Deep eutectic solvents (DESs) have attracted an increasing attention in the fields of biocatalysis and biopolymer processing. In this study, papain immobilized on choline chloride- lactic acid (ChCl-Lac) DES-treated chitosan exhibited excellent thermostability as compared to the free enzyme. The properties of native or DES-treated chitosan and immobilized enzyme were characterized by FT-IR, SEM, surface area and pore property analysis. Like the common enzyme immobilization, papain immobilized on DES-treated chitosan resulted in a lower catalytic efficiency and a higher thermostability than the free enzyme due to the restricted diffusion. The results also revealed that DES could control the active group content, thus achieving the appropriate microporous structure of immobilized enzyme. Meanwhile, it could also help to construct the optimal microenvironment by hydrogen-bonding interaction between enzyme, chitosan, and residual DES, which are benefit for maintaining an active conformation and subsequently a high thermostability of papain. Moreover, it was found that trace DES (10 mM) significantly promoted the activity of free papain (145%). Deactivation thermodynamics study showed that the DES could enhance the thermostability of papain especially at high temperature (half-life of 7.4 vs. 3.5 h) because of the increased Gibbs free energy of denaturation. Secondary structure analysis by circular dichroism spectroscopy (CD) agreed well with the activity and thermostability data, further confirming the formation of rigid conformation induced by a specific amount of DES. This work provides a new way of enzyme immobilization synergistically intensified by solvents and supporting materials to achieve better microporous structure and catalytic microenvironment.

中文翻译：

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通过固定在具有最佳微孔结构和催化微环境的深共晶溶剂处理的壳聚糖上提高木瓜蛋白酶的热稳定性

深共熔溶剂（DESs）在生物催化和生物聚合物加工领域引起了越来越多的关注。在这项研究中，与游离酶相比，固定在氯化胆碱-乳酸 (ChCl-Lac) DES 处理的壳聚糖上的木瓜蛋白酶表现出优异的热稳定性。通过FT-IR、SEM、表面积和孔性质分析表征天然或DES处理的壳聚糖和固定化酶的性质。与常见的酶固定化一样，由于扩散受限，固定在 DES 处理的壳聚糖上的木瓜蛋白酶比游离酶具有更低的催化效率和更高的热稳定性。结果还表明DES可以控制活性基团的含量，从而实现固定化酶的适当微孔结构。同时，它还可以通过酶、壳聚糖和残留 DES 之间的氢键相互作用来构建最佳微环境，这有利于维持木瓜蛋白酶的活性构象和随后的高热稳定性。此外，发现痕量 DES (10 mM) 显着促进了游离木瓜蛋白酶的活性 (145%)。失活热力学研究表明，DES 可以提高木瓜蛋白酶的热稳定性，尤其是在高温下（半衰期为 7.4 小时与 3.5 小时），因为变性的吉布斯自由能增加。通过圆二色光谱 (CD) 进行的二级结构分析与活性和热稳定性数据非常吻合，进一步证实了由特定量的 DES 诱导的刚性构象的形成。