title: Efficient production of (R)-o-chloromandelic acid by deracemization of o-chloromandelonitrile with a new nitrilase mined from Labrenzia aggregata.
abstract: (R)-o-Chloromandelic acid is the key precursor for the synthesis of Clopidogrel®, a best-selling cardiovascular drug. Although nitrilases are often used as an efficient tool in the production of α-hydroxy acids, there is no practical nitrilase specifically developed for (R)-o-chloromandelic acid. In this work, a new nitrilase from Labrenzia aggregata (LaN) was discovered for the first time by genomic data mining, which hydrolyzed o-chloromandelonitrile with high enantioselectivity, yielding (R)-o-chloromandelic acid in 96.5% ee. The LaN was overexpressed in Escherichia coli BL21 (DE3), purified, and its catalytic properties were studied. When o-chloromandelonitrile was used as the substrate, the V(max) and K(m) of LaN were 2.53 μmol min⁻¹ mg⁻¹ protein and 0.39 mM, respectively, indicating its high catalytic efficiency. In addition, a study of substrate spectrum showed that LaN prefers to hydrolyze arylacetonitriles. To relieve the substrate inhibition and to improve the productivity of LaN, a biphasic system of toluene-water (1:9, v/v) was adopted, in which o-chloromandelonitrile of 300 mM (apparent concentration, based on total volume) could be transformed by LaN in 8 h, giving an isolated yield of 94.5%. The development of LaN makes it possible to produce (R)-o-chloromandelic acid by deracemizing o-chloromandelonitrile with good ee value and high substrate concentration.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People's Republic of China.
authors.forename: Chen-Sheng
authors.initials: CS
authors.lastname: Zhang
authors.forename: Zhi-Jun
authors.initials: ZJ
authors.lastname: Zhang
authors.forename: Chun-Xiu
authors.initials: CX
authors.lastname: Li
authors.forename: Hui-Lei
authors.initials: HL
authors.lastname: Yu
authors.forename: Gao-Wei
authors.initials: GW
authors.lastname: Zheng
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Xu
mesh_terms: D000619:Aminohydrolases / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D008333:Mandelic Acids / Q000737:chemistry / Q000378:metabolism*; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism*; D041902:Rhodobacteraceae / Q000201:enzymology* / Q000235:genetics
chemical_list: D008333:Mandelic Acids; D009570:Nitriles; D000619:Aminohydrolases; C021867:nitrilase