title: Asymmetric synthesis of D-glyceric acid by an alditol oxidase and directed evolution for enhanced oxidative activity towards glycerol.
abstract: Glycerol as a by-product of biodiesel production is an attractive precursor for producing d-glyceric acid. Here, we demonstrate the successful production of d-glyceric acid based on glycerol via glyceraldehyde in a two-step enzyme reaction with the FAD-dependent alditol oxidase from Streptomyces coelicolor A3(2). The hydrogen peroxide generated in the reaction can be used in detergent, food, and paper industry. In order to apply the alditol oxidase in industry, the enzyme was subjected to protein engineering. Different strategies were used to enhance the substrate specificity towards glycerol. Initial attempts based on rational protein design in the active site region were found unsuccessful to increase activity. However, through directed evolution, an alditol oxidase double mutant (V125M/A244T) with 1.5-fold improved activity for glycerol was found by screening 8,000 clones. Further improvement of activity was achieved by combinatorial experiments, which led to a quadruple mutant (V125M/A244T/V133M/G399R) with 2.4-fold higher specific activity towards glycerol compared to the wild-type enzyme. Through studying the effects of mutations created, we were able to understand the importance of certain amino acids in the structure of alditol oxidase, not only for conferring enzymatic structural stability but also with respect to their influence on oxidative activity.
authors.affiliation: Dept. of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, University of Greifswald, Felix-Hausdorff-Strasse 4, 17487 Greifswald, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Gerstenbruch
authors.forename: Hauke
authors.initials: H
authors.lastname: Wulf
authors.forename: Nina
authors.initials: N
authors.lastname: Mussmann
authors.forename: Timothy
authors.initials: T
authors.lastname: O'Connell
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Maurer
authors.forename: Uwe T
authors.initials: UT
authors.lastname: Bornscheuer
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution; D005988:Glyceric Acids / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D015202:Protein Engineering / Q000379:methods; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D005988:Glyceric Acids; D050505:Mutant Proteins; C042971:glyceric acid; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C515743:alditol oxidase (FAD), Streptomyces coelicolor