Improving alkaline stability of α-l-rhamnosidase from Aspergillus niger through computational strategy combines with folding free energy and binding free energy

Abstract

It is difficult to improve pH stability of enzymes. In the present study, a semi-rational strategy was validated to improve the alkaline stability of an α-L-rhamnosidase. The strategy combined the prediction of folding free energy and binding free energy. Through alanine scanning and virtual saturation mutation, 9 mutations were selected as mutant candidates. The mutant R307Y was confirmed with higher pH stability than the WT. In addition, the mutant also showed an increase of enzyme activity. The structure simulation and molecular dynamics analysis indicated that the interactions, especially hydrogen bonds, hydrophobic interactions and the local structural stability were enhanced by introducing aromatic residues, which play an important role in increasing the rigidity and maintaining the alkaline pH stability. The study not only provides a α-L-rhamnosidase mutant with better pH stability and enzyme activity, but also provides an effective strategy for improving enzyme pH stability.