Department of Chemistry, University of Delhi, Delhi 110 007, India.
Department of Chemistry, Sri Venkateswara College, University of Delhi, Delhi 110 007, India.
Int J Biol Macromol. 2023 Sep 30;249:125988. doi: 10.1016/j.ijbiomac.2023.125988. Epub 2023 Jul 25.
As a new generation of 'green solvents' deep eutectic solvents (DESs) represents a promising alternative to the conventional solvents. Their environmental-benign nature and designer properties promote their utility in biocatalysis. Enzymes are marginally stable when exposed to physical/chemical disturbances. One such enzyme is cellulase which is a propitious catalyst for the depolymerization of cellulose under mild conditions. Therefore, their stability is a prerequisite condition to match demands of biorefineries. To address this issue of low stability, activity and thermal denaturation of cellulase, there is a need to find a sustainable and suitable co-solvent that is biocompatible with enzymes ultimately to facilitate their application in bio-industries. In this regard, we synthesized three choline-based DESs, choline chloride (ChCl)-glycerol, ChCl-ethylene glycol and ChCl-lactic acid and employed them to analyze their suitability for cellulase. The present study systematically evaluates the influence of the mentioned DESs on stability, activity and thermal stability of cellulase with the help of various spectroscopic techniques. The spectroscopic analysis revealed that the structural stability and activity of the enzyme were improved in presence of ChCl-glycerol and ChCl-ethylene glycol. The thermal stability was also very well maintained in both the DESs. Interestingly, the relative activity of cellulase was >80 % even after incubation at 50 °C after 48 h for both the DESs. This activity preservation behaviour was more pronounced for ChCl-ethylene glycol than ChCl-glycerol. Moreover, temperature variations studies also reveal promising results by maintain conformational intactness. On the other side, ChCl-lactic acid showed a deleterious effect on the enzyme both structurally as well as thermally. The dynamic light scattering (DLS) analysis provides more specific information about the negative influence of ChCl-lactic acid towards cellulase native structure. This DES induces unavoidable alterations in the enzyme structure which leads to the unfolding of enzyme, ultimately, destabilizing it. Overall, our results present a physical insight into how the enzyme stability and activity depend on the nature of DES. Also, the findings will help to facilitate the development and application of DESs as biocatalytic process.
作为新一代“绿色溶剂”，深共晶溶剂 (DESs) 代表了传统溶剂的一种很有前途的替代物。其环境友好的性质和设计特性促进了它们在生物催化中的应用。当暴露于物理/化学干扰时，酶的稳定性会略有下降。纤维素酶就是这样一种酶，它在温和条件下是纤维素解聚的有利催化剂。因此，其稳定性是满足生物精炼厂需求的前提条件。为了解决纤维素酶稳定性差、活性和热变性的问题，需要找到一种可持续且合适的共溶剂，与酶具有生物相容性，最终促进其在生物工业中的应用。在这方面，我们合成了三种基于胆碱的深共晶溶剂，氯化胆碱 (ChCl)-甘油、ChCl-乙二醇和 ChCl-乳酸，并利用它们来分析它们对纤维素酶的适用性。本研究利用各种光谱技术系统地评估了所提到的深共晶溶剂对纤维素酶稳定性、活性和热稳定性的影响。光谱分析表明，在 ChCl-甘油和 ChCl-乙二醇存在的情况下，酶的结构稳定性和活性得到了提高。在这两种深共晶溶剂中，热稳定性也得到了很好的维持。有趣的是，即使在 50°C 下孵育 48 小时后，两种深共晶溶剂中纤维素酶的相对活性仍>80%。对于 ChCl-乙二醇，这种活性保持行为比 ChCl-甘油更为明显。此外，温度变化研究也通过保持构象完整提供了有希望的结果。另一方面，ChCl-乳酸对酶的结构和热稳定性都有不利影响。动态光散射 (DLS) 分析提供了关于 ChCl-乳酸对纤维素酶天然结构的负面影响的更具体信息。这种深共晶溶剂会对酶结构产生不可避免的改变，导致酶的展开，最终使其不稳定。总的来说，我们的研究结果提供了关于酶稳定性和活性如何取决于深共晶溶剂性质的物理见解。此外，这些发现将有助于促进深共晶溶剂作为生物催化过程的开发和应用。
