Purification and Characterization of a Microbial Dehydrogenase A Vanillin:NAD(P)⁺ Oxidoreductase

Abstract

Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native Mr value estimated by gel-filtration chromatography gave a value of 210,000. The enzyme made use of NAD+ less effectively than NADP+. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD+ or NADP+. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl2. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD+ and NADP+ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD+ or NADP+. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases.

Investigation of the Amycolatopsis sp. Strain ATCC 39116 Vanillin Dehydrogenase and Its Impact on the Biotechnical Production of Vanillin

“…This is in agreement with both the calculated molecular mass of the vdh translational product and the sizes of other enzymes exhibiting VDH activity (18,32,35,36). The recombinant protein carrying a His 6 tag was purified to apparent homogeneity, which was confirmed by immunodetection.…”

“…Besides the in silico analyses, the specific vanillin dehydrogenase activity in cells of Amycolatopsis sp. ATCC 39116 was investigated after cultivating the strain with or without vanillin and structurally related compounds like ferulic acid as the carbon source, since previous investigations with Pseudomonas showed an inducing effect (27,32). Amycolatopsis sp.…”

Molecular cloning and characterization of vanillin dehydrogenase from Streptomyces sp. NL15-2K

“…5b ), which indicated that VDH from NL15-2K is a tetramer. This was in agreement with previous reports that VDHs from P. fluorescens BTP9 [ 34 ] and Micrococcus sp. TA1 [ 35 ] are tetrameric enzymes.…”

Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum

“…The VDH enzyme assay was performed based on described methods 32 33 34 in 100mM potassium phosphate buffer (pH 7.0). The activity of vanillin dehydrogenase was monitored spectrophotometrically by measuring the rate of decrease in absorption at 340nm due to oxidation of NAD(P)H and was calculated with an extinction coefficient of 6,220M 1 cm 1 .…”

“…HR199 24 , VDH of Amycolatopsis sp. ATCC 39116 21 , VDH of Pseudomonas fluorescens 32 44 , aldehyde dehydrogenase (NAD + ) of Pseudonocardia sp. P1(ZP_08121488.1), benzaldehyde dehydrogenase (NAD + ) of Saccharopolyspora erythraea NRRL 2338(YP_001105347.1), benzaldehyde dehydrogenase (NAD + ) of Burkholderia cenocepacia AU 1054(YP_621190.1), and aldehyde dehydrogenase of Amycolatopsis mediterranei U32 (AMED_0881).…”