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) which allows the production of pure phenylacetaldehydes (>70 mmol L) 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). 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). 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.

On the Enigma of Glutathione-Dependent Styrene Degradation in Gordonia rubripertincta CWB2

“…The 1-ml assay mixture contained 0.5 mM phenylacetaldehyde or 2-phenylethanol in 10 mM Tris-HCl (pH 7.5), 1 mM NAD ϩ , and 50 l of protein-containing sample, respectively. Recombinant PAD activity was assayed as previously described (61).…”

“…Expression of GCWB2_21620 did not yield active protein, as already mentioned. Preparation of StyD and AldH1 was done according to the method of Zimmerling et al(61).…”

A Review: The Styrene Metabolizing Cascade of Side-Chain Oxygenation as Biotechnological Basis to Gain Various Valuable Compounds

“…This is also the reason for a widespread occurrence of PAD genes in many organisms, but only a few studies are available about this enzyme class ( Tischler, 2015 ). Until now, only a few microbial PADs have been investigated including these of P. fluorescens ST ( Beltrametti et al, 1997 ), Pseudomonas putida S12 ( Crabo et al, 2017 ), E. coli K-12 ( Parrott et al, 1987 ; Ferrández et al, 1997 ; Hanlon et al, 1997 ; Zimmerling et al, 2017 ), Arthrobacter globisformis ( Shimizu et al, 1993 ), Xanthobacter sp. 124X ( Hartmans et al, 1989 ), Rhodococcus rhodochrous ( Hartmans et al, 1990 ) and the yeast-like fungus Exophiala jeanselmei ( Cox et al, 1996 ).…”

“…Focused on the above mentioned PADs, the substrate spectrum of these previously studied PAD enzymes is restricted on differently substituted phenylacetaldehydes like 4-chloro-, 4-fluoro, 4-hydroxy- und 3,4-dihydroxy phenylacetaldehyde – similar as described for SOIs ( Ferrández et al, 1997 ; Hanlon et al, 1997 ; Rodríguez-Zavala et al, 2006 ; Zimmerling et al, 2017 ). Between these enzymes, different cofactor dependencies were revealed.…”

“…The E. coli and fungus enzymes prefer strongly NAD + ( Ferrández et al, 1997 ; Rodríguez-Zavala et al, 2006 ; Zimmerling et al, 2017 ), whereas the PAD of Arthrobacter globisformis showed only 2% activity using NAD + instead of NADP + ( Shimizu et al, 1993 ). Due to problems concerning the purity, availability and maximum applicable concentrations of the phenylacetaldehydes during the studies dealing with FeaB of E. coli K-12 ( Hanlon et al, 1997 ; Ferrández et al, 1997 ), an enhanced assay has been reported which allows the in-situ -production of phenylacetaldehydes by using the well characterized and highly active SOI of R. opacus 1CP and styrene oxides as substrates ( Zimmerling et al, 2017 ). This assay serves as distinguished approach for the characterization for phenylacetaldehyde metabolizing enzymes and opens new possibilities for further studies on PADs and related dehydrogenases.…”