(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)
(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)
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.