【作者】 陈艳；

【导师】 孙建义；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2004， 硕士

【摘要】 淀粉液化芽孢杆菌（Bacillus amyloliquefaciens）是一种嗜热菌，其产生的中性植酸酶具有广阔的应用前景。本研究克隆了淀粉液化芽孢杆菌植酸酶基因，采用表达载体pET-30a（+）Vector、pPIC9K Vector及其相应的宿主菌E. coli 21和Pichia pastoris GS115对其进行表达，并对表达产物的活性和耐热性能进行了系统性评价。主要研究结果如下： 通过对植酸酶基因序列的同源性比较，根据其保守区域设计上游引物5BAP01和下游引物3BAP01。以提取的淀粉液化芽孢杆菌BA基因组为模板，利用TD-PCR技术克隆了淀粉液化芽孢杆菌植酸酶基因BAP，并构建了重组质粒pGEM-T Easy BAP Vector。测序表明，其长度为1167 bp，ORF为11bp～1159 bp，编码383个氨基酸，5’端有一个编码26个氨基酸的信号肽序列，其全长理论分子量为41.9 kDa。 淀粉液化芽孢杆菌植酸酶基因和已报道的杆菌植酸酶序列具较高同源性，如与AF029053（B. subtillus）、AF453255（B. amylotiquefaciens）、U85968（Bacillus sp.DS11）、AY220075（B. subtillus）、AY055220（B. amyloliquefaciens）、AY518208（Bacillus sp.）的DNA序列的同源性分别为100％、96％、92％、92％、92％、92％，所编码蛋白同源性分别达到100％、97％、94％、94％、94％、93％。 通过带限制性内切酶位点的引物（5BAP01带BamHI位点，3BAP01带XhoI位点），将淀粉液化芽孢杆菌编码的中性植酸酶基因定向插入到原核表达载体pET-30a（+）上，得到重组表达质粒BAP-pET-30a（+）Vector。在大肠杆菌BL21（DE3）中得以表达，SDS-PAGE凝胶电泳显示在50.2 kDa处有一特异条带，比推导出的分子量41.9 kDa大。单位发酵液的酶活力为480.6 U／ml，表达量占到了大肠杆菌可溶性蛋白的33.5％，为出发菌株酶活的1.27倍。采用金属Ni²⁺亲和层析对基因表达产物进行纯化，产物具有正常的生物学功能。 根据酵母表达载体pPIC9K的多克隆位点和已克隆的中性植酸酶基因限制性内切酶图谱，设计具有SnaBI位点的上游引物5BAPK01和具有NotI位点的下游引物3BAPK01。采用亚克隆技术，将淀粉液化芽孢杆菌植酸酶基因以N-端融合的方式正确插入到酵母表达载体pPIC9K上的α-因子信号肽编码序列的3’硕士学位论文淀粉液化芽抱杆菌中性植酸酶基因在大肠杆菌和毕赤酵母的高效表达端，构成重组表达载体BAP一pPICgK。用电击法将经BgiH单酶切线性化的BAP一PPICgK转化入毕赤酵母Gslls菌株，从而整合到酵母染色体DNA中得到重组转化子。筛选到的基因工程菌经培养后，上清液的发酵活力为4120 U/ml，为出发菌株的10.9倍。sDs一PAGE凝胶电泳检测，表达的植酸酶的分子量约为45 kDa，这表明植酸酶基因在毕赤酵母得到了高效表达g 对两种表达系统的植酸酶生物学特性研究显示，云动11 BAPE菌株与Piohia pastori:BAPK菌株表达的淀粉液化芽饱杆菌的植酸酶具有相似性，最适反应温度均为65一70’C，属嗜热性酶。其最适反应pH为7.0一7.5，在6.。一7.5这一范围内，酶活性均在70%以上。pH值稳定性结果表明，植酸酶BAPE和BAPK在pHS.0一9，O之间极为稳定，残余酶活在85%以上，而pH低于5.0时，酶活性迅速下降，到pH为4.0以下，已无酶活性。热稳定性研究揭示，重组酵母植酸酶BAPK的耐热性优于大肠杆菌植酸酶BAPE，BAPK在70℃和80℃处理2 min，残余酶活为80.54%和68.42%，处理10 min的残余酶活分别为70.03%和50，69%;而植酸酶BAPE在70℃和80℃处理2 min，残余酶活为71.02%和40.34%，处理10 min后，残余酶活为4823%和20.12%。由此可以看出，同种基因经两种不同系统表达得到的植酸酶均适合在动物的肠道内发挥作用，重组酵母植酸酶BAPK耐热性更佳。更多还原

【Abstract】 Bacillus amyloliquefaciens is one of thermophilic bacteria. The phytase found in B. amyloliquefaciens has shown the extremely thermostat)lity and considerable potential in large-scale production. In this study the phytase gene BAP from Bacillus amyloliquefaciens was cloned and fused in the expression plasmid pET-30a(+). then transferred into the host E.coli BL21.Further, the BAP with origin signal peptide sequence was cloned into the expression plasmid pPIC9K and had been overexpressed in Pichia pastoris GS115.Then the biochemical properties of two expressed phytase were investigated. The main results obtained are as follows:Bacillus amyloliquefaciens BA could produce phytase. Its genome was used as template for gene cloning, the phytase gene of BA was amplified by the touchdown polymerase chain reaction (TD-PCR) with primers (5BAP01 and 3BAP01) designed according to the conserved regions of the phytase gene in Genbank.The DNA fragment obtained was cloned into pGEM?T Easy Vector, generating pGEM?T Easy BAP Vector. Sequencing result showed that its nucleotide sequence was 1167 bp, containing an Open Reading Frame(ORF) from 11 bp to 1159 bp,encoding a peptide of 383 amino acids . with a signal peptide of 26 amino acids. The deduced molecular weight of enzyme was 41.9 kDa.Comparison to this sequence with phytase genes of the other Bacillus sources. The nucleotide sequence and amino acid sequence identity were 100% and 100% for AF029053 (B.subtillus); 96% and 97% for AF453255(5. amyloliquefaciens); 92% and 94% for m5968(Bacillus sp.DSU); 92% and 94% for AY220075(5. subtillus); 92% and 94% for AYQ55220(B.amyloliquefaciens); 92% and 93% for AYS\82Q8(Bacillus sp.), respectively.Based on two primers 5BAP01 containing a BamHI site and 3BAP01 containing a Xhol site, the pGEM?T Easy BAP Vector was double digested with BamHI and Xhol, and then cloned into the expression vector pET-30a(+) previously digested with the same enzymes. The resulting plasmid BAPpET30a(+)Vector was introduced into E.coli BL21(DE3). The expressed enzyme has a molecular mass of 50.2 kDa dectected by SDS-PAGE, which was larger than the 41.9 kDa deduced from amino acid sequence. The fused protein was purified by immobilizing metal affinity chromatography. The expression phytase has normal bioactivity, which was up to 480.6 U/ml (1.27 times as that of B. amyloliquefaciens), over 33.5% the total soluble protein of E. coli.A pair of expression primers, the sense primer 5BAPK01 containing a SnaBI site and antisense primer 3BAPK01 containing a NotI site were designed according to the Multiple Cloning Sites(MCS) of expression vector pPIC9K and the Open Reading Frame of BA gene. The PCR fragment were cloned into pGEM?T Easy Vector, generating subcloned vector of pGEM?T Easy BAPK Vector. After the vector was double digested with SnaBI and NotI it was cloned to pPIC9K, an expression vector double digested with the same restriction enzymes, and the recombinat plasmid BAP-pPIC9K was obtained. The plasmid was linearized with Bgl II ,then, introduced into the host Pichia pastoris GS115 by electroporation. The Pichia pastoris recombinants for phytase overexpression were screened by enzyme activity analysis and SDS-PAGE. It had an apparent molecular weight of 45 kDa as determined by SDS-PAGE.The result revealed that the phytase was overexpressed and secreted into the medium supernatant, its activity was up to 4120 U/ml, which was 10.9 times as that of B. amyloliquefaciens.The biochemical properties of the phytases produced by E.coli BAPE and Pichia pastoris BAPK showed that both of them had an optimum temperature of 65-70 ?and pH of 7.0-7.5 respectively, and it has 70% activity at pH 6.0-7.5. Effects of pH on stability of phytase showed that phytase BAPE and BAPK retained more than 85% treated containing for 1 h at pH 5.0-9.0. The thermostability of different phytase activities varied, the phytase BAPK retained 80.54% and 68.42% treated with containing for 2 min at 70?and 80 癈, and retained 70.03% and 50.69% treating with containing for 10 min at 70 ?and更多还原