本文關(guān)鍵詞： 豆豉溶栓酶 解淀粉芽孢桿菌 菌種鑒定 液體發(fā)酵優(yōu)化 分離純化　出處：《華中科技大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:豆豉溶栓酶是由從豆豉中分離出的細(xì)菌所分泌的一種胞外酶,和日本的納豆激酶具有相似的特性。與臨床上常用的纖溶藥物相比,具有許多優(yōu)點(diǎn):效價(jià)高、半衰期長(zhǎng)、無抗原性、安全無毒、成本低廉等,有望開發(fā)成新一代溶栓藥物,本課題擬對(duì)全國不同地方收集的豆豉樣品進(jìn)行分離篩選,得到可以產(chǎn)生纖溶酶的菌株,并對(duì)該菌株及其產(chǎn)生的酶進(jìn)行一些基礎(chǔ)研究,包括菌種鑒定、液體發(fā)酵探討以及酶的分離純化。 方法:1.酪蛋白平板初篩,纖維蛋白平板復(fù)篩,得到產(chǎn)溶栓酶的細(xì)菌。2.對(duì)該菌株進(jìn)行生理生化及16SrRNA鑒定,確定種屬。3.采用單因素和正交實(shí)驗(yàn)相結(jié)合的方法,探討液體發(fā)酵的最佳培養(yǎng)基和發(fā)酵條件。4.經(jīng)過硫酸銨分段鹽析,透析脫鹽,G-50柱層析,分離得到酶的純品并冷凍干燥保存。5.纖維蛋白平板法測(cè)定酶活,考馬斯亮藍(lán)G-250法測(cè)定蛋白質(zhì)含量,計(jì)算整個(gè)提純過程中酶活的回收率和比酶活。6.SDS-PAGE電泳,考馬斯亮藍(lán)R-250染色,確定酶的分子量。 結(jié)果:1.篩選到8株具有溶栓活性的菌株,以活性最高的一株菌XY-1作為研究對(duì)象。2.經(jīng)生理生化和16S rRNA鑒定后,確定該菌為芽孢桿菌屬的解淀粉芽孢桿菌(Bacillus amyloliquefaciens)。3.培養(yǎng)基的最佳組成為: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。最佳發(fā)酵條件為:溫度35℃、裝液量50ml/250ml、接種量500μl/50ml、發(fā)酵時(shí)間24h。4.經(jīng)過整個(gè)提純周期,豆豉溶栓酶的酶活回收率達(dá)到37%,蛋白質(zhì)回收率19.8%,比酶活為1388.27 U/mg,純化倍數(shù)為1.87倍。5.真空冷凍干燥得到粉末狀酶制品,上SDS-PAGE電泳顯示兩條帶,分子量約為12k和20k。 結(jié)論:我國傳統(tǒng)食品豆豉中同樣可以分離到產(chǎn)溶栓酶的細(xì)菌,經(jīng)生理生化和16SrRNA鑒定后確定為解淀粉芽孢桿菌。采用單因素和正交實(shí)驗(yàn)結(jié)合的方法優(yōu)化液體發(fā)酵,效果較好,使產(chǎn)酶量由235.2U/ml提高到2089.3U/ml。經(jīng)過整個(gè)分離提純過程,酶活回收率雖較高,但純化倍數(shù)不高,樣品中含有兩種蛋白質(zhì),對(duì)分離純化方法有待于進(jìn)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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R378

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