豆豉纤溶酶的纯化、生化特性研究及其体内外溶栓效果的评价
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摘要
近年来,血栓性疾病的发病率和死亡率居高不下,已成为严重威胁人类健康的心血管系统疾病。针对血栓病的治疗最有效和常规的方法是通过口服或注射纤溶剂来溶解血栓中的主要蛋白成分-纤维蛋白,从而引起血栓的溶解。现有溶栓剂存在价格昂贵、出血风险高、半衰期短、副作用大等问题,亟需开发新的溶栓药物来克服这些问题。本研究以我国传统大豆发酵食品-豆豉为研究对象,对豆豉纤溶酶产生菌的分离鉴定及液体发酵条件优化、豆豉纤溶酶提取纯化及基础生化特性研究、豆豉纤溶酶体内外抗栓效果评价等内容进行研究,目的在于从豆豉中分离出纤溶酶产生菌,提取并纯化耐热、耐酸碱、比活性高、特异性高、出血风险小、安全价廉的纤溶酶,为其开发成为新一代溶栓药物提供科学依据。
第一部分豆豉纤溶酶产生菌的分离鉴定及液体发酵条件的优化
目的：从不同豆豉样品中分离出产纤溶酶的菌株,并筛选出产量最高的一株,鉴定其种属。对其优化发酵条件和培养基成分,以获得最高产酶量。
方法：以纤维蛋白平板法初步筛选出产纤溶酶的细菌,再定量测定其发酵液的酶活性,选择酶活性最大的一株菌进行研究。对该菌株进行生理生化及16SrDNA序列研究,确定种属。以单因素实验方法筛选出该菌株液体发酵产酶所需的最佳条件(温度、初始pH、接种量、装液量)和最佳碳、氮源。根据Plackett-Burman设计确定影响产酶的关键培养基成分,再采用响应面法优化各关键成分的比例。最后根据优化结果拟合出多元回归方程,计算最佳培养基组成。按优化出的发酵条件和培养基,绘制该菌株的生长和产酶曲线。
结果：筛选到8株产溶栓酶菌株,命名产酶活性最高的一株菌为XY-1,该菌所产纤溶酶命名为豆豉纤溶酶(Douchi Fibrinolytic Enzyme, DFE)。经生理生化试验及16SrDNA序列鉴定,确定XY-1为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。最佳发酵条件为：温度40℃、装液量50ml/250ml、接种量4%、初始pH8.0。最佳碳、氮源为蔗糖和蛋白胨。培养基的最佳组成为：蛋白胨1.14%,蔗糖0.5%,MgSO40.05%, NaCl0.1%。DFE产量的实际最大值达到了21.33FU/ml,相当于最大预测值的107.84%,与未优化的培养基相比,增加了79.55%。细胞外酶活性远大于胞内酶活。该菌的生长迟缓期很短,不到4小时,然后进入对数生长期,16小时后,细菌生长进入稳定期。DFE的产量随着细菌数量的增多而增加,在24小时左右时达到最大值。
结论：从豆豉中分离到产纤溶酶的解淀粉芽孢杆菌XY-1(Bacillus amyloliquefaciens),其培养基的最佳组成为：蛋白胨1.14%,蔗糖0.5%,MgSO40.05%, NaCl0.1%,最佳发酵条件为：温度40℃、装液量50ml/250ml,接种量4%、初始pH8.0。DFE的产量在24小时左右时达到最大值。
第二部分豆豉纤溶酶的提取、纯化及基础生化特性研究
目的：探讨从菌株XY-1的发酵液中提取、纯化DFE的方法和条件,并在获得纯品DFE的基础上,研究该酶的生化特点。
方法：发酵液经过硫酸铵分段盐析、透析脱盐后,上SP sepharose fast flow阳离子层析柱提取纯化,并计算在此过程中酶活性和蛋白含量的变化。比较不同pH和温度对酶反应活性的影响,以及不同pH、温度、金属离子和化学抑制剂对DFE稳定性的影响。比较DFE在含有或不含纤溶酶原平板上的溶圈差异,鉴别其纤溶方式。根据Lineweaver-Burk法,以纤维蛋白作为底物,测定DFE的K氏反应常数Km和Vmax。将DFE纯品处理后,经HPLC-MS/MS检测,并将碎片结构与NCBI中非冗余蛋白质数据库的质谱信息进行比对,鉴定DFE类别。
结果：纯化的DFE经SDS-PAGE鉴定仅有一条带,达到电泳纯,分子量为27000Da。DFE被纯化了24.3倍,回收率为19.9%,比酶活为2647.3FU/mg。最佳反应pH为9.0,在pH7.0～10.0范围内相当稳定。DFE在40℃时活性最强,在40℃以下时活性较稳定。Ba2+和Co2+对DFE活性有明显促进作用,而Pb2+,Fe3+和Hg+会显著抑制DFE活性,Ca2+和Cu2+轻微抑制其活性。Na+,K+和Mg2+并未表现出对DFE有明显的抑制或促进作用。EDTA和PMSF完全抑制该酶活性,而β-巯基乙醇对其几乎无影响。DFE可以直接降解纤维蛋白,而不需要先激活纤溶酶原。Km值为6.98e-08mmol, Vmax为232.5FU。该酶N-末端的15个氨基酸残基序列为APALHSQGYTGSNVK,与纳豆激酶一致。
结论：经一系列提取纯化方法获得DFE纯品,该酶是一种丝氨酸蛋白酶,可以耐高温和耐碱,与纤维蛋白的亲和力非常高。
第三部分豆豉纤溶酶体内外抗栓效果评价
目的：评价DFE的溶栓作用,在体外研究其溶解血凝块的作用,在体内评价对大鼠血栓模型的抗栓效果。
方法：将DFE配制成不同浓度的溶液。从大鼠体内抽血,自主凝固后形成血凝块,切成若干份。分别加入高、中、低剂量的DFE溶液、生理盐水和UK溶液后,37℃孵育16小时,比较反应前后血凝块重量的变化。选取尾长大于15cm的S-D大鼠,分成DFE高、中、低剂量组,空白对照组、阴性对照组和UK组,先经一侧尾静脉注入各受试药物。30分钟后,结扎尾部,经另一侧尾静脉给予1mg/ml的卡拉胶(κ-carrageenan)溶液,立即冰浴1分钟,20分钟后除去结扎6小时后,测量尾部血栓长度,麻醉大鼠,经腹主动脉取血,制备血浆,测量APTT、TT、PT和D-dimer水平。
结果：生理盐水对照组,体外血凝块溶解率只有6.5%,尿激酶组溶解了47.9%的血凝块,DFE中、高剂量组血凝块的溶解率明显升高(59%vs.6.5%,p<0.01；71.7%vs.6.5%,p<0.01),有统计学意义,并呈剂量-反应关系。与空白组进行比较,阴性对照组大鼠尾部血栓平均长度为14.19±0.51cm。UK组的大鼠尾部血栓长度明显小于阴性对照组(10.45±3.02cm,p<0.01)。在DFE中(10.25±3.24cm,p<0.05)、高(8.31±3.32cm,p<0.01)剂量组,大鼠尾部血栓长度均明显短于生理盐水对照组,并呈剂量-反应关系。给药组的大鼠除了尾部血栓长度明显下降外,血栓处的红肿和炎症程度也不如阴性对照组明显。对于APTT,仅DFE中剂量组有统计学意义的增加(p<0.01)。对于PT,尿激酶组和DFE组均明显延长,并具有统计学意义。对于TT,不同组之间不具有统计学差异。阴性对照组的D-dimer水平明显升高,差异有统计学意义(p<0.01)。尿激酶组(p<0.05)和DFE高剂量组(p<0.01),其D-dimer水平均显著高于阴性对照组。
结论：DFE在体外溶解血凝块效果明显,在体内也有显著的抑制血栓形成的作用。
In recent years, the morbility and mortality of thrombotic diseases has been increasing, and has become one of the most serious cardiovascular diseases that threat to human health. For the treatment of thrombosis, the most effective and conventional method is by dissolving fibrin-the major protein component of blood clots orally or intravenously to further cause the collapse of thrombus. However, new thrombolytic agents need to be developed for the various shortages of existing thrombolytic agents, such as:high cost, hemorrhage risk, short high-life and side effects.
In this study, we investigated Douchi, a traditional Chinese fermented soybean food, from the following aspects:isolating and identifying a fibrinolytic enzyme-producing strain, optimization of liquid fermention, purifying enzyme and studying its biochemical and molecular structure, in vivo and in vitro evaluating anti-thrombolytic effect. We aimed to isolate a high-fibrinolytic enzyme-producing strain from Douchi and extract fibrinolytic enzyme with heat and alkaline tolerance, high specific activity, high specificity, little risk of hemorrhage, safe and low cost. All of the above will provide scientific evidence for developing new thrombolytic drugs.
Part Ⅰ
Isolation, Identification of Douchi Fibrinolytic Enzyme Producing Strain and Optimization of Liquid Fermentation Conditions
Objective:This section aimed to isolate a high fibrinolytic enzyme producing strain from different Douchi samples. Then, its species was identified and the fermentation condition and culture medium was optimized to increase enzyme yield.
Methods:The fibrin plate method was applied to screen fibrinolytic enzyme strains, then the strain which produced the most amount enzyme was chosed by comparing their enzyme activity. The strain was identified according to physio-biochemical characteristics. The optimal nitrogen and carbon source and fermentation conditions including temperature, primary pH, inoculate volume and medium volume were determined using single factor method. The key components of culture medium for enzyme production were screened by Plackett-Burman design and its concentration were further optimized by response surface method. The optimal culture medium was calculated according to the multiple regression equation that based on the above results. Cell growth and enzyme production curve were drawed following the optimal fermentation condition and culture medium.
Results:Eight strains that produce fibrinolytic enzyme were screened, and the strain which produced the most enzyme was named XY-1and selected for further investigation. The enzyme produced by XY-1was name as DFE (Douchi Fibrinolytic Enzyme). Strain XY-1was identified as Bacillus amyloliquefaciens. The optimal fermentation condition were:temperature40℃, medium volume50ml/250ml, inoculate volume4%, primary pH8.0. the optimal nitrogen and carborn source were sucrose and peptone. The optimal culture medium were:peptone1.14%, sucrose0.5%, MgSO40.05%, NaCl0.1%. The actual maximal DFE yield was21.33FU/ml, equal to107.84%of the predicted maximal value, and had increased79.55%comparing to non-optimized culture medium。Intracellular enzyme activity was much higher than extracellular. The growth retardation period of XY-1was short, no more than4hours, then it came to exponential growth phase. After16hours, cell growth came to stationary phase. The production of DFE increased with the growth of bacteria, and reached the maximal value at24hour.
Conclusion:The high-fibrinolytic enzyme-producing strain XY-1(Bacillus amyloliquefaciens) was isolated from Douchi. The optimal fermentation condition were:temperature40℃, medium volume50ml/250ml, inoculate volume4%, primary pH8.0. The optimal culture medium were:peptone1.14%, sucrose0.5%, MgSO40.05%, NaCl0.1%. The production of DFE reached the maximal value at24hour.
Part II
Extraction and Purification of Douchi Fibrinolytic Enzyme and Its Biochemical Characteristics
Objective:To research how to extract and purify DFE from the fermentation liquid of XY-1, and further investigate the biochemical characteristics of DFE.
Methods:The purified DFE was acquired by ammonium sulfate segmenting salting-out, dialysis desalination and SP sepharose fast flow chromatography. The change of enzyme activity and protein concentration during the process was calculated. The effect of pH, temperature, metal ions and chemical inhibitors on enzyme activity and stability were evaluated. The fibrinolytic manner of DFE was identified according to the difference of dissolving circle on fibrin plate with or without plasminogen. Following the method of Lineweaver-Burk, the K's constant(Km, Vmax) of DFE was determined with fibrin as substrate. DFE was identified by HPLC-MS/MS, and the fragmented structure was compared with mass spectral information of NCBI non-redundant protein database.
Results:The samples purified by above extraction steps were identified by SDS-PAGE, and only one band existing indicated it was pure with molecular weight of27000Da. DFE had been purified24.3-folds with recovery rate of19.9%and specific activity of47.3FU/mg. It was stable at pH7.0-10.0and the optimal pH was 9.0. DFE showed the highest activity at40℃and was stable below40℃. Ba2+and Co2+promoted DFE activity, while Pb2+, Fe3+and Hg+inhibited its activity. Ca2+and Cu2+inhibited its activity slightly, and Na+, K+and Mg2+didn't exhibit significant promotion and inhibition. EDTA and PMSF inhibited the enzyme activity completely, while P-mercaptoethanol almost had no effect. DFE could degrade fibrin directly, but not activate plasminogen first. Km and Vmax of DFE was6.98e-08mmol and232.5FU ml-1. The first15N-terminal amino acids of DFE was APALHSQGYTGSNVK, which is consistent with nattokinase.
Conclusion:Pure DFE was acquired after steps of extraction methods. According to its biochemical property, the enzyme was identified as a serine protease. It could tolerate high temperature and alkaline and had high afinity with fibrin.
Part Ⅲ
In vitro and vivo Anti-thrombolytic Evaluation of Douchi Fibrinolytic Enzyme
Objective:To evaluate the thrombolytic effect of DFE by in vitro blood clot dissolving test and in vivo anti-thrombolytic activity on a rat thrombus model.
Methods:DFE was purified following the methods in part II and prepared in different concentrations. Blood was drawed from a rat and clotted spontaneously. High, middle and low dose DFE solution, UK solution and physiology saline were added in blood clots, then the clots were incubated at37℃for16hours. The weight of blood clots before and after incubation were determined. S-D rats with tails more than15cm were selected for thrombus modeling. Agents for testing were injected through left caudal vein at first. After30min, the tails were ligated and lmg/ml κ-carrageenan were injected through right caudal vein. The tails were immersed in icy bath for1min, and after20min, the ligation was removed. The tail thrombosis length were measured6hours after κ-carrageenan were injected. Rat blood were collected from abdominal aorta under anesthesia and the plasma level of APTT, TT, PT and D-dimer were determined.
Results:in normal saline group, the dissolution rate of blood clots was6.5%, while UK group dissolved about47.9%. In middle and high dose DFE group, the dissolution rate of blood clots increased significantly with a dose-response manner(59%vs.6.5%, p<0.01;71.7%vs.6.5%,p<0.01). Compared to negative control group(14.19±0.51cm), the average tail thrombosis length of UK group were significantly short(10.45±3.02cm,/p<0.01), so were the middle and high dose DFE group(10.25±3.24cm,/p<0.05;8.31±3.32cm,p<0.01). besides, the red swelling and inflammation degree of thrombosis in agents treated groups were also not obvious as negative control group. Only DFE middle group showed significant increase for APTT value(p<0.01). PT value in UK and DFE group were prolonged significantly. No differences were observed between groups for TT value. Compared with blank group, the level of D-dimer in negative control group increased significantly(p<0.01). In UK(p<0.05) and high dose DFE group(p<0.01), the D-dimer level were significantly higher than negeative control group.
Conclusion:In vitro dissolving effect of DFE on blood clots was obviouse and it could also inhibit the formation of thrombus in vivo significantly.
第一部分豆豉纤溶酶产生菌的分离鉴定及液体发酵条件的优化
目的：从不同豆豉样品中分离出产纤溶酶的菌株,并筛选出产量最高的一株,鉴定其种属。对其优化发酵条件和培养基成分,以获得最高产酶量。
方法：以纤维蛋白平板法初步筛选出产纤溶酶的细菌,再定量测定其发酵液的酶活性,选择酶活性最大的一株菌进行研究。对该菌株进行生理生化及16SrDNA序列研究,确定种属。以单因素实验方法筛选出该菌株液体发酵产酶所需的最佳条件(温度、初始pH、接种量、装液量)和最佳碳、氮源。根据Plackett-Burman设计确定影响产酶的关键培养基成分,再采用响应面法优化各关键成分的比例。最后根据优化结果拟合出多元回归方程,计算最佳培养基组成。按优化出的发酵条件和培养基,绘制该菌株的生长和产酶曲线。
结果：筛选到8株产溶栓酶菌株,命名产酶活性最高的一株菌为XY-1,该菌所产纤溶酶命名为豆豉纤溶酶(Douchi Fibrinolytic Enzyme, DFE)。经生理生化试验及16SrDNA序列鉴定,确定XY-1为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。最佳发酵条件为：温度40℃、装液量50ml/250ml、接种量4%、初始pH8.0。最佳碳、氮源为蔗糖和蛋白胨。培养基的最佳组成为：蛋白胨1.14%,蔗糖0.5%,MgSO40.05%, NaCl0.1%。DFE产量的实际最大值达到了21.33FU/ml,相当于最大预测值的107.84%,与未优化的培养基相比,增加了79.55%。细胞外酶活性远大于胞内酶活。该菌的生长迟缓期很短,不到4小时,然后进入对数生长期,16小时后,细菌生长进入稳定期。DFE的产量随着细菌数量的增多而增加,在24小时左右时达到最大值。
结论：从豆豉中分离到产纤溶酶的解淀粉芽孢杆菌XY-1(Bacillus amyloliquefaciens),其培养基的最佳组成为：蛋白胨1.14%,蔗糖0.5%,MgSO40.05%, NaCl0.1%,最佳发酵条件为：温度40℃、装液量50ml/250ml,接种量4%、初始pH8.0。DFE的产量在24小时左右时达到最大值。
第二部分豆豉纤溶酶的提取、纯化及基础生化特性研究
目的：探讨从菌株XY-1的发酵液中提取、纯化DFE的方法和条件,并在获得纯品DFE的基础上,研究该酶的生化特点。
方法：发酵液经过硫酸铵分段盐析、透析脱盐后,上SP sepharose fast flow阳离子层析柱提取纯化,并计算在此过程中酶活性和蛋白含量的变化。比较不同pH和温度对酶反应活性的影响,以及不同pH、温度、金属离子和化学抑制剂对DFE稳定性的影响。比较DFE在含有或不含纤溶酶原平板上的溶圈差异,鉴别其纤溶方式。根据Lineweaver-Burk法,以纤维蛋白作为底物,测定DFE的K氏反应常数Km和Vmax。将DFE纯品处理后,经HPLC-MS/MS检测,并将碎片结构与NCBI中非冗余蛋白质数据库的质谱信息进行比对,鉴定DFE类别。
结果：纯化的DFE经SDS-PAGE鉴定仅有一条带,达到电泳纯,分子量为27000Da。DFE被纯化了24.3倍,回收率为19.9%,比酶活为2647.3FU/mg。最佳反应pH为9.0,在pH7.0～10.0范围内相当稳定。DFE在40℃时活性最强,在40℃以下时活性较稳定。Ba2+和Co2+对DFE活性有明显促进作用,而Pb2+,Fe3+和Hg+会显著抑制DFE活性,Ca2+和Cu2+轻微抑制其活性。Na+,K+和Mg2+并未表现出对DFE有明显的抑制或促进作用。EDTA和PMSF完全抑制该酶活性,而β-巯基乙醇对其几乎无影响。DFE可以直接降解纤维蛋白,而不需要先激活纤溶酶原。Km值为6.98e-08mmol, Vmax为232.5FU。该酶N-末端的15个氨基酸残基序列为APALHSQGYTGSNVK,与纳豆激酶一致。
结论：经一系列提取纯化方法获得DFE纯品,该酶是一种丝氨酸蛋白酶,可以耐高温和耐碱,与纤维蛋白的亲和力非常高。
第三部分豆豉纤溶酶体内外抗栓效果评价
目的：评价DFE的溶栓作用,在体外研究其溶解血凝块的作用,在体内评价对大鼠血栓模型的抗栓效果。
方法：将DFE配制成不同浓度的溶液。从大鼠体内抽血,自主凝固后形成血凝块,切成若干份。分别加入高、中、低剂量的DFE溶液、生理盐水和UK溶液后,37℃孵育16小时,比较反应前后血凝块重量的变化。选取尾长大于15cm的S-D大鼠,分成DFE高、中、低剂量组,空白对照组、阴性对照组和UK组,先经一侧尾静脉注入各受试药物。30分钟后,结扎尾部,经另一侧尾静脉给予1mg/ml的卡拉胶(κ-carrageenan)溶液,立即冰浴1分钟,20分钟后除去结扎6小时后,测量尾部血栓长度,麻醉大鼠,经腹主动脉取血,制备血浆,测量APTT、TT、PT和D-dimer水平。
结果：生理盐水对照组,体外血凝块溶解率只有6.5%,尿激酶组溶解了47.9%的血凝块,DFE中、高剂量组血凝块的溶解率明显升高(59%vs.6.5%,p<0.01；71.7%vs.6.5%,p<0.01),有统计学意义,并呈剂量-反应关系。与空白组进行比较,阴性对照组大鼠尾部血栓平均长度为14.19±0.51cm。UK组的大鼠尾部血栓长度明显小于阴性对照组(10.45±3.02cm,p<0.01)。在DFE中(10.25±3.24cm,p<0.05)、高(8.31±3.32cm,p<0.01)剂量组,大鼠尾部血栓长度均明显短于生理盐水对照组,并呈剂量-反应关系。给药组的大鼠除了尾部血栓长度明显下降外,血栓处的红肿和炎症程度也不如阴性对照组明显。对于APTT,仅DFE中剂量组有统计学意义的增加(p<0.01)。对于PT,尿激酶组和DFE组均明显延长,并具有统计学意义。对于TT,不同组之间不具有统计学差异。阴性对照组的D-dimer水平明显升高,差异有统计学意义(p<0.01)。尿激酶组(p<0.05)和DFE高剂量组(p<0.01),其D-dimer水平均显著高于阴性对照组。
结论：DFE在体外溶解血凝块效果明显,在体内也有显著的抑制血栓形成的作用。
In recent years, the morbility and mortality of thrombotic diseases has been increasing, and has become one of the most serious cardiovascular diseases that threat to human health. For the treatment of thrombosis, the most effective and conventional method is by dissolving fibrin-the major protein component of blood clots orally or intravenously to further cause the collapse of thrombus. However, new thrombolytic agents need to be developed for the various shortages of existing thrombolytic agents, such as:high cost, hemorrhage risk, short high-life and side effects.
In this study, we investigated Douchi, a traditional Chinese fermented soybean food, from the following aspects:isolating and identifying a fibrinolytic enzyme-producing strain, optimization of liquid fermention, purifying enzyme and studying its biochemical and molecular structure, in vivo and in vitro evaluating anti-thrombolytic effect. We aimed to isolate a high-fibrinolytic enzyme-producing strain from Douchi and extract fibrinolytic enzyme with heat and alkaline tolerance, high specific activity, high specificity, little risk of hemorrhage, safe and low cost. All of the above will provide scientific evidence for developing new thrombolytic drugs.
Part Ⅰ
Isolation, Identification of Douchi Fibrinolytic Enzyme Producing Strain and Optimization of Liquid Fermentation Conditions
Objective:This section aimed to isolate a high fibrinolytic enzyme producing strain from different Douchi samples. Then, its species was identified and the fermentation condition and culture medium was optimized to increase enzyme yield.
Methods:The fibrin plate method was applied to screen fibrinolytic enzyme strains, then the strain which produced the most amount enzyme was chosed by comparing their enzyme activity. The strain was identified according to physio-biochemical characteristics. The optimal nitrogen and carbon source and fermentation conditions including temperature, primary pH, inoculate volume and medium volume were determined using single factor method. The key components of culture medium for enzyme production were screened by Plackett-Burman design and its concentration were further optimized by response surface method. The optimal culture medium was calculated according to the multiple regression equation that based on the above results. Cell growth and enzyme production curve were drawed following the optimal fermentation condition and culture medium.
Results:Eight strains that produce fibrinolytic enzyme were screened, and the strain which produced the most enzyme was named XY-1and selected for further investigation. The enzyme produced by XY-1was name as DFE (Douchi Fibrinolytic Enzyme). Strain XY-1was identified as Bacillus amyloliquefaciens. The optimal fermentation condition were:temperature40℃, medium volume50ml/250ml, inoculate volume4%, primary pH8.0. the optimal nitrogen and carborn source were sucrose and peptone. The optimal culture medium were:peptone1.14%, sucrose0.5%, MgSO40.05%, NaCl0.1%. The actual maximal DFE yield was21.33FU/ml, equal to107.84%of the predicted maximal value, and had increased79.55%comparing to non-optimized culture medium。Intracellular enzyme activity was much higher than extracellular. The growth retardation period of XY-1was short, no more than4hours, then it came to exponential growth phase. After16hours, cell growth came to stationary phase. The production of DFE increased with the growth of bacteria, and reached the maximal value at24hour.
Conclusion:The high-fibrinolytic enzyme-producing strain XY-1(Bacillus amyloliquefaciens) was isolated from Douchi. The optimal fermentation condition were:temperature40℃, medium volume50ml/250ml, inoculate volume4%, primary pH8.0. The optimal culture medium were:peptone1.14%, sucrose0.5%, MgSO40.05%, NaCl0.1%. The production of DFE reached the maximal value at24hour.
Part II
Extraction and Purification of Douchi Fibrinolytic Enzyme and Its Biochemical Characteristics
Objective:To research how to extract and purify DFE from the fermentation liquid of XY-1, and further investigate the biochemical characteristics of DFE.
Methods:The purified DFE was acquired by ammonium sulfate segmenting salting-out, dialysis desalination and SP sepharose fast flow chromatography. The change of enzyme activity and protein concentration during the process was calculated. The effect of pH, temperature, metal ions and chemical inhibitors on enzyme activity and stability were evaluated. The fibrinolytic manner of DFE was identified according to the difference of dissolving circle on fibrin plate with or without plasminogen. Following the method of Lineweaver-Burk, the K's constant(Km, Vmax) of DFE was determined with fibrin as substrate. DFE was identified by HPLC-MS/MS, and the fragmented structure was compared with mass spectral information of NCBI non-redundant protein database.
Results:The samples purified by above extraction steps were identified by SDS-PAGE, and only one band existing indicated it was pure with molecular weight of27000Da. DFE had been purified24.3-folds with recovery rate of19.9%and specific activity of47.3FU/mg. It was stable at pH7.0-10.0and the optimal pH was 9.0. DFE showed the highest activity at40℃and was stable below40℃. Ba2+and Co2+promoted DFE activity, while Pb2+, Fe3+and Hg+inhibited its activity. Ca2+and Cu2+inhibited its activity slightly, and Na+, K+and Mg2+didn't exhibit significant promotion and inhibition. EDTA and PMSF inhibited the enzyme activity completely, while P-mercaptoethanol almost had no effect. DFE could degrade fibrin directly, but not activate plasminogen first. Km and Vmax of DFE was6.98e-08mmol and232.5FU ml-1. The first15N-terminal amino acids of DFE was APALHSQGYTGSNVK, which is consistent with nattokinase.
Conclusion:Pure DFE was acquired after steps of extraction methods. According to its biochemical property, the enzyme was identified as a serine protease. It could tolerate high temperature and alkaline and had high afinity with fibrin.
Part Ⅲ
In vitro and vivo Anti-thrombolytic Evaluation of Douchi Fibrinolytic Enzyme
Objective:To evaluate the thrombolytic effect of DFE by in vitro blood clot dissolving test and in vivo anti-thrombolytic activity on a rat thrombus model.
Methods:DFE was purified following the methods in part II and prepared in different concentrations. Blood was drawed from a rat and clotted spontaneously. High, middle and low dose DFE solution, UK solution and physiology saline were added in blood clots, then the clots were incubated at37℃for16hours. The weight of blood clots before and after incubation were determined. S-D rats with tails more than15cm were selected for thrombus modeling. Agents for testing were injected through left caudal vein at first. After30min, the tails were ligated and lmg/ml κ-carrageenan were injected through right caudal vein. The tails were immersed in icy bath for1min, and after20min, the ligation was removed. The tail thrombosis length were measured6hours after κ-carrageenan were injected. Rat blood were collected from abdominal aorta under anesthesia and the plasma level of APTT, TT, PT and D-dimer were determined.
Results:in normal saline group, the dissolution rate of blood clots was6.5%, while UK group dissolved about47.9%. In middle and high dose DFE group, the dissolution rate of blood clots increased significantly with a dose-response manner(59%vs.6.5%, p<0.01;71.7%vs.6.5%,p<0.01). Compared to negative control group(14.19±0.51cm), the average tail thrombosis length of UK group were significantly short(10.45±3.02cm,/p<0.01), so were the middle and high dose DFE group(10.25±3.24cm,/p<0.05;8.31±3.32cm,p<0.01). besides, the red swelling and inflammation degree of thrombosis in agents treated groups were also not obvious as negative control group. Only DFE middle group showed significant increase for APTT value(p<0.01). PT value in UK and DFE group were prolonged significantly. No differences were observed between groups for TT value. Compared with blank group, the level of D-dimer in negative control group increased significantly(p<0.01). In UK(p<0.05) and high dose DFE group(p<0.01), the D-dimer level were significantly higher than negeative control group.
Conclusion:In vitro dissolving effect of DFE on blood clots was obviouse and it could also inhibit the formation of thrombus in vivo significantly.
引文
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