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.

Soluble and functional expression of a recombinant enantioselective amidase from Klebsiella oxytoca KCTC 1686 in Escherichia coli and its biochemical characterization

“…When the recombinant proteins were expressed in E. coli, a large number of inclusion bodies were formed and the recombinant proteins had very low activity (Table 3). These results are similar to the findings of other scholars [19,25].…”

“…With benzamide as substrate, the V max and K m of the purified native enzyme were 22.6 ± 3.54 mol/(min mg protein) and 1.1 ± 0.09 mM, respectively. These results are similar to those obtained for the amidase from R. erythropolis AJ270 [25]. High-level expression of amidases in E. coli has often been disappointing owing to the formation of inclusion bodies or inactive protein.…”

“…First, different heterologous expression strategies have been investigated, such as using Bacillus subtilis as a heterologous expression host [23], expressing the amidase gene together with upstream and downstream flanking sequences [24], and using pET22b as the expression vector to add a sixhistidine (6 × His)-tag to the C-terminal end of the protein [25]. Second, some studies have focused on the optimization of expression conditions, for example by using auto-induction media [25,26], adding ethanol to the culture medium [27], lowering the growth temperature to 29 • C, and cultivating cells without IPTG induction [28]. Although these strategies improved expression efficiency to a certain extent, they did not eliminate the problems of low expression level and formation of inclusion bodies.…”

Molecular dynamic analysis of mutant Y195I α-cyclodextrin glycosyltransferase with switched product specificity from α-cyclodextrin to γ-cyclodextrin

“…The purification process was based on the method of Xue [28]. Briefly, both wild-type and mutant α-CGTases were harvested, and the supernatant was obtained as crude enzyme by centrifugation at 8,000 rpm for 30 min at 4°C.…”

A novel amidase from Brevibacterium epidermidis ZJB-07021: gene cloning, refolding and application in butyrylhydroxamic acid synthesis

“…Although some AS amidases have been cloned and overexpressed in E. coli [11,23,39,41], high-level expression of amidases often leads to formation of insoluble inclusion bodies (IBs) in the cytoplasm [18,25,37]. In many cases, optimization of culture conditions, such as temperature, pH and autoinduction media could not eliminate the formation of IBs, and the amidase expression efficiency was unsatisfactory [32,35,42]. Therefore, it is necessary to establish an efficient refolding protocol to obtain bioactive amidases.…”