Studies On β-mannanase From Aureobasidium Pullulans G7b

Theβ-1,4-mannan glycosidic bond could be hydrolyzed byβ-mannanase, which could be categorized into one kind of hemicellulase. Theβ-mannanase has derived from Microorganism had many advantages such as higher enzyme activity, lower cost and easier extraction. Moreover, it had a wider range of reaction pH and temperatures and the specifity of its substrate is much stronger, all of which served as some significant characters for microbe derived P-mannanase. Therefore this enzyme have been widely used in food, medication, feed, papermaking, textile and oil industry.In this thesis,17 marine yeast strains that could produceβ-mannanase were isolated from the culture collection of our lab. A much higher enzyme activity could be detected from the strain named G7b, which was isolated from the sea water sample of the South Sea and indentified to be Aureobasidium pullulans. The optimal P-mannanase activity condition was pH 5.0 and 60 centigrade. After an optimization, the most appropriate medium was locust been gum 0.6%(w/v), yeast extract 0.5%(w/v), ammonium sulfate 0.5%(w/v), potassium dihydrogen phosphate 0.2%(w/v) in sea water, the initial pH should be adjusted to 6.0. After shaking incubation at 28 centigrade and 180 rpm for 48 hour, the P-mannanase activity of the supernatant could reach 10.5U/ml. After dealing substrate with gross P-mannanase, the product was mannan and little manno-oligosaccharides.Thisβ-mannanase was purified and its character was further studied. A 66kDa purified enzyme was obtained using SephadexTM G-75 gel filtration chromatography and DEAE Sepharose Fast Flow anion-exchange chromatography. The optimal function pH of purified P-mannanase is 5.0, and it would stay stable between pH 5.0-7.0.The best reaction temperature is 60 centigrade and is more stable below 50 centigrade. Mg2+ and Zn2+ could stimulate this enzyme whereas the Hg2+, Mn2+ and Cu2+ could suppress it. All the protein inhibitors could somehow suppress the purified β-mannanase, of which SDS was the most intense one. The Km and Vmax for the substrate of purified enzyme was 2945.1μg/ml和27.9μmol/(min* ml), respectively. At last, the result of thin-layer chromatography has illustrated that the main product of locust been gum with purifiedβ-mannanase was mannan and little manno-oligosaccharides, which ensured that this enzyme belonged to endo-β-1,4-mannanase family and the authenticity of this experiment can be testified.