Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 95, Issue 1, Pages 91-99Publisher
SPRINGER
DOI: 10.1007/s00253-012-3993-4
Keywords
Nitrilase; Labrenzia aggregate; Enantioselective hydrolysis; (R)-o-Chloromandelic acid; Biphasic system
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Funding
- National Natural Science Foundation of China [20902023]
- Ministry of Science and Technology, People's Republic of China [2011CB710800, 2011AA02A210]
- Shanghai Committee of Science and Technology [11431921600]
- Shanghai Municipal Education Commission [11CXY24]
- China National Special Fund [2060204]
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(R)-o-Chloromandelic acid is the key precursor for the synthesis of ClopidogrelA (R), a best-selling cardiovascular drug. Although nitrilases are often used as an efficient tool in the production of alpha-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 mu mol 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.
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