Improving stability of nitrile hydratase by bridging the salt-bridges in specific thermal-sensitive regions

The regions and types suitable mutations for bridging salt-bridges to intensify enzyme stability are identified in this study. Using nitrile hydratase (NHase) as the model enzyme, three deformation-prone thermal-sensitive regions (A1, A2 and A3 in β-subunit), identified by RMSF calculations of the thermophilic NHase 1V29 from Bacillus SC-105-1 and 1UGQ from Pseudonocardia thermophila JCM3095, were determined and the stabilized salt-bridge interactions were transferred into the corresponding region of industrialized mesophilic NHase-TH from Rhodococcus ruber TH. Three types of salt bridges—active-center-adjacent (in A1), internal neighboring-residue-bridged (in A2) and C-terminal-residue-bridged (A3)—were constructed in NHase-TH. The engineered NHase-TH-A1 showed reduced expression of β-subunit, reduced activity and irregular stability. NHase-TH-A2 exhibited a enhanced expression of β-subunit but complete loss of activity; while NHase-TH-A3 exhibited not only a slightly enhanced expression of β-subunit and enzyme activity, but also a 160% increase in thermal stability, a 7% enhanced product tolerance and a 75% enhanced resistance to cell-disruption by ultrasonication. The molecular dynamic (MD) simulation revealed that NHase-TH-A3, with a moderate RMSD value, generates 10 new salt bridges in both internal-subunit and interfacial-subunit, confirming that a C-terminal salt-bridge strategy is powerful for enzyme stability intensification through triggering global changes of the salt bridge networks.

► Three deformation-prone thermal-sensitive regions of NHase were identified by RMSF calculation.
► The stabilized salt-bridge interactions in thermophilic NHase were transferred into NHase-TH.
► The three salt bridges showed different results in expression, enzyme activity and stability.
► Salt bridge network changes were further modeled by molecular dynamic simulation.
► The C-terminal salt-bridge is powerful for stability intensification with unreduced activity.