根霉12^#发酵产生纤溶酶的酶学性质

Characteristics of a New Fibrinolytic Enzyme Produced from Rhizopus chinensis 12 #

溶栓疗法是血栓性疾病安全有效的治疗手段 ,开发新型纤溶酶具有实际应用意义。分离自南方小酒药的根霉 12 #以豆粕和麸皮为原料可产生纤溶酶。已采用盐析 ,疏水层析、离子交换层析和凝胶层析方法对纤溶酶分离提纯。提纯的纤溶酶比活力 2 14 3u mg(尿激酶单位 ) ,有直接溶解血栓和激活纤溶酶原的双重溶栓作用 ,降解纤维蛋白α、β和γ肽链速度快 ;最适作用温度 4 5℃ ,适宜作用pH范围 6 . 8～ 8. 8;等电聚焦方法测定该酶等电点 8 .5± 0. 1;只分解生色底物N_Succinyl_Ala_Ala_Pro_Phe_pNA ,其米氏常数Km 为 0 .2 3mmol L ,酶转换数Kcat为 16 36s- 1 ;Molish实验和甲苯胺蓝实验均证明该酶为糖蛋白 ,地衣酚_硫酸法测得该酶含糖量 4 70 % ;EDTA、PMSF、PCMB对该纤溶酶有抑制作用 ,说明活性中心含有巯基、金属和丝氨酸 ;N端 12个氨基酸序列为NH2 _Ser_Val_Ser_Glu_Ile_Gln_Leu_Met_His_Asn_Leu_Gly,与其它生物来源的纤溶酶相比较没有同源性。根霉 12 #产生的纤溶酶为新型纤溶酶 ,有希望开发成溶栓药物。

As a therapeutic agent in thrombosis the fibrinolytic e nzymes are of interest and the search for a new enzyme continues. A novel fibrin olytic enzyme was produced from Rhizopus chinensis 12#, which w as screened from the starter for brewing rice wine in the South of China, by sol id fermentation, and purified through ammonium sulfate precipitation, hydrophobi c interaction, ionic exchange and gel filtration chromatographies. The purified enzyme hydrolyzed fibrin, it cleaved the α-, β- and γ-ch ains of fibrinogen simultaneously, and it also activated plasminogen to plasmin. The enzyme hydrolyzed N-Succinyl-Ala-Ala- Pro-Phe-pNA, and K\-m was 0.23mm ol/L and K cat 16.36 s -1. The optimal temperature of the enzy me for hydro lying fibrin was 45℃, and the optimal pH range of 6.8～8.8. The isoelectric point of the enzyme estimated by isoelectric focusing electrophoresis was 8.5± 0.1. The enzyme was a glycoprotein. EDTA、PCMB、PMSF inhibited the activety of the en zyme, and SBTI、Lys、TPCK、Aprotinine had none obvious inhibition, which suggest ed that the activity centre of the enzyme had hydrosulfuryl, metal and serine. T he first 12 amino acids of the N-termimal sequence of the enzyme were NH 2- Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly,and had none homology with that of other fibrinolytic enzyme from other microbes. The novel fibrinolytic enzyme from Rhizopus chinensis 12# has potential to become a therapeu tic agent in thrombosis.