Thermostability improvement of the glucose oxidase from Aspergillus niger for efficient gluconic acid production via computational design

Gluconic acid (GA) and its alkali salts are extensively used in the food, feed, beverage, textile, pharmaceutical and construction industries. However, the cost-effective and eco-friendly production of GA remains a challenge. The biocatalytic process involving the conversion of glucose to GA is catalysed by glucose oxidase (GOD), in which the catalytic efficiency is highly dependent on the GOD stability. In this study, we used in silico design to enhance the stability of glucose oxidase from Aspergillus niger. A combination of the best mutations increased the apparent melting temperature by 8.5 °C and significantly enhanced thermostability and thermoactivation. The variant also showed an increased optimal temperature without compromising the catalytic activity at lower temperatures. Moreover, the combined variant showed higher tolerance at pH 6.0 and 7.0, at which the wild-type enzyme rapidly deactivated. For GA production, an approximate 2-fold higher GA production yield was obtained, in which an almost complete conversion of 324 g/L d-glucose to GA was achieved within 18 h. Collectively, this work provides novel and efficient approaches for improving GOD thermostability, and the obtained variant constructed by the computational strategy can be used as an efficient biocatalyst for GA production at industrially viable conditions.