Structure-Guided Engineering of a Protease to Improve Its Activity under Cold Conditions

Bacillus proteases commonly exhibitremarkablyreduced activity under cold conditions. Herein, we employed a tailoredcombination of a loop engineering strategy and iterative saturationmutagenesis method to engineer two loops for substrate binding atthe entrance of the substrate tunnel of a protease (bcPRO) from Bacillus clausii to improve its activity under coldconditions. The variant MT6 (G95P/A96D/S99W/S101T/P127S/S126T) exhibitedan 18.3-fold greater catalytic efficiency than the wild-type (WT)variant at 10 & DEG;C. Molecular dynamics simulations and dynamictunnel analysis indicated that the introduced mutations extended thesubstrate-binding pocket volume and facilitated extra interactionswith the substrate, promoting catalysis through binding in a morefavorable conformation. This study provides insights and strategiesrelevant to improving the activities of proteases and supplies a novelprotease with enhanced activity under cold conditions for the foodindustry to maintain the initial flavor and color of food and reduceenergy consumption.

Automated summary

In this study, a tailored combination of loop engineering strategy and iterative saturation mutagenesis method was used to improve the activity of a protease from Bacillus clausii under cold conditions. An engineered variant called MT6 exhibited 18.3-fold higher catalytic efficiency than the wild-type variant at 10°C. Molecular dynamics simulations and dynamic tunnel analysis revealed that the introduced mutations increased the substrate-binding pocket volume and enhanced interactions with the substrate, leading to improved catalysis. This research provides insights and strategies for improving protease activity and offers a novel protease with enhanced activity under cold conditions for the food industry.