碱性蛋白酶交联聚集体的制备及其催化性能研究  

Preparation and Characteristics of Cross-Linked Enzyme Aggregates of Alkaline Protease

作　　者：区晓阳 曾英杰 彭飞 倪子富 熊隽 宗敏华[1] 娄文勇[1,2] OU Xiaoyang;ZENG Yingjie;PENG Fei;NI Zifu;XIONG Jun;ZONG Minhua;LOU Wenyong(School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China;Innovation Center of Bioactive Molecule Development and Application, South China Institute of CollaborativeInnovation, Dongguan 221116, China)

机构地区：[1]华南理工大学食品科学与工程学院,广东广州510640 [2]华南协同创新研究院生物活性分子开发与应用创新中心,广东东莞221116

出　　处：《食品科学技术学报》2019年第3期33-40,共8页Journal of Food Science and Technology

基　　金：国家自然科学基金资助项目(21878105; 21676104);国家重点研发计划项目(2018YFC1603400; 2018YCF1602100)

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33mmol/L,交联时间为6h),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(4.3min-1)比游离酶(3.7min-1)更高。尽管交联酶最大反应速度Vmax(9.8mg/(mL·min))低于游离酶(13.3mg/(mL·min)),但交联酶对底物的亲和力Km(2.3mg/mL)比游离酶(3.6mg/mL)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。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/(mL ·min)) was lower than that of free enzyme(13.3 mg/(mL ·min)), the substrate affinity of CLEAs(2.3 mg/mL)increased compared with the free enzyme (3.6 mg/mL). 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.

关 键 词：碱性蛋白酶 无载体固定化 交联酶聚集体 酶学性质 重复使用稳定性 

分 类 号：TS201.3[轻工技术与工程—食品科学] Q814[轻工技术与工程—食品科学与工程]