*Article* **Deep Eutectic Solvents as New Reaction Media to Produce Alkyl-Glycosides Using Alpha-Amylase from** *Thermotoga maritima*

**Alfonso Miranda-Molina** †**,**‡**, Wendy Xolalpa** †**, Simon Strompen §, Rodrigo Arreola-Barroso, Leticia Olvera, Agustín López-Munguía, Edmundo Castillo and Gloria Saab-Rincon \***

Departamento Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos 62250, Mexico; alfonso\_itz@yahoo.com.mx (A.M.-M.); wxolalpa@ibt.unam.mx (W.X.); simon.strompen@basf.com (S.S.); rarreolb@ibt.unam.mx (R.A.-B.); lolvera@ibt.unam.mx (L.O.); agustin@ibt.unam.mx (A.L.-M.); edmundo@ibt.unam.mx (E.C.)

**\*** Correspondence: gsaab@ibt.unam.mx


§ Present address: BASF Personal Care and Nutrition GmbH, 40589 Duesseldorf-Holthausen, Germany

Received: 17 August 2019; Accepted: 25 September 2019; Published: 31 October 2019

**Abstract:** 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:** Enzymatic glycosylation; alkyl glycosides (AG)s; Deep eutectic solvents (DES); Amy A; alcoholysis; hydrolysis; methanol; circular dichroism; protein stability; alpha-amylase
