Salt bridges are pivotal for the kinetic stability of GH26 endo-mannanase (ManB-1601)

摘要

Hitherto, how salt bridges contribute towards the structure-function of endo-mannanases has not been demonstrated. In the present study, we revealed that ManB-1601 (GH26 endo-mannanase from Bacillus sp.) has eight salt bridges which are highly conserved among GH26 endo-mannanases from Bacillus spp. Disruption of salt bridges do not alter overall structure, optimum pH and temperature of ManB-1601. Among the salt bridges, elimination of K95/E156 and El 71/R221 pair decreased the substrate affinity and catalytic efficiency of ManB-1601. Differential scanning calorimetry and isothermal equilibrium denaturation studies suggested that salt bridges do not markedly contribute towards thermal (Delta T-m 0 to -5 degrees C) and conformational stability (Delta Delta G -037 to -1.25 Kcal mol(-1)) of ManB-1601. Interestingly, salt bridges were found to prominently contribute towards kinetic stability of ManB-1601 as salt bridge mutants exhibited drastic reduction in half-life of enzyme inactivation (T-1/2) at 66 degrees C (1.1 to 6-fold) and 70 degrees C (4.09 to 22.5-fold). Molecular dynamic simulations studies showed that salt bridges contribute towards maintaining the biological activity against thermal denaturation by rigidifying the active site. Our study on ManB-1601 suggest that even in the case salt bridges do not confer thermal and conformational stabilities they may serve as crucial structural elements for enzyme functioning by contributing towards kinetic stability. (C) 2019 Elsevier B.V. All rights reserved.