Heterologous expression, purification, and biophysical characterisation of the cobalt-dependent nitrile hydratase from Rhodococcus rhodochrous ATCC BAA-870

Abstract

Nitrile hydratases (NHases) are cobalt-dependent metalloenzymes that catalyse the hydration of nitriles to amides and are widely employed as industrial biocatalysts in the sustainable production of pharmaceuticals, polymers, and fine chemicals. Despite their biotechnological importance, detailed biophysical characterisation of many NHases remains limited, constraining rational enzyme engineering and process optimisation. Here, we present the first comprehensive biophysical characterisation of the NHase from Rhodococcus rhodochrous ATCC BAA-870. The enzyme was recombinantly co-expressed in E. coli T7 Express, purified to high homogeneity, and yielded ∼10.4 mg L -1 of culture. A hydroxamate-based colorimetric assay was successfully adapted and optimised for NHase activity measurements, revealing a specific activity of 212.51 U mg -1 toward acetonitrile and confirming robust catalytic competence. Spectroscopic analyses demonstrated a predominantly α-helical structure with a compact tertiary fold and hydrophobic pockets near the catalytic region associated with substrate interaction. Mass photometry revealed a concentration-dependent equilibrium between the αβ heterodimer and α 2 β 2 heterotetramer, with tetramer populations increasing from 63.3% at 35 nM to 77.7% at 100 nM. Differential scanning fluorimetry showed biphasic thermal transitions at ∼45 °C and ∼54 °C, consistent with tetramer dissociation followed by unfolding of the catalytic αβ unit. Together, these findings provide new insight into NHase structural dynamics and establish a foundation for future structure-guided engineering of NHases with enhanced industrial performance.