Biochemical characteristics of a fibrinolytic enzyme purified from a marine bacterium, Bacillus subtilis HQS-3

Abstract

A fibrinolytic enzyme isolated from marine Bacillus subtilis HQS-3 was purified to electrophoretic homogeneity using ammonium sulphate precipitation, alkaline solution treatment, membrane concentration, dialysis, ion exchange, and gel filtration chromatography. SDS-PAGE and gel filtration chromatography showed that it was a monomeric protein with an apparent molecular weight of 26 kDa. The purified enzyme was active at pH 6.0-10.0 with an optimum pH of 8.0. It was stable at temperatures ranging from 25 to 37 °C, exhibiting maximum activity between 45 °C and 50 °C. The isoelectric point of the enzyme was 9.0-9.2, which was higher than those of other known fibrinolytic enzymes from Bacillus species. PMSF, EDTA, Cu(2+), Zn(2+), and Co(2+) inhibited the enzyme activity significantly. This enzyme did not cause hemolysis in vitro and preferred direct degradation of fibrin in the following order: α, β, and γ-γ chains. Thus, these results suggest that the marine-derived enzyme is a plasmin-like serine metalloprotease, which is distinct from other fibrinolytic enzymes from genus Bacillus.

Keywords: Bacillus subtilis; Fibrinolytic activity; Plasmin-like serine metalloprotease.