Abstract
A potent fibrinolytic serine protease (Subtilisin CIR) of B. subtilis CIR 110 isolated from fermented food was covalently immobilized onto porous cellulose beads and polyurethane (PU) surfaces, in an attempt to develop a new and useful antithrombogenic materials. The immobilization methods were based on N-hydroxysuccinimide activation for cellulose beads and the surface treatment with maleic anhydridemethyl vinyl ether copolymer (MAMEC) as an enzyme carrier for PU surfaces, respectively. The immobilized Subtilisin CIR retained its caseinolytic and fibrinolytic activity to a significant extent, digesting fibrinogen and fibrin irrespective of the presence of plasminogen. It was stable up to 60°C, and demonstrated better storage stability at 37°C, and highly increased resistance to plasma protease inhibitors, such as a2-macroglobulin, compared to soluble Subtilisin CIR. In the preliminary studies of in vitro test with human whole blood and ex vivo rabbit A-V shunt experiment for Subtilisin CIR immobilized PU surfaces, it was demonstrated that the coagulation times were much prolonged and fewer blood cells adhered on the Subtilisin CIR immobilized PU surfaces, as compared with the control MAMEC treated PU surface. This indicates that Subtilisin CIR immobilized surfaces possess good blood compatibility as well as high thrombolytic activity. Thus, the immobilization of Subtilisin OR provides promise for potential applications in antithrombogenic biomaterials used in blood-contacting medical devices and artificial organs.
