Improved activity and thermostability of glycosylating biocatalysts acting in neat natural deep eutectic solvents: Pushing boundaries of enzymes.

Non-aqueous solvents are valuable tools for improving enzyme properties. Natural Deep Eutectic Solvents (NaDES) recently emerged as an alternative non-aqueous green media, however, further information is required to understand the structure-function changes induced in enzymes. The behavior of a Levansucrase (SacB) and a β-fructofuranosidase (Invertase) was studied in near-anhydrous NaDES. We compare their catalytic activity, optimum temperature (T), and thermostability (T and t) observed in NaDES with their properties in acetate buffer. The NaDES were selected and designed using the COSMO-RS model based on their tunable nature. Both enzymes showed activity in NaDES at 50 °C, with the highest activity in sugar-polyol NaDES. The changes observed in Fructose-Sorbitol-Water (F-S-W) showed a radical functional transformation: Activities increased by up to 742 % and 580 %, with a Topt of 70 °C and 80 °C for SacB and Invertase, respectively, transferring both enzymes to a thermophilic classification. Both enzymes showed activity above 100 °C, and their t increased 23,100 times for SacB and 2500 times for Invertase. While the thermodynamic DSC analyses in F-S-W showed increases in their T, 31.2 °C for SacB and 3.2 °C for Invertase, the spectroscopic CD experiments suggested that this surprising enzyme behavior resulted from a significant increase in β-strand structures. When pre-equilibrated in F-S-W, the enzymes showed the highest increase in β-strand structure. This new perspective on using NaDES in biocatalysis becomes a worthwhile alternative for thermostabilizing and preserving proteins.