Laccase Activation in Deep Eutectic Solvents

Abstract

The research on alternative solvents and co-solvents is a relevant aspect when envisioning the improvement of biocatalytic reactions. Among these solvents and co-solvents, deep eutectic solvents (DES) may be considered as customizable new reaction media for biocatalysis. Accordingly, in this work, sixteen DES aqueous solutions, as well as of the individual DES components at the same conditions have been investigated in laccasecatalyzed reactions. Cholinium-and betaine-based DES formed with polyols at different molar ratio and concentrations were evaluated. The results reported show that in presence of most DES the laccase activity is preserved and, with a particular DES, enhanced up to 200%. Molecular docking studies demonstrated that while most DES components establish hydrogen-bonds with the enzyme amino acids, those that establish stronger interactions with the enzyme (expressed by absolute values of docking affinity energies) lead to an enhanced laccase activity. Finally, the laccase stability was evaluated in additional tests under extreme storage temperatures (60 ºC and -80 ºC). Although no significant protection to high temperatures was afforded by DES, an enhanced laccase activity when stored at low temperatures was found, at least up to 20 days. Combining experimental results and molecular docking this work shows that DES can be designed as co-solvents to improve biocatalytic reactions.

Enhancing the Thermostability of Papain by Immobilizing on Deep Eutectic Solvents-Treated Chitosan With Optimal Microporous Structure and Catalytic Microenvironment

“…Interestingly, DESs not only can stabilize the enzyme at high temperature, but some of them also have been reported to increase the laccase stability at low temperature. The author concluded that the DES aqueous solution has a strong effect on the crystallization/freezing and melting process of water, which can reduce the number of ice crystals and hence ice crystal damage in enzyme ( Toledo et al, 2019 ). This finding also proved that DES may be helpful for providing an ease environment together with the environmental matrix (water, immobilization materials, etc.…”

Designing a biocatalytic process involving deep eutectic solvents

“…10 The influence of DESs on reactions catalysed by isolated enzymes Observed increase in enzyme activity has been linked to involvement of the DES in the reaction mechanism itself. 10,40 For lipasecatalysed synthesis of butyl acetate by esterification, Cvjetko Bubalo and co-workers proposed that the favourable enzyme activity in Ch-based DES containing EG as an HBD could be explained by the increase in nucleophilicity of the hydroxyl group of the active site serine residue, but also by the fact that DES supports stabilisation of tetrahedral intermediates through H-bonding, leading to a decrease in the activity energy barrier and, therefore, an increase in reaction rate. 10 Recently, molecular docking studies to better understand the molecular-level effect of several DESs on laccase activity demonstrated that, while most DES components establish hydrogen bonds with the enzyme amino acids, those that establish stronger interactions with the enzyme (expressed by absolute values of docking affinity energies) enhance laccase activity.…”

“…10 Recently, molecular docking studies to better understand the molecular-level effect of several DESs on laccase activity demonstrated that, while most DES components establish hydrogen bonds with the enzyme amino acids, those that establish stronger interactions with the enzyme (expressed by absolute values of docking affinity energies) enhance laccase activity. 40 There are numerous publications that have suggested that DESs are great stabilisers of enzymes. The Villeneuve group reported long-term stability in the evaluation of immobilised C. antarctica B at 50°C in Ch:U and Ch:Gly.…”

“…When studying a chemoenzymatic method for the production of epoxidised vegetable oils, Zhou and co-workers showed that lipase G exhibited comparably poor stability in buffer only (halflife time of t ≈ 2 h). 42 In the presence of the co-substrate H 2 O 2 , 40 One of the first works that has led to a deeper understanding of the stability of an enzyme (C. antarctica lipase B) in a solvent with a high content of denaturing agents, such as urea (in nonaqueous DES ChU (1:2) with about 66% urea), by using molecular dynamics simulation was published by Monhemi et al 44 . The enzyme showed particularly good conformational stability in DES, in contrast to complete enzyme denaturation in water containing only 12% urea (urea interrupts the intra-chain H-bonds).…”

“…Observed increase in enzyme activity has been linked to involvement of the DES in the reaction mechanism itself 10,40 . For lipase‐catalysed synthesis of butyl acetate by esterification, Cvjetko Bubalo and co‐workers proposed that the favourable enzyme activity in Ch‐based DES containing EG as an HBD could be explained by the increase in nucleophilicity of the hydroxyl group of the active site serine residue, but also by the fact that DES supports stabilisation of tetrahedral intermediates through H‐bonding, leading to a decrease in the activity energy barrier and, therefore, an increase in reaction rate 10 .…”