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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

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

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 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 )-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.