本文選題：纖維素酶　切入點(diǎn)：枯草芽孢桿菌　出處：《河南科技大學(xué)》2017年碩士論文

【摘要】：纖維素酶是一種常見(jiàn)的生物降解酶,能夠破壞β-1,4葡萄糖甘鍵,將纖維素降解為葡萄糖。在畜牧養(yǎng)殖方面,畜禽體內(nèi)微生物對(duì)纖維素的消化吸收效果很那達(dá)到養(yǎng)殖者的要求,但是纖維素降解后的產(chǎn)物對(duì)畜禽的身體發(fā)育卻能夠產(chǎn)生積極的效果。為了追求纖維素在畜禽生產(chǎn)中的高利用率,本試驗(yàn)構(gòu)建一株能夠降解纖維素的重組菌,對(duì)擴(kuò)大飼料原料來(lái)源具有參考意義。本試驗(yàn)選取纖維素酶CelKg基因?yàn)槟康幕?以野生型枯草芽胞桿菌(Bacillus subtilis,B.subtilis)LN基因組中M1、M2為同源片段,以啟動(dòng)子P43基因作為啟動(dòng)子構(gòu)建整合載體,將其轉(zhuǎn)入野生型B.subtilis LN中,以期獲得能夠高效表達(dá)纖維素酶的重組菌,并對(duì)其進(jìn)行酶活性分析。主要研究如下:選擇質(zhì)粒pGEM-T Easy為載體,利用T4連接酶將纖維素酶Cel Kg基因、啟動(dòng)子P43基因和同源片段M1、M2連接在一起構(gòu)建重組質(zhì)粒pGEM-Kmpgmt,將其轉(zhuǎn)化后,通過(guò)藍(lán)白斑篩選、聚合酶鏈?zhǔn)椒磻?yīng)(PCR)驗(yàn)證,雙酶切驗(yàn)證挑選重組大腸桿菌Kpg。在野生型B.subtilis LN感受態(tài)細(xì)胞的制備過(guò)程中,分別添加體積比為1%、2%、3%、4%和5%的吐溫-80、甲醇和丙酮,統(tǒng)計(jì)感受態(tài)細(xì)胞的復(fù)活菌數(shù)。將外源質(zhì)粒pGEM-kmpgt通過(guò)化學(xué)轉(zhuǎn)化的方式轉(zhuǎn)入制備好的B.subtilis感受態(tài)細(xì)胞中,以P4326F/P4326R為引物,利用PCR驗(yàn)證轉(zhuǎn)化結(jié)果,并計(jì)算轉(zhuǎn)化效率。試驗(yàn)結(jié)果顯示,在野生型B.subtilis LN感受態(tài)細(xì)胞的制備過(guò)程中添加4%的甲醇,所制備的感受態(tài)細(xì)胞復(fù)活數(shù)最多為546個(gè)/μL,經(jīng)轉(zhuǎn)化后最多可獲得52個(gè)轉(zhuǎn)化子/μg,轉(zhuǎn)化效率最高。利用spizizen轉(zhuǎn)化法,將重組質(zhì)粒pGEM-Kmpgmt轉(zhuǎn)入野生型B.subtilis LN感受態(tài)細(xì)胞,以雙交換的形式將纖維素酶基因CelKg轉(zhuǎn)移至野生型B.subtilis LN基因組中,通過(guò)PCR驗(yàn)證目的片段成功整合到野生型B.subtilis LN中,獲得重組菌B.subtilis Kpg。剛果紅染色結(jié)果顯示重組菌對(duì)羧甲基纖維素鈉有降解作用。通過(guò)對(duì)重組菌菌落和菌液的觀察,符合B.subtilis菌落和菌液的形態(tài)特征。在1000×倍的顯微鏡下觀察重組菌,菌體呈桿狀,均有芽孢出現(xiàn),和野生型B.subtilis LN形態(tài)相同。對(duì)重組菌B.subtilis Kpg的最適培養(yǎng)時(shí)間、其表達(dá)的纖維素酶的最佳反應(yīng)溫度和最佳反應(yīng)環(huán)境進(jìn)行分析,結(jié)果顯示重組菌B.subtilis Kpg在37℃條件下?lián)u瓶培養(yǎng),生長(zhǎng)至18 h時(shí)上清液中纖維素酶活力最大為55.22 U/mL,與野生型B.subtilis LN相比提高了115%。B.subtilis Kpg纖維素酶的最適反應(yīng)溫度為60℃,此時(shí)纖維素酶活性最大為108.45 U/mL,反應(yīng)液pH值為6時(shí),纖維素酶活性達(dá)到97.72 U/mL。將重組菌B.subtilis Kpg添加到麩皮中進(jìn)行發(fā)酵,測(cè)定其表達(dá)的纖維素酶對(duì)纖維素的實(shí)際降解能力。研究結(jié)果發(fā)現(xiàn)重組菌B.subtilis Kpg能夠顯著降低麩皮中的纖維素含量,同時(shí)發(fā)現(xiàn)重組菌B.subtilis Kpg和野生型B.subtilis LN均能將無(wú)機(jī)氮轉(zhuǎn)化為真蛋白,增加麩皮中的真蛋白量。通過(guò)對(duì)重組菌B.subtilis Kpg的酶學(xué)性質(zhì)研究和對(duì)發(fā)酵麩皮含量的分析,初步確定了重組菌B.subtilis Kpg對(duì)纖維素的降解能力,以及在發(fā)酵麩皮的利用價(jià)值,為B.subtilis工程菌的構(gòu)建和實(shí)際應(yīng)用提供部分的理論支撐。
[Abstract]:Cellulase is a common biological degradation enzyme, can destroy the -1,4 beta glucoside bond, the degradation of cellulose into glucose. In animal husbandry, livestock and poultry in microorganisms on cellulose digestion and absorption effect is reached that farmers requirements, but the products of cellulose degradation after livestock body development can have a positive effect. In the pursuit of cellulose in livestock and poultry production utilization rate, this experiment to construct the recombinant strain capable of degrading cellulose, to expand the source of feedstuff is of great reference significance. This study selected cellulase CelKg gene in wild Bacillus subtilis (Bacillus subtilis, B.subtilis) LN genome M1, M2 homologous fragment, with promoter P43 gene as promoter construct integration vector, and transformed into wild type B.subtilis in LN, in order to obtain the efficient expression of Cellulase The recombinant strains, and the enzyme activity analysis. The main research is as follows: the choice of plasmid pGEM-T Easy vector by T4 ligase Cel cellulase Kg gene promoter, P43 gene and homologous fragments M1, M2 connected together to construct the recombinant plasmid pGEM-Kmpgmt, transformed, through the blue white screening, polymerase chain reaction (PCR) verification, double enzyme digestion verification of selected recombinant Escherichia coli Kpg. in wild-type B.subtilis LN competent cell preparation process, were added to the volume ratio of 1%, 2%, 3%, 4% and 5% Twain -80, methanol and acetone, statistical competent cells. The number of bacteria resurrection plasmid pGEM-kmpgt by chemical transformation of the way into the prepared B.subtilis competent cells, using P4326F/P4326R as primers, the transformation results by PCR test, and calculate the conversion efficiency. The test results show that the sense of the preparation process of the competent cells in wild type B.subtilis LN Adding 4% methanol, the competent cells were raised for a maximum of 546 / L, after conversion to a maximum of 52 transformants / g, the highest transformation efficiency. By using the method of spizizen transformation, the recombinant plasmid pGEM-Kmpgmt of wild-type B.subtilis LN competent cells by double exchange. The form will be transferred to the cellulase gene CelKg of wild type B.subtilis LN genome, verified by PCR fragment was successfully integrated into the wild type B.subtilis LN, the recombinant bacteria B.subtilis Kpg. Congo red staining showed that the recombinant bacteria degradation of sodium carboxymethyl cellulose. Through the observation of the recombinant bacteria colony and the morphological features of B.subtilis colonies and bacteria. Recombinant bacteria in 1000 x times under a microscope, cells were rod-shaped, spore were the same, and wild type B.subtilis LN form. The optimum culture of recombinant B.subtilis Kpg Time, the expression of the optimum reaction temperature of cellulase and the optimum reaction conditions of the analysis, the results showed that the recombinant bacteria B.subtilis Kpg under the condition of 37 DEG C in shake flask culture, the maximum growth at 18 h in the supernatant of cellulase activity was 55.22 U/mL, compared with the wild type B.subtilis LN improves the optimum 115%.B.subtilis Kpg cellulase reaction temperature at 60 degrees, the cellulase activity was 108.45 U/mL, the reaction solution pH value is 6, the cellulase activity reached 97.72 U/mL. recombinant B.subtilis Kpg was added to the bran fermentation, the actual capacity determination on the expression of cellulase degradation of cellulose. Results showed that recombinant B.subtilis Kpg can significantly reduce the content of cellulose in wheat bran. At the same time found that the recombinant B.subtilis Kpg and wild type B.subtilis LN can be transformed into inorganic nitrogen true protein, wheat bran increased in real eggs White. Through the study on enzymatic properties of recombinant B.subtilis Kpg and analysis of fermented bran content, determined the degradation ability of recombinant B.subtilis Kpg of cellulose, and use value in the fermentation of wheat bran, B.subtilis for the construction of engineering bacteria and application to provide theoretical support for the part.

【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S816.3

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