An effective computational-screening strategy for simultaneously improving both catalytic activity and thermostability of α-L-rhamnosidase

Abstract

Catalytic efficiency and thermostability are the two most important characteristics of enzymes. However, it is always tough to improve both catalytic efficiency and thermostability of enzymes simultaneously. In the present study, a computational strategy with double-screening steps was proposed to simultaneously improve both catalysis efficiency and thermostability of enzymes; and a fungal α-L-rhamnosidase was used to validate the strategy. As the result, by molecular docking and sequence alignment analysis within the binding pocket, seven mutant candidates were predicted with better catalytic efficiency. By energy variety analysis, three among the seven mutant candidates were predict with better thermostability. The expression and characterization results showed the mutant D525N had significant improvements in both enzyme activity and thermostability. Molecular dynamics simulations indicated that the mutations located within the 5 Å range of the catalytic domain, which could improve RMSD, electrostatic, Van der Waal interaction and polar salvation values, and formed water bridge between the substrate and the enzyme. The study indicated that the computational strategy based on the binding energy, conservation degree and mutation energy analyses was effective to develop enzymes with better catalysis and thermostability, providing practical approach for developing industrial enzymes.

Engineering of reaction specificity, enantioselectivity, and catalytic activity of nitrilase for highly efficient synthesis of pregabalin precursor

“…Similarly, it was found that deletion of C‐terminus of arylacetonitrilase from Pseudomonas fluorescens EBC191 led to increased amide formation ability, but loss of enantioselectivity and significantly reduced enzyme activity (Kiziak et al, 2007). Although the stability‐activity trade‐offs were overcome by several groups in recent years (L. Li, Li, et al, 2021; S. F. Li, Xie, et al, 2021; Liu et al, 2019), the compromises among three or more enzyme properties have seldom been addressed to date.…”