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Background  Dicarboxylic acids offer several applications in detergent builder and biopolymer fields. One of these acids, 4- O -methyl  d -glucaric acid, could potentially be produced from glucuronoxylans, which are a comparatively underused fraction of wood and agricultural biorefineries.    Results  Accordingly, an enzymatic pathway was developed that combines AxyAgu115A, a GH115 α-glucuronidase from  Amphibacillus xylanus , and GOOX, an AA7 gluco-oligosaccharide oxidase from  Sarocladium strictum , to produce this bio-based chemical from glucuronoxylan. AxyAgu115A was able to release almost all 4- O -methyl  d -glucuronic acid from glucuronoxylan while a GOOX variant, GOOX-Y300A, could convert 4- O -methyl  d -glucuronic acid to the corresponding glucaric acid at a yield of 62%. Both enzymes worked effectively at alkaline conditions that increase xylan solubility. Given the sensitivity of AxyAgu115A to hydrogen peroxide and optimal performance of GOOX-Y300A at substrate concentrations above 20 mM, the two-step enzyme pathway was demonstrated as a sequential, one-pot reaction. Additionally, the resulting xylan was easily recovered from the one-pot reaction, and it was enzymatically hydrolysable.    Conclusions  The pathway in this study requires only two enzymes while avoiding a supplementation of costly cofactors. This cell-free approach provides a new strategy to make use of the underutilized hemicellulose stream from wood and agricultural biorefineries.

Enzymatic production of 4-O-methyl d-glucaric acid from hardwood xylan