Enzyme Activity Assays

To measure 2,3-butanediol dehydrogenase activities in B. licheniformis MW3 and B. licheniformis MW3 (ΔbudCΔgdh), the cells of the strains were grown for 12 h, harvested, washed twice and resuspended in 67 mM phosphate buffer (pH 7.4), and disrupted by sonication with a Sonics sonicator (500 W; 20 kHz). Cell debris was removed through centrifugation (12,000 g, 15 min) and the resulting supernatants were used as the crude extracts for enzyme activity assays.

Activities of 2,3-butanediol dehydrogenase were spectrophotometrically assayed by measuring the change in absorbance at 340 nm corresponding to the oxidation of NADH or reduction of NAD⁺ at 30°C using a UV/visible spectrophotometer (Ultrospec 2100 pro, Amersham Biosciences, United States). For the reduction reaction, the reaction solution contains 0.2 mM of NADH and 5 mM of acetoin or diacetyl in 67 mM phosphate buffer (pH 7.4). For oxidation reactions, the reaction solution contains 10 mM (2R,3R)-2,3-butanediol, (2S,3S)-2,3-butanediol, and meso-2,3-butanediol and 1 mM NAD⁺ in 67 mM phosphate buffer (pH 7.4). One unit of activity was defined as the amount of enzyme that consumed or formed 1 μmol of NADH per min. The protein concentration in crude extract was measured by the Lowry method, with bovine serum albumin as the standard (Hartree, 1972).