白蚁及其肠道微生物来源木质纤维素酶基因的克隆与表达
发布时间：2018-08-19 10:55
【摘要】：随着能源危机和温室效应问题的日益严重,使用可再生的木质纤维素材料生产生物燃料受到越来越多国家的关注。白蚁具有高效的木质纤维素消化能力,是生物质能源生产的重要模型,也是一些新型木质纤维素酶的潜在来源。本文的研究对象为高等培菌白蚁黄翅大白蚁Macrotermes barneyi,实验室前期对其肠道各部分进行比较转录组测序,发现了很多注释为木质纤维素酶的基因,包括漆酶、β-葡萄糖苷酶、内切葡聚糖酶等。在中肠表达量较高的β葡萄糖苷酶(MbBG)和内切葡聚糖酶(MbEG)已经成功异源表达,并对酶活性质进行了研究,但是对于白蚁漆酶的相关研究还较少。另外实验室前期在黄翅大白蚁后肠分离得到了具有木聚糖酶活性以及具有漆酶活性的微生物,对于这些微生物来源的木聚糖酶和漆酶所起的作用我们还不清楚。为了研究这些蛋白在白蚁木质纤维素降解中所发挥的作用,我们进行了以下研究:1.M.barney 自身由来漆酶基因的克隆与表达。对前期M.brneyi转录组测序数据中被注释为漆酶基因(MbLac)的序列进行比对和进化树分析,推测该基因为M.bareyi自身来源。我们以M.baneyi唾液腺-前肠的cDNA为模板,PCR扩增得到了MbLa 基因,并将该基因在E.coliJM109、E.coliBL21(DE3)以及毕赤酵母GS115中异源表达。结果显示,在E.coli JM109和E.coliBL21(DE3)中均可重组表达MbLac,但重组蛋白无漆酶活性,可能因为大肠杆菌不具备对蛋白进行二级结构修饰的能力。MbLac在毕赤酵母GS115中不能表达,Western blotting检测不到信号,可能由于密码子偏好性使得白蚁漆酶不能在酵母中表达。2.M.barney 后肠微生物由来漆酶基因的克隆与表达。从M.barneyi中分离到一株具有漆酶活性的高地芽孢杆菌(Bacillusaltitudinis CMC2)。从中克隆漆酶CotA基因,全长为1533 bp,编码510个氨基酸和一个终止密码子。将该基因在E.coli JM109中异源表达和纯化。得到的重组蛋白,以ABTS为底物时最适温度和pH分别为70 ℃和5.0。该酶在偏碱性的环境中比较稳定。重组酶对ABTS的Km值为0.278 mM,最大反应速率为555.55 U/mg。重组CotA可以对靛蓝、结晶紫和孔雀绿进行脱色,脱色3小时后脱色率可达到80%。3.M.barneyi后肠微生物由来木聚糖酶基因的表达。将M.barneyi后肠类芽孢杆菌(Paenibacillus sp.Mb1)来源的XylMb1基因在E.coli JM109中异源表达,使用Ni-NTA柱纯化得到大量重组蛋白。以Birchwood xylan为底物,测得重组酶的比活力为3203.12 U/mg。4.白蚁木质纤维素酶的协同作用。将M.barneyi自身的β-葡萄糖苷酶突变体BGDS-5和来自恒春新白蚁的内切葡聚糖酶突变体EG71在E.coliBL21中进行了共表达,两个蛋白都可表达,且可以协同作用降解滤纸和磷酸处理的微晶纤维素(PASC)。另外我们研究了重组CotA、XylMb1、BGDS-5和EG71对滤纸和PASC的协同降解作用,四种酶降解滤纸和PASC时的协同因子分别为1.63和1.4375。总之,本文首先克隆表达了黄翅大白蚁自身的漆酶基因MbLac以及其肠道微生物来源的木聚糖酶基因XylMb1和漆酶基因CotA,然后研究了白蚁及共生微生物来源的木质纤维素酶(CotA、XylMb1、BGDS-5和EG71)对滤纸和PASC的协同降解作用。本文加深了我们对于白蚁木质纤维素降解机制的理解,同时也为白蚁及其微生物木质纤维素降解酶的应用奠定了理论基础。
[Abstract]:With the energy crisis and greenhouse effect becoming more and more serious, the use of renewable lignocellulose materials to produce biofuels has attracted more and more attention in many countries. Termites have high digestibility of lignocellulose, which is an important model for biomass energy production and a potential source of some new lignocellulases. Macrotermes barneyi, a higher culture strain of termite yellow-winged termite, was selected for the study. The comparative transcriptome sequencing of the intestinal tract showed that many genes annotated lignocellulase, including laccase, beta-glucosidase, endoglucanase and so on, were found in the early stage of the laboratory. Xylanase (MbEG) has been successfully expressed heterologously and its enzyme activity has been studied. However, there are few studies on termite laccase. In addition, microorganisms with xylanase activity and laccase activity were isolated from the hindgut of the yellow-winged termite in the early stage of the laboratory. To investigate the role of these proteins in termite lignocellulose degradation, we have carried out the following studies: 1. Cloning and expression of the laccase gene from M. Barney itself. Sequence alignment and evolutionary tree analysis of the laccase gene (MbLac) annotated in the transcriptome sequencing data of M. brneyi We amplified the MbLa gene from the salivary gland-foregut cDNA of M. baneyi and expressed it heterologously in E. coli JM109, E. coli BL21 (DE3) and Pichia pastoris GS115. The results showed that MbLac could be recombined and expressed in E. coli JM109 and E. coli BL21 (DE3), but no recombinant protein was found. Laccase activity may be due to the inability of Escherichia coli to modify the secondary structure of proteins. MbLac could not be expressed in Pichia pastoris GS115. Western blotting could not detect the signal. It may be due to codon preference that termite laccase could not be expressed in yeast. 2. Cloning and expression of laccase gene from backgut microorganisms of M. Barney Bacillus altitudinis CMC2 with laccase activity was isolated from M. barneyi. The CotA gene of laccase was cloned from M. barneyi. It was 1 533 BP in length and encoded 510 amino acids and a termination codon. The recombinant protein was heterologously expressed and purified in E. coli JM109. The optimum temperature was ABTS as substrate. The Km value of recombinant enzyme to ABTS was 0.278 mM, and the maximum reaction rate was 555.55 U/mg. Recombinant CotA could decolorize indigo, crystal violet and malachite green, and the decolorization rate could reach 80% after decolorization for 3 hours. 3. XylMb1 gene from Paenibacillus sp. Mb1 was heterologously expressed in E. coli JM109 and purified by Ni-NTA column. Using Birchwood xylan as substrate, the specific activity of the recombinant enzyme was determined to be 3203.12 U/mg.4. Glycosidase mutant BGDS-5 and endoglucanase mutant EG71 from Hengchun termite were co-expressed in E.coli BL21. Both proteins could be expressed and degraded PASC synergistically. In addition, we studied the synergistic degradation of filter paper and PASC by recombinant CotA, XylMb1, BGDS-5 and EG71. In summary, the laccase gene MbLac and the xylanase gene XylMb 1 and laccase gene CotA from the intestinal microorganism of the yellow-winged termite were cloned and expressed, and then the lignocellulase (CotA, Xy) from the termite and the symbiotic microorganism were studied. The synergistic degradation effect of lMb1, BGDS-5 and EG71 on filter paper and PASC was studied in this paper. Our understanding of the degradation mechanism of termite lignocellulose was deepened, and the theoretical basis was laid for the application of termite and its microbial lignocellulose degrading enzyme.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q936
本文编号：2191455
[Abstract]:With the energy crisis and greenhouse effect becoming more and more serious, the use of renewable lignocellulose materials to produce biofuels has attracted more and more attention in many countries. Termites have high digestibility of lignocellulose, which is an important model for biomass energy production and a potential source of some new lignocellulases. Macrotermes barneyi, a higher culture strain of termite yellow-winged termite, was selected for the study. The comparative transcriptome sequencing of the intestinal tract showed that many genes annotated lignocellulase, including laccase, beta-glucosidase, endoglucanase and so on, were found in the early stage of the laboratory. Xylanase (MbEG) has been successfully expressed heterologously and its enzyme activity has been studied. However, there are few studies on termite laccase. In addition, microorganisms with xylanase activity and laccase activity were isolated from the hindgut of the yellow-winged termite in the early stage of the laboratory. To investigate the role of these proteins in termite lignocellulose degradation, we have carried out the following studies: 1. Cloning and expression of the laccase gene from M. Barney itself. Sequence alignment and evolutionary tree analysis of the laccase gene (MbLac) annotated in the transcriptome sequencing data of M. brneyi We amplified the MbLa gene from the salivary gland-foregut cDNA of M. baneyi and expressed it heterologously in E. coli JM109, E. coli BL21 (DE3) and Pichia pastoris GS115. The results showed that MbLac could be recombined and expressed in E. coli JM109 and E. coli BL21 (DE3), but no recombinant protein was found. Laccase activity may be due to the inability of Escherichia coli to modify the secondary structure of proteins. MbLac could not be expressed in Pichia pastoris GS115. Western blotting could not detect the signal. It may be due to codon preference that termite laccase could not be expressed in yeast. 2. Cloning and expression of laccase gene from backgut microorganisms of M. Barney Bacillus altitudinis CMC2 with laccase activity was isolated from M. barneyi. The CotA gene of laccase was cloned from M. barneyi. It was 1 533 BP in length and encoded 510 amino acids and a termination codon. The recombinant protein was heterologously expressed and purified in E. coli JM109. The optimum temperature was ABTS as substrate. The Km value of recombinant enzyme to ABTS was 0.278 mM, and the maximum reaction rate was 555.55 U/mg. Recombinant CotA could decolorize indigo, crystal violet and malachite green, and the decolorization rate could reach 80% after decolorization for 3 hours. 3. XylMb1 gene from Paenibacillus sp. Mb1 was heterologously expressed in E. coli JM109 and purified by Ni-NTA column. Using Birchwood xylan as substrate, the specific activity of the recombinant enzyme was determined to be 3203.12 U/mg.4. Glycosidase mutant BGDS-5 and endoglucanase mutant EG71 from Hengchun termite were co-expressed in E.coli BL21. Both proteins could be expressed and degraded PASC synergistically. In addition, we studied the synergistic degradation of filter paper and PASC by recombinant CotA, XylMb1, BGDS-5 and EG71. In summary, the laccase gene MbLac and the xylanase gene XylMb 1 and laccase gene CotA from the intestinal microorganism of the yellow-winged termite were cloned and expressed, and then the lignocellulase (CotA, Xy) from the termite and the symbiotic microorganism were studied. The synergistic degradation effect of lMb1, BGDS-5 and EG71 on filter paper and PASC was studied in this paper. Our understanding of the degradation mechanism of termite lignocellulose was deepened, and the theoretical basis was laid for the application of termite and its microbial lignocellulose degrading enzyme.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q936
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