食神鞘氨醇杆菌(Sphingo spiritivorum)肝素酶的制备和性质研究

Preparation and Characterization of Novel Heparinase from Sphingo spiritivorum

筛选新型肝素酶产生菌,制备新型肝素酶并对其降解肝素能力进行测试。采用定向富集方法对土壤中的菌群进行培养,筛选分离产肝素酶的新种菌株,利用微生物发酵和蛋白纯化技术制备肝素酶,考察新型肝素酶的理化性质和酶切位点,之后使用新型肝素酶降解肝素并测试产物特征。筛选分离得到1株新的肝素酶产生菌,属食神鞘氨醇杆菌(Sphingo spiritivorum)。发酵后得到的粗酶,经多步柱层析纯化后得到的新型肝素酶SShep I和SShep II,用得到的酶降解肝素得到两种新型酶制低分子肝素。利用食神鞘氨醇杆菌中分离纯化得到的两种新型肝素酶SShep I和SShep II制备的新型酶制低分子肝素,有望开发成抗凝血效果更佳或具有新临床功能的低分子肝素药物。

This work aims to screen new type of strain producing novel heparinase,and to prepare novel heparinase and test its ability ofdegrading heparin. The bacteria in soil were cultured using the directional enrichment method,and strains producing heparinase were isolated.Microbial fermentation and purification technology were adopted to prepare novel heparinase,and to study the physical and chemical propertiesand the cleavage site of novel heparinase. Then,the heparinase were applied to degrade heparin,and the obtained low molecular weightheparins（LMWHs）were characterized. A new heparinase-producing strain belonging to the genus of Sphingo spiritivorum was isolated. Thecrude enzyme after fermentation was purified with multi-step column chromatography and novel heparinase SShep I and SShep II were obtained,with which two kinds of novel low molecular weight heparins were generated by the degradation of heparin. Conclusively,novel heparinaseSShep I and SShep II isolated and purified from a strain of S. spiritivorum are adopted to degrade heparin for preparing low molecular weightheparins,which are expected to be developed into LMWH drugs with better anticoagulant effect or new clinical function.