【作者】 刘辉；

【导师】 龚月生； 杨明明；

【作者基本信息】 西北农林科技大学 ， 动物营养与饲料科学， 2007， 硕士

【摘要】 半纤维素是仅次于纤维素含量第二丰富的可利用自然资源,其主要成分是木聚糖(xylan)。木聚糖的来源不同,分枝程度不同,其主链和支链带有多种不同的取代基团。由于木聚糖结构的复杂性,它的完全降解需要多种水解酶的参与共同作用完成。β-1,4-内切木聚糖酶(EC. 3.2.1.8)能够以内切方式作用于木聚糖主链产生不同长度的木寡糖和少量的木糖,因此它是木聚糖降解酶中最关键的酶。本试验采用刚果红染色的方法筛选出一株耐碱性木聚糖酶高产菌株,对其产酶条件及酶学性质进行研究,并通过基因克隆手段得到编码木聚糖酶的基因,实现该基因在大肠杆菌中的高效表达,整个试验分三部分进行。1.根据木聚糖酶分解木聚糖底物生成的寡糖不能被刚果红染色,从而在菌落周围形成透明圈这一特性,对从武汉晨鸣纸厂采集的碱性纸浆进行生孢子处理,根据不同菌株所形成的透明圈的有无,决定需要复筛的菌株,经产酶基本培养基复筛后,筛选到一株碱性木聚糖酶高产菌株,对该菌株进行普通染色、革兰氏染色、形态学特征和16SrDNA序列分析,初步确定该菌株为枯草芽孢杆菌(Bacillus. Subtilis),并且命名为BYG6-6。2.对其生长条件和产酶条件研究结果表明,该菌适宜在pH7.0、温度33℃下生长。为了给菌体提供有效的营养,对培养基中的氮源和碳源进行了优化,结果表明,应用桦木木聚糖作为碳源、牛肉膏作为氮源的培养基,能够使细菌很好的生长,并表现良好的产酶能力。改变木聚糖酶和底物作用时的温度和pH值,确定该酶的最适值及其耐受性。结果显示:该酶最适反应温度为酶反应的最适温度为55℃。在65℃时酶活仍有73.07%,证明该酶属于嗜中温酶类,不具有高温耐受性;酶反应最适pH值为6.74,在pH为7.22时剩余木聚糖酶的活力为91.8%,即使反应pH值升高到9.0和10.0时,剩余酶活力仍在70%以上,说明该菌株所产酶具有较强的耐碱性。3.根据已经报到的枯草芽胞杆菌木聚糖酶基因,设计特异性引物从BYG6-6基因组中扩增得到一个基因片段,连接到pGEM -T Vector载体上测序得到该基因序列,经在线比对分析该基因与已报道的木聚糖酶基因的同源性达到99%以上。借助软件分析表明:该基因全长642个碱基,基因序列为一个完整的阅读框,起始密码为ATG,终止密码TAG连续编码214个氨基酸,分子大小约为24kDa。我们利用引物B3-up和B3-down,从高产木聚糖酶的枯草芽孢杆菌BYG6-6中克隆木聚糖酶基因,接着将其构建在表达载体PET-28(a)中,用Escherichia coli BL21 (DE3)进行电转化,经过筛选获得重组质粒,将含有该质粒的重组菌株命名为PYG-6。在37℃、诱导物浓度为1.0mmol/L、诱导后5-6小时时,该木聚糖酶基因得到了很好的表达,表达产物表现出的酶活比原始菌种分泌的木聚糖酶活提高了10倍左右。这也说明该基因能够在E.coli中实现异源表达,并且在T7启动子控制下合成有活性的木聚糖酶,同时,我们也可以通过SDS-PAGE和酶谱证明该基因确实是编码木聚糖酶基因。更多还原

【Abstract】 Second only to cellulose in natural abundance, xylan is a major component of hemicellulose fraction of the plant cell walls. Depending on the sources, its backbone may carry various substitutents like arabinose and glucuronic acid. Typically, backbone depolymerization is accomplished by the action of endo-xylanases andβ-xylosidases. Xylanase can hydrolyzeβ-1,4-glycosidic linkages of the xylan backbone to produce short chain xylo-oligosaccharides of various length, hence Endo-β-xylanase is the crucial enzyme components of microbial xylanolytic systems.The aim of this experiment is that we sceen a high-xylanase producing bacterium according to the method of Kongo red dye, reaserching the optimization of xylanase production conditions and characterization of xylanase, obtaining the genes coding the xylanase and achieving high expression of the xylanase in E.coli, This thesis was described in the following three sections.1. It is based on the oligoses produced by the xylanases breaking down xylan substrates can’t be dyed by Kongo red, consequently, lots of transparent circle will be formed around the bacterium, a bacteria strain BYG6-6 which overproduced alkali-tolerant xylanase was isolated from a waste water treatment plant of pulp paper. It was preliminary identified as Bacillus.Subtilis according to the common dye, Gram staining, its morpHology characters and 16S rDNA sequence.2. Researching in the optimization living conditions of BYG6-6 and enzyme characterization producing by strain BYG6-6, The results showed: the optimization living environments was pH7.0 and temperature 33℃.In order to supply the effiency nutrition for the bacterium, the nitrogen source and carbon source in the culture medium were optimized. When used Birchwood-xylan as carbon source and beef extract as nitrogen source, The activity of the xylanase was relatively higher than before.The further research was performed on the properties of xylanase, we got through changed temperature and PH values when xylanase reacted with the relevant substrate, in order to determine the optimal reaction temperature and PH optimal, The activities of the crude enzymes were measured at different temperature and various PH, The result demonstrated that the optimal temperature of xylanase is at 55℃. And still retained about 73.07% enzyme activity at 65℃, this pHenomenon is proved that bacteria strain BYG6-6 belonged to midtemperature-resistant enzymatic classify, however, when it existed in a high temperature, enzymatic activities dropped dramatically. The optimal PH is at 6.74, and still retained about 91.8% activity when PH is 7.22, even if the PH rise to 9.0 and 10.0, the remain of enzyme activity is approximately 70% upwards, This results revealed that bacteria strain BYG6-6 was slightly more alkaline resistant.3. Amplified a DNA fragment from B. subtilis BYG6-6 genome DNA through designed primers, which was designed according to the reported xylanase gene of Bacillus subtilis. Ligated this fragment to pGEM -T Vector, then sequenced. The homology of this sequence and other reported endo-1,4-xylanase gene was reached 99% through blast online. This xylanase gene was consist of 642 base pairs, and was an integral reading frame, start codon was ATG, and terminal codon was TAG, coding 214 amino acids continuously, the molecular weight is approximately 25kDa.We have cloned a gene that encodes xylanase enzyme from Bacillus.sp. BYG6-6 by using primers B3-up and B3-down, then the gene was cloned into a PET-28(a) expression vector and transformation of Escherichia coli BL21 (DE3), This recombined strain was denominated PYG-6 in our experiment after screening. In 37℃, the xylanase gene was expressed in E. coli after inducement. The xylanase activity of E. coli was ten times higher than that of wide type bacterium. such a pHenomenon showed that the gene realized to heterogenous expression in Escherichia coli and synthesized xylanase which have the activity of the enzyme under the control of the T7 promoter. At the same time we also can prove that the gene was indeed belonging to encoding xylanase gene via SDS-PAGE and zymogram.更多还原