Cloning of Clostridium acetobutylicum ATCC 824 acetyl coenzyme A acetyltransferase (thiolase; EC 2. 3. 1. 9) gene

Journal Article · Sun Sep 01 00:00:00 EDT 1991 · Applied and Environmental Microbiology; (United States)

OSTI ID:5910085

Petersen, D J; Bennett, G N ^[1]

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1. Rice Univ., Houston, TX (United States)

Thiolase (acetyl coenzyme A acetyltransferase; EC 2.3.1.9) from Clostridium acetobutylicum is a key enzyme in the production of acid and solvents in this organism. The purification and properties of the enzyme have already been described. The thl gene encoding the thiolase has been cloned by using primary antibodies raised to the purified enzyme. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was prepared and screened by immunoblots with the antithiolase antibodies. Phage DNA was purified from positive plaques, and restriction enzyme digests identified an {approximately} 4.8-kb AccI fragment common to all positive plaques. A corresponding fragment was also found in AccI digests of C. acetobutylicum chromosomal DNA. The fragment was purified and EcoRI linkers were attached before subcloned into pUC19. Maxicell analysis showed the production of an {approximately} 42-kDa protein, whose size corresponded to the molecular size of the purified thiolase, from the clostridial insert. Enzyme activity assays and Western blot (immunoblot) analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell extracts of Escherichia coli harboring the cloned thl confirmed the presence of the thiolase encoded within the cloned DNA.

• OSTI ID: 5910085
• Journal Information: Applied and Environmental Microbiology; (United States), Journal Name: Applied and Environmental Microbiology; (United States) Vol. 57:9; ISSN 0099-2240; ISSN AEMID
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BACTERIOPHAGES
CLONING
CLOSTRIDIUM
CLOSTRIDIUM ACETOBUTYLICUM
DNA
DNA HYBRIDIZATION
DNA-CLONING
ENZYME ACTIVITY
ENZYMES
GENES
HYBRIDIZATION
METHANOGENIC BACTERIA
MICROORGANISMS
MOLECULAR BIOLOGY
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PARASITES
PROTEINS
TRANSFERASES
VIRUSES