Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1997-02-27
1999-06-01
Prouty, Rebecca E.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
435190, C07H 2104, C12N 904
Patent
active
059089245
ABSTRACT:
The adhB gene encoding Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (2.degree. Adh) was cloned, sequenced, and expressed in Escherichia coli. The 1056 bp gene encodes a homotetrameric recombinant enzyme consisting of 37.7 kDa subunits. The purified recombinant enzyme is optimally active above 90.degree. C. with a half-life of approximately 1.7 h at 90.degree. C. An NADP(H)-dependent enzyme, the recombinant 2.degree. Adh has 1400-fold greater catalytic efficiency in propan-2-ol versus ethanol oxidation. The enzyme was inactivated by chemical modification using dithionitrobenzoate (DTNB) and diethylpyrocarbonate, indicating that Cys and His residues are involved in catalysis. Zinc was the only metal enhancing 2.degree. Adh reactivation after DTNB modification, implicating the involvement of a strongly bound zinc in catalysis. Arrhenius plots for the oxidation of propan-2-ol by the native and recombinant 2.degree. Adhs were linear from 25.degree. C. to 90.degree. C. when the enzymes were incubated at 55.degree. C. prior to assay. Discontinuities in the Arrhenius plots for propan-2-ol and ethanol oxidations were observed, however, when the enzymes were preincubated at 0.degree. C. or 25.degree. C. The observed Arrhenius discontinuity, therefore, resulted from a temperature dependent, catalytically significant 2.degree. Adh structural change. Hydrophobic Cluster Analysis comparisons of both mesophilic versus thermophilic 2.degree. Adh and 1.degree. versus 2.degree. Adh amino acid sequences were performed. These comparisons predicted that specific proline residues may contribute to 2.degree. Adh thermostability and thermophilicity, and that the catalytic Zn ligands are different in 1.degree. Adhs (two Cys and a His) and 2.degree. Adhs (Cys, His, and Asp).
REFERENCES:
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Burdette, Douglas, et al., "Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2.degree. Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase," Biochem. J., 302:163-170 (1994).
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Burdette Douglas S.
Zeikus Joseph G.
Michigan State University
Prouty Rebecca E.
Stole Einar
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