Gene cloning, expression and characterization of 2,3-butanediol dehydrogenase from Taiwanofungus camphorata
| Autor: | Wei-Wei Tsai, 蔡薇薇 |
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| Rok vydání: | 2012 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 100 The purpose of this thesis is to study NAD(P)H-dependent 2,3-butanediol dehydrogenase (2,3-Bdh) from Taiwanofungus camphorata, a medium chain dehydrogenase/reductase (MDR) is the main enzyme catalyzing the reduction of acetoin to 2,3-butanediol. It contains an open reading frame of 1219 bp which encodes a protein of 408 amino acid, molecular mass is 49.3 kDa and has the highly conserved GXGXXG sequence found in the MDR family and residues found in the coenzyme-binding pocket. To characterize the 2,3-Bdh, the coding region of Bdh was introduced into an expression vector pET-20b(+), and transform into Escherichia coli C43 (DE3) and S. cerevisiae. The purified enzyme showed two bands on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited TcBdh activity via acetoin assay. The Michaelis constant (Km) values for acetoin was 8.46 mM. The enzyme has a half-life of 5.3 min at 45℃ with a thermal inactivation rate constant kd of 3.5 × 10-2 min-1. The enzyme was active in pH 6. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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