关键词: 葡萄糖氧化酶, 绿色木霉, 纯化, 性质

Abstract: Glucose oxidase from Trichoderma viride WX24 (TvGOD) was isolated, purified and characterized to provide a theoretical basis for its application. Purified TvGOD was obtained from the cultured broth of T. viride WX24 by using ammonium sulfate precipitation, dialysis, DEAE-Sepharose Fast Flow anion exchange chromatography, Phenyl Sepharose 6 Fast Flow hydrophobic chromatography and Sephadex G-75 molecular sieve gel chromatography and its enzymatic properties were studied. SDS-PAGE revealed one single band with a molecular mass of about 93 kDa, indicating that the enzyme was purified to electrophoretic homogeneity. The specific activity of TvGOD was 426.67 U/mg; a 14.36-fold purification was achieved with 19.15% yield. The optimum temperature and pH were 60 ℃ and pH 6.0, respectively. The enzyme was stable at temperatures below 70 ℃ and at pH ranging from 4.0 to 7.0. Among the metal ions studied, Mg2+ and Ca2+ had activation effects on TvGOD activity and Na+ and K+ had no effect, while Fe2+, Cu2+ and Zn2+ showed somewhat inhibitory effects and the heavy metals Hg2+ and Pb2+ almost completely inhibited the enzyme. The enzyme was markedly inhibited by PMSF and EDTA. TvGOD was stable in the presence of detergents (Triton X-100, SDS, CTAB and Tween -80). When glucose was used as a substrate, its Km and Vmax values were 11.62 μmol/L and 8.244 mmol/(L·min), respectively. The excellent physicochemical properties including high thermostablity and acidic stability and excellent stability to detergents and high affinity for glucose make TvGOD promising in the fields of flour processing, food and beverage, the feed industry, biosensors and biotechnological industries that require high GOD activity under high temperature and acid conditions.

Key words: glucose oxidase, Trichoderma viride, purification, enzymatic properties