Gene Cloning and Heterologous expression of xylanase from Hypocrea orientalis EU7-22

木聚糖是一种多聚五碳糖，是半纤维素的主要成份，广泛的分布于植物细胞壁中，是除纤维素外自然界中最为丰富的可再生资源多糖，约占植物生物质总量的15%~35%。木聚糖主链通过β-1，4-糖苷键将木糖残基连接而成，侧链上带有不同的取代基。由于木聚糖的复杂结构，它的完全降解需要多步酶促反应完成。内切β-1，4-木聚糖酶（EC3.2.1.8）是最重要的酶之一，作用于木聚糖主链内β-1，4-糖苷键，生成木寡糖和木糖。由于木聚糖酶具有广泛的工业应用前景，已成为国内外科研的热点，不同微生物来源的木聚糖酶基因已被克隆并进行异源表达。为获得优质的低聚木糖资源，就必须获得性状优良的产木聚糖酶工程菌株。 本文以本实验...Xylan is a kind of polysaccharides, which is widely distributed in plants cell wall. In addition to cellulose, xylan is the most abundant renewable polysaccharides in nature. Accounting for plant biomass wet weight of 15% to 35%. The main chain of xylan is composed of D-xylose with β-1,4-glycoside linkage and the branch chain contains different short substituents. Due to xylan complex structure, i...学位：理学硕士院系专业：能源研究院_能源化学学号：3242010115210

Gene cloning and bioinformatics analysis of Xylanases and Xylosidase from Hypocrea orientalis

东方肉座菌Eu7-22具有高产半纤维素酶的能力。根据已报道的同属里氏木霉及绿色木霉木聚糖酶,木糖苷酶相关基因序列,设计PCr引物扩增出东方肉座菌内切木聚糖酶(XynⅠ,XynⅡ)及β-木糖苷酶(β-bXl)基因。序列经nCbIblAST分析:东方肉座菌XynⅠ基因与里氏木霉Xyn1基因(X69573.1)的同源性最高达到91%;XynⅡ基因与绿色木霉Xyn2基因(Ef079061)同源性最高达到93%;β-bXl基因与里氏木霉β-bXl1基因(z69257.1)的同源性最高达到94%。生物信息学分析表明内切木聚糖酶Ⅰ和Ⅱ均属于糖基水解酶家族11,n末端前19个氨基酸均为信号肽。内切木聚糖酶Ⅰ分子量为24.13kd,等电点为7.87,含有3个糖基化位点;内切木聚糖酶Ⅱ分子量为24.44kd,等电点为4.86,含有1个糖基化位点;β-木糖苷酶属于糖基水解酶家族3,分子量为87.27kd,等电点为5.49,n末端前20个氨基酸为信号肽,含有8个糖基化位点。利用SWISS-MOdEl对木聚糖酶,木糖苷酶蛋白质三级结构进行了预测和模拟。对木聚糖酶和木糖苷酶基因及其编码蛋白质的生物信息学分析,为进一步研究这些基因的表达与调控、构建高效利用纤维素组份的工程菌株奠定基础。Hypocrea orientalis EU7-22 had a high potential to yield hemicellulase.Two endo-xylanases genes (xyn Ⅰ, xyn Ⅱ) and one β-xylosidase gene (β-bxl) were successfully cloned by PCR, according to the reported xylanases and β-xylosidase gene sequences of Trichoderma reesei and Trichoderma viride.The xyn I and β-bxl gene from H.orientalis showed the highest nucleotide homology of 91% and 94% with the corresponding gene from T.reesei, respectively.While the xyn II gene from H.orientalis showed the highest of 93% nucleotide homology with the corresponding gene from T.viride.The bioinformatics analysis indicated that the enzyme XYN I and XYN II belonged to the glycosyl hydrolase family 11 with the molecular weight of 24.13 kD and 24.44 kD, respectively.The first 19 AA of N-terminal of XYN I and XYN II were the signal peptide sequence.The enzyme XYN I and XYN II contained three and one N-glycosylation site, respectively.The isoelectric point of enzyme XYN I and XYN II were identified as 7.87 and 4.86, respectively.The enzyme β-BXL belonged to glycosyl hydrolase family 3 with molecular weight of 87.27 kD and isoelectric point of 5.49.The first 20 AA of N-terminal of β-BXL belonged to signal peptide sequence.The enzyme contained 8 N-glycosylation sites.By using SWISS-Model, the tertiary structure of the three enzyme proteins were predicted and simulated.These genes cloning and bioinformatics analysis would help to the further research on mechanism of expression and regulation of hemicellulase.国家重点基础研究发展计划(973计划)(NO.2010CB732201); 中央高校基本科研业务费专项资金(NO.201112G026); 国家自然科学基金(NO.31170067