High-Level Expression of Nitrile Hydratase in Escherichia coli for 2-Amino-2,3-Dimethylbutyramide Synthesis

Abstract

In the synthesis of imidazolinone herbicides, 2-Amino-2,3-dimethylbutyramide (ADBA) is an important intermedium. In this study, the recombinant production of nitrile hydratase (NHase) in Escherichia coli for ADBA synthesis was explored. A local library containing recombinant NHases from various sources was screened using a colorimetric method. NHase from Pseudonocardia thermophila JCM3095 was selected, fused with a His-tag and one-step purified. The enzymatic properties of recombinant NHase were studied and indicated robust thermal stability and inhibition of cyanide ions due to substrate degradation. After systematic optimization of fermentation conditions, the OD600 (optical density at 600 nm), enzyme activity and specific activity of recombinant strain E. coli BL21(DE3)/pET-28a+NHase reached 19.4, 3.72 U/mL and 1.04 U/mg protein at 42 h, representing 5.86-, 26.6- and 4-fold increases, respectively. These results offered an efficient recombinant whole-cell biocatalyst for ADBA synthesis.

Effects of His-Tag Length on the Soluble Expression and Selective Immobilization of D-Amino Acid Oxidase from Trigonopsis variabilis: A Preliminary Study

“…They are routinely attached to the terminus of proteins, with the assumption that the fusion will not significantly affect the structure and function of the target proteins due to their small molecular weight [3]. The fusion position of His-tags can be varied to improve protein expression and purification [3][4][5][6]. D-amino acid oxidase (DAO) (E.C.…”