Computational protocol: Cloning, expression and characterization of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium strain BKM-F-1767

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Protocol publication

[…] Enzymatic activity of Pc Aad1p was determined spectrophotometrically using an Agilent HP 8453 UV-visible spectrophotometer (Agilent Technologies, Massy, France). Unless otherwise specified, all assays were carried out at 30°C in 1 mL reaction mixtures using 1 cm optical path length microcuvettes. Reactions were initiated by substrate addition and were monitored by recording the absorption at 355 nm. At this wavelength, the molar extinction coefficients of the substrate compounds could be considered as negligible (less than 4%) compared to that of NAD(P)H (ε355 = 5.12 The effect of pH was studied at 30°C, using 25 mM MES (pH 5.5 − 6.4), 50 mM HEPES (pH 6.9 − 8.2), 25 mM Tris–HCl (pH 8.8) or 100 mM Glycine-KOH (pH 9.0 − 10.7) as buffers. The temperature dependence was evaluated in 50 mM MES buffer (pH 6.1) in the presence of 0.2 mM 3,4-Dimethoxybenzaldehyde and 0.2 mM NADPH and the reaction was started by adding 9.0 μg of the enzyme. The substrate specificity towards a range of substrates (Table ) and the kinetic parameters determinations (Table ) were determined in 50 mM MES buffer (pH 6.1) using 0.3 mM NADPH and 1 mM substrate in the reduction sense, or in 100 mM Glycine-KOH buffer (pH 10.3) using 0.3 mM NADP+ and 10 mM substrate (except for Octanol where 1 mM was used, and for 2-Chlorobenzyl alcohol and 4-Chlorobenzyl alcohol where 3 mM were used) for the oxidation sense. The specific activity towards 3,4-Dimethoxybenzaldehyde (5.1 μmol·min-1·mg-1) and to 3,4-Dimethoxybenzyl alcohol (2.0 μmol·min-1·mg-1) were taken as 100% for the reduction and oxidation reactions, respectively (Table ).The kinetic parameters KM, kcat and Ki for aldehyde and alcohol substrates (Table ) were computed by fitting initial reaction rates, measured as a function of substrate concentration, to the Michaelis-Menten equation (Equation 1) or, when substrate inhibition was observed, to the uncompetitive substrate inhibition equation (Equation 2) with the non-linear regression Enzyme Kinetics 1.3 module of the SigmaPlot 11.0 package (Systat Software, IL, USA): (1) V = V max S / K M + S (2) V = V max S / K M + S + S 2 / K i where V represents the reaction rate, Vmax is the limiting reaction rate, S is the substrate concentration, KM is the Michaelis constant and Ki is the substrate inhibition constant. The catalytic constant kcat of the enzyme for the different substrates was derived from kcat=Vmax/E. The total enzyme concentration [E] was evaluated using a protein molecular mass of 74.2 kDa. The enzyme kinetic parameters for NAD(P)H and NAD(P)+ + were determined with 0.2 mM 3,4-Dimethoxybenzaldehyde and 10 mM 3,4-Dimethoxybenzyl alcohol, respectively. Results are the mean ± SEM from at least three separate experiments. […]

Pipeline specifications

Software tools EMBOSS, SigmaPlot
Application Miscellaneous
Organisms Escherichia coli
Chemicals Alcohols, Aldehydes, Carbon Dioxide, NAD, NADP