文献类型：期刊文献

英文题名：Characterization of a new nitrilase from Hoeflea phototrophica DFL-43 for a two-step one-pot synthesis of (S)-β-amino acids

作者：Zhang, Zhi-Jun[1,2];Cai, Rui-Feng[1,2];Xu, Jian-He[1,2]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Lab Biocatalysis & Synthet Biotechnol, 130 Mcilong Rd, Shanghai 200237, Peoples R China;[2]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, 130 Mcilong Rd, Shanghai 200237, Peoples R China

年份：2018

卷号：102

期号：14

起止页码：6047

外文期刊名：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

收录：;EI(收录号：20181905177999);WOS:【SCI-EXPANDED(收录号:WOS:000435986500019)】；

基金：This study was funded by National Natural Science Foundation of China (nos. 21406067 and 21536004), Natural Science Foundation of Shanghai (no. 18ZR1408400), Fundamental Research Funds for the Central Universities (no. 22221818014) and Shanghai Commission of Science and Technology (no. 15JC1400403).

语种：英文

外文关键词：Biocatalysis; Nitrilase; omega-Transaminase; Cascade reaction; (S)-beta-Phenylalanine

摘要：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 A degrees 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 (M) of 4.2 mM and a k (cat) of 170 s(-1), 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)-beta-phenylalanine from benzoylacetonitrile was developed by coupling HpN with an omega-transaminase from Polaromonas sp. JS666 in toluene-water biphasic reaction system using beta-alanine as an amino donor. Various (S)-beta-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)-beta-phenylalanine in the future.