【作者】 刘柱；

【导师】 江波；

【作者基本信息】 江南大学 ， 食品科学， 2009， 硕士

【摘要】 血栓栓塞性疾病主要包括脑血栓、心肌梗死、静脉血栓等,是目前临床上死亡率最高的疾病之一,并且在世界范围内的发病率正逐年上升,因此人们一直都在研制开发新型的溶血栓药物。本文从亚洲传统发酵食品——虾酱中筛选到一株产纤溶酶能力较强的菌株,由中国典型培养物保藏中心定名为Bacillus sp.nov.SK006（CCTCC NO.:M 205071）,已经证明该菌株能产生四种不同的纤溶酶,本文在优化该菌产酶条件的基础上,主要对芽孢杆菌Bacillus sp.nov.SK006产生的其中一种纤溶酶（SPFE-Ⅲ）作了深入的研究。首先在摇瓶水平上考察了培养基组成及培养条件对Bacillus sp.nov.SK006发酵产纤溶酶能力的影响,结果表明该菌株产酶最佳的培养基组分为:葡萄糖20 g/L,胰蛋白胨30g/L,Na₂HPO₄·12H₂O 15g/L,NaH₂PO₄·2H₂O 1.3g/L,MgSO₄·7H₂O 0.5g/L,CaCl₂0.1g/L;最佳培养条件:种龄18h,接种量3%,发酵周期24h,初始pH为7.0,摇床转速180r/min,发酵温度37℃,在此条件下发酵液纤溶活性（以plasmin为标准）高达2.63 U/mL,是优化前0.70 U/mL的3.76倍。芽孢杆菌Bacillus sp.nov.SK006的发酵液经乙醇沉淀、离子交换层析（DEAE-Sepharose CL-6B）、凝胶过滤层析（Superdex 75）后分离纯化出一种电泳纯的纤溶酶SPFE-Ⅲ,分子量约为42.8 kDa,酶活为7.1 U/mg（以plasmin为标准）。SPFE-Ⅲ对纤维蛋白原的降解过程为最先降解α链,其次是γ链,而对β链的降解最缓慢;利用加热纤维蛋白平板法研究纤溶酶SPFE-Ⅲ对纤维蛋白的作用方式,结果发现该酶对纤维蛋白有直接降解作用,而对纤溶酶原的敏感性较低。其次研究了纯化后纤溶酶SPFE-Ⅲ的酶学性质:最适反应温度为30-40℃,最适反应pH范围为7.0-8.0,在40℃下保温1h剩余酶活力约为60%,而在50℃保温1h剩余酶活力只有30%左右,纤溶酶SPFE-Ⅲ对高温较为敏感。金属离子实验表明:在实验选择的范围内,除Ba²⁺外其它金属离子对纤溶酶SPFE-Ⅲ纤溶活性均有一定程度的抑制作用,其中Ca²⁺、Mn²⁺、Fe³⁺和Zn²⁺的抑制作用较强,抑制率为50%左右。抑制剂实验表明:蛋白酶抑制剂对纤溶酶SPFE-Ⅲ有不同程度的抑制作用,EDTA和β-巯基乙醇对纤溶酶SPFE-Ⅲ都有较强的抑制作用。最后分别选择了三种人工合成的发光底物D-Val-Leu-Arg-pNA、D-Val-Leu-Lys-pNA和N-Succ-Ala-Ala-Pro-Phe-pNA与纤溶酶SPFE-Ⅲ反应。SPFE-Ⅲ对底物N-Succ-Ala-Ala-Pro-Phe-pNA具有最强的专一性,其酰胺水解活性是75.77AU,该底物是胰凝乳蛋白酶和枯草杆菌蛋白酶的最适底物。对N-Succ-Ala-Ala-Pro-Phe-pNA的米氏常数K_m为0.239 mmol/L,酶反应转换数k_cat为475.5s^-1,表明纤溶酶SPFE-Ⅲ对该底物的亲和性较好,反应速度较高。更多还原

【Abstract】 The incidence of thrombotic disorder including cerebral stroke, myocardial infarction, and venous thromboembolism are rapidly increasing throughout the world. A tremendous amount of research has been done in the area of prevention and the treatment of the diseases. An extracellar fibrinolytic strain, isolated from fermented shrimp paste, a popular seasoning in Asian countries, was shown to have a strong fibrinolytic activity. It was named as Bacillus sp. nov. SK006 by CCTCC. The strain was proved to produce four fibrinolytic isoenzymes. In this study, one fibrinolytic enzyme （SPFE-Ⅲ） from Bacillus sp. nov. SK006 was intensively investigated.The medium composition and fermentation conditions for fibrinolytic enzyme production were optimized in the research. The maximal yield of fibrinolytic enzyme （2.63 U/mL） could be obtained in the medium with concentrations of 20 g/L glucose, 30g/L tryptone, 15 g/L Na₂HPO₄·12H₂O, 1.3 g/L NaH₂PO₄·2H₂O, 0.5 g/L MgSO₄·7H₂O, 0.1 g/L CaCl₂, respectively, under the optimized cultivation conditions of pH 7.0, rotation speed 180 r/min and inoculation size 3% in 50 mL of culture medium in 250 mL shake flasks at 37℃for 24 h. The fibrinolytic enzyme production of Bacillus sp. nov. SK006 was 3.76 times of the control.A fibrinolytic enzyme （SPFE-Ⅲ） which produced from Bacillus sp. nov. SK006, was isolated using a combination of ethanol precipitation, DEAE-Sepharose CL-6B ion exchange chromatography, and Superdex 75 gel filtration chromatography. The purified SPFE-Ⅲthat has a molecular weight of 42.8 kDa was assessed homogeneous by SDS-PAGE. The specific activity was determined to be 7.1 U/mg using plasmin as a standard. SPFE-Ⅲeffectively degraded fibrin clots by direct fibrinolysis. During the degradation process of fibrinogen,α-subunits of fibrinogen were cleaved first, followed by slower release of theγ-chains.β-chains were resistant to the enzyme digestion.The enzyme had an optimal pH of 7.0-8.0. It had an optimal reaction temperature of 30℃-40℃, and kept 60% and 30% of the initial activity after heating at 40℃and 50℃for 1 hour. In addition, barium ion stimulated the enzyme activity whereas Zn²⁺, Ca²⁺, Fe³⁺, Mn²⁺ and Cu²⁺ caused deactivation. SPFE-Ⅲwas completely inhibited either by 10 mmol/L EDTA or 1 mmol/L 2-mercaptoethanol. The fibrinolytic activity was partially inhibited by other protease inhibitor.SPFE-Ⅲhad the highest affinity for N-Succ-Ala-Ala-Pro-Phe-pNA, which is a well-known substrate for subtilisin or chymotrypsin. SPFE-Ⅲalso degraded D-Val-Leu-Lys-pNA （for plasmin）. However, it showed a lower activity for D-Val-Leu-Arg-pNA （for kallikrein） compared with other investigated fibrinolytic enzymes. K_m and k_cat for N-Succ-Ala-Ala-Pro-Phe-pNA were 0.239 mmol/L and 475.5 s^-1, respectively. In conclusion, SPFE-Ⅲ, a new type of fibrinolytic enzyme, has some potential for practical application as a source of functional foods and new therapeutic agents to treat thrombosis.更多还原