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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 ( KM) 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. [ABSTRACT FROM AUTHOR]

Ascorbate peroxidase (Apx) plays important roles both as a reductant and as a H2O2 scavenger via ascorbate (AsA). In this paper, we discuss how a ClApx cDNA (1,068 bp, GQ465430) encoding a putative Apx was cloned from lemon (Citrus limon). The deduced amino acid sequence is similar to the Apxes from other plant species. A 3-D structural model of ClApx was constructed based on the crystal structure of Pisum sativum Apx (PDB code 1APX). To characterize the ClApx protein, the coding region was subcloned into an expression vector pYEX-S1 and transformed into Saccharomyces cerevisiae. The recombinant His6-tagged ClApx was overexpressed and purified by Ni2+-nitrilotriacetic acid Sepharose. The purified enzyme showed two prominent bands on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Michaelis constant (KM) values of the recombinant enzyme for AsA and H2O2 were 0.40 and 0.11 mM, respectively. The enzyme was active from pH range 6 to 8. The thermal inactivation of the enzyme showed a half-life of 6.5 min at 45°C, and its inactivation rate constant Ki was 1.1 × 10-1 min-1. The enzyme retained 35% activity after chymotrypsin digestion at pH 8 and 37°C for 40 min. [ABSTRACT FROM AUTHOR]