Glucose oxidase (GOx) catalyzes the oxidation of D-glucose to D-glucono-1,5-lactone and has a wide range of applications in various industries. However, the strict substrate specificity of GOx hampers its application in the conversion of other abundant sugars such as D-xylose. In this study, the substrate preference of GOx from Aspergillus niger (AnGOx) was engineered using a semi-rational design approach. The mutant T110V/F414L exhibited a 5.7-fold increase in D-xylose oxidation activity compared to that of the wild-type enzyme, which was attributed to its enhanced affinity for the substrate. Molecular dynamics simulations indicated that the T110V and F414L mutations may mitigate the non-productive binding of D-xylose at the entrance of the substrate-binding pocket, and therefore, are beneficial for providing access of its C1 hydroxyl group to the catalytic residues. Moreover, the mutant simultaneously oxidized D-xylose and D-glucose in the corncob hydrolysate to the corresponding aldonic acids when coupled with catalase. These findings provide new insights into substrate recognition by GOx and offer a new method for the utilization of D-xylose from lignocellulosic feedstocks.

中文翻译：

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设计葡萄糖氧化酶的底物偏好以进行木糖的酶氧化

葡萄糖氧化酶（GOx）催化D-葡萄糖氧化为D-葡萄糖酸-1,5-内酯，在各行业有着广泛的应用。然而，GOx 严格的底物特异性阻碍了其在其他丰富糖（如D-木糖）转化中的应用。在本研究中，使用半理性设计方法对黑曲霉(AnGOx)的 GOx 的底物偏好进行了改造。与野生型酶相比，突变体T110V/F414L的D-木糖氧化活性增加了5.7倍，这归因于其对底物的亲和力增强。分子动力学模拟表明，T110V 和 F414L 突变可能会减轻D-木糖在底物结合袋入口处的非生产性结合，因此有利于提供其 C1 羟基与催化残基的接触。此外，当与过氧化氢酶偶联时，突变体同时将玉米芯水解产物中的D-木糖和D-葡萄糖氧化成相应的醛糖酸。这些发现为 GOx 的底物识别提供了新的见解，并提供了从木质纤维素原料中利用D-木糖的新方法。