Active Site Engineering of a Glycerol Dehydrogenase Improves its Oxidative Activity and Scope Toward Glycerol Derivatives.

Regioselective oxidation of glyceryl alkyl ethers is of utmost importance for the fabrication of substituted hydroxy ketones and enantiopure 1,2-diols as green solvents and pharmaceutical building blocks, respectively. An engineered glycerol dehydrogenase from Bacillus stearothermophilus was described to perform the regioselective oxidation of alkyl glycerol ethers, identifying position 252 as key for accepting larger substrates than glycerol. In this work, we further engineer that position through partial saturation mutagenesis to broaden the substrate scope toward other glycerol derivatives, improving enzyme kinetics and minimizing product inhibition. In particular, the BsGlyDH-L252S variant becomes the most efficient biocatalyst for the deracemization of alkyl glyceryl ethers in a two-step, one-pot immobilized system. The discovery and use of these alternative mutants of GlyDH opens the road to more applications and increases the enzymatic toolbox for the modifications of glyceryl ethers.