绿色木霉耐高温新型葡萄糖氧化酶的特性分析

摘要/Abstract

摘要： 从绿色木霉(Trichoderma viride)WX24菌株发酵中分离、纯化葡萄糖氧化酶（TvGOD），研究其酶学特性，为葡萄糖氧化酶的应用提供理论基础。将绿色木霉胞外产酶发酵液经硫酸铵沉淀、透析、DEAE-Sepharose F F阴离子交换层析、Phenyl Sepharose 6 Fast Flow疏水层析和Sephadex G-75分子筛凝胶过滤层析纯化，并研究酶学性质。由SDS-PAGE法测得TvGOD表观分子量93 kDa，达到电泳纯。经纯化，TvGOD的比活力426.67 U/mg，纯化倍数为14.36，活力回收率19.15%。TvGOD最适pH6.0，最适反应温度60 ℃。70 ℃以下，pH7.0～7.0之间，TvGOD稳定性好。所测金属离子中，Na+和K+对酶活无影响，Fe2+、Cu2+和Zn2+有轻微抑制作用，Mg2+和Ca2+对TvGOD有显著激活作用，而重金属离子Hg2+和Pb2+几乎完全抑制酶活性。丝氨酸蛋白酶抑制剂PSFM和金属螯合剂EDTA对TvGOD抑制作用较为强烈。TvGOD对表面活性剂SDS、CTAB、Triton X-100和吐温80表现出强稳定性。以葡萄糖为底物，酶的Km和Vmax分别为11.62 μmol/L和8.244 mmol/(L?min)。基于TvGOD在高温、酸性下的高活性与高稳定性以及对表面活性剂的高耐受性、对底物葡萄糖高亲和性等优良特性，TvGOD很有希望在面粉加工、食品饮料、饲料及生物传感器领域以及需要在高温、高酸环境下使用的生物技术工业中应用。本研究是国内外首次从绿色木霉中分离葡萄糖氧化酶并对酶学性质研究。

关键词: 葡萄糖氧化酶, 绿色木霉（Trichoderma viride)）, 纯化, 性质

Abstract: The glucose oxidase (TvGOD) from Trichoderma viride WX24 was isolated, purified and characterized to provide theoretical basis for enzyme application. The TvGOD was got from the Trichoderma viride WX24 culture by using ammonium sulfate precipitation, dialysis, DEAE-Sepharose FF anion exchange chromatography, Phenyl Sepharose 6 Fast Flow hydrophobic chromatography and Sephadex G-75 molecular sieve gel chromatography and the enzymatic properties were studied． SDS-PAGE revealed one single band corresponding to a molecular weight of about 93 kDa and also indicated the enzyme was purified to electrophoretic homogeneity. The specific activity of the TvGOD was 426.67U/mg; purification folds are 14.36 and the yields is 19.15%. The optimum temperature and optimum pH are 60 ℃ and pH6.0, respectively. The enzyme is stable below 70 ℃ and ranging from pH4.0 to pH7.0. Among the metal ions studied, Mg2+ and Ca2+ had activation effects on TvGOD activity, Na+ and K+ did not affect, Fe2+, Cu2+ and Zn2+ showed somewhat inhibitory effects, while heavy metals like Hg2+ and Pb2+ strongly 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, the Km and Vmax values were 11.62 μmol/L and 8.244 mmol/(L?min), respectively. The excellent physical and chemical properties including high thermostable and acidic activity stability and excellent stability with detergents and high affinity for glucose make TvGOD suitable in the fields of processing, food and beverage, feed industry, biosensor and biotechnological industries that require high activity at high temperature and acid conditions. This is the first report on the study of purification of TvGOD from Trichoderma viride and properties.

Key words: Glucose oxidase, Trichoderma viride, Purification, Properties

中图分类号:

Q939.97

高兆建王先凤尚业成许祥张铁柱张抗震焦魏. 绿色木霉耐高温新型葡萄糖氧化酶的特性分析[J]. 食品科学, 0, (): 0-0.

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