pH and heat-dependent behaviour of glucose oxidase down to single molecule level by combined fluorescence spectroscopy and molecular modelling
pH and heat-dependent behaviour of glucose oxidase down to single molecule level by combined fluorescence spectroscopy and molecular modelling
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DOI：
10.1002/jsfa.7296
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发表时间：
2016-04-01
影响因子：
4.1
通讯作者：
Aprodu, Iuliana
中科院分区：
文献类型：
--
作者：
Dumitrascu, Loredana;Stanciuc, Nicoleta;Aprodu, Iuliana
背景：在食品工业中，葡萄糖氧化酶(GOX)被用来延长食品原料的保质期。利用荧光光谱和分子动力学模拟方法定量研究了黑曲霉葡萄糖氧化酶在pH和热诱导下的构象变化。结果：相图显示了一个全有或无的转变过程，表明pH和温度对GOx的构象状态有很大影响。当蛋白质遇到较低的pH(pH 4.0)时，Trp，Tyr的最大波长发生了移动，这表明生色团分子周围的极性发生了显著的变化。KI猝灭实验表明，Trp和黄素腺嘌呤二核苷酸在加热或改变pH值时的猝灭常数较大，这主要与蛋白质基质上的构象变化有关。结论：GOX蛋白的构象和结构与所用pH和热处理有关。分子动力学模拟表明，在60℃以上的温度下，底物结合区发生了显著的变化，这可能会影响酶的活性。此外，在高温下，负责氧活化的His(516)正电残基所在的小口袋的一个重要变化似乎是明显的。(C)2015年化学工业学会
BACKGROUND: In the food industry, glucose oxidase (GOX) is used to improve the shelf life of food materials. The pH- and heat-induced conformational changes of glucose oxidase from Aspergillus niger were quantified by means of fluorescence spectroscopy and molecular dynamics simulations.RESULTS: The phase diagram showed an all-or-none transition process, indicating that pH and temperature largely influence the conformational state of GOX. Shifts in maximum wavelength of Trp, Tyr were registered as the protein encounters a lower pH (pH 4.0), suggesting significant changes of the polarity around the chromophore molecule. Quenching experiments using KI showed higher quenching constants of Trp and flavin adenine dinucleotide upon heating or by changing pH value, and were mainly correlated with the conformational changes upon protein matrix. Finally, valuable insights into the thermal behaviour of GOX were obtained from molecular modelling results.CONCLUSIONS: The conformation and structure of GOX protein is dependnt upon the pH and heat treatment applied. Molecular dynamics simulation indicated significant changes in the substrate binding region at temperatures over 60 degrees C that might affect enzyme activity. Moreover, an important alteration of the small pocket hosting the positively charged His(516) residue responsible for oxygen activation appears evident at high temperatures. (C) 2015 Society of Chemical Industry