Betaine aldehyde chloride

Betaine aldehyde chloride, the diethylacetal of APAL and NAD⁺ were obtained from Sigma-Aldrich Química, S.A. de C.V. (Toluca, México). APAL was freshly prepared by hydrolyzing its diethylacetal form as described by Flores and Filner [49 (link)]. The exact concentration of the resulting free APAL was determined in each experiment by the amount of NADH produced after its complete oxidation in the reaction catalyzed by SoBADH in the presence of an excess of NAD⁺.

Betaine aldehyde chloride was obtained from Sigma-Aldrich (St Louis, MO). The diethylacetals of AB-ald and AP-ald were obtained from Sigma-Aldrich and Tokyo Kasei Kogyo (Tokyo), respectively. TMAB-ald and TMAP-ald were distributed by Professor Nobuhiro Mori (Tottori University, Japan). They were hydrolyzed as described previously (Fujiwara et al. 2008 (link)) and neutralized by adding equivalent volume of KOH. Aldehyde dehydrogenase activities were determined as described previously (Fujiwara et al. 2008 (link)). One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 μmol of NADH per min.
The effect of NaCl on the recombinant BADHs was determined by incubating purified enzymes (2 μg) in 50 mM HEPES buffer (pH 8.0), 0.5 mM NAD⁺ and the indicated concentrations of NaCl for 5 min at 30°C. After pre-incubation, 5 mM betaine aldehyde was added and the activity was measured.