Immobilized enzymes have encountered two main challenges: Reduced enzyme activity compared to free enzymes and exhausted immobilized enzymes due to reusability. Herein, we suggested a promising activity enhancement strategy to overcome these challenges. The emission from upconversion nanoparticles (UCNPs) under near-infrared (NIR) excitation can increase the activity of PEG--ASNase due to Förster Resonance Energy Transfer. For this purpose, UCNPs were initially synthesized using the hydrothermal method. Subsequently, these UCNPs were functionalized with a polycationic polymer, branched polyethyleneimine (PEI), and the immobilization of PEG--ASNase was achieved through adsorption. We preliminarily explored the parameters such as enzyme concentration, incubation time, pH, temperature, reusability, storage stability, and kinetic study, etc. Further, the biocompatibility, hemolytic behavior, and anticancer activity of the produced UCNPs were also analyzed as crucial parameters. The results showed the pH durance, thermal and storage stability of the immobilized PEG--ASNases were enhanced. The immobilized PEG--ASNases maintained their activity to ≥55 % after 20 cycles. Enzyme immobilization led to a decrease in Km and Vmax compared to PEG--ASNase. assays revealed that immobilized enzyme further reduced the proliferation of human leukemia cell line (HL-60) upon NIR irradiation exposure but did not cause toxicity. This research may provide a new strategy to promote the catalytic activity of -ASNase and demonstrates its potential application on human leukemia cells. Finally, these outcomes are valuable for the use of NIR induction in enzymatic reactions.

中文翻译：

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生物催化剂设计中的近红外诱导支持：增强酶活性的有用且通用的工具

固定化酶遇到了两个主要挑战：与游离酶相比酶活性降低以及由于可重复使用而耗尽固定化酶。在此，我们提出了一种有前景的活动增强策略来克服这些挑战。由于福斯特共振能量转移，上转换纳米颗粒 (UCNP) 在近红外 (NIR) 激发下的发射可以增加 PEG-ASNase 的活性。为此，最初使用水热法合成了 UCNP。随后，这些UCNPs被聚阳离子聚合物、支化聚乙烯亚胺(PEI)功能化，并通过吸附实现了PEG-ASNase的固定化。我们初步探讨了酶浓度、孵育时间、pH、温度、可重复使用性、储存稳定性和动力学研究等参数。此外，还对所生产的 UCNP 的生物相容性、溶血行为和抗癌活性作为关键参数进行了分析。结果表明，固定化PEG--ASNase的pH持久性、热稳定性和储存稳定性均得到提高。固定化的 PEG-ASNase 在 20 个循环后仍保持其活性≥55%。与 PEG-ASNase 相比，酶固定化导致 Km 和 Vmax 降低。测定表明，固定化酶进一步减少了近红外辐射暴露后人白血病细胞系（HL-60）的增殖，但不会引起毒性。这项研究可能为促进-ASNase的催化活性提供新的策略，并展示其在人类白血病细胞中的潜在应用。最后，这些结果对于在酶反应中使用近红外感应很有价值。