Enzymology of acetone-butanol-isopropanol formation. Progress report, June 16, 1990--June 15, 1993
During the current project period, alcohol dehydrogenases (ADH) and acetoacetyl-CoA-reacting enzymes of C. beijerinckii were purified to homogeneity. Structural and catalytic properties of the purged enzymes were determined. A range of conditions was used to investigate the activity and stability of each enzyme. This information will facilitate the selection of differential assays and handling conditions for the unequivocal determination of the activity of a specific enzyme in crude extracts. In genetic studies, crude extracts are often the most practical material for the monitoring of specific enzyme activities. A selective assay is especially important when the relative levels of interfering enzymes change during physiological or genetic manipulations. The results from our study of ADH and aldehyde dehydrogenase (ALDH) demonstrate the technical difficulties associated with the measurement of these enzyme activities in crude extracts. First of all, because the two enzymes catalyze sequential reactions, the NAD(P)H-linked activities of ADH and ALDH in crude extracts are easily over or underestimated or masked. Secondly, the presence of multiple ADHs with overlapping coenzyme specificities in the same cell makes it difficult to assign the measured activity to a specific isozyme. Lastly, these enzymes are especially oxygen-sensitive in crude extracts, which, necessitates the use of good anaerobic techniques. We have determined the N-terminal amino acid sequence (the first 30-45 residues) the primary/secondary ADH the ALDH, the 3-hydroxybutyryl-CoA dehydrogenase, and both subunits of the CoA-transferase. The amino acid sequences will allow us to design oligonucleotide probes for the cloning of their structural genes and then to study the organization and regulation of these genes. Using this approach, we have cloned and sequenced the ADH gene encoding the primary/secondary ADH.
- Research Organization:
- Virginia Polytechnic Inst. and State Univ., Blacksburg, VA (United States)
- Sponsoring Organization:
- USDOE, Washington, DC (United States)
- DOE Contract Number:
- FG05-85ER13368
- OSTI ID:
- 10156463
- Report Number(s):
- DOE/ER/13368-10; ON: DE94013037; BR: KC0600000
- Resource Relation:
- Other Information: PBD: Jun 1993
- Country of Publication:
- United States
- Language:
- English
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