Purification and Properties of Bacillus subtilis SA-22 Endo-1,4-β-D-mannanase

mannanase (EC 3 2 1 78) from Bacillus subtilis SA 22 was purified successively by ammonium sulfate precipitation, hydroxyapatite chromatography, Sephadex G 75 gel filtration and DEAE 52 anion exchange chromatography. Through these steps, the enzyme was concentrated 30 75 fold with a recovery rate of 23 43%, with a specific activity of 34780 56 u/mg. Molecular weight of the enzyme was determined to be 38kD by SDS PAGE and 34kD by gel filtration. The results revealed that the optimal pH value for the enzyme was 6 5 and the optimal temperature was 70℃. The enzyme is stable between pH 5 to 10. The enzyme remained most of its activity after a treatment of 4h at 50℃, but lost 25% of activity at 60℃ for 4h, lost 50% of activity at 70℃ for 3h. The enzyme activity was strongly inhibited by Hg 2+ . The Michaelis constants ( K m) were measured as 11 30 mg/mL for locust bean gum and 4 76 mg/mL for konjac powder, while V max for these two polysaccharides were 188 68 (μmol·mL -1 ·min -1 ) and 114 94 (μmol·mL -1 ·min -1 ) , respectively.