嗜热真菌热稳定性β-葡萄糖苷酶的克隆表达、纯化及酶学性质

Cloning,Expression,Purification and Analysis of Enzymic Properties of Thermostable β-glucosidase from the Thermophilic Fungus

从嗜热子囊菌光孢变种(Thermoascus aurantiacus var.levisporus)中克隆出β-葡萄糖苷酶基因bglII的全长序列,GenBank注册号为EU263992。将该基因插入巴斯德毕赤酵母Pichia pastoris分泌型表达载体pPIC9K中以获得重组质粒,将重组质粒转导入毕赤酵母中,大量筛选后获得高效表达β-葡萄糖苷酶的酵母工程菌株。经甲醇诱导6d,培养基中β-葡萄糖苷酶的活力可达0.23U/mg。该酶的最适反应pH为5.0,最适反应温度为50℃。通过DEAE-Sepharose Fast Flow阴离子层析纯化了该重组蛋白质。SDS-PAGE测得该重组蛋白质分子量约为118kDa。

The β-glucosidase bgIⅡ gene was cloned from thermophilic fungus Thermoascus aurantiacus var. levisporus. The accession number in GenBank is EU263992. The gene was ligated with the Pichia pastoris expression vector pPIC9K, resulting in the recombinant plasmid. The recombinant plasmid was transformed into P. pastoris. Highly efficient β-glucosidases from project strain were achieved through massive screening. The project strain was induced with methanol in 6 d. The β-glucosidase was expressed and secreated into the culture with activity of 0.23 U/mg. The β-glucosidase exhibited optimum catalytic activity at 50 ℃ and pH 5.0. The recombinant β-glucosidase was purified by using DEAE-Sepharose Fast Flow chromatography. A molecular mass of the purified enzyme is 118 kDa determined by SDS-PAGE.