匍枝根霉组成型内切葡聚糖酶基因的筛选和表达

摘要

The constitutive endoglucanase genes (zeg1 and zeg2) from the cDNA library of Rhizopus stolonifer were screened and ex-pressed in E.coli BL21 (DE3).zeg1 and zeg2 have 86%similarity, having endoglucanase gene with similar catalytic activity area by CDD ( conservative sequence database) analysis.The protein belongs to glycoside hydrolase family 5 compared with the PDB ( protein structure database) key catalytic residues in the fifth family.The two proteins were predicted with the 147th glutamic acid (E) and the 199th tryptophan acid (W) for the protein catalytic residues.The activities of zeg1 and zeg2 were peaked at 0.422 IU/mL after 20 h fermentation and 0.509 IU/mL after 24 h fermentation, respectively.Enzymatic properties of both two recombinant strains revealed the optimal temperature and the optimal pH at 50℃and 5.0, respectively.The molecular weights of zeg1 (55 kD) and zeg2 (58 kD) were measured by CMC-SDS-PAGE electrophoresis.%从匍枝根霉cDNA文库中筛选得到组成型内切葡聚糖酶基因（ zeg1和zeg2），经比对zeg1和zeg2相似度为86％，有相似的催化活性区域，经CDD（保守序列数据库）分析，该蛋白归属于糖苷水解酶第5家族，对比PDB（蛋白结构数据库）中第五家族的关键催化残基，预测该两个蛋白的第147位的谷氨酸（ E）及199位的色氨酸（ W）为该蛋白的关键催化残基。重组zeg1发酵至20 h时达到最高酶活为0.422 IU/mL；重组zeg2发酵至24 h达到最高酶活为0.509 IU/mL。酶学性质研究表明重组zeg1和zeg2的最适温度均为50℃，最适pH值均为5.0。通过CMC-SDS-PAGE电泳，复性后染色，测得重组zeg1和zeg2的分子量分别约为55 kD和58 kD。