Deep Eutectic Solvents (DES) were investigated as new reaction media for the synthesis of alkyl glycosides catalyzed by the thermostable α-amylase from Thermotoga maritima Amy A. The enzyme was almost completely deactivated when assayed in a series of pure DES, but as cosolvents, DES containing alcohols, sugars, and amides as hydrogen-bond donors (HBD) performed best. A choline chloride:urea based DES was further characterized for the alcoholysis reaction using methanol as a nucleophile. As a cosolvent, this DES increased the hydrolytic and alcoholytic activity of the enzyme at low methanol concentrations, even when both activities drastically dropped when methanol concentration was increased. To explain this phenomenon, variable-temperature, circular dichroism characterization of the protein was conducted, finding that above 60 °C, Amy A underwent large conformational changes not observed in aqueous medium. Thus, 60 °C was set as the temperature limit to carry out alcoholysis reactions. Higher DES contents at this temperature had a detrimental but differential effect on hydrolysis and alcoholysis reactions, thus increasing the alcoholyisis/hydrolysis ratio. To the best of our knowledge, this is the first report on the effect of DES and temperature on an enzyme in which structural studies made it possible to establish the temperature limit for a thermostable enzyme in DES.
Keywords: Amy A; Deep eutectic solvents (DES); Enzymatic glycosylation; alcoholysis; alkyl glycosides (AG)s; alpha-amylase; circular dichroism; hydrolysis; methanol; protein stability.