【摘要】 碱性蛋白酶在食品、医药、酿造、丝绸、皮革等行业中发挥着重要作用。制备了碱性蛋白酶交联体,并对其催化性能进行研究。在最佳制备条件下（90%叔丁醇作为沉淀剂,沉淀时间为15min,交联剂浓度为33 mmol/L,交联时间为6 h）,交联酶的酶活回收率为22. 6%。与游离酶相比,交联酶的最适pH值向碱性方向变化,由7. 5变为8. 0,最适温度由60℃变成65℃。酶动力学研究表明,交联酶对酪蛋白的催化水解能力(4. 3 min^-1)比游离酶(3. 7 min^-1)更高。尽管交联酶最大反应速度V_max（9. 8 mg/（m L·min））低于游离酶（13. 3 mg/（m L·min））,但交联酶对底物的亲和力Km（2. 3 mg/m L）比游离酶（3. 6 mg/m L）有所增加,而且其热稳定性和酸碱稳定性都得到一定程度的提高。另外,在磷酸盐缓冲液中重复使用5和8批次后,交联酶还能保持82. 5%和56. 5%的酶活性。更多还原

【Abstract】 Cross-linked enzyme aggregates（ CLEAs） of alkaline protease play a crucial role in food,medicine, brewing, silk, leather, and other industries CLEAs were successfully prepared and characterized in this paper. Under the optimum preparation conditions（ 90% tert-butanol as the precipitant,precipitated time of 15 min,glutaraldehyde concentration of 33 mmol/L and cross-linking time of 6 h）,the activity recovery of CLEAs recorded 22. 6%. In addition,the CLEAs displayed a shift in optimal pH towards the alkaline side from 7. 5 to 8. 0,and their optimal temperature was also improved to a certain extent compared to free enzyme from 60 ℃ to 65 ℃. The enzymatic kinetics studies indicated that the CLEAs( 4. 3 min^-1) were more efficient than the free enzyme( 3. 7 min^-1) in catalyzing casein hydrolysis. Although the V_max of CLEAs（ 9. 8 mg/（ m L·min）） was lower than that of free enzyme（ 13. 3 mg/（ m L·min））,the substrate affinity of CLEAs（ 2. 3 mg/m L） increased compared with the free enzyme（ 3. 6 mg/m L）. The CLEAs also enhanced the thermal and pH stability of alkaline protease. Moreover,after being used repeatedly for 5 and 8 batches in phosphate buffer,CLEAs retained 82. 5% and 56. 5%of their initial activity.更多还原