【作者】 连惠芗；

【导师】 汪世华；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 木聚糖酶(E.C 3.2.1.8)是一类以内切方式降解木聚糖分子中β-1,4-木糖苷键的酶系。它对自然界中大量存在的半纤维素起着重要的作用,该酶广泛应用于造纸、食品、饲料添加等方面。其中耐热木聚糖酶由于其耐热性好,被广泛运用于工业生产特别是造纸工业,因此已受到广泛的关注。本研究以高产木聚糖酶的黑曲霉C3486(Aspergillus niger C3486)的基因组为模板,通过PCR扩增出木聚糖酶的特异性条带,连接到原核表达载体pET32a(+),转化E. coli BL21(DE3),IPTG诱导表达后得到目的蛋白,纯化后所得蛋白Xyn-1不具有木聚糖酶活性。而后将木聚糖酶基因连接到真核表达载体pPIC9K,转化至毕赤酵母Pichia pastoris GS115中,纯化所得的木聚糖酶Xyn-2具有一定的酶活性。然后再以Aspergillus niger C3486总RNA为摸板,进行RT-PCR后,重新转化至大肠杆菌中,所得的木聚糖酶Xyn-3也具有一定的酶活性。通过木聚糖酶DNA与cDNA的序列比对,发现该酶的的基因组中且一个大小约为50 bp的内含子。通过生物信息学分析表明,该酶含有187个氨基酸,具有信号肽。并且具有典型的木聚糖酶保守区域,利用在线预测该酶进行三级结的预测,结果表明该酶是一个以β-折叠为主为的蛋白酶,形成一个紧密的球状蛋白。通过Error PCR和DNA Shuffling体外分子进化技术,将来自于黑曲霉与里氏木霉的木聚糖酶基因进行共同进化,获得了耐热性进一步提高的木聚糖酶。更多还原

【Abstract】 Xylanase (E.C 3.2.1.8) is a widespread group of enzyme which can catalyze the endohydrolysis of 1,4-β-D-xylosidic linkages in xylan. Xylanse has a wide application in industrial processes. It can be used in biobleachin of kraft pulps,fruit and vegetable processing, animal feeding and so on. The thermotable xylanase has atttacted widely attention because of its thermostabilization.In this study, xylanase gene was obtained from Aspergillus niger C3486 by PCR. To achieve secretive expression, pET32a (+) was used as expression vector, and the cloned gene was expressed in E. coli BL21 (DE3). After induction by IPTG, a large quantity of xylanase was produced but without enzyme activity. And some progress have made in the fugal expression system: the xylanase gene was then transformed into the Pichia.pastoris GS115, the enzyme produced from the Pichia.pastoris GS115 showed xylanase activity.Moreover, the total RNA was obtained, after RT-PCR, the gene was transformed into the pET28a as well, and the enzyme named Xyn-3 produced from the E.coli BL21 (DE3) also showed xylanase activity, and through the compare between the DNA and cDNA sequence, we know that the xylanase DNA contains an intron about 50 bp.With the analysis of bioinformatics, this xylanase contains 187 amino acids with a preceding peptide. Moreover, it has a typical conserve domain of xylanase. Perdicted by web site on line. The xylanase is globulin, and its super structure contains manyβ-sheet.And then, the xylanase gene from A.niger and T.reesei was mixed for co-evolution by the technology of Error PCR and DNA Shuffling, and the xylanase with higher thermostability were obtained.更多还原