Purification, characterization, and anticoagulant mechanism of an anticoagulant protein from marine Bacillus velezensis B01.

Anticoagulants are the primary drugs for the prevention and treatment of thromboembolism. However, most currently available anticoagulants have certain limitations, and there is an urgent need for novel anticoagulants. Marine microorganisms represent a valuable reservoir for the discovery of novel drugs. A marine microorganism capable of producing an anticoagulant protein was isolated from the mangrove mudflat sediment in Beihai City, China. The strain was classified and identified based on 16S rRNA gene sequencing and sequence alignment, combined with phenotypic characteristics. The anticoagulant protein was isolated and purified by ammonium sulfate precipitation, DEAE Sepharose Fast Flow anion exchange chromatography, and Sepharose CL-6B gel filtration chromatography. Its molecular weight was determined by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) and COSMOSIL 5Diol-120-II gel filtration chromatography. Physicochemical properties, hemolytic safety, and the effect on erythrocyte aggregation were also examined. To evaluate its anticoagulant effect, in vitro experiments including inhibition of fibrin formation and fibrinogen degradation were performed. The results showed that the strain was identified as Bacillus velezensis and designated as B01. The purified anticoagulant protein had a molecular weight of 27.2 kDa. Its optimal reaction temperature was 50 °C, and it exhibited good temperature stability in the range of 25-37 °C. The optimal pH was 9.0, and it remained stable under alkaline conditions, particularly at pH 9.0-10.0. Cu2+ and Fe3+ significantly inhibited the protein's activity, whereas Zn2+, Ca2+, and Mn2+ promoted its anticoagulant activity. The protein showed no hemolytic activity and did not induce erythrocyte aggregation. It degraded fibrinogen with a chain degradation order of γ > Bβ > Aα, a pattern that is relatively rare. This anticoagulant protein has the potential to become a novel anticoagulant agent.