Semi-Rational\nEngineering of Aldoxime Dehydratase\nfor Conducting a Chemoenzymatic Sequence to Prepare 2‑Furonitrile\nfrom Xylose

Exploiting efficient methods for converting biomass-based\nresources\ninto high-value-added chemicals has attracted extensive interest in\n sustainable development in the chemical industry. Here, we have developed\na chemoenzymatic sequence for synthesizing 2-furonitrile (2-FN) from\nxylose, a biobased five-carbon monosaccharide derived from agricultural\nwaste. Firstly, a 1,2-dichloroethane (DCE)/H<sub>2</sub>O biphasic\nsystem (1:1, v/v) was adopted to produce 2-furaldehyde oxime (2-FOx)\nfrom xylose by integrating two steps of dehydration and oximation\nin a one-pot sequence using a temporal compartmentalization strategy,\nresulting in a yield of 2-FOx from xylose over 78%. Secondly, the\ncatalytic efficiency of aldoxime dehydratase from <i>Pseudomonas\nputida</i> F1 (OxdF1) was significantly improved by engineering\nthe substrate access tunnel and a distal residue. The activity of\nan optimal mutant L318I–N266S has reached 3.94 U·mg<sup>–1</sup> towards 2-FOx, approximately 6 times higher than\nthat of the wild-type OxdF1 (0.65 U·mg<sup>–1</sup>).\nConsequently, 2-FN was prepared in a 400 mL reaction mixture at room\ntemperature using a continuous feeding strategy. After 1.5 h, 100\nmM 2-FOx was completely converted to 2-FN with a space-time yield\nof 6.2 g·L<sup>–1</sup>·h<sup>–1</sup>. The\nchemoenzymatic process proposed an alternative strategy for synthesizing\n2-FN from biomass-based materials under mild conditions without using\nhighly toxic cyanide.