Properties of a 2,3-butanediol dehydrogenase from taiwanofungus camphorata

2,3-Butanediol dehydrogenase (Bdh) plays important roles in reduction of acetoin to 2,3-butanediol, an important platform chemical with many industrial applications. Here, a TcBdh cDNA (1348 bp, GenBank accession JF896462) encoding a putative Bdh was cloned from Taiwanofungus camphorata. The deduced amino acid sequence is similar to the Bdhs from other species. A 3-D structural model of TcBdh has been constructed based on the known structure of Pseudomonas putida formaldehyde dehydrogenase (PpFdh, PDB code 1KOL). To characterize the TcBdh protein, the coding region was subcloned into an expression vector pYEX-S1 and transformed into Saccharomyces cerevisiae. The recombinant His6-tagged TcBdh was expressed and purified by Ni²⁺-nitrilotriacetic acid Sepharose. The purified enzyme showed a single band of 49 kDa on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Michaelis constant (K_M) value of the recombinant enzyme for acetoin was 8.5 mM. The enzyme's optical pH was 6. The thermal inactivation of the enzyme showed a half-life of 5.3 min at 45°C.