Insights into the impact of deep eutectic solvents on horseradish peroxidase: Activity, stability and structure

• 1st study about DES effects on performance and structure of a same enzyme.
• DES affects horseradish peroxidase (HRP) by affecting its protein structure.
• Affecting factors include the salt, the HBD, and the molar ratio between the two.
• DES can induce a significant stabilization effect on HRP.
• DESs help to improve the HRP-mediated phenolic wastewater treatment.

As a new type of ionic fluids that are inexpensive and environmentally friendly, deep eutectic solvents (DESs) have recently attracted a broad interest in many fields including biocatalysis. In this study, 24 DESs were prepared by mixing two cholinium salts (cholinium chloride (ChCl) and cholinium acetate (ChAc)) with four H-bond donors (HBDs) (urea, glycerol, acetamide, ethylene glycol) at three molar ratios, and their effects on the activity, stability and structure of horseradish peroxidase (HRP) have been investigated. The ChCl-based DESs have been found to be superior to the ChAc-based ones in terms of promoting the HRP activity. For the DESs composed of the same salt and same HBD, an increase in the HRP activity has been observed with a higher salt/HBD molar ratio of 1:2 < 1:1 < 2:1. Structural studies with fluorescence and circular dichroism spectroscopy have agreed well with the activity data, suggesting that DESs capable of providing the enzyme with a higher α-helix content and a slightly more relaxed tertiary structure may facilitate the HRP activity. All the 24 DESs were able to highly stabilize the enzyme. Addition of DESs may help to improve the HRP-mediated phenolic wastewater treatment. Our experiments have also supported the idea that the extensive H-bonding network throughout the DES is sufficiently strong to prevent the DES from dissociation in aqueous solution.

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