重组毕赤酵母葡萄糖氧化酶的纯化和性质

Purification and Characterization of Recombinant Glucose Oxidase from Pichia pastoris

目的:探讨重组毕赤酵母葡萄糖氧化酶(glucose oxidase,GOD)的分离纯化过程及其性质。方法:摇瓶发酵得到的粗酶液经Q-Sepharose Fast Flow层析纯化,用聚丙烯酰胺凝胶电泳(PAGE)和变性聚丙烯酰胺凝胶电泳(SDS-PAGE)确定其分子质量,用紫外和荧光分光光度计研究其光谱特征。结果:获得GOD的纯品,酶的纯化倍数为1.35倍,酶活回收率为92.7%,天然分子质量为150kD,亚基分子质量为75kD;酶活力在pH5.0～8.0和55℃以下稳定,最适pH值为6.0,最适温度为40℃;以葡萄糖为底物的酶学动力学常数Km值为21.06mmol/L;Hg2+、Ag+、Cu2+、Fe2+对其有强烈的抑制作用;该酶在275nm波长处有最大紫外光吸收,在344nm波长处有最大荧光吸收峰;液体状态下该酶在4℃条件下放置6个月活性没有损失,常温下可保存3～5d。结论:重组毕赤酵母葡萄糖氧化酶纯化成本低,收率高,有利于大规模纯化,得到的纯品稳定性好,具有较高的利用价值。

Purpose： To explore the purification and properties of glucose oxidase（GOD） from recombinant Pichia pastoris.Methods： The crude glucose oxidase was purified by Q-Sepharose Fast Flow chromatography.Polyacrylamide gel electrophoresis（PAGE） and sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） were used to determine its molecular weight.Ultraviolet and fluorescence absorption spectroscopy were used to observe its spectral characteristics.Results： Purified GOD with a purification factor of 1.35 and a recovery of 92.7% was obtained.This enzyme exhibited a dimeric form with a molecular mass of 150 kD.The GOD showed the highest activity at pH 6.0 and 40 ℃ and was stable in a broad pH range of 5.0-8.0 and at 55 ℃ or below.The Km of the GOD enzyme was 21.06 mmol/L with glucose as the substrate.The enzyme was inhibited intensively by Hg2＋,Fe2＋,Ag＋ and Cu2＋ and showed a maximum ultraviolet and fluorescence absorption peak at 275 nm and 344 nm,respectively.The lyophilized enzyme was stable at 4 ℃ over a period of 6 months and could be stored for 3-5 d at normal temperature.Conclusion： The recombinant GOD has simple purification procedure,high recovery,good stability and promising application potential.