Kalisz H M, Hendle J, Schmid R D
GBF-Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Germany.
Appl Microbiol Biotechnol. 1997 May;47(5):502-7. doi: 10.1007/s002530050963.
Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety. A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE) for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not-been published for glucose oxidase from P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M (NH4)2SO4, which stabilised glucose oxidase 100- to 300-fold at 50 degrees C and pH 7-8, and 2 M KF, which stabilised the enzyme up to 36-fold at 60 degrees C and pH 6. In sodium acetate buffer, changes in pH (4-6) affected the affinity for glucose but had no effect on the Vmax of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in Vmax and a 2-fold decrease in K(m) were observed.
通过离子交换色谱法将来自尼崎青霉的葡萄糖氧化酶纯化至同质，并使用内切糖苷酶H进行去糖基化。基于气相色谱和十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS-PAGE）分析，蛋白质结合的高甘露糖型碳水化合物部分相当于糖基化葡萄糖氧化酶分子量的13%。根据N-乙酰葡糖胺含量确定每个二聚体共有六个N-糖基化位点。酶促去糖基化的酶所含的碳水化合物部分不到原来的5%。去糖基化酶的二聚体分子量测定为130 kDa（凝胶过滤）和133 kDa（天然PAGE），单体分子量为67 kDa（SDS-PAGE）。尼崎青霉葡萄糖氧化酶的N端序列迄今尚未发表，与黑曲霉葡萄糖氧化酶的N端同源性不到50%，去糖基化后其氨基酸组成未发生改变。去糖基化也不影响葡萄糖氧化动力学或pH和温度最佳值。它也没有增加酶对蛋白水解降解的敏感性。然而，去糖基化的葡萄糖氧化酶表现出pH和热稳定性降低。两种酶的热稳定性均显示取决于缓冲液浓度，并可通过某些添加剂增强，特别是1 M硫酸铵，其在50℃和pH 7-8下可将葡萄糖氧化酶稳定100至300倍，以及2 M氟化钾，其在60℃和pH 6下可将酶稳定高达36倍。在醋酸钠缓冲液中，pH值变化（4-6）影响对葡萄糖的亲和力，但对反应的Vmax没有影响。相比之下，在pH 8的TRIS缓冲液中，观察到Vmax降低10倍，Km降低2倍。
