Purification And Characterizations Of β-mannanase From Bacillus Subtilis WY45

B-manannase is an enzyme capable of hydrolyzing B-l,4-D-manno-pyanosyl linkages of glucomannans, yielding manno-oligosaccharides that have the marked effect on the proliferation of Bifidobacteria. Bacillus subtilis WY45 was isolated from soil samples, which can produce high activity of extraceellular B-manannase. The main research contents of this thesis include the following: optimizing the culture conditions of the fermentation, purifying the B-manannase produced by Bacillus subtilis WY45 and finding out the main characterizations of the purified enzyme. The results showed that:1. Bacillus subtilis WY45 grew best when cultured at 50 ℃ with the medium composed of 4% konjac powder as carbon source, 1.33% soya peptone as nitrogen source, and adjusting pH to 5.5. After 96 hours culturing, the enzyme could show the highest activity as 2800 u/ml and it is the highest value as far as now.2. The B-manannase was purified to homogeneity by ammonium sulfate(60-90%) precipitation, Sephacryl S-100 gel filtration chromatography and the Q-sepharose ion exchange chromatogramphy. In SDS-PAGE a single protein strip can be seen. The molecular weight of the purified enzyme was 44.47kDa.3. The enzyme was sensitive to the temperature and pH, and the maximum enzyme activity existed at 65℃, and the optimized pH was 6.0. It was stable at pH 5.5-6.5, below 50℃. Most mental ions could enhance the enzyme activity, especially Co²⁺ and Ni⁺.