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将黑曲霉葡萄糖氧化酶 (GOD)基因重组进大肠杆菌 酵母穿梭质粒pPIC9,转化甲基营养酵母Pichiapasto risGS115 ,构建出GOD的高产酵母工程菌株。在酵母α Factor及AOX1基因启动子和终止信号的调控下 ,黑曲霉GOD在甲基酵母中大量表达并分泌至胞外 ,经甲醇诱导 3～ 4d ,发酵液中的GOD活力可达 30～ 40u mL。SDS PAGE证实GOD在培养物上清中的含量显著高于其它杂蛋白 ,约占胞外蛋白总量的 6 0 %～ 70 % ,经QSepharoseTM FastFlow离子交换柱一步纯化即达电泳纯。重组酵母GOD比活达 42 6 6 3u mg蛋白 ,是商品黑曲霉GOD的 1 6倍。动力学性质分析表明 ,重组酵母GOD的Km 和kcat分别为 38 2 5mmol L和 34 92 6 6s- 1 ,与商品黑曲霉GOD相比 ,具有更高的催化效率。重组酵母GOD的高活力特性可有效提高葡萄糖传感器的线性检测范围。
The Aspergillus niger glucose oxidase (GOD) gene was recombined into E. coli yeast shuttle plasmid pPIC9 and transformed into methylotrophic yeast Pichia pastoris risGS115 to construct a high-yield yeast engineering strain of GOD. Under the control of yeast α Factor and AOX1 promoter and stop signal, GOD of Aspergillus niger was highly expressed in the yeast and secreted extracellularly. After induced by methanol for 3-4 days, the GOD activity in the fermentation broth reached 30-40u mL. SDS PAGE confirmed that the content of GOD in the culture supernatant was significantly higher than other hybrid proteins, accounting for 60% to 70% of the total extracellular proteins, purified by QSepharoseTM FastFlow ion exchange column in one step. The specific activity of recombinant yeast GOD was 42 6 6 3u mg protein, which was 16 times higher than that of commercial Aspergillus niger GOD. Kinetic analysis showed that the Km and kcat of the recombinant yeast GOD were 38 2 5 mmol L and 34 92 6 6s-1, respectively, which showed higher catalytic efficiency than that of the commercial Aspergillus niger GOD. The high activity of recombinant yeast GOD can effectively improve the linear detection range of glucose sensor.