發(fā)布時間：2018-03-08 05:27

  本文選題：纖維化纖維菌　切入點：克隆表達　出處：《東北師范大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：木聚糖是植物半纖維素的主要成分,在自然界中含量豐富,是自然界中最主要的可再生資源之一,在造紙、食品、醫(yī)藥等行業(yè)應(yīng)用廣泛。木聚糖生物質(zhì)的徹底降解需要一個完善的水解酶系,包括木聚糖酶和β-木糖苷酶等。其中,β-木糖苷酶是木聚糖降解的關(guān)鍵酶之一,與木聚糖酶協(xié)同作用將木聚糖徹底降解為木糖。因此,獲得高活性的β-木糖苷酶并進行穩(wěn)定制備,用其與木聚糖酶協(xié)同降解木聚糖,是木聚糖降解領(lǐng)域研究重點之一,具有重要的理論研究意義和實際應(yīng)用價值,也是本論文的研究重點。本論文的主要研究成果如下:1.本研究從纖維化纖維菌中克隆得到一個編碼糖苷水解酶家族3(GH3)蛋白的基因ccxyl3a,將該基因與pET-28a(+)載體連接,得到的重組質(zhì)粒pET-28a/ccxyl3a轉(zhuǎn)化到大腸桿菌BL21(DE3)感受態(tài)細胞中,得到重組菌株BL21/pET-28a/ccxyl3a。2.重組菌株BL21/pET-28a/ccxyl3a在16℃條件下、0.5 mM IPTG誘導(dǎo)20 h,實現(xiàn)了重組蛋白CcXyl3A的過表達。將重組酶CcXyl3A從大腸桿菌細胞裂解液上清中通過Ni sepharose fastflow親和柱層析進行純化,得到純度大于90%的重組酶CcXyl3A。3.對純化的重組酶CcXyl3A進行酶學(xué)性質(zhì)研究。SDS-PAGE結(jié)果表明,CcXyl3A的分子量為95 KDa。以對硝基苯基-β-D-木糖苷(pNPβXyl)為底物,測定CcXyl3A的酶活力。結(jié)果表明,CcXyl3A的最適pH值為8.5,最適反應(yīng)溫度為45 ℃。離子耐受實驗結(jié)果表明K+和Na+能夠促進CcXyl3A的酶活性,在50 mM K~+或Na~+緩沖體系中,CcXyl3A酶活力分別提高到132.2%和132.6%。底物專一性結(jié)果表明CcXyl3A能水解對硝基苯基-β-D-木糖苷(pNPβXyl)、對硝基苯基-β-D-葡萄糖苷(pNPβGlc)和對硝基苯基-α-L-呋喃阿拉伯糖苷(pNPαAraf)。其中,CcXyl3A對水解pNPβXyl的酶活力最高,對pNPαAraf和pNPβGlc的水解活性較低。以寡聚木糖為水解底物時,CcXyl3A能夠完全水解木二糖、木三糖、木四糖和木六糖,生成唯一產(chǎn)物木糖。4.對重組酶CcXyl3A和真菌木聚糖酶聯(lián)合降解木聚糖的能力進行研究,結(jié)果表明CcXyl3A和噬熱真菌木聚糖酶能夠協(xié)同作用,降解櫸木木聚糖。其中,噬熱真菌木聚糖酶將櫸木木聚糖降解為木寡糖和木二糖,CcXyl3A進而將其完全降解成木糖。本研究為纖維化纖維菌的糖苷酶在半纖維素降解中的應(yīng)用奠定了理論基礎(chǔ)。
[Abstract]:Xylan is the main component of hemicellulose in plants, rich in nature, is one of the most important renewable resources in nature, in paper, food, The complete degradation of xylan biomass requires a complete hydrolase system, including xylanase and 尾 -xylosidase. Among them, 尾 -xylosidase is one of the key enzymes in the degradation of xylan. Therefore, it is one of the important research points in the field of xylan degradation to obtain high activity 尾 -xylosidase and prepare it stably, and use it to co-degrade xylan with xylanase. Has the important theory research significance and the practical application value, The main research results of this thesis are as follows: 1. A gene ccxyl3aencoding glycoside hydrolase family 3 (GH3) was cloned from Fibrofibria and linked with pET-28a () vector. The recombinant plasmid pET-28a/ccxyl3a was transformed into Escherichia coli BL21DE3) competent cells. The recombinant strain BL21 / pET-28a / ccxyl3a.2. the recombinant strain BL21/pET-28a/ccxyl3a was induced by 0.5 mm IPTG at 16 鈩，

本文編號：1582601