海洋来源褐藻胶裂解酶及其基因B1SM的克隆和表达

Cloning and expression of the alginate lyase gene B1SM from marine Pseudoalteromonas sp. B1

从海洋来源的假交替单胞菌(Pseudoalteromonas sp.) B1基因组中克隆1个褐藻胶裂解酶基因,并在大肠杆菌中实现异源表达,分离纯化重组酶并研究其酶学性质。通过Touch down PCR与热不对称交错PCR(thermal asymmetric interlaced PCR,TAIL-PCR)从假交替单胞菌B1基因组中扩增到1个褐藻胶裂解酶基因(B1SM),将目的基因插入到p GEX-4T-1载体,转化到宿主大肠杆菌(Escherichia coli,E. coli) BL21 (DE3)。结果显示,从菌株B1克隆得到褐藻胶裂解酶基因B1SM,序列长度为1 005 bp,编码334个氨基酸,理论等电点为8. 51,编码蛋白质的理论分子质量为37. 13 k Da。重组褐藻胶裂解酶B1SM的最适pH和温度分别为8. 0和25℃。该褐藻胶裂解酶在pH 7. 0～9. 0范围内,25℃下保温1 h,仍剩余60%以上活力,pH稳定性较好。在最适pH 8. 0和30℃下保温1 h重组酶仍剩余60%以上活力。该研究为褐藻胶裂解酶基因B1SM在E. coli BL21(DE3)中实现了异源表达,为褐藻胶裂解酶的制备和研究奠定基础。

The recombinant alginate lyase B1SM was isolated and purified,and its enzymatic properties were characterized.The alginate lyase gene B1SM was amplified from the genome of Pseudomonas aeruginosa B1 by Touch down polymerase chain reaction(PCR)and Thermal Asymmetric Interlaced PCR(TAIL-PCR).The gene B1SM was inserted into a pGEX-4T-1 expression vector and expressed in Escherichia coli BL21(DE3).The results showed that the optimal pH and temperature for B1SM were 8.0 and 25℃,respectively.Moreover,B1SM maintained more than 60%of its relative enzyme activity after incubating at 25℃for 1 h at pH=7.0-9.0,indicating that it had a good pH stability.Furthermore,it maintained its relative enzyme activity to more than 60%after incubating at 30℃and at pH=8.0 for 1 h.It was concluded that heterologous expression of alginate lyase gene B1SM was achievable in E.coli BL21(DE3),which laid a foundation for preparing and further study of alginate lyase.