Structure-Guided Engineering of a Protease to Improve Its Activity under Cold Conditions
Fenghua Wang, Xiangyang Ma, Ying Sun, Enping Guo, Chaoshuo Shi, Zhaoting Yuan, Yu Li, Qinggang Li, Fuping Lu, Yihan Liu
Abstract
Bacillus proteases commonly exhibit remarkably reduced activity under cold conditions. Herein, we employed a tailored combination of a loop engineering strategy and iterative saturation mutagenesis method to engineer two loops for substrate binding at the entrance of the substrate tunnel of a protease (bcPRO) from Bacillus clausii to improve its activity under cold conditions. The variant MT6 (G95P/A96D/S99W/S101T/P127S/S126T) exhibited an 18.3-fold greater catalytic efficiency than the wild-type (WT) variant at 10 °C. Molecular dynamics simulations and dynamic tunnel analysis indicated that the introduced mutations extended the substrate-binding pocket volume and facilitated extra interactions with the substrate, promoting catalysis through binding in a more favorable conformation. This study provides insights and strategies relevant to improving the activities of proteases and supplies a novel protease with enhanced activity under cold conditions for the food industry to maintain the initial flavor and color of food and reduce energy consumption.