| ?-galactosidase is a kind of hydrolytic enzyme which can cleaves ?-1-6-linked D-galactosyl residues from a wide range of substrates such as raffinose, stachyose and other derivatives. So?-galactosidase is used to degrade raffinose-family oligosaccharides in food and feed industry. This enzyme widely distributed in plants, animals and microorganisms. In our research, we detected?-galactosidase from a variety of edible mushroom and choose the fruting body and fermentation broth of mushroom with high enzyme activity as research object. We purified this enzyme and studied its enzymology properties and degradation effect of oligosaccharide. We also studied the feasibility of mushroom residue with ?-galactosidase activity as feed. The main research results as follows:First of all, we purified an ?-galactosidase from the fruiting body of Termitomyces albuminosus with a 146-fold of purification and a specific activity of 3135 U/mg. This enzyme was a monomeric protein with molecular mass of 72 kDa. The optimum pH and temperature was 5.0 and 60? which was also stable in acidic condition and exhibited good thermostability retaining 90% of the original activity at 60? for 2 h. The heavy metal ion Cd2+ and Hg2+ strongly inhibited the enzyme activity. The activity of ?-galactosidase was also inhibited by NBS signifying the pivotal role played by tryptophan in the catalytic activity of the enzyme. The enzyme completely hydrolyzed oligosaccharides mixture (10 mg/mL) to galactose at 50? within 6 hour detected by TLC and DNS method. These properties make?-galactosidase from T. albuminosus can be used in food and feed industry for releasing raffinose substrate, be helpful for human and animal health.T. albuminosus can not be cultivated and its mycelium is also difficult to cultivate, so we selected Trametes versicolor as research material to study ?-galactosidase of fermentation broth. ?-galactosidase activity was induced in the fermentation broth when added soybean cake powder to culture medium. We optimized the fermentation condition for producing the enzyme from T. versicolor through orthogonal experiment and determined the best fermentation medium components was soybean cake powder 2%,galactose 1%, MgSO4 0.1%, KH2PO4 0.05%. Triangular flask (250 mL) contained 50 mL culture medium for shaking culture about 7 days at 26?/180 rpm. Then we purified this ?-galactosidase from the fermentation broth of T. versicolor. The molecular mass was 70 kDa according to FPLC and SDS-PAGE results. The optimum pH was 3.0 and acid-tolerance,the optimum temperature was 60?and the activity was very stable lower than 50?. The enzyme activity was strongly inhibited by Pb2+and Fe3+, the higher concentration, more obvious inhibition. Cd2+, Cu2+ and Hg2+ make the enzyme activity decreased about 20%-50% while other metal ion had no effect on the activity of ?-galactosidase.The fermentation broth of T. versicolor had excellent degradation effect on guar gum, because there was a lot of degradation enzyme such as ?-galactosidase and mannase synergism in fermented liquid.With corn cob as carbon source, soybean cake power as nitrogen source which was also as inducer,we cultivated two mushrooms T. versicolor and Pleurotus ostreatus to study ?-galactosidase changes in mushroom residue and fruiting bodies. We also evaluated its security as forage. The enzyme activity in fruiting body and mushroom residue of P. ostreatus with soybean cake power was higher than that of P.ostreatus without soybean cake power. The a-galactosidase activity can reach 303 U/g in mushroom bran of T. versicolor. We did not detect aflatoxin B1 in mushroom bran and the content of heavy metal mercury, cadmium, lead, arsenic and fluorine also meet Feed hygiene standards.The above experimental results show that the a-galactosidase from mushroom fruiting body and fermented liquid was acid-tolerance, heat-resisting and had good degradation effect on galactose nucleoside substance. The enzyme activity was very high in mushroom residue adding soybean cake power. Pleurotus ostreatus was a large number of cultivation mushrooms and the production of mushroom residue was very high. If we use mushroom residue with a-galactosidase activity as feed or feed additive which can improve the utilization rate of feed, also can avoid environment pollution because of a large number of discarded mushroom residue. |
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