Purification And Characteristics Of Cellulases From Eisenia Foetida

Cellulose is the most abundant renewable resource in nature. Cellulose could be degraded into simple sugar under the catalysis of cellulases, then could be fermented into clean energy sources, such as ethanol. Earthworms take plants for food in the soil, that relate to the effects of cellulases in their bodies. Study on properties of earthworms cellulases was signality for environmental protection and soil toxicology.The crude enzyme solution from Eisenia foetida was extracted, and the activities of three kinds of cellulases were analyzed by DNS method. Three endocellulase components (also called Cx enzymes) were identified by Congo red staining, named as Cx enzymeⅠ, Cx enzymeⅡand Cx enzymeⅢrespectively.DEAE-Cellulose-52 ion exchange chromatography was used to separate Cx enzymes, it was found that the effluent of 0.20 mol/L NaCl contained the active components. The effluent of 0.20 mol/L NaCl was loaded on Sephadex G-100 column to separate Cx enzymes, the three Cx enzymes couldn’t be separated, showing their similar molecular weight. The three Cx enzymes were obtained by preparative electrophoresis, their molecular weights were 33 400 Da, 28 800 Da and 28 800 Da respectively, which determined by SDS-PAGE. The optimal temperature of endocellulases in the crude enzyme solution measured by DNS method was 40℃,and the optimal pH was pH 7.0.Enzymatic hydrolysate was analyzed by thin layer chromatography (TLC), it showed that the absorbent cotton and CMC could be hydrolyzed to cello-oligosaccharide and glucose by crude enzyme solution, and the two kinds of substrates also could be hydrolyzed to cello-oligosaccharide by the three Cx enzymes, indicating the Cx enzymes had both endocellulase and exocellulase (C1 enzyme) activity. Oneβ- glucosidase component was preliminarily purified by preparative electrophoresis with molecular weight of 60 330 Da.The earthworms were induced by cellulose type food, it was found that the earthworms didn’t grow very well, but the activities of three kinds of cellulases were obviously enhanced.