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

Alfonso Miranda-Molina,Rodrigo Arreola-Barroso,Agustín López-Munguía,Simon Strompen,Gloria Saab-Rincon,Leticia Olvera,Edmundo Castillo,Wendy Xolalpa

doi:10.3390/ijms20215439

Alfonso Miranda-Molina, Rodrigo Arreola-Barroso + Show 6 more

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https://doi.org/10.3390/ijms20215439

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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.

Highlights

Alkyl glycosides (AGs) are a class of non-ionic surfactants prepared from renewable agricultural resources, namely starch and fats or their derivatives [1]
Cholinium (Ch+), a natural Generally Recognized as Safe (GRAS) compound, commonly used as a food additive, is the preferred hydrogen bond acceptor (HBA) [24]
Achieving an anhydrous medium that would eliminate the hydrolysis reaction that competes with the production of alkyl glycosides is not possible, indicating that some hydration is needed to form an aqueous shell around the protein that allows it to fold and the necessary lubrication in the active site to work, as has been observed by others [33]; this contrasts with lipases for which Deep Eutectic Solvents (DES) seem sufficient to mimic the water layer covering active enzymes in alternative reaction media to water [21,22]

Summary

Introduction

Alkyl glycosides (AGs) are a class of non-ionic surfactants prepared from renewable agricultural resources, namely starch and fats or their derivatives [1]. AGs contain a carbohydrate head group such as glucose, galactose, maltose, xylose, α-cyclodextrin, and a hydrocarbon tail, usually a primary alcohol of different chain length of saturated or unsaturated nature [2,3] Their high biodegradability and a low toxicity make them attractive for many applications such as cosmetics, foods, the extraction of organic dyes, membrane protein research, and pharmaceuticals [4]. The industrial production of alkyl glycosides has been typically achieved by traditional organic chemical procedures This strategy requires multiple protection, deprotection and activation steps, the preparation of an anomerically-pure AG requiring high temperature and pressure, as well as the use of toxic catalysts, harmful chemicals, dangerous solvents, and other harsh conditions that adversely impact the environment and human health [4,5]. Reports dealing with α-amylases are scarce [1,16,17,18]

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