【作者】 肖竞；

【导师】 孙建义；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2003， 硕士

【摘要】 本研究克隆了环状芽孢杆菌木聚糖酶基因，采用表达载体pET-30a（+）Vector、pGEX-4T-3 Vector和宿主菌E.coli BL21对其进行了表达，并研究了表达产物的酶学特性。主要结果如下： 本试验以具有木聚糖酶活性的环状芽孢杆菌BC（Bacillus circulans BC）为出发菌株，通过对GenBank上登录的其它来源β-1，4-木聚糖酶基因序列同源性分析，根据其保守区设计了5｀端引物XP51和3｀端引物XP31。采用TD-PCR技术克隆了环状芽孢杆菌木聚糖酶基因（己被GenBank收录，其收录号为AF490980），该基因序列与pGEM^?-T easy Vector连接，获得了重组克隆子pGEM^?-T easy BCX Vector。测序结果表明，其长度为702bp，具有一个629bp的开放阅读框（open reading frame,ORF），编码213个氨基酸的酶蛋白，其理论分子量为23.2KDa。 经同源性比较发现，该环状芽孢杆菌木聚糖酶基因与GenBank上登录的其它芽孢杆菌木聚糖酶基因如AF490979（B.subtilis-2）、X07723（B.circulans）、M36648（B.subtilis）、X59058（Bacillus sp.）、Z34519（B.subtilis 168）、AF441773（Bacillus sp.NBL420）和U51675（Bacillus sp.）具很较高同源性，其核苷酸序列同源性分别为98％、96％、96％、97％、96％、92％和91％；氨基酸序列同源性分别为97％、97％、97％、97％、97％、94％和94％；经分析发现该木聚糖酶含有一个28个氨基酸的信号肽。 根据表达载体多克隆位点（MCS）和已经克隆的木聚糖酶基因开放阅读框设计了一对表达引物，其中上游引物XP52 5｀端含有BamHI限制性酶切位点，下游引物XP32 5｀端含有XhoI限制性酶切位点。以pGEM（?）-T easy BCX Vector为模板，采用TD-PCR方法得到一条能编码木聚糖酶且两端带有限制性酶切位点的DNA片段。该片段与pGEM^?-T easy Vector连接得到亚克隆重组质粒pGEM^?-T easy BCXE Vector。用BamHI和XhoI双酶切亚克隆重组质粒，回收目的片段并分别与经双酶切的表达质粒pET-30a（+）Vector和pGEX-4T-3 Vector进行连接，将获得的重组表达质粒pET-BCXE和pGEX-BCXE分别导入E.coliBL21中，利用转化子在RBB一木聚糖平板上形成的透明圈，分别筛选到含有重组表达质粒pET一BCxE和重组表达质粒pGEX一BcXE的阳性表达子E.coliBCES和E.coli BCEI。菌株发酵活力为1 35.4lU/ml和123.5U/ml，分别为出发菌株的2.33倍和2.13倍。 对两种表达系统表达的木聚糖酶生物学特性研究显示，它们的最适反应温度均为50一60℃，在40℃以下相对稳定;最适反应pH均为为5.0，在pH3，。⁹.0之间相对稳定，并与出发菌株的酶学特性基本一致。用SePhadexG一25、sephadexG一1 00和Source 3oQ等对E.eoli BCES菌株的表达产物进行了分离纯化。SDS.PAGE凝胶电泳结果显示纯化的酶蛋白分子量为20.3kDa，与成熟酶蛋白的理论值相符，表明纯化的酶蛋白为成熟木聚糖酶，同时也表明来自环状芽抱杆菌BC的木聚糖酶中确实存在28个氨基酸的信号肤序列。更多还原

【Abstract】 In this study the xylanase gene of B. circulans BC was cloned and expressed in the E. coli BL21 by using pET-30a(+) and pGEX-4T-3 as expression vectors, then the biochemical properties of expression products were analyzed. The main results are as follows:Bacilus circulans BC could produce xylanase, and its genome was used as template for xylanase gene cloning. The homology of nucleotide sequences of J3 -1,4-xylanase genes from different microbes in the GenBank were analyzed, and a pair of primers, XP51 for 5’ end fragment and XP31 for 3’ end fragment, were designed based on their conserved regions. The xylanase gene from Bacillus circulans BC was obtained by touchdown polymerase chain reaction (TD-PCR), then was cloned into pGEM?T easy Vector, generating pGEM?T easy BCX Vector. Sequencing results showed that the nucleotide sequence was 702bp, and it had an open reading frame(ORF) of 639bp encoding a polypeptide of 213 amino acids with a theoretical molecular weight of 23.2kDa. The accession number of the xylanase gene sequence is AF490980 in the GenBank.Strong homology of the nucleotide sequence and amino acid sequence of the xylanase was found between Bacillus circulans BC and other Bacillus sources. The nucleotide sequence and amino acid sequence identity were 98% and 97% for AF490979 (B. subtilis-2); 96% and 97% for X07723 (B. circulans); 96% and 97% for M36648(5. subtilis); 97% and 97% for X59058 (Bacillus sp.); 96% and 97% for Z34519 (B. subtilis 168); 92% and 94% for AF441773 (Bacillus sp. NBL420); 91% and 94% for U51675 (Bacillus sp.), respectively. Comparison of thesequences indicated that there is a putative 28-amino-acid signal peptide in front of the amino terminus of the mature enzyme.Based on the multiple cloning sites(MCS) of expression vectors, two expression primers, upstream primer XP52 containing a BamHI restriction site and downstream primer XP32 including a Xhol restriction site, were designed corresponding to open reading frame of the cloned xylanase gene. By using TD-PCR method a DNA fragment, which comprised of the xylanase encoding region flanked with restriction enzyme sites at both ends, was amplified using pGEM?T easy BCX Vector as template. The PCR fragment was ligated with pGEM?T easy Vector, generating subcloned vector of pGEM?T easy BCXE Vector. The subcloned vector was double digested with restriction endonucleases, and a target fragment was recovered. Then it was inserted into pET-30a(+) vector and pGEX-4T-3 vector at BamHI and Xhol restriction sites, respectively. The recombinant expression plasmids pET-BCXE and pGEX-BCXE were obtained, and then they were transformed into E. coli BL21. Two positive recombinants, E. colt BCE5 containing pET-BCXE vector and E. coli BCE1 harboring pGEX-BCXE vector, were obtained, which forming zones of clearing on RBB-xylan plates. The levels of xylanase activity expressed by E. coli BCE5 and E. coli BCE1 were up to 135.41U/ml (2.33 times as that of B. circulans BC ) and 123.5U/ml (2.13 times as that of B. circulans BC ), respectively.The biochemical properties of the xylanases produced by E.coli BCE5 and E.coli BCE1 showed that both of them had an optimum temperature of 50~60癈 and pH of 5.0, and they were stable at 40 癈 and pH3.0-9.0, these characterizations were similar to that of enzyme produced by B. circulans BC. The xylanase expressed by E. coli BCE5 was purified by ion exchange and gel permeation chromatography, it had an apparent molecular weight of 20.3kDa as determined by SDS-PAGE, which is consistent with that calculated mass from the amino acid analysis of the mature xylanase. It was suggested that the purified xylanase was mature xylanase and there was indeed a signal peptide of 28 amino acids in the xylanase from B. circulans BC.更多还原