Abstract

The invertase enzyme has been immobilized onto various support materials to enhance operational stability, shelf life, and reusability compared to free ones. Among the immobilization methods, carrier-free immobilization, such as cross-linked enzyme aggregates (CLEAs), attracts attention. However, recovery of the CLEAs from the reaction environment by centrifugation or filtration is a great challenge. The use of magnetic nanoparticles (Fe₃O₄) can overcome these separation limitations of the aggregates. For this aim, in this study, the surface of magnetite nanoparticles was functionalized with a significant number of free amino groups through silanization reaction. After precipitation of these aggregates and then cross-linking to the functionalized structure, the resulting magnetite cross-linked invertase aggregates (MCLIAs) were obtained, having different enzyme and glutaraldehyde concentrations. Magnetic nanoparticles were characterized by SEM, VSM, and FTIR analysis. The assay parameters, both constructional and experimental ones on the activity of MCLIAs, were practically determined by a personal glucose meter (PGM). The results indicate that magnetite nanoparticles with superparamagnetic behaviour were successfully functionalized with amino groups. The activity results demonstrated that experimental parameters were more effective than constructional parameters. MCLIAs also exhibited maximum activity at pH 5. Furthermore, 30-min incubation time and 35 °C were the best activity assay conditions.

摘要

与游离酶相比，转化酶已固定在各种支持材料上，以提高操作稳定性、保质期和可重复使用性。在固定化方法中，无载体固定化，例如交联酶聚集体（CLEAs），引起了人们的关注。然而，通过离心或过滤从反应环境中回收 CLEA 是一个巨大的挑战。磁性纳米粒子（Fe ₃ O ₄) 可以克服聚集体的这些分离限制。为此，在本研究中，磁铁矿纳米颗粒的表面通过硅烷化反应被大量的游离氨基官能化。在这些聚集体沉淀，然后交联到功能化结构后，得到了具有不同酶和戊二醛浓度的磁铁矿交联转化酶聚集体 (MCLIA)。通过 SEM、VSM 和 FTIR 分析对磁性纳米颗粒进行了表征。测定参数，包括关于 MCLIA 活性的结构参数和实验参数，实际上是由个人血糖仪 (PGM) 确定的。结果表明具有超顺磁性行为的磁铁矿纳米粒子成功地被氨基官能化。活动结果表明，实验参数比结构参数更有效。MCLIA 在 pH 5 时也表现出最大活性。此外，30 分钟的孵育时间和 35 °C 是最佳的活性测定条件。