白蟻及其腸道微生物來源木質纖維素酶基因的克隆與表達
發(fā)布時間：2018-08-19 10:55
【摘要】：隨著能源危機和溫室效應問題的日益嚴重,使用可再生的木質纖維素材料生產生物燃料受到越來越多國家的關注。白蟻具有高效的木質纖維素消化能力,是生物質能源生產的重要模型,也是一些新型木質纖維素酶的潛在來源。本文的研究對象為高等培菌白蟻黃翅大白蟻Macrotermes barneyi,實驗室前期對其腸道各部分進行比較轉錄組測序,發(fā)現了很多注釋為木質纖維素酶的基因,包括漆酶、β-葡萄糖苷酶、內切葡聚糖酶等。在中腸表達量較高的β葡萄糖苷酶(MbBG)和內切葡聚糖酶(MbEG)已經成功異源表達,并對酶活性質進行了研究,但是對于白蟻漆酶的相關研究還較少。另外實驗室前期在黃翅大白蟻后腸分離得到了具有木聚糖酶活性以及具有漆酶活性的微生物,對于這些微生物來源的木聚糖酶和漆酶所起的作用我們還不清楚。為了研究這些蛋白在白蟻木質纖維素降解中所發(fā)揮的作用,我們進行了以下研究:1.M.barney 自身由來漆酶基因的克隆與表達。對前期M.brneyi轉錄組測序數據中被注釋為漆酶基因(MbLac)的序列進行比對和進化樹分析,推測該基因為M.bareyi自身來源。我們以M.baneyi唾液腺-前腸的cDNA為模板,PCR擴增得到了MbLa 基因,并將該基因在E.coliJM109、E.coliBL21(DE3)以及畢赤酵母GS115中異源表達。結果顯示,在E.coli JM109和E.coliBL21(DE3)中均可重組表達MbLac,但重組蛋白無漆酶活性,可能因為大腸桿菌不具備對蛋白進行二級結構修飾的能力。MbLac在畢赤酵母GS115中不能表達,Western blotting檢測不到信號,可能由于密碼子偏好性使得白蟻漆酶不能在酵母中表達。2.M.barney 后腸微生物由來漆酶基因的克隆與表達。從M.barneyi中分離到一株具有漆酶活性的高地芽孢桿菌(Bacillusaltitudinis CMC2)。從中克隆漆酶CotA基因,全長為1533 bp,編碼510個氨基酸和一個終止密碼子。將該基因在E.coli JM109中異源表達和純化。得到的重組蛋白,以ABTS為底物時最適溫度和pH分別為70 ℃和5.0。該酶在偏堿性的環(huán)境中比較穩(wěn)定。重組酶對ABTS的Km值為0.278 mM,最大反應速率為555.55 U/mg。重組CotA可以對靛藍、結晶紫和孔雀綠進行脫色,脫色3小時后脫色率可達到80%。3.M.barneyi后腸微生物由來木聚糖酶基因的表達。將M.barneyi后腸類芽孢桿菌(Paenibacillus sp.Mb1)來源的XylMb1基因在E.coli JM109中異源表達,使用Ni-NTA柱純化得到大量重組蛋白。以Birchwood xylan為底物,測得重組酶的比活力為3203.12 U/mg。4.白蟻木質纖維素酶的協(xié)同作用。將M.barneyi自身的β-葡萄糖苷酶突變體BGDS-5和來自恒春新白蟻的內切葡聚糖酶突變體EG71在E.coliBL21中進行了共表達,兩個蛋白都可表達,且可以協(xié)同作用降解濾紙和磷酸處理的微晶纖維素(PASC)。另外我們研究了重組CotA、XylMb1、BGDS-5和EG71對濾紙和PASC的協(xié)同降解作用,四種酶降解濾紙和PASC時的協(xié)同因子分別為1.63和1.4375�？傊�,本文首先克隆表達了黃翅大白蟻自身的漆酶基因MbLac以及其腸道微生物來源的木聚糖酶基因XylMb1和漆酶基因CotA,然后研究了白蟻及共生微生物來源的木質纖維素酶(CotA、XylMb1、BGDS-5和EG71)對濾紙和PASC的協(xié)同降解作用。本文加深了我們對于白蟻木質纖維素降解機制的理解,同時也為白蟻及其微生物木質纖維素降解酶的應用奠定了理論基礎。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q78;Q936
本文編號：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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q78;Q936
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