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Abstract：
This study aimed was to covalently immobilize β-galactosidase from Aspergillus oryzae and protease from Bacillus licheniformis on amino-functionalized multi-walled carbon nanotubes. In this study, a two-level factorial design was employed to investigate the impact of seven continuous variables (activation pH, glutaraldehyde molarity, activation time (0-8 h), buffer solution pH (8-0), buffer solution molarity, MWCNT-NH 2 -glutaraldehyde quantity, and stabilization time (0-180 h)) on the immobilization efficiency and enzymatic activity of protease and β-galactosidase. Furthermore, the effect of time on the percentage of enzymatic activity was examined during specific intervals (24, 48, 72, 96, and 120 h) of the immobilization process. The analysis of variance results for protease enzymatic activity revealed a notable influence of the seven variables on immobilization efficiency and enzymatic activity. Additionally, the findings indicate that activation time, buffer pH, MWCNT-NH 2 -glutaraldehyde quantity, and stabilization time significantly affect the activity of the protease enzyme. The interplay between buffer pH and stabilization time is also significant. Indeed, both activation time and the quantity of MWCNT-NH 2 -glutaraldehyde exert a reducing effect on enzyme activity. Notably, the influence of MWCNT-NH 2 -glutaraldehyde quantity is more significant (p < 0.05). In terms of beta-galactosidase enzymatic activity, the study results highlight that among the seven variables considered, only the glutaraldehyde molarity, activation time, and the interplay of activation time and the quantity of MWCNT-NH 2 -glutaraldehyde can exert a statistically significant positive impact on the enzyme\'s activity (p < 0.05). The combination of activation time and buffer solution molarity, as well as the interactive effect of buffer pH and MWCNT-NH2-glutaraldehyde, can lead to a significant improvement in the stabilization efficiency of the protease of carbon nanotubes. The analysis of variance results demonstrated that the efficiency of covalently immobilizing β-galactosidase from Aspergillus oryzae on amino-functionalized multi-walled carbon nanotubes is influenced by the molarity of glutaraldehyde, buffer pH, stabilization time, and the interplay of activation time + buffer pH, buffer pH + activation time, activation time + buffer molarity, and glutaraldehyde molarity + MWCNT-NH 2 -glutaraldehyde (p < 0.05). Through the optimization and selection of optimal formulations, the obtained results indicate enzyme activities and stabilization efficiencies of 64.09 % ± 72.63 % and 65.96 % ± 71.77 % for protease and beta-galactosidase, respectively. Moreover, increasing the enzyme stabilization time resulted in a reduction of enzyme activity. Furthermore, an increase in pH, temperature, and the duration of milk storage passing through the enzyme-immobilized carbon nanotubes led to a decrease in enzyme stabilization efficiency, and lactose hydrolysis declined progressively over 8-h. Hence, the covalent immobilization of β-galactosidase from Aspergillus oryzae and protease from Bacillus licheniformis onto amino-functionalized multi-walled carbon nanotubes is anticipated to be achievable for milk applications.
摘要：
本研究旨在将米曲霉β-半乳糖苷酶和地衣芽孢杆菌蛋白酶共价固定在氨基功能化多壁碳纳米管上。在这项研究中,采用两级阶乘设计来研究七个连续变量(活化pH，戊二醛摩尔浓度，激活时间（0-8小时），缓冲溶液pH（8-0），缓冲溶液摩尔浓度,MWCNT-NH2-戊二醛量，和稳定时间（0-180h））对蛋白酶和β-半乳糖苷酶的固定化效率和酶活性。此外，在固定过程的特定间隔（24、48、72、96和120h）中检查了时间对酶活性百分比的影响。蛋白酶酶活性的方差分析结果揭示了七个变量对固定化效率和酶活性的显着影响。此外,研究结果表明，激活时间，缓冲液pH值,MWCNT-NH2-戊二醛量，和稳定时间显著影响蛋白酶的活性。缓冲液pH和稳定时间之间的相互作用也是显著的。的确,活化时间和MWCNT-NH2-戊二醛的数量都对酶活性有降低作用。值得注意的是,MWCNT-NH2-戊二醛量的影响更为显著(p<0.05)。在β-半乳糖苷酶酶活性方面，研究结果强调，在所考虑的七个变量中，只有戊二醛的摩尔浓度，激活时间,活化时间和MWCNT-NH2-戊二醛数量的相互作用可以对酶的活性产生统计学上显著的正向影响(p<0.05)。活化时间和缓冲溶液摩尔浓度的组合，以及缓冲液pH和MWCNT-NH2-戊二醛的相互作用效应，可以显著提高碳纳米管蛋白酶的稳定效率。方差分析结果表明,米曲霉β-半乳糖苷酶在氨基功能化多壁碳纳米管上的共价固定效率受戊二醛摩尔浓度的影响,缓冲液pH值,稳定时间,以及活化时间+缓冲液pH的相互作用，缓冲液pH+活化时间，激活时间+缓冲液摩尔浓度，戊二醛摩尔浓度+MWCNT-NH2-戊二醛(p<0.05)。通过优化和选择最佳配方，结果表明，蛋白酶和β-半乳糖苷酶的酶活性和稳定效率分别为64.09％±72.63％和65.96％±71.77％，分别。此外，增加酶稳定时间导致酶活性降低。此外，pH值升高,温度,通过酶固定化碳纳米管的牛奶储存时间导致酶稳定效率降低，乳糖水解在8小时内逐渐下降。因此，预期将来自米曲霉的β-半乳糖苷酶和来自地衣芽孢杆菌的蛋白酶共价固定到氨基官能化的多壁碳纳米管上对于牛奶应用是可实现的。
