蜡状芽孢杆菌ZJB-07112酰胺酶的分离纯化及其酶学性质

用一株蜡状芽孢杆菌新菌株ZJB-07112 (Bacillus cereus ZJB-07112)发酵生产酰胺酶，经超声波细胞破碎、High Q阴离子色谱、Phenyl-Sepharose疏水色谱等步骤获得了凝胶电泳均一的酰胺酶。用12.5% SDS-PAGE测得该酶的分子量约为60.6 kD。其N端氨基酸序列为ATIRPDDKAI。该酶水解反应的最适pH和最适温度分别为7.5和35 ℃。在pH 7.5条件下，该酶在50 ℃以上容易失活，60 ℃保温30 min后，仅保留10.8%的酶活。除了Hg⁺ 、Ag⁺等重金属离子和尿素外，其他金属离子和EDTA对该酶的活性影响不大。以丙烯酰胺为底物时，该酶的K_m和V_max值分别为2.64 mmol·L^-1和0.6 μmol·min^-1·ml^-1。

Purification and characterization of amidase from Bacillus cereus ZJB-07112

An amidase from a strain Bacillus cereus ZJB-07112 was purified to homogeneity by using sonication, anion-exchange chromatography, phenyl-sepharose chromatography.The molecular weight of amidase was estimated to be 60.6×10³ by 12.5% SDS-PAGE. Its N-terminal sequence was ATIRPDDKAI. The optimum pH and temperature of the amidase for acrylamide were pH 7.5 and 35℃, respectively. The enzyme was unstable at a temperature over 50℃ and only 10.8% activity was retained after exposure to 60℃ for 30 min. Most of metal ions and EDTA had no significant effect on the enzyme activity, whereas Hg⁺，Ag⁺ and urea caused obvious inhibition.The K _m and V _max values of the amidase for acrylamide were 2.64 mmol·L^-1 and 0.6 μmol·min^-1·ml^-1, respectively.