| 主权项 |
1. A method for producing laccase by liquid fermentation of Lentinus edodes, comprises the following steps of:
(1) inoculating Lentinus edodes into a PD liquid fermentation medium to perform activation culture and obtain a seed liquid; (2) inoculating the seed liquid into a liquid fermentation medium for liquid fermentation culture at temperature between 25 and 30° C. for 1 to 5 days, then adding an expansin solution at a concentration of 1.0˜3.0 mg/mL for further culture for 4 to 8 days, then separating the supernatant to obtain a ferment solution; (3) extracting laccase from the ferment solution obtained in step (2). wherein the liquid fermentation medium in step (2) has a pH between 5.0 and 7.0, and contains corn powder, bran, sucrose, white sugar, peptone, MgSO4.7H2O, KH2PO4; wherein the expansin solution in step (2) is prepared by sterilizing a broad bean or cucumber seed with mercury chloride (HgCl2) for 4 to 6 minutes, washing with running water for 5 to 7 hours, culturing in the darkroom for 4 to 6 days at the temperature between 15 and 28° C.; precooling seedling hypocotyl apices for 0.5 hours at −20 ° C., adding a homogenate buffer solution that is pre-cooled to 4° C., filtering with a nylon net having an aperture of 70 μm after homogenate, washing the filter residue with a homogenate buffer solution, adding the filter residue in the homogenate buffer solution, settling for 1 to 3 hours to obtain a settled solution, adding an extracting solution in the settled solution for 44 to 50 hours at 4° C., adding 0.3 to 0.5 g/mL of ammonium sulfate ((NH4)2SO4) in the filtrate while stirring to prevent a partial supersaturation of the (NH4)2SO4, settling for 25 to 30 hours, centrifuging for 5 to 10 minutes at 4° C., dissolving the sediment with an acid buffer solution, dialyzing in a dialysis bag with a molecular weight of 3000 Da at 4° C., centrifuging the dialyzate at 20000 g for 10 minutes, and taking the supernatant to obtain the expansin solution. |
