Selective oxidation of glycerol to 1,3-dihydroxyacetone by covalently immobilized glycerol dehydrogenases with higher stability and lower product inhibition

• Covalent immobilization stabilizes glycerol dehydrogenase from different sources.
• Orientation through the most acid area of GlyDH minimizes its product inhibition.
• Covalent post-immobilization cross-linking rather improves enzyme properties.

Glycerol dehydrogenase (GlyDH) catalyzes the regioselective oxidation of glycerol to yield 1,3-dihydroxyacetone (DHA); an important building block in chemical industry. Three recombinant GlyDHs from Geobacillus stearothermophilus, from Citrobacter braakii and from Cellulomonas sp. were stabilized by covalent immobilization. The highest activity recoveries (40–50%) of the insoluble preparations were obtained by immobilizing these enzymes in presence of polyethylene glycol (PEG). Noteworthy, these immobilized preparations were more stable and less inhibited by DHA than their soluble counterparts. In particular, GlyDH from G.stearothermophilus immobilized on agarose activated with both amine and glyoxyl groups and crosslinked with dextran aldehyde was 3.7-fold less inhibited by DHA than its soluble form and retained 100% of its initial activity after 18 h of incubation at 65 °C and pH 7. This is one of the few examples where the same immobilization protocol has minimized enzyme product inhibition and maximized thermal stability.