Description of the methods used to collect and generate the data - The oxidase activity of CBM3-8BxHMFO was evaluated using two complementary assays: (i) Oxygen consumption assay, performed with a Pyro Science robust oxygen probe coupled to a FireSting-PRO oxygen meter. Reactions were carried out in 2.5 mL of 50 mM Tris-HCl buffer (pH 8.0) at 30 °C and 300 rpm with 60 mM HMF as substrate. (ii) Vanillyl alcohol spectrophotometric assay, conducted in 96-well plates at 25 °C using a SPECTROstar® Nano plate reader (BMG LABTECH, Germany). Each well contained 10 µL of enzyme sample and 190 µL of 3 mM vanillyl alcohol in 50 mM Tris-HCl buffer (pH 8.0), and absorbance was recorded at 340 nm using an extinction coefficient of 23.1 mM⁻¹ cm⁻¹. *Protein concentration in soluble and immobilized fractions was determined at 595 nm using the Bradford assay (Coomassie® Protein Assay Kit) and a BSA calibration curve (0–2 mg mL⁻¹). *The melting temperature (Tm) of soluble and immobilized CBM3-8BxHMFO was determined using SYPRO Orange dye and a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories). Melt curves were recorded from 10 °C to 95 °C in 0.5 °C increments. *The enzymatic reactions, the oxidation of HMF to FDCA was performed at 30 °C and pH 8.0 in 50 mM Tris-HCl buffer with continuous air bubbling (0.6 vvm) and catalase supplementation (97.7 U mL⁻¹). Soluble and immobilized enzyme derivatives were tested at substrate concentrations between 6 mM and 125 mM. *HMF and oxidation products (FDCA, DFF, FFCA) were quantified by HPLC (Agilent 1220 Infinity II, UV detector at 264 nm) using a SUPELCOGEL C-610H column with 5 mM H₂SO₄ as mobile phase (0.5 mL min⁻¹, 30 °C). Information about instruments, calibration and standards - Pyro Science FireSting-PRO oxygen meter, SPECTROstar® Nano plate reader (BMG LABTECH), CFX96 Touch Real-Time PCR System (Bio-Rad Laboratories), Agilent 1220 Infinity II HPLC with SUPELCOGEL C-610H column, Varian Cary 50 Bio UV-Vis Spectrophotometer, Calibration curves prepared using HMF, DFF, FFCA, FDCA and BSA standards. Environmental or experimental conditions - All enzymatic reactions were conducted at 30 °C, pH 8.0, and 500 rpm agitation within a controlled laboratory environment. Bioreactor experiments were performed under continuous air bubbling at 0.6 vvm and monitored pH control.

Dataset related to the enzymatic production of FDCA, including enzyme immobilization, process optimization, and determination of the enzyme Tm. Additionally, the evaluation of the reactions under industrially relevant conditions using crude HMF was included. The data include the results of: CBM3-8BxHMFO immobilization FDCA production using pure and crude HMF FDCA purification Melting temperature analysis of CBM3-8BxHMFO

Bio-Rad CFX96 Touch, null

Microsoft Excel, null