title: Process development for oxidations of hydrophobic compounds applying cytochrome P450 monooxygenases in-vitro.
abstract: Cytochrome P450 monooxygenases are a unique family of enzymes that are able to catalyze regio- and stereospecific oxidations for a broad substrate range. However, due to limited enzyme activities and stabilities, hydrophobicity of substrates, as well as the necessity of a continuous electron and oxygen supply the implementation of P450s for industrial processes remains challenging. Aim of this study was to point out key aspects for the development of an efficient synthesis concept for cytochrome P450 catalyzed oxidations. In order to regenerate the natural cofactor NADPH, a glucose dehydrogenase was applied. The low water soluble terpene α-ionone was used as substrate for the model reaction system. The studies reveal that an addition of surfactants in combination with low volumetric amounts of co-solvent can significantly increase substrate availability and reaction rates. Furthermore, these additives facilitated a reliable sampling procedure during the process. Another key factor for the process design was the oxygen supply. Based on various investigations, a bubble-aerated stirred tank reactor in batch mode represents a promising reactor concept for P450 oxidations. Main restriction of the investigated reaction system was the low process stability of the P450 monooxygenase, characterized by maximum total turnover numbers of ∼4100molα-ionone/molP450.
authors.affiliation: Hamburg University of Technology, Institute of Technical Biocatalysis, Denickestr. 15, 21073 Hamburg, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Brummund
authors.affiliation: DSM Chemical Technology R&D B.V., Urmonderbaan 22, P.O. Box 18, 6160 MD Geleen, The Netherlands.
authors.forename: Monika
authors.initials: M
authors.lastname: Müller
authors.affiliation: DSM Chemical Technology R&D B.V., Urmonderbaan 22, P.O. Box 18, 6160 MD Geleen, The Netherlands.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schmitges
authors.affiliation: DSM Chemical Technology R&D B.V., Urmonderbaan 22, P.O. Box 18, 6160 MD Geleen, The Netherlands.
authors.forename: Iwona
authors.initials: I
authors.lastname: Kaluzna
authors.affiliation: DSM Chemical Technology R&D B.V., Urmonderbaan 22, P.O. Box 18, 6160 MD Geleen, The Netherlands.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mink
authors.affiliation: Hamburg University of Technology, Institute of Technical Biocatalysis, Denickestr. 15, 21073 Hamburg, Germany.
authors.forename: Lutz
authors.initials: L
authors.lastname: Hilterhaus
authors.affiliation: Hamburg University of Technology, Institute of Technical Biocatalysis, Denickestr. 15, 21073 Hamburg, Germany. Electronic address: liese@tuhh.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Liese
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D060847:Metabolic Engineering / Q000379:methods*; D008954:Models, Biological; D009249:NADP / Q000378:metabolism; D045792:Norisoprenoids / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D045792:Norisoprenoids; D011994:Recombinant Proteins; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; C011879:alpha-ionone; D010100:Oxygen
keywords: Biocatalytic oxidations; Cofactor regeneration; Cytochrome P450 monooxygenases; Hydrophobic substrates; Oxidative enzyme stability; Oxygen supply