本文關(guān)鍵詞： 半纖維素 水解作用 木聚糖酶 乙酰木聚糖酯酶 β-木糖苷酶　出處：《青島大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：農(nóng)林業(yè)廢棄物主要由纖維素、半纖維素、木質(zhì)素等大分子化合物構(gòu)成,木聚糖作為其組成成分中含量最多的半纖維素,對其完全酶解可產(chǎn)生木糖,這不僅能夠減少其焚燒帶來的環(huán)境污染,也實現(xiàn)了廢物額資源化利用。天然木聚糖結(jié)構(gòu)中通常含有不同的側(cè)鏈取代基(如阿拉伯糖、O-乙酰基、阿魏酸和4-O-甲基葡萄糖醛酸等),所以,對其完全降解需要一系列酶系協(xié)同作用。通常可降解半纖維素的酶制劑主要有木聚糖酶、β-木糖苷酶、乙酰木聚糖酯酶、阿拉伯呋喃糖苷酶和阿魏酸酯酶等。本文通過基因組序列注釋、比對以及蛋白結(jié)構(gòu)預(yù)測的方法,分別在嗜熱厭氧微生物熱解纖維素菌屬Caldicellulosiruptor sp.F32基因組中發(fā)現(xiàn)一個熱穩(wěn)定性良好的乙酰木聚糖脂酶Axe7以及在同菌屬Caldicellulosiruptor saccharolyticus基因組中發(fā)現(xiàn)兩個β-木糖苷酶CsXyl39A和CsXyl39B,利用基因克隆、質(zhì)粒構(gòu)建以及在大腸桿菌中表達目標(biāo)蛋白并純化等實驗方法,獲得了這幾種酶的重組蛋白并分別用不同方法對其進行酶學(xué)性質(zhì)測定并探究了在實際中的應(yīng)用。Axe7是Caldicellulosiruptor sp.F32所表達的具有最適溫度高,熱穩(wěn)定性好、酶催化效率高等諸多優(yōu)點的、具有一定商業(yè)化潛力的乙酰木聚糖脂酶。以4-甲基乙酸傘形酯(4-Methylumbelliferyl-acetate)作為底物時,Axe7的最適反應(yīng)pH在6.5-7.0之間,最適反應(yīng)溫度為85°C,在最適的溫度和pH條件下,Axe7活性半衰期(Half-life)超過4 h。不同金屬離子對其酶活有一定的抑制影響。通過測定酶動力學(xué)發(fā)現(xiàn)Axe7對底物的催化效率較好。眾多優(yōu)良特性使得Axe7為木質(zhì)纖維素的高溫糖化和生物煉制提供了一個可工業(yè)化的潛在選擇。CsXyl39A(GenBank登錄號為Csac_2404)和CsXyl39B(GenBank登錄號為Csac_2409)為從熱解纖維素菌Caldicellulosiruptor saccharolyticus中獲得的β-木糖苷酶,該兩種酶位于C.saccharolyticus基因組中與半纖維素水解有關(guān)的同一基因簇中,但序列相似性有很大差異。經(jīng)酶學(xué)性質(zhì)研究表明,兩者均為熱穩(wěn)定的β-木糖苷酶,最適溫度和pH較為相似,CsXyl39A具有很強的有機溶劑耐受性,而CsXyl39B則有極強的木糖以及葡萄糖耐受性。在實際應(yīng)用過程中,CsXyl39A經(jīng)大孔樹脂進行固定化后發(fā)現(xiàn),固定化并未明顯改變其熱穩(wěn)定性,但進一步提升了其對有機溶劑的耐受性,另外,該酶可應(yīng)用于水解三七皂苷R1中,反應(yīng)產(chǎn)物經(jīng)HPLC分析確定生成了藥理作用更好的人參皂苷Rg1。而CsXyl39B與木聚糖酶有較好的協(xié)同作用,兩者共同反應(yīng)能夠明顯提高木聚糖的水解效率。這些優(yōu)良特性使這兩種β-木糖苷酶在工業(yè)化應(yīng)用中潛力巨大。
[Abstract]:Agricultural and forestry wastes are mainly composed of cellulose, hemicellulose, lignin and other macromolecular compounds. Xylan is the most abundant hemicellulose in its composition, and its complete enzymatic hydrolysis can produce xylose. This can not only reduce the environmental pollution caused by incineration, but also realize the recycling of waste. Natural xylan structures usually contain different side chain substituents (such as arabinose O- acetyl). Ferulic acid and 4-O- methylglucuronic acid, etc., therefore, the complete degradation of hemicellulose requires a series of enzymatic synergism. Usually, xylanase and 尾 -xylanase are the main enzymes that can degrade hemicellulose. Acetylxylanesterase, arabinofuran glucosidase, ferulic esterase, etc. In this paper, the methods of genome sequence annotation, alignment and protein structure prediction were used. In addition to thermophilic anaerobic microorganism pyrolytic cellulose bacteria Caldicellulosiruptor. The discovery of a thermostable acetylxylanlipase Axe7 in sp.F32 genome and its presence in synbacteria Caldicellulosiruptor. Two 尾 -xylosidase CsXyl39A and CsXyl39B were found in saccharolyticus genome. The methods of gene cloning, plasmid construction, expression and purification of the target protein in Escherichia coli were used. The recombinant proteins of these enzymes were obtained, and their enzymatic properties were determined by different methods, and the application of .Axe7 to Caldicellulosiruptor was explored. The expression of sp.F32 has the optimum temperature. Good thermal stability, high enzyme catalytic efficiency and many other advantages. Acetylxylanlipase with certain commercial potential. Using 4-Methylumbelliferyl-acetate4-Methylumbelliferyl-acetate4-Methylumbelliferyl-acetate4-Methylumbelliferyl-acetate4 as substrate. The optimum reaction pH of Axe7 is between 6.5-7.0 and the optimum reaction temperature is 85 擄C. Axe7 active half-life. More than 4. The enzyme activity was inhibited by different metal ions. Through the determination of enzyme kinetics, it was found that the catalytic efficiency of Axe7 to substrate was better. Many excellent properties made Axe7 saccharify lignocellulose at high temperature. Bio-refining offers a potential option for industrialization. CsXyl39A (. GenBank login number Csacs 2404) and CsXyl39B(GenBank login number Csacs 2409). 尾 -xylosidase obtained from pyrolytic cellulose strain Caldicellulosiruptor saccharolyticus. The two enzymes are located in the same gene cluster related to hemicellulose hydrolysis in the genome of C. saccharolyticus, but the sequence similarity is very different. Both of them were thermostable 尾 -xylosidase, and the optimum temperature and pH were similar to those of CsXyl39A. However, CsXyl39B had strong tolerance to xylose and glucose. In practical application, CsXyl39A was immobilized with macroporous resin. The immobilization did not change its thermal stability, but further enhanced its tolerance to organic solvents. In addition, the enzyme could be used in the hydrolysis of Panax notoginsenoside R1. The reaction product was determined by HPLC analysis to produce a better pharmacological effect of ginsenoside Rg1.And CsXyl39B and xylanase had a better synergistic effect. These excellent properties make these two 尾 -xylosidases have great potential in industrial application.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q936

【參考文獻】

相關(guān)期刊論文 前10條

1 吳紅麗;薛勇;劉健;甘禮惠;龍敏南;;乙酰木聚糖酯酶研究進展[J];中國生物工程雜志;2016年03期

2 英瑜;孟冬冬;李福利;;解糖熱解纖維素菌F32降解未預(yù)處理秸稈及產(chǎn)纖維素酶特性分析[J];微生物學(xué)通報;2014年02期

3 田倩倩;宋萍;陳晨;林明;江凌;李霜;;枯草芽胞桿菌乙酰木聚糖酯酶的鑒定及誘導(dǎo)劑對酯酶活力的影響[J];生物加工過程;2013年01期

4 岳昌海;薄德臣;李憑力;;醋酸水解玉米芯中木聚糖的動力學(xué)[J];化學(xué)工程;2011年09期

5 毛連山;余世袁;;木聚糖酶在紙漿漂白中應(yīng)用的研究現(xiàn)狀[J];中國造紙學(xué)報;2006年03期

6 蘇二正,夏濤,張正竹,黃繼軫,胡潁蕙;共固定化單寧酶和β-葡萄糖苷酶對茶飲料增香和除混效果的研究[J];食品與發(fā)酵工業(yè);2005年05期

7 賈鵬翔,劉雪瑩,劉京龍,焦利敏,湯克勇;固定化酶及其在化工等領(lǐng)域中的應(yīng)用[J];皮革科學(xué)與工程;2004年05期

8 謝占玲,吳潤;纖維素酶的研究進展[J];草業(yè)科學(xué);2004年04期

9 毛連山,尤紀(jì)雪,宋向陽,勇強,余世袁;內(nèi)切木聚糖酶與木糖苷酶預(yù)處理對麥草漿漂白性能的影響[J];林產(chǎn)化學(xué)與工業(yè);2003年02期

10 劉瑞田,曲音波,姜英輝,高培基;假單胞菌堿性木聚糖酶的純化及性質(zhì)[J];微生物學(xué)報;1999年02期

，

本文編號：1453245