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Supplementary materials Method 1: liquid chromatography for Enzyme purification The dialyzed ammonium sulfate fraction (200 ml) was applied to a Fractogel DEAE-650M column (2.5 × 30 cm). The enzyme was then eluted with buffer A (flow rate, 90 ml/h). Ammonium sulfate was added to the pooled active fractions to a final 2-M concentration. The enzyme solution was then applied to a Toyoperal Phenyl-650M column (2.5 × 15 cm) pre-equilibrated with buffer A containing 2 M ammonium sulfate. After washed with 500 ml buffer A containing 2 M ammonium sulfate, the column was eluted with a 1-L linear gradient of 0–2 M ammonium sulfate in buffer A (flow rate, 85 ml/h). Ammonium sulfate was added to the pooled active fractions to a 3.5-M final concentration. The enzyme solution was then applied to a Toyoperal MX-Trp-650M column (1.5 × 15 cm) pre-equilibrated with buffer A containing 3.5 M ammonium sulfate. After the column was washed with 500-ml buffer A containing 3.5 M ammonium sulfate, it was eluted with a 1-L linear gradient of 3.5–1 M ammonium sulfate in buffer A (flow rate, 70 ml/h). To concentrate the pooled active fraction (250 ml), ammonium sulfate was added to a 2-M final concentration, recharged to a small Toyoperal Phenyl-650M column (1.5 × 15 cm) pre-equilibrated with buffer A containing 2 M ammonium sulfate, and eluted directly within 20-ml buffer A. The concentrated enzyme solution was applied to a Fractogel HW-50 column (2.5 × 115 cm). The column was eluted with 10 mM phosphate (pH 7.5) (flow rate, 15 ml/h). The pooled active fractions were further purified using an Ultragel-HA column (2.5 × 15 cm) pre-equilibrated with 10 mM phosphate (pH 7.5). After washing with 10 mM phosphate buffer, the column was eluted with a 500-ml linear gradient of 10–200 mM phosphate buffer (flow rate, 30 ml/h). The active fractions were pooled and stored at −20°C. 1 2 75 KDa 73 KDa sFig. 1 SDS-PAGE analysis of purified laccase. purified laccase is shown in lane 2. The 73-kDa band representing Lane 1: protein marker. SFig. 2 Cyclic voltammetry of borate–fructose complex (upper) and ABTS (lower). (A) (B) sFig. 3 Optimal temperature (A) and thermostability (B) of laccase. Relative activities assay was performed by mixing ABTS, borate buffer, fructose, and suitably diluted enzyme in 1-ml cuvettes as described in 1.4. sFig. 4 pH stability of laccase. The four different pH regions using the following buffers are examined (without substrate or mediator) for relative activities: CH3COOH-CH3COONa (pH 4.5-5.5) (□), KH2PO4-NaHPO4 (pH 5.5-7.0) (○), Tris-HCl (pH 7.0-9.0) (Δ), and Na2CO3-NaHCO3 (pH 9.0-10.0) ( ). Relative activities assay was performed by mixing ABTS, borate buffer, fructose, and suitably diluted enzyme in 1-ml cuvettes as described in 1.4. H H H H H O OH O H + H2O OH O HO B OH H H O + H+ OH OH O H OH O H HO BOH sFigure 5 The structure and acid–base reaction of borate-fructose complex. The complex resulting in a six-membered heterocyclic compound is similar to phenol, not only structure but also size. It is suggest that the borate–fructose complex can be a good substrate, serve as a mediator, and provided a hydrogen buffer on laccase activity. sTable 1 purification of laccase from strain Edenia sp. TS-76 Total volume (ml) Total protein (mg) Total activity (U) Specific activity (U/mg) Crude extract 800 1,268 2,576 2.03 1.0 100 Precipitation 80 659 1,555 2.36 1.2 60 180 359 2,267 6.31 3.1 88 525 311 1,748 5.62 2.8 68 325 218 1,749 8.02 4.0 68 104 55 1,311 23.84 11.7 51 120 12 818 68.2 33.6 32 Fractogel DEAE-650M Toyopearl phenyl-650M Toyoperal MX-Trp-650M Fractogel HW 50 gel filtration Ultragel-HA Purification Yield fold (%)