GPC Analysis

The obtained enzyme lignin was first acetylated (10 mg) in 0.5 mL of pyridine:acetate anhydride (2:1, v/v; 10 mL) at room temperature for 12 h. Acetylated maize lignin was obtained after coevaporation of the solvent with ethanol under reduced pressure at 45°C until pyridine and acetic anhydride were completely removed. The acetylated product was dissolved in N,N-dimethylformamide (0.1 m lithium bromide) for GPC acquisition. Analytical GPC was performed on a Shimadzu LC20 device with a photodiode array detector (SPD-M20A). Lithium bromide (0.1 m) in N,N-dimethylformamide was used as an eluent at 40°C with a flow rate of 0.3 mL min⁻¹. Polystyrene (low molecular) Standard ReadyCal Set M(p) 250−70,000 (P/N 76552; Fluka) was used to calibrate and calculate the M_r distribution of the lignin samples. Separation was performed using a series of three columns, first a TSKgel guard column with α-stationary phase (6 mm × 4 cm, 13 μm; TOSOH Bioscience), then a TSKgel Alpha-2500 column (7.8 mm × 30 cm, 13 μm; TOSOH Bioscience), followed by a TSKgel Alpha-M column (7.8 mm × 30 cm, 13 μm; TOSOH Bioscience).