Purification of acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli

Journal Article · Wed Oct 31 23:00:00 EST 1990 · Applied and Environmental Microbiology; (USA)

OSTI ID:5922022

Petersen, D J; Bennett, G N ^[1]

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1. Rice Univ., Houston, TX (USA)

In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone. We report here the purification of the enzyme from C. acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein. Phage DNA from positive plaques was analyzed by Southern hybridization. Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an {approximately}2.1-kb EcoRI/BglII fragment. A polypeptide with a molecular weight of {approximately}28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. Although the expression of the gene is tightly regulated in C. acetobutylicum, it was well expressed in E. coli, although from a promoter sequence of clostridial origin.

• OSTI ID: 5922022
• Journal Information: Applied and Environmental Microbiology; (USA), Journal Name: Applied and Environmental Microbiology; (USA) Vol. 56:11; ISSN 0099-2240; ISSN AEMID
• Country of Publication: United States
• Language: English

Related Subjects

550400* -- Genetics
59 BASIC BIOLOGICAL SCIENCES
ACETONE
BACTERIA
BACTERIOPHAGES
BIOCONVERSION
CARBON-CARBON LYASES
CARBOXY-LYASES
CLONING
CLOSTRIDIUM
DNA
DNA HYBRIDIZATION
DNA-CLONING
ENZYMES
ESCHERICHIA COLI
FERMENTATION
GENETIC ENGINEERING
GENETIC MAPPING
HYBRIDIZATION
KETONES
LYASES
MAPPING
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PARASITES
PURIFICATION
RECOMBINANT DNA
VIRUSES