| 黑曲霉MAN1基因在大肠杆菌中的表达及酶学性质分析 |
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| 引用本文： | 张旭,李璐,曹雨婷,徐欣,李杰.黑曲霉MAN1基因在大肠杆菌中的表达及酶学性质分析[J].东北农业大学学报,2011,42(12):91-96. |
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| 作者姓名： | 张旭 李璐 曹雨婷 徐欣 李杰 |
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| 作者单位： | 东北农业大学生命科学学院,哈尔滨,150030 |
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| 基金项目： | 山东大学微生物技术国家重点实验室开放项目 |
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| 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PE...
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| 关 键 词： | β-甘露聚糖酶 MAN1 大肠杆菌 克隆 |
Expression of Aspergillus MAN1 gene in E.coli and its enzymatic characterization analysis |
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| ZHANG Xu,LI Lu,CAO Yuting,XU Xin,LI Jie.Expression of Aspergillus MAN1 gene in E.coli and its enzymatic characterization analysis[J].Journal of Northeast Agricultural University,2011,42(12):91-96. |
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| Authors: | ZHANG Xu LI Lu CAO Yuting XU Xin LI Jie |
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| Institution: | (College of Life Sciences,Northeast Agricultural University,Harbin 150030,China) |
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| Abstract: | 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.coli 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 of β-mannanase to explore new ways. |
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| Keywords: | β-mannanase MAN1 E coli DH5 cloning |
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