Overexpression of a recombinant amidase in a complex auto-inducing culture: purification,biochemical characterization,and regio- and stereoselectivity |
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Authors: | Zhiquan Xue Yapeng Chao Dexian Wang Meixiang Wang Shijun Qian |
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Institution: | (1) State Key Laboratories of Transducer Technology, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, 100101 Chaoyang District, Beijing, China;(2) Graduate School of the Chinese Academy of Sciences, 100039 Beijing, China;(3) Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Molecular Recognition and Function, Institute of Chemistry, Chinese Academy of Sciences, 100190 Beijing, China;(4) The Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Ministry of Education, Department of Chemistry, Tsinghua University, 100084 Beijing, China; |
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Abstract: | Rhodococcus erythropolis AJ270 metabolizes a wide range of nitriles via the two-step nitrile hydratase/amidase pathway. In this study, an amidase
gene from R. erythropolis AJ270 was cloned and expressed in Escherichia coli BL21 (DE3). The activity reached the highest level of 22.04 U/ml in a complex auto-inducing medium using a simplified process
of fermentation operation. The recombinant amidase was purified to more than 95% from the crude lysate using Ni-NTA affinity
chromatography and Superose S10-300 gel filtration. The V
max and K
m values of the purified enzyme with acetamide (50 mM) were 6.89 μmol/min/mg protein and 4.12 mM, respectively, which are similar
to those of the enzyme from the wild-type cell. The enzyme converted racemic α-substituted amides, O-benzylated β-hydroxy amides, and N-benzylated β-amino amides to the corresponding (S)-acids with remarkably high enantioselectivity. The ionic liquid BMIm]PF6] (1-butyl-3-methylimidazolium hexafluorophosphate) enhanced the activity by 1.5-fold compared with water. The adequate expression
of the enzyme and excellent enantioselectivity of the recombinant amidase to a broad spectrum of amides suggest that the enzyme
has prospective industrial-scale practical applications in pharmaceutical chemistry. |
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