食品科学 ›› 2019, Vol. 40 ›› Issue (14): 153-159.doi: 10.7506/spkx1002-6630-20180721-267

• 生物工程 • 上一篇    下一篇

绿色木霉耐高温葡萄糖氧化酶的特性分析

高兆建,王先凤,尚业成,许 祥,李宝林,张抗震,焦 魏   

  1. 1.徐州工程学院食品(生物)工程学院,江苏 徐州 221018;2.江苏智荟生物科技有限公司,江苏 徐州 221018;3.江苏太合食品有限公司,江苏 徐州 221018;4.邳州市金大地肥料有限公司,江苏 徐州 221100
  • 出版日期:2019-07-25 发布日期:2019-07-23
  • 基金资助:
    江苏省重点研发计划项目(BE2016316);江苏省苏北科技计划项目(BC2013417;BN2015021);徐州市科技计划项目(XF13C027);2014年度国家星火计划项目(2014GA690014)

Properties of a Heat-Resistant Glucose Oxidase from Trichoderma viride

GAO Zhaojian, WANG Xianfeng, SHANG Yecheng, XU Xiang, LI Baolin, ZHANG Kangzhen, JIAO Wei   

  1. 1. College of Food (Biological) Engineering, Xuzhou Institute of Technology, Xuzhou 221018, China; 2. Jiangsu Zhihui Biological Technology Co. Ltd., Xuzhou 221018, China; 3. Jiangsu Taihe Food Co. Ltd., Xuzhou 221018, China; 4. Pizhou City Golden Earth Fertilizer Co. Ltd., Xuzhou 221100, China
  • Online:2019-07-25 Published:2019-07-23

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(T. viride glucose oxidase,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 Fast Flow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。由十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测得TvGOD表观分子质量93 kDa,达到电泳纯。经纯化,TvGOD比活力为426.67 U/mg,纯化倍数为14.36,活力回收率19.15%。TvGOD最适pH 6.0,最适反应温度60 ℃。70 ℃以下、pH 4~7之间,TvGOD稳定性好。所测金属离子中,Na+和K+对酶活力无影响,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+几乎完全抑制酶活力;丝氨酸蛋白酶抑制剂苯甲基磺酰氟和金属螯合剂乙二胺四乙酸二钠对TvGOD抑制作用较为强烈;TvGOD对表面活性剂十二烷基硫酸钠、十六烷基三甲基溴化铵、Triton X-100和吐温80表现出强稳定性。以葡萄糖为底物,酶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。

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

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