title: Overexpression of a recombinant amidase in a complex auto-inducing culture: purification, biochemical characterization, and regio- and stereoselectivity.
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.
authors.affiliation: State Key Laboratories of Transducer Technology, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, 100101 Chaoyang District, Beijing, China.
authors.forename: Zhiquan
authors.initials: Z
authors.lastname: Xue
authors.forename: Yapeng
authors.initials: Y
authors.lastname: Chao
authors.forename: Dexian
authors.initials: D
authors.lastname: Wang
authors.forename: Meixiang
authors.initials: M
authors.lastname: Wang
authors.forename: Shijun
authors.initials: S
authors.lastname: Qian
mesh_terms: D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006836:Hydro-Lyases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology*; D013696:Temperature
chemical_list: C412621:1-butyl-3-methylimidazolium hexafluorophosphate; D007093:Imidazoles; D011994:Recombinant Proteins; D000581:Amidohydrolases; C019665:amidase; D006836:Hydro-Lyases; C048831:nitrile hydratase