Abstract This study focuses on well-known but commonly overlooked or unreported factors in the preparation of cross-linked enzyme nano-aggregates (nano-CLEAs). The parameters including the ionic strength of the protein solution, protein, precipitant and cross-linker concentrations, pH and addition order of the reagents were fine-tuned for nanoaggregate preparation without the need of non-protein support material, special equipment or sophisticated procedures. For this purpose, precipitation as nano-aggregates and then cross-linking while maintaining submicron size distribution were studied independently. Moreover, nano-aggregate formation from reverse micellar solutions was also investigated to improve the scope of the method to membrane-bound enzymes. Five different precipitation agents together with three different cross-linkers were investigated for immobilization of the Trametes versicolor laccase as cross-linked nano-aggregates. Although complete activity recovery was possible for micro-aggregates, the best activity results for nano-aggregates were 40.5%±5.0. The Km values of the immobilized enzymes were slightly lower than the Km values of the free counterparts which indicate little or no mass transfer limitation due to the nano-immobilization process. However, Vmax values were also lower. The activity loss and Vmax reduction upon immobilization were found to mainly result from the modification of the amine groups instead of excess crosslinking. The thermal stabilities of the crosslinked laccase nano-aggregates were significantly higher (∼10–30 fold at 60 °C) compared to free laccase and the nano-CLEAs retained up to 30% of their initial activities upon 7 consequent usages. The sizes of the obtained immobilized enzyme products were found to be greatly variable depending on the cross-linker type. The smaller particles (∼200 nm radius) were obtained when EDAC was used as the cross-linker. The larger products (∼600 nm radius) were prepared when the cross-linker was dextran poly-aldehyde. The addition order of the reagents was found to be effective on particle size and thermal stability values.

中文翻译：

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交联漆酶纳米聚集体的微调制备

摘要 本研究侧重于制备交联酶纳米聚集体 (nano-CLEAs) 中众所周知但通常被忽视或未报告的因素。包括蛋白质溶液的离子强度、蛋白质、沉淀剂和交联剂浓度、pH 值和试剂添加顺序在内的参数经过微调，用于纳米聚集体制备，无需非蛋白质支持材料、特殊设备或复杂程序。为此，独立研究了作为纳米聚集体的沉淀，然后在保持亚微米尺寸分布的同时进行交联。此外，还研究了从反胶束溶液形成纳米聚集体，以扩大该方法对膜结合酶的范围。研究了五种不同的沉淀剂和三种不同的交联剂，用于将变色栓菌漆酶固定为交联纳米聚集体。尽管微团聚体可以完全恢复活性，但纳米团聚体的最佳活性结果为 40.5%±5.0。固定化酶的 Km 值略低于游离酶的 Km 值，这表明由于纳米固定化过程很少或没有传质限制。然而，Vmax 值也较低。发现固定后的活性损失和 Vmax 降低主要是由于胺基团的修饰而不是过度交联。与游离漆酶相比，交联漆酶纳米聚集体的热稳定性显着更高（60°C 时约 10-30 倍），并且纳米 CLEA 在 7 次后续使用后保留了其初始活性的 30%。发现所获得的固定化酶产物的大小随交联剂类型的不同而变化很大。当 EDAC 用作交联剂时，获得较小的颗粒（约 200 nm 半径）。当交联剂为葡聚糖聚醛时，制备出较大的产物（半径约 600 nm）。发现试剂的添加顺序对粒度和热稳定性值有效。发现所获得的固定化酶产物的大小随交联剂类型的不同而变化很大。当 EDAC 用作交联剂时，获得较小的颗粒（~200 nm 半径）。当交联剂为葡聚糖聚醛时，制备出较大的产物（半径约 600 nm）。发现试剂的添加顺序对粒度和热稳定性值有效。发现所获得的固定化酶产物的大小随交联剂类型的不同而变化很大。当 EDAC 用作交联剂时，获得较小的颗粒（约 200 nm 半径）。当交联剂为葡聚糖聚醛时，制备出较大的产物（半径约 600 nm）。发现试剂的添加顺序对粒度和热稳定性值有效。