State Key Laboratories of Transducer Technology, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, 100101 Chaoyang District, Beijing, China.
J Ind Microbiol Biotechnol. 2011 Dec;38(12):1931-8. doi: 10.1007/s10295-011-0979-7. Epub 2011 May 12.
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.
红平红球菌 AJ270 通过两步腈水合酶/酰胺酶途径代谢广泛的腈。在这项研究中，从红平红球菌 AJ270 中克隆并在大肠杆菌 BL21 (DE3) 中表达了一种酰胺酶基因。在使用简化发酵操作的复杂自动诱导培养基中，活性达到了 22.04 U/ml 的最高水平。通过 Ni-NTA 亲和层析和 Superose S10-300 凝胶过滤从粗裂解物中纯化重组酰胺酶，纯度超过 95%。用乙酰胺（50 mM）测定的纯化酶的 Vmax 和 K m 值分别为 6.89 μmol/min/mg 蛋白和 4.12 mM，与野生型细胞中的酶相似。该酶以显著的高对映选择性将外消旋α取代酰胺、O-苄基-β-羟基酰胺和 N-苄基-β-氨基酰胺转化为相应的（S）-酸。与水相比，离子液体[BMIm][PF₆]（1-丁基-3-甲基咪唑六氟磷酸盐）将酶的活性提高了 1.5 倍。酶的充分表达和重组酰胺酶对广泛的酰胺的优异对映选择性表明，该酶在药物化学方面具有广阔的工业规模实际应用前景。
