Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
Appl Microbiol Biotechnol. 2018 Jul;102(14):6047-6056. doi: 10.1007/s00253-018-9057-7. Epub 2018 May 10.
A nitrilase from Hoeflea phototrophica DFL-43 (HpN) demonstrating excellent catalytic activity towards benzoylacetonitrile was identified from a nitrilase tool-box, which was developed previously in our laboratory for (R)-o-chloromandelic acid synthesis from o-chloromandelonitrile. The HpN was overexpressed in Escherichia coli BL21 (DE3), purified to homogeneity by nickel column affinity chromatography, and its biochemical properties were studied. The HpN was very stable at 30-40 °C, and highly active over a wide range of pH values (pH 6.0-10.0). In addition, the HpN could tolerate against several hydrophilic organic solvents. Steady-state kinetics indicated that HpN was highly active towards benzoylacetonitrile, giving a K of 4.2 mM and a k of 170 s, the latter of which is ca. fivefold higher than the highest record reported so far. A cascade reaction for the synthesis of optically pure (S)-β-phenylalanine from benzoylacetonitrile was developed by coupling HpN with an ω-transaminase from Polaromonas sp. JS666 in toluene-water biphasic reaction system using β-alanine as an amino donor. Various (S)-β-amino acids could be produced from benzoylacetonitrile derivatives with moderate to high conversions (73-99%) and excellent enantioselectivity (> 99% ee). These results are significantly advantageous over previous studies, indicating a great potential of this cascade reaction for the practical synthesis of (S)-β-phenylalanine in the future.
从本实验室先前开发的腈水解酶工具盒中，鉴定到一种来自 Hoeflea phototrophica DFL-43（HpN）的腈酶，它对苯甲酰乙腈具有出色的催化活性，可用于（R）-邻氯扁桃酸的合成，原料是邻氯扁桃腈。HpN 在大肠杆菌 BL21（DE3）中过表达，通过镍柱亲和层析进行纯化至均相，并研究了其生化性质。HpN 在 30-40°C 时非常稳定，在很宽的 pH 值范围内（pH 6.0-10.0）都具有高活性。此外，HpN 能够耐受几种亲水性有机溶剂。稳态动力学表明，HpN 对苯甲酰乙腈具有高活性，K 值为 4.2mM，k 值为 170s，后者比迄今为止报道的最高记录高约五倍。通过将 HpN 与来自 Polaromonas sp. JS666 的ω-转氨酶偶联，在甲苯-水两相反应体系中，以β-丙氨酸为氨基供体，从苯甲酰乙腈合成光学纯（S）-β-苯丙氨酸的级联反应被开发出来。各种（S）-β-氨基酸可以从苯甲酰乙腈衍生物中获得，转化率（73-99%）和对映选择性（>99%ee）都很高。这些结果明显优于以前的研究，表明该级联反应在未来（S）-β-苯丙氨酸的实际合成中具有很大的潜力。
