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Characterization of Aldehyde Dehydrogenases Applying an Enzyme Assay with In Situ Formation of Phenylacetaldehydes

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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−1 protein) 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−1 protein). 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.

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Abbreviations

adh/ADH:

Gene of/gene product aldehyde dehydrogenase

adh-/ADH-CWB2:

Gene of/gene product aldehyde dehydrogenase of Gordonia rubripertincta CWB2

ADH-Sc:

Aldehyde dehydrogenase of Saccharomyces cerevisiae

AlDH-Sc:

Alcohol dehydrogenase of Saccharomyces cerevisiae

DH:

Dehydrogenases

feaB/FeaB:

Gene of/gene product phenylacetaldehyde dehydrogenase

feaB-/FeaB-K-12:

Gene of/gene product phenylacetaldehyde dehydrogenase of Escherichia coli K-12

PAD:

Phenylacetaldehyde dehydrogenase

PMS:

Phenazine methosulfate

SOI:

Styrene oxide isomerase of Rhodococcus opacus 1CP

styC :

Gene of styrene oxide isomerase of Rhodococcus opacus 1CP

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Acknowledgments

Juliane Zimmerling and Michel Oelschlägel were supported by fellowships from the Deutsche Bundesstiftung Umwelt and Dirk Tischler by a grant from the European Social Fund and the Saxonian Government (GETGEOWEB: 100101363).

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  1. Interdisciplinary Ecological Center, Environmental Microbiology Group, TU Bergakademie Freiberg, Leipziger Str. 29, 09599, Freiberg, Germany

    Juliane Zimmerling, Dirk Tischler, Carolin Großmann, Michael Schlömann & Michel Oelschlägel

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  1. Juliane Zimmerling
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Correspondence to Juliane Zimmerling or Dirk Tischler.

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Zimmerling, J., Tischler, D., Großmann, C. et al. Characterization of Aldehyde Dehydrogenases Applying an Enzyme Assay with In Situ Formation of Phenylacetaldehydes. Appl Biochem Biotechnol 182, 1095–1107 (2017). https://doi.org/10.1007/s12010-016-2384-1

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