Stabilized 5-Hydroxymethylfurfural Oxidase and Kinetics-Guided Feeding Mitigate Substrate Inhibition in Resting-Cell 2,5-Furandicarboxylic Acid Production

HMFO enables O2-dependent conversion of 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA) without soluble cofactor regeneration, but prolonged operation is limited by HMF substrate inhibition, pH drift, and enzyme deactivation. Here, these limitations were addressed through a combination of kinetic analysis, enzyme stabilization, and process optimization. High HMF and FDCA concentrations reduced apparent HMFO activity; pH control relieved the FDCA-related acidification effect, whereas HMF caused direct substrate inhibition, favoring controlled feeding over high initial loading. 8B×HMFO was therefore stabilized by computation-guided mutagenesis and disulfide engineering. 8B×HMFO-5M increased apparent melting temperature by 17 °C and extended the 30 °C half-life from 29.1 to 98.5 h while retaining similar apparent kinetic behavior. E. coli resting cells expressing 8B×HMFO-5M served as a simplified catalyst format, and CAT–HMFO fusion improved FDCA formation. Kinetic feeding incorporating HMF substrate inhibition, catalyst deactivation, and a whole-cell effectiveness factor converted 231.5 mM cumulative HMF input to 214.8 mM FDCA in a 3 L stirred-tank reactor over 72 h, giving a 92.8% molar yield. An E-factor of 4.47 kg·kg−1 including crude FDCA isolation demonstrated the material efficiency of this HMFO-based route.