黑曲霉葡萄糖氧化酶基因的克隆及其在酵母中的高效表达

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Pichia pastoris GS115，构建出GOD的高产酵母工程菌株。在酵母αFactor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60%～70%，经Q SepharoseTMFast Flow离子交换柱一步纯化即达电泳纯。重组酵母GOD比活达426.63u/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K_m和K_cat分别为38.25mmol/L和3492.66s-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。

Cloning and Expression of Aspergillus niger Glucose Oxidase Gene in Methylotrophic Yeast

The DNA fragment encoding A. niger glucose oxidase was amplified by PCR using A. niger genomic DNA as template, and was cloned into vector of pPIC9 for expression in Pichia pastoris. When transformed into methylotrophic yeast Pichia pastoris GS115, The constructed plasmid pPICGOD1 directed the synthesis and secretion of functionally active GOD. After induction in MM medium for 4 days, the GOD activity in the medium reached 30-40 u/mL. SDS-PAGE revealed that recombinant yeast GOD was expressed up to 60%-70% of the total soluble protein, and the secreted GOD could be purified to electrophoretic homogeneity with one purification step using Q Sepharose Fast Flow ion exchange chromatography. The recombinant yeast GOD had very high catalytic activity, showed about 1.6-fold increase of specific activity over the commercial A. niger GOD. Kinetic analysis clearly demonstrated that recombinant yeast GOD showed similar substrate affinity for glucose to A. niger GOD, but the turnover number of the GOD from yeast was determined to be much higher than that of A. niger GOD. In addition, the linear range of glucose electrode made with recombinant yeast GOD was efficiently widened due to the high catalytic activity of yeast GOD.