Pseudomonas fluorescens Mannitol‐2‐Dehydrogenase as an NAD + Recycling Enzyme in the Conversion of Ethanol to Acetaldehyde

Abstract

Bioethanol is predominantly produced by fermentation of corn feedstocks in the United States. In recent years, market saturation has reduced the value of ethanol, threatening the viability of bioethanol plants throughout the Midwest. The overall goal of this project is to develop enzymatic and catalytic methods for converting ethanol into higher value chemical derivatives that enhance the economic sustainability of bioethanol plants. Acetaldehyde has a market price 2.5 times greater than ethanol. Alcohol dehydrogenases may be used to oxidize ethanol to acetaldehyde, but they require the cofactor NAD+, which must be regenerated in a recycling reaction. In this study, we examined a mannitol-2-dehydrogenase from Pseudomonas fluorescens (PfM2DH) as a candidate recycling enzyme to regenerate NAD+. PfM2DH catalyzes the reversible, NADH-dependent reduction of fructose to mannitol. Fructose is a readily available substrate at bioethanol plants as it is derived from corn starches. The efficacy of PfM2DH in an industrial setting will depend on the effect temperature, time (enzyme durability), pH and potential inhibitors (high ethanol or acetaldehyde concentration) have on PfM2DH activity. We have purified PfM2DH using published methods and have investigated the factors above using continuous assays measuring NADH oxidation by UV-Vis spectrophotometry. We are currently developing methods to assay the coupled production of acetaldehyde by gas chromatography. Our results demonstrate that PfM2DH retains activity from pH 7.5 up to pH 9.0, which is valuable for coupling with alcohol dehydrogenases that have pH optimums between 8.0 and 9.0. PfM2DH shows modest inhibition by acetaldehyde and ethanol at high concentrations and retains activity at room temperature for several days. These data are guiding our design of coupled enzyme reactions to efficiently generate acetaldehyde as a higher value derivative of ethanol. The success of this project will lay the groundwork for the future industrial valorization of bioethanol.