Discovery and biochemical characterization of thermostable glycerol oxidases

Alditol oxidases are promising tools for the biocatalytic oxidation of glycerol to more valuable chemicals. By integrating in
silico bioprospecting with cell-free protein synthesis and activity screening, an effective pipeline was developed to rapidly
identify enzymes that are active on glycerol. Three thermostable alditol oxidases from Actinobacteria Bacterium, Strepto-
myces thermoviolaceus, and Thermostaphylospora chromogena active on glycerol were discovered. The characterization of
these three flavoenzymes demonstrated their glycerol oxidation activities, preference for alkaline conditions, and excellent
thermostabilities with melting temperatures higher than 75 °C. Structural elucidation of the alditol oxidase from Actino-
bacteria Bacterium highlighted a constellation of side chains that engage the substrate through several hydrogen bonds, a
histidine residue covalently bound to the FAD prosthetic group, and a tunnel leading to the active site. Upon computational
simulations of substrate binding, a double mutant targeting a residue pair at the tunnel entrance was created and found to
display an improved thermal stability and catalytic efficiency for glycerol oxidation. The hereby described alditol oxidases
form a valuable panel of oxidative biocatalysts that can perform regioselective oxidation of glycerol and other polyols.