Complementary Distant and Active Site Mutations Simultaneously Enhance Catalytic Activity and Thermostability of α-Galactosidase

Zhuangzhuang Huang, Junru Zhou, Jialing Wang, Sheng Xu, Cheng Cheng, Jiangfeng Ma, Zhen Gao

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

The industrial applications of enzymes are limited due to the activity-stability trade-off, which implies that the improvement of thermostability often accompanies decreased activity. This study presents a dual-strategy approach to simultaneously improve the catalytic efficiency and thermostability of α-galactosidase galV from Anoxybacillus vitaminiphilus WMF1. Our integrated method combines computational analysis with enzyme property prediction to selectively target and modify the catalytic region and residues that are distant from the active site. We identified and experimentally validated mutations that improve activity without compromising stability and further increased thermostability through additional distant-site mutations. The resulting mutant enzyme variant N549Q/T550N/Y634F demonstrated a 6.2-fold increase in catalytic efficiency and a 3.2-fold improvement in the half-life at 65 °C. Molecular dynamics (MD) simulations supported the structural basis for the observed enhancements. This approach offers a refined strategy for engineering α-galactosidases with improved industrial applicability, overcoming the traditional trade-offs between enzyme activity and stability. Hydrolytic activity toward raffinose family oligosaccharides (RFOs) was validated using soymilk as a model substrate, demonstrating significant practical potential.

Original languageEnglish
Pages (from-to)3635-3644
Number of pages10
JournalJournal of Agricultural and Food Chemistry
Volume73
Issue number6
DOIs
StatePublished - 12 Feb 2025

Keywords

  • activity-stability trade-off
  • computer-aided design
  • distant residues
  • substrate-binding pocket
  • α-galactosidase

Fingerprint

Dive into the research topics of 'Complementary Distant and Active Site Mutations Simultaneously Enhance Catalytic Activity and Thermostability of α-Galactosidase'. Together they form a unique fingerprint.

Cite this