The activity was measured by decreasing the A340 nm based on the transformation of vanillin to vanillic acid. Influence on latent absorbance by the production of NAD(P)H was adjusted by molar extinction coefficients. Calculations were based on molar extinction coefficients
of vanillin (10.6 × 103; pH 7) and NAD(P)H (6.30 × 103) at 340 nm. The reaction mixture comprised 100 mM KPB, 5 mM NAD(P)+, and 0.25 mM vanillin at the final concentration. The other method investigated the enzyme’s substrate specificity and the effect of pH or temperature on it. AC220 Enzyme activity was determined by quantification of corresponding oxidized products from aromatic aldehydes by HPLC as described above. The reaction mixture (1.0 mL) comprised 100 mM appropriate buffer, 5 mM NAD(P)+, 2.5 mM substrates, and 0.23 and
0.26 U of purified VDH from Micrococcus sp. TA1 and B. cepacia TM1, respectively. The reaction mixture was incubated for 20 min at an appropriate temperature, after which the reaction was stopped by adding 0.2 mL of Lapatinib manufacturer 0.5 N HCl. The acid produced was quantified by HPLC. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the transformation of 1 μmol of vanillin to vanillic acid. The enzyme from Micrococcus sp. TA1 that catalyzes the dehydrogenation of vanillin was purified from cells as follows: after cultivation on 2 g L−1 vanillin as a carbon source with each liquid medium as described in Materials and methods, cells were harvested by centrifugation (10 000 g, 10 min, 4 °C). The pellet was resuspended in 50 mM KPB (pH 7.5) and the suspension was treated 5-Fluoracil purchase with an ultrasonicator (201 M sonicator; Kubota, Tokyo, Japan) at 9 KHz and 170 W for 15 min; the utmost care was taken to maintain the temperature below 5 °C. The lysate was then centrifuged at 16 000 g for 20 min. The resulting supernatant was used as the cell extract. The cell extract dialyzed against 10 mM Tris-HCl buffer (pH 8.5) was applied to a 100 mL DEAE-Sepharose FF column (GE Healthcare Bio-Science, Buckinghamshire, UK). The enzyme was eluted with a
linear gradient of increasing NaCl concentration (0–0.5 M, total volume 1.5 L). The fractions with VDH activity were pooled. NaCl (2 M) was added with gentle stirring to the pooled fraction, and this solution was applied to a 30-mL butyl-Sepharose column (GE Healthcare Bio-Science). The enzyme was eluted using a gradient of decreasing NaCl concentration (2–0 M, total volume 600 mL). The active fractions were dialyzed against 10 mM Tris-HCl buffer (pH 8.5). The dialyzed enzyme solution was applied to a 1-mL Resource Q column (GE Healthcare Bio-Science). The enzyme was eluted with a linear gradient of increasing NaCl concentration (0–0.5 M, total volume 100 mL). The enzyme from B. cepacia TM1 was purified from the cell extract as follows: the cell extract of B.