Biocatalytic Furfuryl Alcohol Production with Ethanol as the Terminal Reductant Using a Single Enzyme

Abstract

Traditionally, furfuryl alcohol (FOL) is produced from biomass-derived furfural (FAL) by hydrogenation using metal-based chemocatalysts. It is challenging due to high metal toxicity, high hydrogen partial pressure, and the need for organic solvents. NAD­(P)­H-dependent yeast alcohol dehydrogenase I (YADH), which offers high atom economy and up to 100% product selectivity at room temperature in an aqueous medium, is used in this study for the biocatalytic production of FOL from FAL using ethanol (EtOH) as the terminal reductant for in situ regeneration of NAD­(P)­H. Up to 74% FAL conversion was observed at pH 8 with 40 and 160 mM initial FAL and EtOH concentrations, respectively. The conversion was determined to be equilibrium-limited. Circular dichroism spectroscopy and differential scanning fluorimetry studies of YADH show a significant change in the secondary structure upon treatment with increasing concentrations of aldehydes resulting in loss of catalytic activity. Benign reaction conditions support efforts toward sustainable processing, but opportunities for further improvement by increasing product titer and catalyst stability have been identified. This study lays the framework for developing the science and process for alternatives to biomass-derived ethanol.

Covalent Immobilization of Yeast Alcohol Dehydrogenase on an Amine-Functionalized Polymeric Resin Enhances Stability for Furfural Hydrogenation to Furfuryl Alcohol Using Ethanol as the Terminal Reductant

“…In our earlier publication, free YADH stability was assessed in a fed-batch experiment with 0.2 mg/mL YADH and relatively lower substrate concentration levels (51.4 mM FAL and 106 mM EtOH) . Overall, the reaction provided only 45% FAL and 18% EtOH conversions due to declining reaction rates with free YADH.…”

“…16−18 Recently, we demonstrated an isolated commercial YADH-catalyzed process with in situ NAD(P)H regeneration for EtOH-dependent reduction of FAL to FOL with AcH as the coproduct (Scheme 1). 19 It was observed that under optimum reaction conditions, 74% FAL conversion with 100% FOL selectivity could be achieved across a broad pH range, and up to 40 mM titer of each coproduct could be obtained when high initial substrate concentrations of 320 mM EtOH and 80 mM FAL were used.…”

“…YADH exhibits several favorable characteristics, including high turnover frequency for EtOH oxidation, stability in a wide range of conditions, and a low Michaelis constant ( K m ) value for NAD+, suggesting it can be efficiently saturated with NAD+ even at low concentrations. − Recently, we demonstrated an isolated commercial YADH-catalyzed process with in situ NAD­(P)H regeneration for EtOH-dependent reduction of FAL to FOL with AcH as the coproduct (Scheme ). It was observed that under optimum reaction conditions, 74% FAL conversion with 100% FOL selectivity could be achieved across a broad pH range, and up to 40 mM titer of each coproduct could be obtained when high initial substrate concentrations of 320 mM EtOH and 80 mM FAL were used.…”

“…22,23 Aldehydes like AcH and FAL can induce significant changes and partial unfolding of enzyme proteins, resulting in the loss of catalytic activity, as observed in YADH. 19 Additionally, the presence of inhibitors, which can be reactants or products at high concentrations, can diminish the kinetic stability of enzymes by forming unreactive intermediates. 24,25 However, these issues have been mitigated in some cases by immobilizing enzymes within an inert matrix, which is crucial to improving the operational performance of enzymes and making a biocatalytic process economically feasible.…”

“…The facile technology of yeast production at EtOH biorefineries can enable low-cost YADH and EtOH, which underscores the need for further research into FAL reduction using EtOH, leveraging the operational and economic advantages of YADH. 19 This work explores the integration of cell-free biocatalysis for biomass-derived chemical production and provides a comprehensive understanding of the scope, merits, and challenges associated with the covalent immobilization of YADH on aminated polymeric resins and the potential for its large-scale application.…”