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 (Topt), and thermostability (Tm and t1/2) 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-Water1:1:5 (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 t1/2 increased 23,100 times for SacB and 2500 times for Invertase. While the thermodynamic DSC analyses in F-S-W showed increases in their Tm, 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.