Marine organisms are a rich source of enzymes that exhibit excellent biological activity and a wide range of applications. However, there has been limited research on the proteases found in marine mudflat organisms. Based on this background, the marine fibrinolytic enzyme FELP, which was isolated and purified from clamworm (Perinereis aibuhitensis), has exhibited excellent fibrinolytic activity. We demonstrated the FELP with a purification of 10.61-fold by precipitation with ammonium sulfate, ion-exchange chromatography, and gel-filtration chromatography. SDS-PAGE, fibrin plate method, and LC–MS/MS indicated that the molecular weight of FELP is 28.9 kDa and identified FELP as a fibrinolytic enzyme-like protease. FELP displayed the maximum fibrinolytic activity at pH 9 (407 ± 16 mm2) and 50 °C (724 ± 27 mm2) and had excellent stability at pH 7–11 (50%) or 30–60 °C (60%), respectively. The three-dimensional structure of some amino acid residues of FELP was predicted with the SWISS-MODEL. The fibrinolytic and fibrinogenolytic assays showed that the enzyme possessed direct fibrinolytic activity and indirect fibrinolysis via the activation of plasminogen; it could preferentially degrade Aα-chains of fibrinogen, followed by Bβ- and γ-chains. Overall, the fibrinolytic enzyme was successfully purified fromPerinereis aibuhitensis,a marine Annelida (phylum), with favorable stability that has strong fibrinolysis activity in vitro. Therefore, FELP appears to be a potent fibrinolytic enzyme with an application that deserves further investigation.Keywords:Perinereis aibuhitensis;plasmin-like protease;amino acid residue sequences;fibrinolysis in vitro
The change of macrofaunal bioturbation potential (BP) under environmental stress has application value in ecological restoration and ecological models. Single and combined toxic effects of metals cadmium and copper on the BP of polychaete Perinereis aibuhitensis were studied. The distribution of tracer sand showed a negative correlation between the transportation capacity of P. aibuhitensis and the Cd concentration; Cu stress indicated a stronger ability of promoting downward transportation, and the linear relationship with concentration was not so obvious as Cd. The toxicity stress of Cd and Cu also showed a significant effect on the oxidation-reduction potential (ORP) in the sediment matrix. There was a certain synergistic effect between cadmium and copper, and toxicity effects were associated with metal concentration and stress duration. In the later stage of the combined experiment, P. aibuhitensis avoided living in depth near the bottom, especially when the concentrations of Cd and Cu were high. For the bioturbation model, it was suggested that the two parameters of mobility and reworking would be reduced by half or one category scale depending on the cadmium and copper concentration and stress duration. The results can be used for ecological restoration prediction and ecological risk assessment ; it is necessary to carry out more studies with a variety of environmental factors and indicators, since a variety of coexisted pollutants would show complex influence on the BP of macrobenthos .
Tuo Song,Xiaozhen Diao,Jun Cheng,Yang Man,Boyu Chen,Haixing Zhang,Wenhui Wu
As an essential thrombolytic agent, the tissue plasminogen activator receives increasing attention due to its longer half-life, lower immunogenicity, and easier administration, which are superior to other thrombolytic agents. In this study, the isolated and purified plasminogen activator from the sandworm (Perinereis aibuhitensis) was expressed inE.coli(Escherichia coli) to investigate its potential for simplifying the development process. The sandworm plasminogen activator was previously successfully cloned and expressed inE.coliwith low yield and activity in the culture supernatant. This low yield and activity prompted us to optimize its DNA sequence. Furthermore, to raise the efficiency in the separation of the target protein, the protein’s solubility was enhanced by fusing it with maltose-binding protein (MBP) tags. Eventually, the fibrinolytic activity was successfully restored after digestion with tobacco etch virus (TEV) protease. This study provides an innovative method of efficiently expressing and purifying plasminogen activators from sandworm inE.coliand broadens its applications in therapeutic treatment of cardiovascular diseases, including thrombosis, stroke, and coronary atherosclerotic heart disease.
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