Characterization of an Intracellular Alkaline Serine Protease from Bacillus velezensis SW5 with Fibrinolytic Activity.

ISP-SW5 is an intracellular alkaline serine protease gene from Bacillus velezensis SW5 that was heterologously expressed in Escherichia coli BL21 (DE3). Sequence analysis indicated that the ISP-SW5 gene has 960 bp open reading frame and encodes a protein of 319 amino acid residues. Three-dimensional structure of ISP-SW5 with the fibrinolytic activity from Bacillus velezensis was predicted by in silico analysis. Gly219 was the most likely active site for the fibrinolytic activity of ISP-SW5. The recombinant enzyme ISP-SW5 was purified by Ni-NTA Superflow Column. SDS-PAGE showed that this enzyme had a molecular mass of 34 kDa. The result of native-PAGE and N-terminal sequencing showed that the N-terminal propeptide of ISP-SW5 was cleaved during the maturation of protease. The optimum pH and temperature were 8.0 and 40 °C, respectively. Enzyme activity was markedly inhibited by PMSF and EDTA but enhanced by 5 mM Ca and 2 mM Zn by up to 143% and 115%, respectively. Additionally, ISP-SW5 retained 93%, 78%, and 49% relative enzyme activity after incubation with 0.5 M, 1 M and 2 M NaCl, respectively, at 4 °C for 12 h. The enzyme activity determined by casein as substrate was 1261 U/mg. ISP-SW5 could degrade fibrin at an activity of 3428 U/mg, and its properties reflect its potential application in developing a novel biological catalyst for efficient fibrin hydrolysis in medical treatment.