Semi-Rational Engineering of Aldoxime Dehydratase for Conducting a Chemoenzymatic Sequence to Prepare 2-Furonitrile from Xylose

Exploiting efficient methods for converting biomass-based resources into high-value-added chemicals has attracted extensive interest in sustainable development in the chemical industry. Here, we have developed a chemoenzymatic sequence for synthesizing 2-furonitrile (2-FN) from xylose, a biobased five-carbon monosaccharide derived from agricultural waste. Firstly, a 1,2-dichloroethane (DCE)/H₂O biphasic system (1:1, v/v) was adopted to produce 2-furaldehyde oxime (2-FOx) from xylose by integrating two steps of dehydration and oximation in a one-pot sequence using a temporal compartmentalization strategy, resulting in a yield of 2-FOx from xylose over 78%. Secondly, the catalytic efficiency of aldoxime dehydratase from Pseudomonas putida F1 (OxdF1) was significantly improved by engineering the substrate access tunnel and a distal residue. The activity of an optimal mutant L318I–N266S has reached 3.94 U·mg^–1 towards 2-FOx, approximately 6 times higher than that of the wild-type OxdF1 (0.65 U·mg^–1). Consequently, 2-FN was prepared in a 400 mL reaction mixture at room temperature using a continuous feeding strategy. After 1.5 h, 100 mM 2-FOx was completely converted to 2-FN with a space-time yield of 6.2 g·L^–1·h^–1. The chemoenzymatic process proposed an alternative strategy for synthesizing 2-FN from biomass-based materials under mild conditions without using highly toxic cyanide.