摘要
利用硫酸铵沉淀、羟基磷灰石柱层析、SephadexG 75凝胶过滤和DEAE 5 2离子交换柱层析的方法 ,将枯草芽孢杆菌SA 2 2 β 甘露聚糖酶纯化了 30 75倍 ,同时 ,该酶比活达到 34780 5 6u mg ,收率达到 2 3 4 3%。利用SDS PAGE凝胶电泳和SephadexG 75凝胶过滤的方法测得枯草芽孢杆菌SA 2 2 β 甘露聚糖酶的分子量分别为 38kD和34kD。实验发现该酶的最适pH为 6 5 ,在pH 5～ 10的范围内稳定 ;该酶最适温度为 70℃ ,在 5 0℃保温 4h后其活力不变 ,在 6 0℃保温 4h后剩余酶活为 74 2 % ,70℃的酶活半衰期为 3h。实验还发现Hg2 + 对酶活力有明显抑制作用。该酶对槐豆胶和魔芋胶的Km 和Vmax值分别为 11 30mg mL ,4 76mg mL和 188 6 8(μmol·mL- 1 ·min- 1 ) ,114 94(μmol·mL- 1 ·min- 1 )
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.
出处
《生物工程学报》
CAS
CSCD
北大核心
2003年第3期327-331,共5页
Chinese Journal of Biotechnology
