Cloning, Expression, and Characterization of budC Gene Encoding meso-2,3-Butanediol Dehydrogenase from Bacillus licheniformis.
The budC gene encoding a meso-2,3-butanediol dehydrogenase (BlBDH) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli BL21(DE3). Sequence analysis reveals that this BlBDH belongs to short-chain dehydrogenase/reductase (SDR) superfamily. In the presence of NADH, BlBDH catalyzes the reduction of diacetyl to (3S)-acetoin (97.3% ee), and further to (2S,3S)-2,3-butanediol (97.3% ee and 96.5% de). Similar to other meso-2,3-BDHs, it shows oxidative activity to racemic 2,3-butanediol whereas no activity toward racemic acetoin in the presence of NAD(+). For diacetyl reduction and 2,3-butanediol oxidation, the pH optimum of BlBDH is 5.0 and 10.0, respectively. Unusually, it shows relatively high activity over a wide pH range from 5.0 to 8.0 for racemic acetoin reduction. BlBDH shows lower K m and higher catalytic efficiency toward racemic acetoin (K m = 0.47 mM, k cat /K m = 432 s(-1)·mM(-1)) when compared with 2,3-butanediol (K m = 7.25 mM, k cat /K m = 81.5 s(-1)·mM(-1)), indicating its physiological role in favor of reducing racemic acetoin into 2,3-butanediol. The enzymatic characterization of BlBDH provides evidence for the directed engineering of B. licheniformis for producing enantiopure 2,3-butanediol.
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
Guo-Chao
GC
Xu
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
Ya-Qian
YQ
Bian
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
Rui-Zhi
RZ
Han
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
Jin-Jun
JJ
Dong
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China. yni@jiangnan.edu.cn.
Ye
Y
Ni
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D000429:Alcohol Oxidoreductases; C019801:butanediol dehydrogenase
Bacillus licheniformis; Characterization; Expression; meso-2,3-Butanediol dehydrogenase