【摘要】：隨著能源危機(jī)和溫室效應(yīng)問(wèn)題的日益嚴(yán)重,使用可再生的木質(zhì)纖維素材料生產(chǎn)生物燃料受到越來(lái)越多國(guó)家的關(guān)注。白蟻具有高效的木質(zhì)纖維素消化能力,是生物質(zhì)能源生產(chǎn)的重要模型,也是一些新型木質(zhì)纖維素酶的潛在來(lái)源。本文的研究對(duì)象為高等培菌白蟻黃翅大白蟻Macrotermes barneyi,實(shí)驗(yàn)室前期對(duì)其腸道各部分進(jìn)行比較轉(zhuǎn)錄組測(cè)序,發(fā)現(xiàn)了很多注釋為木質(zhì)纖維素酶的基因,包括漆酶、β-葡萄糖苷酶、內(nèi)切葡聚糖酶等。在中腸表達(dá)量較高的β葡萄糖苷酶(MbBG)和內(nèi)切葡聚糖酶(MbEG)已經(jīng)成功異源表達(dá),并對(duì)酶活性質(zhì)進(jìn)行了研究,但是對(duì)于白蟻漆酶的相關(guān)研究還較少。另外實(shí)驗(yàn)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(duì)于這些微生物來(lái)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(zhì)纖維素降解中所發(fā)揮的作用,我們進(jìn)行了以下研究:1.M.barney 自身由來(lái)漆酶基因的克隆與表達(dá)。對(duì)前期M.brneyi轉(zhuǎn)錄組測(cè)序數(shù)據(jù)中被注釋為漆酶基因(MbLac)的序列進(jìn)行比對(duì)和進(jìn)化樹(shù)分析,推測(cè)該基因?yàn)镸.bareyi自身來(lái)源。我們以M.baneyi唾液腺-前腸的cDNA為模板,PCR擴(kuò)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(dá)。結(jié)果顯示,在E.coli JM109和E.coliBL21(DE3)中均可重組表達(dá)MbLac,但重組蛋白無(wú)漆酶活性,可能因?yàn)榇竽c桿菌不具備對(duì)蛋白進(jìn)行二級(jí)結(jié)構(gòu)修飾的能力。MbLac在畢赤酵母GS115中不能表達(dá),Western blotting檢測(cè)不到信號(hào),可能由于密碼子偏好性使得白蟻漆酶不能在酵母中表達(dá)。2.M.barney 后腸微生物由來(lái)漆酶基因的克隆與表達(dá)。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(zhǎng)為1533 bp,編碼510個(gè)氨基酸和一個(gè)終止密碼子。將該基因在E.coli JM109中異源表達(dá)和純化。得到的重組蛋白,以ABTS為底物時(shí)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(duì)ABTS的Km值為0.278 mM,最大反應(yīng)速率為555.55 U/mg。重組CotA可以對(duì)靛藍(lán)、結(jié)晶紫和孔雀綠進(jìn)行脫色,脫色3小時(shí)后脫色率可達(dá)到80%。3.M.barneyi后腸微生物由來(lái)木聚糖酶基因的表達(dá)。將M.barneyi后腸類芽孢桿菌(Paenibacillus sp.Mb1)來(lái)源的XylMb1基因在E.coli JM109中異源表達(dá),使用Ni-NTA柱純化得到大量重組蛋白。以Birchwood xylan為底物,測(cè)得重組酶的比活力為3203.12 U/mg。4.白蟻木質(zhì)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(lái)自恒春新白蟻的內(nèi)切葡聚糖酶突變體EG71在E.coliBL21中進(jìn)行了共表達(dá),兩個(gè)蛋白都可表達(dá),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(duì)濾紙和PASC的協(xié)同降解作用,四種酶降解濾紙和PASC時(shí)的協(xié)同因子分別為1.63和1.4375�？傊�,本文首先克隆表達(dá)了黃翅大白蟻?zhàn)陨淼钠崦富騇bLac以及其腸道微生物來(lái)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(lái)源的木質(zhì)纖維素酶(CotA、XylMb1、BGDS-5和EG71)對(duì)濾紙和PASC的協(xié)同降解作用。本文加深了我們對(duì)于白蟻木質(zhì)纖維素降解機(jī)制的理解,同時(shí)也為白蟻及其微生物木質(zhì)纖維素降解酶的應(yīng)用奠定了理論基礎(chǔ)。
[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(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78;Q936

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 寧娜;張倪;吳燕;倪金鳳;;臺(tái)灣乳白蟻和黃翅大白蟻消化道主要木質(zhì)纖維素降解酶活性比較[J];應(yīng)用與環(huán)境生物學(xué)報(bào);2015年04期

2 余小霞;劉曉青;田健;伍寧豐;;來(lái)源于枯草芽孢桿菌的漆酶cotA基因克隆與表達(dá)及其酶學(xué)性質(zhì)研究[J];中國(guó)農(nóng)業(yè)科技導(dǎo)報(bào);2015年01期

3 司靜;李偉;崔寶凱;戴玉成;;真菌漆酶性質(zhì)、分子生物學(xué)及其應(yīng)用研究進(jìn)展[J];生物技術(shù)通報(bào);2011年02期

相關(guān)碩士學(xué)位論文 前4條

1 于孟蘭;黃翅大白蟻中腸木質(zhì)纖維素降解相關(guān)酶類的序列分析與表達(dá)研究[D];山東大學(xué);2014年

2 王巍;白蟻腸道木質(zhì)素分解菌的分離鑒定及PY12 LiP基因的克隆與序列分析[D];吉林農(nóng)業(yè)大學(xué);2013年

3 劉建明;細(xì)菌纖維素外切酶基因的篩選及耐熱纖維素酶基因在枯草桿菌系統(tǒng)里的表達(dá)[D];華東理工大學(xué);2011年

4 周剛;白蟻內(nèi)生菌的分離及其纖維素酶、木質(zhì)素酶高產(chǎn)菌株的鑒定[D];黑龍江大學(xué);2006年

，

本文編號(hào)：2191455