title: Engineering Cyclohexanone Monooxygenase for the Production of Methyl Propanoate.
abstract: A previous study showed that cyclohexanone monooxygenase from Acinetobacter calcoaceticus (AcCHMO) catalyzes the Baeyer-Villiger oxidation of 2-butanone, yielding ethyl acetate and methyl propanoate as products. Methyl propanoate is of industrial interest as a precursor of acrylic plastic. Here, various residues near the substrate and NADP+binding sites in AcCHMO were subjected to saturation mutagenesis to enhance both the activity on 2-butanone and the regioselectivity toward methyl propanoate. The resulting libraries were screened using whole cell biotransformations, and headspace gas chromatography-mass spectrometry was used to identify improved AcCHMO variants. This revealed that the I491A AcCHMO mutant exhibits a significant improvement over the wild type enzyme in the desired regioselectivity using 2-butanone as a substrate (40% vs 26% methyl propanoate, respectively). Another interesting mutant is the T56S AcCHMO mutant, which exhibits a higher conversion yield (92%) and kcat(0.5 s-1) than wild type AcCHMO (52% and 0.3 s-1, respectively). Interestingly, the uncoupling rate for the T56S AcCHMO mutant is also significantly lower than that for the wild type enzyme. The T56S/I491A double mutant combined the beneficial effects of both mutations leading to higher conversion and improved regioselectivity. This study shows that even for a relatively small aliphatic substrate (2-butanone), catalytic efficiency and regioselectivity can be tuned by structure-inspired enzyme engineering.
authors.affiliation: Molecular Enzymology Group, University of Groningen , Nijenborgh 4, 9747AG Groningen, The Netherlands.
authors.forename: Hugo L
authors.initials: HL
authors.lastname: van Beek
authors.affiliation: Molecular Enzymology Group, University of Groningen , Nijenborgh 4, 9747AG Groningen, The Netherlands.
authors.forename: Elvira
authors.initials: E
authors.lastname: Romero
authors.affiliation: Molecular Enzymology Group, University of Groningen , Nijenborgh 4, 9747AG Groningen, The Netherlands.
authors.forename: Marco W
authors.identifier: 0000-0001-6346-5014
authors.initials: MW
authors.lastname: Fraaije
mesh_terms: D016954:Acinetobacter calcoaceticus / Q000201:enzymology; D002074:Butanones / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism*; D011422:Propionates / Q000378:metabolism*; D015202:Protein Engineering; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature
chemical_list: D002074:Butanones; D011422:Propionates; D011993:Recombinant Fusion Proteins; C005222:methylethyl ketone; D006861:Hydrogen Peroxide; D010105:Oxygenases; C019961:cyclohexanone oxygenase; D009247:NADH, NADPH Oxidoreductases; C429539:NAD phosphite oxidoreductase