and k(cat) of wild-type and Mut3 for mandelonitrile were 20.64 mM, 33.74 μmol mg(-1) min(-1), 24.45 s(-1), and 9.24 mM, 47.68 μmol mg(-1) min(-1), and 34.55 s(-1), respectively. A homology modeling and molecular docking study showed that the diameter of the catalytic tunnel of Mut3 became longer and that the tunnel volume was smaller. These structural changes are proposed to improve the hydrolytic activity and pH stability of Mut3. Mut3 has the potential for industrial applications in the upscale production of (R)-(-)-mandelic acid.
腈水合酶作为立体选择性合成羧酸的生物催化剂，近年来受到了广泛关注。为了提高活性，根据同源建模和以前关于突变的报道，选择来自粪产碱杆菌的腈水合酶通过基因定点饱和突变（GSSM）方法进行进一步修饰。突变后，采用基于产物形成和 pH 指示剂的两步高通量筛选方法结合 HPLC 方法，对阳性突变体进行筛选。经过三轮 GSSM，选择了活性和对底物的耐受性最高的突变体 Mut3（Gln196Ser/Ala284Ile）。与野生型 A. faecalis 腈水合酶相比，Mut3 的比活性提高了 154%。Mut3 在 pH 6.5 孵育 6 小时后仍保留 91.6%的残余活性。在以 800 mM 扁桃腈为底物的分批补料反应中，转化 7.5 小时后（R）-（-）-扁桃酸的累积产量达到 693 mM，对映体过量为 99%，Mut3 的时空产率比野生型腈水合酶高 21.50 倍。野生型和 Mut3 对扁桃腈的 Km、Vmax 和 k(cat)分别为 20.64 mM、33.74 μmol mg(-1) min(-1)、24.45 s(-1)和 9.24 mM、47.68 μmol mg(-1) min(-1)、34.55 s(-1)。同源建模和分子对接研究表明，Mut3 的催化隧道直径变长，隧道体积变小。这些结构变化被认为提高了 Mut3 的水解活性和 pH 稳定性。Mut3 有望在（R）-（-）-扁桃酸的规模化生产中得到应用。
