Protein engineering, expression optimization, and application of alkaline protease from Alkalihalobacillus clausii FYX.

Alkaline protease has been commercially used in the areas of detergents, food and agriculture, and improving the performance and production of alkaline protease serves as an important role in promoting its market expansion. Here, an alkaline protease AE0 from Alkalihalobacillus clausii FYX was firstly characterized in Bacillus licheniformis DW2△aprE, the optimal temperature and pH of AE0 were 60 °C and 10.5, the K and K values for casein were 17.25 mg/mL and 60.51 s, respectively, as well as the specific activity was 21,365.93 U/mg. Subsequently, six mutants (G113I, H118D, T141Y, S151A, N167S and Q185S) were obtained through semi-rational design, and G113I exhibited the most optimal performance with a specific activity of 28,150.64 U/mg. Furthermore, the double mutant AE0 and triple mutant AE0 were attained, and their specific activities reached 31,026.32 U/mg and 31,868.56 U/mg, respectively. Concurrently, through the evaluation of thermal stability and measurement of reaction kinetic parameters, G113I was advantageous for enhancing the thermal stability of AE0, while H118D and N167S were more beneficial for enhancing the catalytic efficiency. In addition, the enzyme activity of AE0 produced by strain DW2△aprE/RC0-AE0 was increased by 178.5 % through promoter engineering, reached 36,685.33 U/mL, which also showed the wonderful performance on enzymatic hydrolysis of soybean meal to enhance its utilization rate. Taken together, this work provided an alkaline protease with improved thermal stability and catalytic efficiency, as well as an efficient expression system of alkaline protease for industrial application.