Assessing the compatibility of choline-based deep eutectic solvents for the structural stability and activity of cellulase: Enzyme sustain at high temperature

Abstract

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.

Keywords: Activity preservation; Cellulase; Deep eutectic solvents; Enzyme stability; Spectroscopic techniques.