Rational design of protease HapR for efficient reduction of harmful compounds from tobacco

Excess tobacco protein facilitates the formation of hazardous compounds, posing risks to human health. Therefore, the development of efficient protein degradation strategies is conducive to decreasing hazardous compounds in tobacco. In this study, protease HapR was successfully modified through sequence alignment, alanine scanning mutagenesis, and virtual saturation mutagenesis. Compared to the wild-type HapR, the tri-mutant T26S/S288A/L302V increased by 169 % and 218 % in fermentation enzymatic activity and specific activity, respectively. Molecular dynamics simulation revealed that the increase in global rigidity, local flexibility, and hydrogen bond number in this tri-mutant jointly improved its catalytic efficiency. The enzymatic kinetics analysis further demonstrated that the tri-mutant exhibited higher affinity and catalytic efficiency than the wild-type. Enzymatic characterization indicated that this tri-mutant had the optimal enzymatic activity at 50°C and pH 9.0, with Ca²⁺ serving as optimal activator. Finally, the application of tri-mutant T26S/S288A/L302V significantly decreased tobacco-specific nitrosamines (TSNAs) and harmful pyrolysis products in tobacco. Overall, this study provides an effective method and an engineering protease for reduction of harmful tobacco compounds.