title: Tailoring a stabilized variant of hydroxynitrile lyase from Arabidopsis thaliana.
abstract: The R-selective hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) cannot be applied for stereoselective cyanohydrin syntheses in aqueous media because of its limited stability at pH<5, which is required in order to suppress the uncatalyzed racemic cyanohydrin formation. To stabilize AtHNL we designed a surface-modified variant incorporating 11 changes in the amino acids on the protein surface. Comparative characterization of the variant and the wild-type enzyme showed a broadened pH optimum towards the acidic range, along with enhancement of activity by up to twofold and significantly increased pH- and thermostabilities. The effect can most probably be explained by a shift of the isoelectic point from pH 5.1 to 4.8. Application of the variant for the synthesis of (R)-cyanohydrins in an aqueous/organic two-phase system at pH 4.5 demonstrated the high stereoselectivity and robustness of the variant relative to the wild-type enzyme, which is immediately inactivated under these conditions.
authors.affiliation: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Okrob
authors.forename: Julia
authors.initials: J
authors.lastname: Metzner
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wiechert
authors.forename: Karl
authors.initials: K
authors.lastname: Gruber
authors.forename: Martina
authors.initials: M
authors.lastname: Pohl
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology*; D001665:Binding Sites; D004795:Enzyme Stability; D006361:Heating; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008958:Models, Molecular; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D009570:Nitriles; D010940:Plant Proteins; C430593:cyanohydrin; D000446:Aldehyde-Lyases; C087495:acetone-cyanohydrin lyase