Enhancing the Stress Resistance of Nitrile Hydratase from Rhodococcus Ruber Via SpyTag/SpyCatcher-mediated Α- and Β- Subunits Ligation

Abstract Nitrile Hydratase (NHase) is one of the most important industrial enzyme widely used in the petroleum exploitation field. The enzyme, composed of two unrelated α- and β-subunits, catalyzes the conversion of acrylonitrile to acrylamide, releasing a significant amount of heat and generating the organic solvent product, acrylamide. Both the heat and acrylamide solvent have an impact on the structural stability of NHase and its catalytic activity. To improve the thermostability and acrylamide tolerance of NHase, the two subunits were fused in vivo using SpyTag and SpyCatcher, which were attached to the termini of each subunit in various combinations. Analysis of the engineered strains showed that the C-terminus of β-NHase is a better fusion site than the N-terminus, while the C-terminus of α-NHase is the most suitable site for fusion with a larger protein. Fusion of SpyTag and SpyCatcher to the C-terminus of β-NHase and α-NHase, respectively, led to improved acrylamide tolerance and a slight enhancement in the thermostability of one of the engineered strains, NBSt. These results indicate that in vivo ligation of different subunits using SpyTag/SpyCatcher is a valuable strategy for enhancing subunit interaction and improving stress tolerance.