碱性蛋白酶交联聚集体的制备及其催化性能研究
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(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)

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Preparation and Characteristics of Cross-Linked Enzyme Aggregates of Alkaline Protease
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(1.School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China;2.Innovation Center of Bioactive Molecule Development and Application, South China Institute of Collaborative Innovation, Dongguan 221116, China)

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    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。

    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 15min, glutaraldehyde concentration of 33mmol/L and cross-linking time of 6h), 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 (4.3min-1) were more efficient than the free enzyme (3.7min-1) in catalyzing casein hydrolysis. Although the Vmax of CLEAs (9.8mg/(mL·min)) was lower than that of free enzyme(13.3mg/(mL·min)), the substrate affinity of CLEAs(2.3mg/mL)increased compared with the free enzyme (3.6mg/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.

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区晓阳,曾英杰,彭飞,倪子富,熊隽,宗敏华,娄文勇.碱性蛋白酶交联聚集体的制备及其催化性能研究[J].食品科学技术学报,2019,37(3):33-40.

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  • 收稿日期:2019-04-15
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  • 在线发布日期: 2019-06-17
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