2.3 Conversion of indican to indoxyl
The indican solution was added to four times the volume of anhydrous ethanol and stood overnight at room temperature for separation of protein, starch and other macromolecules that are insoluble in ethanol. The suspension was centrifuged at 11,510 × g for 5 min. The ethanol was removed and the indican solution was concentrated using a rotary evaporator. Cellulase was separated from Acremonium cellulolyticus which has the activity of β-d-glucosidase was added to concentrate for converse indican to indoxyl at 45 ℃ for 30 min.