黑曲霉MAN1基因在大肠杆菌中的表达及酶学性质分析  

Expression of Aspergillus MAN1 gene in E.coli and its enzymatic characterization analysis

作　　者：张旭[1] 李璐[1] 曹雨婷[1] 徐欣[1] 李杰[1] 

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030

出　　处：《东北农业大学学报》2011年第12期91-96,共6页Journal of Northeast Agricultural University

基　　金：山东大学微生物技术国家重点实验室开放项目

摘　　要：为了通过基因工程的方法在大肠杆菌中生产β-甘露聚糖酶。以β-甘露聚糖酶生产菌黑曲霉为材料,通过RT-PCR扩增获得β-甘露聚糖酶基因MAN1的cDNA片段,将该片段克隆连至pMD18-T并测序。Blast比对结果表明,该序列与GenBank中编号XM001400053的序列相似性为100%,将pMD18-MAN1与PET-32b进行双酶切后连接,构建原核表达载体pET-MAN1。酶切鉴定正确后,将该载体导入大肠杆菌BL21。重组菌株经IPTG诱导表达,将表达产物进行SDS-PAGE,并用DNS法的测定不同温度、不同pH下该融合蛋白的甘露聚糖酶活性。结果表明,经IPTG诱导,转化得到约62 ku的融合蛋白条带,IPTG最佳诱导表达时间是6 h,融合蛋白质以可溶组分形式存在。经DNS测定,重组甘露聚糖酶的最适温度为55℃,最适pH为5.5,在此条件下,甘露聚糖酶活力为48 IU.mL-1。研究结果以期为甘露聚糖酶的工业化生产探索新的途径。Through genetic engineering fermentation in E.Coli β-mannanase. Though β-manganase producing strain of Aspergillums niger as the material, Aspergillums niger obtained the cDNA of MAN1 fragment by RT-PCR amplification of β-manganase gene. The fragment was connected with PMD18-T and sequenced. Blast comparisons results showed the sequence number XM001400053 of GenBank have 100% similarity. PMD18-MAN1 and PET-32b were double ligated to construct the prokaryotic expression vector PET-MAN1. When restriction endonuclease analysis is correct, the vector was introduced into E.co/i BL21. Recombinant strains are induced by IPTG, the expressed products were SDS-PAGE and though DNS method in different temperatures and different pH determinate the mannan fusion protein activity. As the results： After IPTG induction, transformed by the fusion protein about 62 ku, it shows that the optimum time was 6 h, fusion proteins form soluble fractions.it shows that the optimum temperature was 55 ℃, the optimum pH of 5.5, in this condition, mannanase activity was 48 IU ·mL^-1. Results to the industrial production ofβ-mannanase to explore new ways.

关 键 词：Β-甘露聚糖酶 MAN1 大肠杆菌 克隆 

分 类 号：Q786[生物学—分子生物学]