碱性果胶裂解酶基因pelA在E.coli中的表达和酶学性质研究

碱性果胶裂解酶基因pelA在E.coli中的表达和酶学性质研究 Study on the Expression of a pelA in E.coli DH5α and Property of the pelA 翟超 ZHAI Chao 1 2 first-author 曹军卫 CAO Jun-wei 1 2 武汉大学生命科学学院 武汉大学生命科学学院 College of Life Sciences, Wuhan University College of Life Sciences, Wuhan University 武汉大学生命科学学院,湖北,武汉,430072 武汉大学生命科学学院,湖北,武汉,430072 根据核苷酸序列分析结果设计引物 ,通过 PCR的方法 ,在嗜碱芽孢杆菌 NTT3 3的总 DNA和 p13 5 0中分别扩增到预计大小的片段 ,证明 p13 5 0中的外源 DNA片段来自于嗜碱芽孢杆菌 NTT3 3 .同时也获得了含有pel A完整 ORF的 DNA片段 .经计算 ,该基因表达的成熟蛋白质的分子量为 3 4 .76× 10 3.构建表达载体 p BV2 2 0 -pel,在 E.coli DH5α中进行表达 .SDS- PAGE检测发现 ,表达的蛋白质分子的大小约为 3 5× 10 3,与预测的相符 .初步研究其酶学性质发现 ,该酶在 p H 9.0～ p H10 .0 ,4 5°C的条件下有较高的活性 ,而且必需 Ca2 + 参与反应 . Two couples of primers were synthesized according to the sequence of the pelA gene. They are used to prove that the pelA gene obtained from NTT33 by PCR. A couple of them were also used to clone the complete ORF of the pelA gene by PCR in order to express it in E.coli DH5α.Thereby, expression plasmid pBV220-pel was constructed. The expression of pelA gene in E.coli DH5α was identified by SDS-PAGE analysis. It was shown that the molecular weight of pelA was about 35×10 3,which agreed with the estimate molecular weight of the matured protein which was 34.76×10 3 after subtracted the signal peptide.The enzyme in E.coli DH5αexhibited optimum activity at pH9.0 p H10.0 and 45°C, and had an absolute requirement of Ca 2+ . 碱性果胶裂解酶 嗜碱芽孢杆菌 PCR 表达 pectate lyase Bacillus alcalophillus PCR expression Q785 2001-04-01 2021-04-01 4