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

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Abstract

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 KM of 4.2 mM and a kcat 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)-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.

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Funding

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).

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  1. 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

    Zhi-Jun Zhang, Rui-Feng Cai & Jian-He Xu

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  1. Zhi-Jun Zhang
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Correspondence to Jian-He Xu.

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Zhang, ZJ., Cai, RF. & Xu, JH. Characterization of a new nitrilase from Hoeflea phototrophica DFL-43 for a two-step one-pot synthesis of (S)-β-amino acids. Appl Microbiol Biotechnol 102, 6047–6056 (2018). https://doi.org/10.1007/s00253-018-9057-7

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