Abstract: In this study, through promoter optimization and host screening, an SplB expression vector with His-tag at the C-terminal was constructed, and SplB was successfully expressed in Bacillus subtilis. The enzymatic properties of the purified recombinant SplB protease were studied, and the application of recombinant SplB protease in the cleavage of recombinant protein Prx with a tag was explored. The results showed that using B. subtilis ATCC6051Δ10 as the host, the recombinant expression activity of SplB mediated by promoter P43 was the highest (10.24 U/mL). The recombinant SplB was purified by affinity chromatography and its enzymatic properties were studied. The optimum temperature was 40 ℃, the optimum pH was 8.5, and the purified SplB had good temperature and pH stability. Low concentrations of Co2+ promoted the activity of SplB, while low concentrations of Mg2+ and K+ did not affect its activity; low concentrations of Cu2+, Zn2+ and Ni+ inhibited the activity of SplB, and sodium dodecyl sulfate (SDS) greatly inhibited the enzyme activity of recombinant SplB. The recombinant SplB protease could concentration-dependently cleave the WELQ peptide tag on the recombinant protein Prx. This study provides support for optimizing the recombinant expression of SplB and its application in the fields of food and medicine.

Key words: chymotrypsin SplB; Bacillus subtilis; WElQ peptide tag