title: Characterization of Aldehyde Dehydrogenases Applying an Enzyme Assay with In Situ Formation of Phenylacetaldehydes.
abstract: Herein, different dehydrogenases (DH) were characterized by applying a novel two-step enzyme assay. We focused on the NAD(P)+-dependent phenylacetaldehyde dehydrogenases because they produce industrially relevant phenylacetic acids, but they are not well studied due to limited substrate availability. The first assay step comprises a styrene oxide isomerase (440 U mg-1protein) which allows the production of pure phenylacetaldehydes (>70 mmol L-1) from commercially available styrene oxides. Thereafter, a DH of interest can be added to convert phenylacetaldehydes in a broad concentration range (0.05 to 1.25 mmol L-1). DH activity can be determined spectrophotometrically by following cofactor reduction or alternatively by RP-HPLC. This assay allowed the comparison of four aldehyde dehydrogenases and even of an alcohol dehydrogenase with respect to the production of phenylacetic acids (up to 8.4 U mg-1protein). FeaB derived from Escherichia coli K-12 was characterized in more detail, and for the first time, substituted phenylacetaldehydes had been converted. With this enzyme assay, characterization of dehydrogenases is possible although the substrates are not commercially available in sufficient quality but enzymatically producible. The advantages of this assay in comparison to the former one are discussed.
authors.affiliation: Interdisciplinary Ecological Center, Environmental Microbiology Group, TU Bergakademie Freiberg, Leipziger Str. 29, 09599, Freiberg, Germany. juliane.zimmerling@ioez.tu-freiberg.de.
authors.forename: Juliane
authors.initials: J
authors.lastname: Zimmerling
authors.affiliation: Interdisciplinary Ecological Center, Environmental Microbiology Group, TU Bergakademie Freiberg, Leipziger Str. 29, 09599, Freiberg, Germany. dirk.tischler@ioez.tu-freiberg.de.
authors.forename: Dirk
authors.initials: D
authors.lastname: Tischler
authors.affiliation: Interdisciplinary Ecological Center, Environmental Microbiology Group, TU Bergakademie Freiberg, Leipziger Str. 29, 09599, Freiberg, Germany.
authors.forename: Carolin
authors.initials: C
authors.lastname: Großmann
authors.affiliation: Interdisciplinary Ecological Center, Environmental Microbiology Group, TU Bergakademie Freiberg, Leipziger Str. 29, 09599, Freiberg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schlömann
authors.affiliation: Interdisciplinary Ecological Center, Environmental Microbiology Group, TU Bergakademie Freiberg, Leipziger Str. 29, 09599, Freiberg, Germany.
authors.forename: Michel
authors.initials: M
authors.lastname: Oelschlägel
mesh_terms: D000079:Acetaldehyde / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D000445:Aldehyde Oxidoreductases / Q000737:chemistry*; D048168:Escherichia coli K12 / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D010648:Phenylacetates / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; D010648:Phenylacetates; D000445:Aldehyde Oxidoreductases; C053855:phenylacetaldehyde dehydrogenase; C025136:phenylacetic acid; D000079:Acetaldehyde; C013192:phenylacetaldehyde
keywords: In situ production of aromatic aldehydes; Isomerization; NAD(P)H formation; Phenylacetaldehyde dehydrogenase; Styrene oxide isomerase