Enzymatic production of acetaldehyde using a Pseudomonas fluorescens mannitol‐2‐dehydrogenase mutant to regenerate NAD+

Abstract

Over the past decade, market saturation has drastically reduced the value of ethanol, which threatens the viability of industrial bioethanol facilities throughout the Midwest. In response, we are exploring enzymatic methods to convert ethanol and other biomass substrates into higher value chemical derivatives, such as acetaldehyde, to enhance the economic sustainability of bioethanol production. Acetaldehyde has a market price 2.5 times greater than ethanol and the volatility of acetaldehyde offers the potential for ready industrial separation. We are currently evaluating a coupled enzyme reaction in which horse liver alcohol dehydrogenase produces acetaldehyde from ethanol while an N191D mutant of Pseudomonas fluorescens mannitol‐2‐dehydrogenase (PfM2DH) is used to produce mannitol from fructose and to oxidize NADH regenerating NAD+. We have chosen the N191D mutant based on work by Klimacek and Nidetsky demonstrating that this mutation converts wild type PfM2DH, which is reversible, into an enzyme selective for fructose reduction (1). In previous work, we have shown that PfM2DH is active under a wide range of pH and temperature conditions making it a promising enzyme for industrial production. We are developing a GC‐MS solid phase microextraction (SPME) assay to evaluate accumulation of acetaldehyde in the head space of our reaction vessel. This assay will allow us to optimize conditions for the industrial stripping of acetaldehyde from a reactor. We are also optimizing a HILIC‐LC‐MS assay to quantify mannitol production in solution. Combining these assays allows the determination of total acetaldehyde production and the acetaldehyde fraction entering the gas phase. In addition to evaluating the viability of our horse liver ADH and PfM2DH_N191D coupled reaction, these assays will allow the characterization of future enzyme pairs in our efforts to valorize bioethanol using an industrial enzymatic process.