Method for producing laccase by means of liquid fermentation of Lentinus edodes

A method for producing laccase by liquid fermentation of Lentinus edodes, comprises the following steps of:

• (1) inoculating Lentinus edodes into a potato dextrose (PD) liquid fermentation medium comprising 200 g peeled potato, 20 g glucose and 1000 ml water to perform activation culture to obtain a seed liquid;
• preparing an expansin solution by sterilizing a broad bean or cucumber seed with mercury chloride (HgCl 2) 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 5g/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 dialysate at 20000 g for 10 minutes, and taking the supernatant to obtain the expansin solution;
• (2) inoculating the seed liquid into a liquid fermentation medium for liquid fermentation culture at a temperature between 25 and 30° C. for 1 to 5 days, then adding the 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 fermentation solution;
• (3) extracting laccase from the fermentation solution obtained in step (2), comprising:
• adding grinded ammonium sulfate (NH4)2SO4 into the fermentation solution obtained in step (2) at a temperature between 0 and 4° C., settling at 4° C. for 6 to 8 hours, centrifuging with a rotation speed of 10000 rpm for 20 minutes at 4° C., discarding the supernatant, dissolving the precipitate into sodium hydrogen phosphate-citrate buffer;
• dialyzing in a dialysis bag with a molecular weight between 10 and 20 kDa at 4° C., ultrafiltrating and concentrating by a dialysis membrane with a molecular weight between 10 and 20 kDa at 4° C., removing small molecule proteins to obtain Lentinus edodes laccase solution,
• 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.