title: Benzoylformate decarboxylase from Pseudomonas putida as stable catalyst for the synthesis of chiral 2-hydroxy ketones.
abstract: The thiamin diphosphate- and Mg2+-dependent enzyme benzoylformate decarboxylase (BFD) from Pseudomonas putida was characterized with respect to its suitability to catalyze the formation of chiral 2-hydroxy ketones in a benzoin-condensation type reaction. Carboligation constitutes a side reaction of BFD, whereas the predominant physiological task of the enzyme is the non-oxidative decarboxylation of benzoylformate. For this purpose the enzyme was obtained in sufficient purity from Pseudomonas putida cells in a one-step purification using anion-exchange chromatography. To facilitate the access to pure BFD for kinetical studies, stability investigations, and synthetical applications, the coding gene was cloned into a vector allowing the expression of a hexahistidine fusion protein. The recombinant enzyme shows distinct activity maxima for the decarboxylation and the carboligation beside a pronounced stability in a broad pH and temperature range. The enzyme accepts a wide range of donor aldehyde substrates which are ligated to acetaldehyde as an acceptor in mostly high optical purities. The enantioselectivity of the carboligation was found to be a function of the reaction temperature, the substitution pattern of the donor aldehyde and, most significantly, of the concentration of the donor aldehyde substrate. Our data are consistent with a mechanistical model based on the X-ray crystallographic data of BFD. Furthermore we present a simple way to increase the enantiomeric excess of (S)-2-hydroxy-1-phenyl-propanone from 90% to 95% by skillful choice of the reaction parameters. Enzymatic synthesis with BFD are performed best in a continuously operated enzyme membrane reactor. Thus, we have established a new enzyme tool comprising a vast applicability for stereoselective synthesis.
authors.affiliation: Institut für Enzymtechnologie, Heinrich-Heine Universität Düsseldorf im Forschungszentrum Jülich, Jülich, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Iding
authors.forename: T
authors.initials: T
authors.lastname: Dünnwald
authors.forename: L
authors.initials: L
authors.lastname: Greiner
authors.forename: A
authors.initials: A
authors.lastname: Liese
authors.forename: M
authors.initials: M
authors.lastname: Müller
authors.forename: P
authors.initials: P
authors.lastname: Siegert
authors.forename: J
authors.initials: J
authors.lastname: Grötzinger
authors.forename: A S
authors.initials: AS
authors.lastname: Demir
authors.forename: M
authors.initials: M
authors.lastname: Pohl
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004795:Enzyme Stability; D007659:Ketones / Q000138:chemical synthesis*; D007700:Kinetics; D008969:Molecular Sequence Data; D016958:Pseudomonas putida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D004274:DNA, Recombinant; D007659:Ketones; D011994:Recombinant Proteins; D002262:Carboxy-Lyases; C019778:benzoylformate decarboxylase