Genome sequencing, purification, and biochemical characterization of a strongly fibrinolytic enzyme from bacillus amyloliquefaciens jxnuwx-1 isolated from Chinese traditional douchi

Abstract

A strongly fibrinolytic enzyme was purified from Bacillus amyloliquefaciens Jxnuwx-1, found in Chinese traditional fermented black soya bean (douchi). The molecular mass of the enzyme, esti-mated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was 29 kDa. The optimal pH and temperature for the enzyme were 7.6 and 41°C, respectively. The enzyme was inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, ethylenediaminetetraacetic acid, Fe3+, and Fe2+. The highest affinity exhibited by the enzyme was towards N-Succinyl-Ala-Ala-Pro-Phe-pNA. These results indicated that it is a subtilisin-like serine metalloprotease. The enzyme degraded both fibrinogen and fibrin, displaying its highest degrading activity towards the Aα-chains followed by Bβ chains and Cγ chains. The enzyme was also activated by plasminogen, indicating its ability to degrade fibrinogen and fibrin in two ways: (a) by activating plasminogen conversion into plasmin, or (b) by direct hydrolysis. It degraded thrombin, sug-gesting that it may act as an anticoagulant to pre-vent thrombosis. Taken together, our results indi-cate the potential of this enzyme in controlling car-diovascular disease.