产纤溶酶微生物的筛

发布时间：2018-01-14 04:15

本文关键词：产纤溶酶微生物的筛选、鉴定及其纤溶酶特性研究　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：近年来,血栓性疾病严重影响着人类的健康生活,且发病率和死亡率逐年增高,导致患者对溶栓药物的需求不断增加。然而,市售的溶栓药物种类较少、特异性差、作用时间短、溶栓能力弱、具有副作用,且价格昂贵。因此,寻找或研制高效、低毒、药效快、特异性高及价廉的溶栓化合物将会对人类健康产生重要意义。本研究对从植物体内分离出的高产纤溶酶的菌株淡紫拟青霉(Paecilomyces lilacinus)及其代谢产生的纤溶酶进行了研究。(1)从山西黄河边、绍兴、长白山等地区采集的245份植物标本分离获得植物内生菌480株。利用脱脂奶粉-琼脂糖平板法从中初步筛选出64株具有分泌胞外蛋白酶能力的菌株。利用纤维蛋白-琼脂糖平板法复筛获得8株分泌纤溶酶的菌株。其中菌株HS-387产酶活力较高,可达266.58U/m L(相当尿激酶酶活力)。该菌株经形态学及分子生物学鉴定为淡紫拟青霉(Paecilomyces lilacinus)。(2)分析发酵条件对菌株HS-387分泌纤溶酶活性的影响,结果显示其最佳发酵条件为培养液装液量为100m L(250m L锥形瓶),培养液(灭菌前)p H值为4.0,真菌接种量为3.0×106CFU,放置28℃,175rpm,培养7天。优化后产生的酶活力为707.97U/m L(相当尿激酶酶活力),最终酶活力增到2.66倍。(3)将硫酸铵分级沉淀、DEAE-弱阴离子交换层析、SP-弱阳离子交换层析、丁基疏水层析等方法相结合,分离、纯化获得经Native-PAGE凝胶电泳验证纯度较高(96%以上)的纤溶酶,纯化后酶活力回收率为37.2%,最终该酶的比活力为6461.75U/mg(以尿激酶为标准)。并对获得的纯度较高的纤溶酶进行QE质谱鉴定、分析。(4)该菌株产生的纤溶酶等电点约为6.6,分子量约为45k Da,p H值5.0-9.0时该酶较稳定,p H3.0以下容易造成酶的失活。该酶在30℃-50℃时均具有较高酶活力,最适温度40℃。典型丝氨酸蛋白酶抑制剂PMSF对该酶的抑制率为60%,推测该酶可能为碱性丝氨酸蛋白酶。该酶水解纤维蛋白原的先后顺序是Aα链Bβ链γ链,对Aα链的降解最迅速;水解纤维蛋白的先后顺序是γ-γ链α链β链,对降解γ-γ链的作用较快。这类似于血纤维蛋白溶酶的水解模式,该酶不仅降解纤维蛋白,对于纤维蛋白的前体纤维蛋白原也有降解作用。研究表明其体外溶栓可能是通过直接降解纤维蛋白的方式发挥作用,不具有或仅有较小的激酶活性。综上所述,本研究从480株植物内生菌中分离筛选获得一株高产纤溶酶的菌株淡紫拟青霉(Paecilomyces lilacinus),目前尚未见有该菌种产生纤溶酶的报道。研究发现其产生的纤溶酶可能为碱性丝氨酸蛋白酶,对纤维蛋白和纤维蛋白原都具有降解作用,推测该酶在溶解血栓的同时还可以防止血栓的继续形成,从而为开发溶栓药物提供了一个潜在的新途径。
[Abstract]:In recent years, thrombotic diseases seriously affect the health of human life, and the incidence and mortality increased year by year, resulting in increased demand for thrombolytic drugs. However, commercially available thrombolytic drugs less species, poor specificity, short action time, thrombolytic ability is weak, has side effects, and the price is expensive. Therefore, find or develop efficient, low toxicity, quick effect, high specificity and cheap thrombolytic compounds will have important significance to human health. The study on high yield strains isolated from purple enzyme in plants of Paecilomyces (Paecilomyces lilacinus) and its metabolite produced by plasmin was studied. (1) from the Shaoxing Shanxi the Yellow River side. 245, Changbai Mountain and other regions of the plant specimens isolated from endophytic bacteria of 480 strains. By agarose plate method from defatted milk powder were screened 64 strains with the secretion of extracellular protease of bacteria Strain. Using fibrin agarose plate method for screening 8 strains. The strain HS-387 secreted plasminogen activator enzyme activity is high, up to 266.58U/m L (the equivalent of urokinase enzyme activity). The strains by morphological and molecular biological identification of p.lilacinus (Paecilomyces lilacinus). (2) analysis of the influence of fermentation conditions of secretion of plasminogen activator activity the strain HS-387, the results showed that the optimum fermentation conditions for cultivating liquid liquid volume was 100m L (250m L, medium (flask) before sterilization) P H 4, fungi inoculation quantity is 3 * 106CFU, placed 28 C, 175rpm, cultured for 7 days. After the optimization of enzyme activity 707.97U/m L (the equivalent of urokinase enzyme activity), the final enzyme activity increased to 2.66 times. (3) the ammonium sulfate precipitation, DEAE- weak anion exchange chromatography, SP- weak cation exchange chromatography, hydrophobic interaction chromatography combination, separation, purification of Native-PAGE by coagulation Gel electrophoresis verification of high purity (more than 96%) of the purified enzyme, enzyme activity recovery was 37.2%, the specific activity of the enzyme is 6461.75U/mg (with urokinase as a standard). And to obtain the high purity of the enzyme was identified, QE mass spectrometry analysis. (4) the Strains Producing Fibrinolytic Enzyme isoelectric point is about 6.6, the molecular weight is about 45k Da, 5.0-9.0 of the enzyme was stable P H P H3.0, the following is likely to cause the inactivation of the enzyme. This enzyme has high enzyme activity at 30 DEG -50 DEG, the optimum temperature is 40 degrees centigrade. The typical serine protease inhibitor PMSF and inhibition of this enzyme was 60%, that the enzyme is alkaline serine protease. The enzyme hydrolyzed fibrinogen alpha chain is the sequence of A B beta gamma chain chain, degradation of A alpha chain most rapidly; in order to hydrolyze fibrin is gamma gamma chain chain alpha beta chain, the degradation of gamma gamma chain function rapidly. This is similar in the blood fibrin dissolving enzyme The enzyme hydrolysis mode, not only the degradation of fibrin, the precursor fibrin raw also degradation. The research showed that the in vitro thrombolysis may play a role in the degradation of fibrin directly, does not have or only small kinase activity. In summary, this study from 480 strains of endophytic bacteria isolated get a strain with high fibrinolytic enzyme strains lilac Paecilomyces (Paecilomyces lilacinus), it has not been reported. The strains of plasmin research showed that the generated plasmin may be alkaline serine protease of fibrin and fibrinogen degradation have speculated that at the same time, the enzyme in dissolving thrombus can be formed to prevent thrombosis thus, it provides a new way for the development of potential thrombolytic drugs.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q936

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