一株溶血栓细菌的鉴定及其溶栓活性成分的研究
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摘要
心脑血管疾病已成为对人类健康的最大威胁之一。近几十年来,该病的发病率逐渐升高,对溶栓药物的需求大增,因此研制溶栓药具有十分重要的意义。本研究对一株来自青藏高原海拔4300 m的河流泥土的纤溶酶高产菌株DR-929进行鉴定,并对该细菌分泌的纤溶酶进行分离纯化和探索其在动物体内的抗栓、溶栓效果。
(1)通过生理生化特征和16S rRNA基因序列分析对菌株DR-929进行了鉴定。生理生化试验结果表明,菌株DR-929与嗜麦芽寡养单胞菌具有高度相似性,其16S rRNA基因序列分析结果显示,菌株DR-929的16S rRNA基因序列与嗜麦芽寡养单胞菌具有同源性,二者在进化分析中被聚为一类,可信度达到99 %。另外,该菌并不产生溶血素,而且其普通肉汤培养物对小鼠也没有毒性。
(2)菌株DR-929分泌纤溶酶的液体发酵优化条件为:可溶性淀粉2.0 %,黄豆粉1.0 %,NaCl 1.0 %, CaCl2 0.02 %,MgSO4 0.05 %,种龄36 h,发酵时间4 d,初始pH 8.0或9.0,温度25℃,装样量30 mL,接种量5 %或6 %,经优化发酵条件后活力达188.66 IU/mL。
(3)经硫酸铵盐析、疏水层析、Q FF阴离子交换层析以及G-75葡聚糖凝胶过滤层析等步骤,分离纯化得到菌株DR-929分泌的纤溶酶,其在SDS-PAGE凝胶电泳胶上呈单一均匀的条带,纯化倍数为271.5倍,回收率为24.5 %,活性为7003.8 IU/mg(以尿激酶为标准)。
(4)菌株DR-929分泌的纤溶酶的分子量为28300 Da,最适反应温度和最适反应pH分别为45℃和pH 8.0,其在45℃温度下稳定,并且在pH 5.0～9.0范围内也比较稳定。另外,该纤溶酶是一种金属蛋白酶,活性能被EDTA完全抑制,而PMSF不影响其活性。该酶也属于纤维蛋白溶解酶类,其不具有纤维蛋白溶酶原激活剂的功能。
(5)采用小鼠抗凝血模型、大鼠静脉血栓形成模型和兔颈动脉血栓模型探索了菌株DR-929分泌的纤溶酶(FA-Ⅰ)分别经口服、肠道给药和静脉注射后在体内的药效。结果显示:FA-Ⅰ明显延长凝血时间(CT)、凝血酶原时间(PT)、活化的部分凝血活酶时间(APTT)和凝血酶时间(TT)(P<0.05或P<0.01),显著降低优球蛋白溶解时间(ELT)和纤维蛋白原(FIG)含量(P<0.01),并且能改善血液流变状态。表明FA-Ⅰ无论静脉注射还是口服均能达到抗栓、溶栓效果。
综上所述,本研究中菌株DR-929分泌的纤溶酶可能是一种新型的纤维蛋白溶解酶,其体内溶栓效果表明不但有望开发成实用的静脉注射溶栓药物,而且可以开发成安全的口服溶栓药物或保健品。
Cardiovascular diseases has been one of dangers for human health. Last decades, its incidence has been increasing gradually, the request for fibrinolytic agents has been also increasing. Therefore, it is very important of reseaching fibrinolytic agents. In this study, a fibrinolytic enzyme-producing microbial strain DR-929 isolated from the sediment of river located in Tibetan Plateau at an elevation of 4300 m was identified. We also purified the fibrinolytic enzyme from strain DR-929 and researched the effect of this fibrinolytic enzyme on thrombosis and thrombolysis.
(1) Strain DR-929 was identified by physiological and biochemical test and 16S rRNA gene sequence evolutional analysis. Results of physiological and biochemical test indicated that strain DR-929 has high similarity of Stenotrophomonas maltophilia. The result of 16S rRNA gene sequence evolutional analysis showed that the 16S rRNA gene sequence of strain DR-929 was homologous with of Stenotrophomonas maltophilia, and those two sequences were clustered together with a high reliability of 99 % when constructing phylogenetic tree. Furthermore, this strain didn’t produced hemolysin and its fermented broth hadn’t pathogenicity to mouse.
(2) The optimization on liquid fermentation of the fibrinolytic enzyme from strain DR-929 was investigated in this paper. The results showed the best fermentation are amidulin 2.0 %, soya flour 1.0 %, NaCl 1.0 %, CaCl2 0.02 %, MgSO4 0.05 %, inoculum of 36 hours, fermental time 4 d, initial pH 8.0 or 9.0, temperature 25℃, volume of media 30 mL(in 150 mL-flask), volume of inoculum 5 % or 6 %. The activity of fermented broth was 188.66 IU/mL after optimized its liquid fermentation.
(3) Via ammonium sulfate salting out, hydrophobic chromatography with Phenyl FF pre-packed column (high sub), ion exchange with HiTrapTM Q FF pre-packed column and gel filtration chromatography with SuperdexTM 75 pre-packed column (10/300 GL), the fibrinolytic enzyme produced by strain DR-929 was isolated and purified, which was showed a single strip in the gel of SDS-PAGE, and purification fold reached 271.5, yield was 24.5 %, and fibrinolytic activity was 7003.8 IU/mg (urokinase is standard).
(4) The fibrinolytic enzyme’s molecular weight was 28300 Da. This enzyme had high fibrinolytic activities at 45℃or pH 8.0, and was stable below 45℃and pH 5.0～9.0. EDTA inhibited its fibrinolytic activity completely and PMSF didn’t inhibit its fibrinolytic activity, which indicated fibrinolytic enzyme from strain DR-929 was a metalloprotease. And it was also a fibrinolytic enzyme, but not a plasminogen activator.
(5) Fibrinolytic enzyme (FA-Ⅰ) is isolated and purified from the metabolite of strain DR-929. In order to study its effect of anticoagulated blood, thrombosis and thrombolysis, investigations were made on the pharmacodynamics in vivo using the model of mouse anticoagulated blood by oral administration, the model of rat venous thrombogenesis inhibition after administered by intestinal route and the model of rabbit carotid thrombosis after treated by venoclysis. The experiment in vivo indicated that clotting time (CT), prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) were obviously prolonged by FA-Ⅰ(P<0.05 or P<0.01). Euglobulin lysis time (ELT) and fibrinogen (FIG) were markedly degraded by FA-Ⅰ(P<0.01). FA-Ⅰalso could improve the hemorheology condition in rabbit. This suggested that both intravenous injection and oral administration of FA-Ⅰcould have a role of thrombosis and thrombolysis
Summarily, the fibrinolytic enzyme produced by strain DR-929 may be a new fibrinolytic enzyme in this paper. The effect of this fibrinolytic enzyme on thrombosis and thrombolysis demonstrated that FA-Ⅰseem to be prospects to develope it not only as a pragmatic venoclysis thrombolytic drug, but also as a new peroral thrombolytic drug.
(1)通过生理生化特征和16S rRNA基因序列分析对菌株DR-929进行了鉴定。生理生化试验结果表明,菌株DR-929与嗜麦芽寡养单胞菌具有高度相似性,其16S rRNA基因序列分析结果显示,菌株DR-929的16S rRNA基因序列与嗜麦芽寡养单胞菌具有同源性,二者在进化分析中被聚为一类,可信度达到99 %。另外,该菌并不产生溶血素,而且其普通肉汤培养物对小鼠也没有毒性。
(2)菌株DR-929分泌纤溶酶的液体发酵优化条件为:可溶性淀粉2.0 %,黄豆粉1.0 %,NaCl 1.0 %, CaCl2 0.02 %,MgSO4 0.05 %,种龄36 h,发酵时间4 d,初始pH 8.0或9.0,温度25℃,装样量30 mL,接种量5 %或6 %,经优化发酵条件后活力达188.66 IU/mL。
(3)经硫酸铵盐析、疏水层析、Q FF阴离子交换层析以及G-75葡聚糖凝胶过滤层析等步骤,分离纯化得到菌株DR-929分泌的纤溶酶,其在SDS-PAGE凝胶电泳胶上呈单一均匀的条带,纯化倍数为271.5倍,回收率为24.5 %,活性为7003.8 IU/mg(以尿激酶为标准)。
(4)菌株DR-929分泌的纤溶酶的分子量为28300 Da,最适反应温度和最适反应pH分别为45℃和pH 8.0,其在45℃温度下稳定,并且在pH 5.0～9.0范围内也比较稳定。另外,该纤溶酶是一种金属蛋白酶,活性能被EDTA完全抑制,而PMSF不影响其活性。该酶也属于纤维蛋白溶解酶类,其不具有纤维蛋白溶酶原激活剂的功能。
(5)采用小鼠抗凝血模型、大鼠静脉血栓形成模型和兔颈动脉血栓模型探索了菌株DR-929分泌的纤溶酶(FA-Ⅰ)分别经口服、肠道给药和静脉注射后在体内的药效。结果显示:FA-Ⅰ明显延长凝血时间(CT)、凝血酶原时间(PT)、活化的部分凝血活酶时间(APTT)和凝血酶时间(TT)(P<0.05或P<0.01),显著降低优球蛋白溶解时间(ELT)和纤维蛋白原(FIG)含量(P<0.01),并且能改善血液流变状态。表明FA-Ⅰ无论静脉注射还是口服均能达到抗栓、溶栓效果。
综上所述,本研究中菌株DR-929分泌的纤溶酶可能是一种新型的纤维蛋白溶解酶,其体内溶栓效果表明不但有望开发成实用的静脉注射溶栓药物,而且可以开发成安全的口服溶栓药物或保健品。
Cardiovascular diseases has been one of dangers for human health. Last decades, its incidence has been increasing gradually, the request for fibrinolytic agents has been also increasing. Therefore, it is very important of reseaching fibrinolytic agents. In this study, a fibrinolytic enzyme-producing microbial strain DR-929 isolated from the sediment of river located in Tibetan Plateau at an elevation of 4300 m was identified. We also purified the fibrinolytic enzyme from strain DR-929 and researched the effect of this fibrinolytic enzyme on thrombosis and thrombolysis.
(1) Strain DR-929 was identified by physiological and biochemical test and 16S rRNA gene sequence evolutional analysis. Results of physiological and biochemical test indicated that strain DR-929 has high similarity of Stenotrophomonas maltophilia. The result of 16S rRNA gene sequence evolutional analysis showed that the 16S rRNA gene sequence of strain DR-929 was homologous with of Stenotrophomonas maltophilia, and those two sequences were clustered together with a high reliability of 99 % when constructing phylogenetic tree. Furthermore, this strain didn’t produced hemolysin and its fermented broth hadn’t pathogenicity to mouse.
(2) The optimization on liquid fermentation of the fibrinolytic enzyme from strain DR-929 was investigated in this paper. The results showed the best fermentation are amidulin 2.0 %, soya flour 1.0 %, NaCl 1.0 %, CaCl2 0.02 %, MgSO4 0.05 %, inoculum of 36 hours, fermental time 4 d, initial pH 8.0 or 9.0, temperature 25℃, volume of media 30 mL(in 150 mL-flask), volume of inoculum 5 % or 6 %. The activity of fermented broth was 188.66 IU/mL after optimized its liquid fermentation.
(3) Via ammonium sulfate salting out, hydrophobic chromatography with Phenyl FF pre-packed column (high sub), ion exchange with HiTrapTM Q FF pre-packed column and gel filtration chromatography with SuperdexTM 75 pre-packed column (10/300 GL), the fibrinolytic enzyme produced by strain DR-929 was isolated and purified, which was showed a single strip in the gel of SDS-PAGE, and purification fold reached 271.5, yield was 24.5 %, and fibrinolytic activity was 7003.8 IU/mg (urokinase is standard).
(4) The fibrinolytic enzyme’s molecular weight was 28300 Da. This enzyme had high fibrinolytic activities at 45℃or pH 8.0, and was stable below 45℃and pH 5.0～9.0. EDTA inhibited its fibrinolytic activity completely and PMSF didn’t inhibit its fibrinolytic activity, which indicated fibrinolytic enzyme from strain DR-929 was a metalloprotease. And it was also a fibrinolytic enzyme, but not a plasminogen activator.
(5) Fibrinolytic enzyme (FA-Ⅰ) is isolated and purified from the metabolite of strain DR-929. In order to study its effect of anticoagulated blood, thrombosis and thrombolysis, investigations were made on the pharmacodynamics in vivo using the model of mouse anticoagulated blood by oral administration, the model of rat venous thrombogenesis inhibition after administered by intestinal route and the model of rabbit carotid thrombosis after treated by venoclysis. The experiment in vivo indicated that clotting time (CT), prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) were obviously prolonged by FA-Ⅰ(P<0.05 or P<0.01). Euglobulin lysis time (ELT) and fibrinogen (FIG) were markedly degraded by FA-Ⅰ(P<0.01). FA-Ⅰalso could improve the hemorheology condition in rabbit. This suggested that both intravenous injection and oral administration of FA-Ⅰcould have a role of thrombosis and thrombolysis
Summarily, the fibrinolytic enzyme produced by strain DR-929 may be a new fibrinolytic enzyme in this paper. The effect of this fibrinolytic enzyme on thrombosis and thrombolysis demonstrated that FA-Ⅰseem to be prospects to develope it not only as a pragmatic venoclysis thrombolytic drug, but also as a new peroral thrombolytic drug.
引文
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