Efficient biodegradation of nitriles by a novel nitrile hydratase derived from Rhodococcus erythropolis CCM2595

Abstract

In our previous study, we firstly cloned the gene of a novel ReNHase (NHase from Rhodococcus erythropolis CCM2595), and the strain was shown to degrade only phenol, hydroxybenzoate, p-chlorophenol, aniline and other aromatic compounds. Here in, we further purified the ReNHase from R. erythropolis CCM2595 and detected its properties of biodegradation. We constructed a plasmid with the gene of ReNHase with His-tag. The encoding ReNHase was cloned and overexpressed in recombinant Escherihia coli and confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The substrate scope of the new heterologous ReNHase was tested and the efficiency of degradation of nitriles by the ReNHase was also investigated by high efficiency liquid chromatography. We also studied the effect of temperature and pH on the catalysis of adiponitrile by the purified ReNHase. The recombinant E.coli showed high catalytic regioselectivity with high substrate affinity towards dinitriles (especially for adiponitrile) whereas lower affinity towards mononitriles. Compared to whole-cell catalysis, the catalytic time was shortened significantly with the purified ReNHase. The enzyme activity of crude recombinant E.coli was 635 U g −1 (DCW), while the specific activity of purified ReNHase was 63.107 U mg −1 . The apparent Km value for the purified ReNHase is 6.6252 mmol L −1 , which revealed the good affinity between the purified ReNHase and adiponitrile. The reaction Kcat is 82.77 s −1 and Kcat/Km is 1.249 × 10 4 (mol −1 L s −1 ), which showed high catalytic activity towards adiponitrile. We propose that this purified ReNHase may be applied for the industry and sewage treatment for environmental protection.

A Preblocking Strategy with Rapid Immobilization of Nitrile Hydratase and Precise Control of Hydrophobic Microenvironment for High Regioselective Amide Transformation

“…ReNHase, sourced from Rhodococcus erythropolis CCM2595, was identified by Liang and colleagues as highly efficient, achieving 100% conversion of adiponitrile within 10 min and producing the main product, 5-cyanovaleramide, with over 90% regioselectivity, as confirmed by HPLC (Figures S1and S2). This enzyme, fused with a his-tag at the N-terminus (Figure S11a), facilitated both purification and site-specific immobilization onto VS-activated resins.…”

“…The preparation method of ReNHase has been reported elsewhere. 58 ReNHase with His-tag was expressed by Escherichia coli Arctic Express (DE3) strain with the constructed plasmid pET24a (+). The protein sequence is provided in the Supporting Information.…”