溶栓酶产生菌的分离筛选鉴定及发酵、提纯研究

发布时间：2018-02-06 07:52

本文关键词： 豆豉溶栓酶解淀粉芽孢杆菌菌种鉴定液体发酵优化分离纯化　出处：《华中科技大学》2009年硕士论文　论文类型：学位论文

【摘要】：目的:豆豉溶栓酶是由从豆豉中分离出的细菌所分泌的一种胞外酶,和日本的纳豆激酶具有相似的特性。与临床上常用的纤溶药物相比,具有许多优点:效价高、半衰期长、无抗原性、安全无毒、成本低廉等,有望开发成新一代溶栓药物,本课题拟对全国不同地方收集的豆豉样品进行分离筛选,得到可以产生纤溶酶的菌株,并对该菌株及其产生的酶进行一些基础研究,包括菌种鉴定、液体发酵探讨以及酶的分离纯化。方法:1.酪蛋白平板初筛,纤维蛋白平板复筛,得到产溶栓酶的细菌。2.对该菌株进行生理生化及16SrRNA鉴定,确定种属。3.采用单因素和正交实验相结合的方法,探讨液体发酵的最佳培养基和发酵条件。4.经过硫酸铵分段盐析,透析脱盐,G-50柱层析,分离得到酶的纯品并冷冻干燥保存。5.纤维蛋白平板法测定酶活,考马斯亮蓝G-250法测定蛋白质含量,计算整个提纯过程中酶活的回收率和比酶活。6.SDS-PAGE电泳,考马斯亮蓝R-250染色,确定酶的分子量。结果:1.筛选到8株具有溶栓活性的菌株,以活性最高的一株菌XY-1作为研究对象。2.经生理生化和16S rRNA鉴定后,确定该菌为芽孢杆菌属的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。3.培养基的最佳组成为:酵母提取物1%、乳糖3%、K2HPO4 0.1%、KH_2PO_4 0.6%、MgSO_4 0.1%、NaCl 0.05%、pH 9.0。最佳发酵条件为:温度35℃、装液量50ml/250ml、接种量500μl/50ml、发酵时间24h。4.经过整个提纯周期,豆豉溶栓酶的酶活回收率达到37%,蛋白质回收率19.8%,比酶活为1388.27 U/mg,纯化倍数为1.87倍。5.真空冷冻干燥得到粉末状酶制品,上SDS-PAGE电泳显示两条带,分子量约为12k和20k。结论:我国传统食品豆豉中同样可以分离到产溶栓酶的细菌,经生理生化和16SrRNA鉴定后确定为解淀粉芽孢杆菌。采用单因素和正交实验结合的方法优化液体发酵,效果较好,使产酶量由235.2U/ml提高到2089.3U/ml。经过整个分离提纯过程,酶活回收率虽较高,但纯化倍数不高,样品中含有两种蛋白质,对分离纯化方法有待于进一步研究。
[Abstract]:Objective: thrombolytic enzyme is a kind of fermented black bean extracellular enzymes secreted by the bacteria isolated from fermented black bean, and Japanese nattokinase has similar properties. Compared with the commonly used fibrinolytic drugs, has many advantages: high titer, long half-life, no antigenicity, non-toxic, low cost, and is expected to be developed a new generation of thrombolytic drugs, this thesis intends to different parts of the country to collect the samples were isolated from fermented black bean, can produce fibrinolytic enzyme strains, and some basic research on the strain and its enzyme, including strain identification, liquid fermentation and purification of the enzyme.
Methods: 1. casein plate screening, fibrin plate screening, bacteria.2. Producing Fibrinolytic Enzyme by physiological and biochemical identification of the strains and 16SrRNA, determine the species of the genus.3. by using the method of single factor and orthogonal experiment combination, to explore the optimum liquid fermentation medium and fermentation conditions of.4. after ammonium sulfate fractionation. Dialysis desalting, G-50 column chromatography, enzyme activity assay enzyme isolated pure and freeze-dried.5. fibrin plate method, determination of protein content of Kaumas blue G-250 recovery method, calculate the enzyme activity and specific activity of.6.SDS-PAGE electrophoresis, Coomassie brilliant blue R-250 staining Kaumas, to determine the molecular weight of the enzyme.
Results: 1. screened 8 strains with thrombolytic activity strains, with the highest activity strain XY-1 as the research object and 16S rRNA.2. by physiological and biochemical identification, to determine the strains of Bacillus amyloliquefaciens bacillus (Bacillus amyloliquefaciens) best group.3. medium: yeast extract 1%, lactose 3% K2HPO4, 0.1%, KH_2PO_4 0.6%, MgSO_4 0.1%, NaCl 0.05%, pH 9.0. the best fermentation conditions: 35 degrees Celsius temperature, liquid volume 50ml/250ml, inoculation amount of 500 l/50ml, fermentation time 24h.4. after the purification cycle, the recovery rate reached 37%. Fermented black bean thrombolytic enzyme enzyme, protein recovery rate was 19.8%, the specific activity was 1388.27 U/mg, the purification factor was 1.87.5. and vacuum freeze drying to obtain powdered enzyme products, SDS-PAGE electrophoresis showed two bands with molecular weight of about 12K and 20k.
Conclusion: the traditional Chinese fermented black bean can also be isolated from food producing fibrinolytic enzyme bacteria by physiological and biochemical and 16SrRNA identified as Bacillus amyloliquefaciens. By using the method of combining single factor and orthogonal experiment to optimize the fermentation liquid, the effect is good, the yield of enzyme from 235.2U/ml up to 2089.3U/ml. after the separation and purification process. The recovery of enzyme activity is higher, but the purification ratio is not high, containing two protein samples, the purification method needs to be further studied.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R378

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