Background: 2R,3R-butanediol dehydrogenase (R-BDH) and other BDHs contribute to metabolism of 3R/3S-Acetoin (3R/3S-AC) and 2,3-butanediol (2,3-BD), which are important bulk chemicals used in different industries. R-BDH is responsible for oxidizing the hydroxyl group at their (R) configuration. Bacillus species is a promising producer of 3R/3S-AC and 2,3-BD. In this study, R-bdh gene encoding R-BDH from Bacillus sp. DL01 was isolated, expressed and identified.

Results: R-BDH exerted reducing activities towards Diacetyl (DA) and 3R/3S-AC using NADH, and oxidizing activities towards 2R,3R-BD and Meso-BD using NAD⁺, while no activity was detected with 2S,3S-BD. The R-BDH showed its activity at a wide range of temperature (25°C to 65°C) and pH (5.0-8.0). The R-BDH activity was increased significantly by Cd²⁺ when DA, 3R/3S-AC, and Meso-BD were used as substrates, while Fe²⁺ enhanced the activity remarkably at 2R,3R-BD oxidation. Kinetic parameters of the R-BDH from Bacillus sp. DL01 showed the lowest K_m, the highest V_max, and the highest K_cat towards the racemic 3R/3S-AC substrate, also displayed low K_m towards 2R,3R-BD and Meso-BD when compared with other reported R-BDHs.

Conclusions: The R-BDH from Bacillus sp. DL01 was characterized as a novel R-BDH with high enantioselectivity for R-configuration. It considered NAD⁺ and Zn²⁺ dependant enzyme, with a significant affinity towards 3R/3S-AC, 2R,3R-BD, and Meso-BD substrates. Thus, R-BDH is providing an approach to regulate the production of 3R/3S-AC or 2,3-BD from Bacillus sp. DL01.