来源于异形曲霉的葡萄糖氧化酶催化活性和热稳定性改良

Improvement of catalytic activity and thermostability of glucose oxidase from Aspergillus heteromorphus

摘要:

葡萄糖氧化酶(glucose oxidase,GOD,EC 1.1.3.4)是一种耗氧脱氢酶，可催化葡萄糖产生葡萄糖酸和过氧化氢，这种特殊的作用机制使其具有良好应用前景。但天然GOD催化活力低、热稳定性差成为制约其工业化生产应用的主要因素。本研究以目前报道的热稳定性好的葡萄糖氧化酶AtGOD为源序列，通过进化分析，以期获得性能优良的GOD。筛选并成功合成了6个基因进行功能验证，其中异形曲霉(Aspergillus heteromorphus)的葡萄糖氧化酶AhGODB在毕赤酵母中表达后表现出较好的热稳定性和催化活性。其最适温度为40℃，比活力为112.2 U/mg，70℃处理5 min后的剩余酶活为47%。为进一步提高其活性和热稳定性，利用定向进化结合理性设计的方法获得多个突变体。其中，突变体T72R/A153P最适温度由野生型酶的40℃提高到50℃，比活力由112.2 U/mg提高到166.1 U/mg，70℃处理30 min后的剩余酶活由野生型酶的完全失活提高到33%。综上所述，本研究获得的葡萄糖氧化酶突变体在催化活性和热稳定性上有所提升，具有一定的应用潜力。

Abstract:

Glucose oxidase (GOD) is an oxygen-consuming dehydrogenase that can catalyze the production of gluconic acid hydrogen peroxide from glucose, and its specific mechanism of action makes it promising for applications, while the low catalytic activity and poor thermostability have become the main factors limiting the industrial application of this enzyme. In this study, we used the glucose oxidase AtGOD reported with the best thermostability as the source sequence for phylogenetic analysis to obtain the GOD with excellent performance. Six genes were screened and successfully synthesized for functional validation. Among them, the glucose oxidase AhGODB derived from Aspergillus heteromorphus was expressed in Pichia pastoris and showed better thermostability and catalytic activity, with an optimal temperature of 40 ℃, a specific activity of 112.2 U/mg, and a relative activity of 47% after 5 min of treatment at 70 ℃. To improve its activity and thermal stability, we constructed several mutants by directed evolution combined with rational design. Compared with the original enzyme, the mutant T72R/A153P showcased the optimum temperature increasing from 40 to 50 ℃, the specific activity increasing from 112.2 U/mg to 166.1 U/mg, and the relative activity after treatment at 70 ℃ for 30 min increasing from 0% to 33%. In conclusion, the glucose oxidase mutants obtained in this study have improved catalytic activity and thermostability, and have potential for application.