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- Title
Characterization of a new nitrilase from Hoeflea phototrophica DFL-43 for a two-step one-pot synthesis of (S)-β-amino acids.
- Authors
Zhang, Zhi-Jun; Cai, Rui-Feng; Xu, Jian-He
- 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.
- Subjects
NITRILASES; BIOCATALYSIS; CHROMATOGRAPHIC analysis; ESCHERICHIA coli; AMINOTRANSFERASES
- Publication
Applied Microbiology & Biotechnology, 2018, Vol 102, Issue 14, p6047
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-018-9057-7