Cloning, expression, and characterization of coenzyme-B₁₂-dependent diol dehydratase from Lactobacillus diolivorans

• Published in: Biotechnology letters Vol. 36; no. 1; pp. 159 - 165
• Main Authors: Wei, Xuqin, Meng, Xiaolei, Chen, Yunlai, Wei, Yutuo, Du, Liqin, Huang, Ribo
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer-Verlag 2014; Springer Netherlands
• Subjects: Applied Microbiology; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; betaine; Biochemistry; Biomedical and Life Sciences; Biotechnology; catalytic activity; Cloning, Molecular; Cobamides; enzyme inactivation; Escherichia coli; glycerol; Glycerol - metabolism; Lactobacillus; Lactobacillus - enzymology; Lactobacillus - genetics; Life Sciences; Microbiology; molecular weight; Original Research Paper; Oxygen - metabolism; Propanediol Dehydratase - chemistry; Propanediol Dehydratase - genetics; Propanediol Dehydratase - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; sorbitol; Characterization; Diol dehydratase; Coenzyme B; Expression; dependent; Glycerol dehydratase
• ISSN: 0141-5492; 1573-6776; 1573-6776
• DOI: 10.1007/s10529-013-1346-8

The three gldCDE genes from Lactobacillus diolivorans, that encode the three subunits of the glycerol dehydratase, were cloned and the proteins were co-expressed in soluble form in Escherichia coli with added sorbitol and betaine hydrochloride. The purified enzyme exists as a heterohexamer (α₂β₂γ₂) structure with a native molecular mass of 210 kDa. It requires coenzyme B₁₂ for catalytic activity and is subject to suicide inactivation by glycerol during catalysis. The enzyme had maximum activity at pH 8.6 and 37 °C. The apparent K ₘ values for coenzyme B₁₂, 1,2-ethanediol, 1,2-propanediol, and glycerol were 1.5 μM, 10.5 mM, 1.3 mM, and 5.8 mM, respectively. Together, these results indicated that the three genes gldCDE encoding the proteins make up a coenzyme B₁₂-dependent diol dehydratase and not a glycerol dehydratase.