枯草芽孢杆菌纤维素酶基因整合载体的构建

发布时间：2018-03-29 06:38

本文选题：纤维素酶　切入点：枯草芽孢杆菌　出处：《河南科技大学》2017年硕士论文

【摘要】：纤维素酶是一种常见的生物降解酶,能够破坏β-1,4葡萄糖甘键,将纤维素降解为葡萄糖。在畜牧养殖方面,畜禽体内微生物对纤维素的消化吸收效果很那达到养殖者的要求,但是纤维素降解后的产物对畜禽的身体发育却能够产生积极的效果。为了追求纤维素在畜禽生产中的高利用率,本试验构建一株能够降解纤维素的重组菌,对扩大饲料原料来源具有参考意义。本试验选取纤维素酶CelKg基因为目的基因,以野生型枯草芽胞杆菌(Bacillus subtilis,B.subtilis)LN基因组中M1、M2为同源片段,以启动子P43基因作为启动子构建整合载体,将其转入野生型B.subtilis LN中,以期获得能够高效表达纤维素酶的重组菌,并对其进行酶活性分析。主要研究如下:选择质粒pGEM-T Easy为载体,利用T4连接酶将纤维素酶Cel Kg基因、启动子P43基因和同源片段M1、M2连接在一起构建重组质粒pGEM-Kmpgmt,将其转化后,通过蓝白斑筛选、聚合酶链式反应(PCR)验证,双酶切验证挑选重组大肠杆菌Kpg。在野生型B.subtilis LN感受态细胞的制备过程中,分别添加体积比为1%、2%、3%、4%和5%的吐温-80、甲醇和丙酮,统计感受态细胞的复活菌数。将外源质粒pGEM-kmpgt通过化学转化的方式转入制备好的B.subtilis感受态细胞中,以P4326F/P4326R为引物,利用PCR验证转化结果,并计算转化效率。试验结果显示,在野生型B.subtilis LN感受态细胞的制备过程中添加4%的甲醇,所制备的感受态细胞复活数最多为546个/μL,经转化后最多可获得52个转化子/μg,转化效率最高。利用spizizen转化法,将重组质粒pGEM-Kmpgmt转入野生型B.subtilis LN感受态细胞,以双交换的形式将纤维素酶基因CelKg转移至野生型B.subtilis LN基因组中,通过PCR验证目的片段成功整合到野生型B.subtilis LN中,获得重组菌B.subtilis Kpg。刚果红染色结果显示重组菌对羧甲基纤维素钠有降解作用。通过对重组菌菌落和菌液的观察,符合B.subtilis菌落和菌液的形态特征。在1000×倍的显微镜下观察重组菌,菌体呈杆状,均有芽孢出现,和野生型B.subtilis LN形态相同。对重组菌B.subtilis Kpg的最适培养时间、其表达的纤维素酶的最佳反应温度和最佳反应环境进行分析,结果显示重组菌B.subtilis Kpg在37℃条件下摇瓶培养,生长至18 h时上清液中纤维素酶活力最大为55.22 U/mL,与野生型B.subtilis LN相比提高了115%。B.subtilis Kpg纤维素酶的最适反应温度为60℃,此时纤维素酶活性最大为108.45 U/mL,反应液pH值为6时,纤维素酶活性达到97.72 U/mL。将重组菌B.subtilis Kpg添加到麸皮中进行发酵,测定其表达的纤维素酶对纤维素的实际降解能力。研究结果发现重组菌B.subtilis Kpg能够显著降低麸皮中的纤维素含量,同时发现重组菌B.subtilis Kpg和野生型B.subtilis LN均能将无机氮转化为真蛋白,增加麸皮中的真蛋白量。通过对重组菌B.subtilis Kpg的酶学性质研究和对发酵麸皮含量的分析,初步确定了重组菌B.subtilis Kpg对纤维素的降解能力,以及在发酵麸皮的利用价值,为B.subtilis工程菌的构建和实际应用提供部分的理论支撑。
[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.

【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S816.3

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