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Engineering a robust glucose oxidase: Enhanced thermostability, acid tolerance and catalytic activity drive
Ying-Zhi Peng1, Xin-Cheng Jiang2, Jing Li3
1Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212100, China; Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu 212100, China.
Abstract:
Gluconic acid (GA) bioproduction with glucose oxidase (GOD) is an eco-friendly catalytic process, yet its practical efficiency is hampered by poor enzymatic stability toward heat and acidic conditions. In this study, a GOD from Aspergillus eucalypticola (AeGOD) was heterologously expressed in Pichia pastoris and engineered via computer-aided rational design. The obtained combinatorial mutant M3 (A12S/L147K/A179V) exhibited significantly enhanced stability, with a 10 °C higher T50 and a 2.6-fold longer half-life at 50 °C compared to AeGOD. Its catalytic efficiency at pH 2.5-3.0 was 1.2- to 3.1-fold higher, and its specific activity increased 1.9-fold. Structural analysis revealed that newly formed hydrogen bonds, salt bridges, and remote charge stabilization introduced by L147K contributed to the improved properties. Under a simulated process condition at 50 °C, M3 produced 161.7 g/L GA within 24 h, which was 70.9% higher than that of AeGOD. This work provides an efficient strategy for application-targeted GOD modification for GA bioproduction.
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