Purification and characterization of the extracellular. alpha. -amylase from Clostridium acetobutylicum ATCC 824

Journal Article · Tue Jan 01 00:00:00 EST 1991 · Applied and Environmental Microbiology; (USA)

OSTI ID:5638300

Paquet, V; Croux, C; Goma, G; Soucaille, P ^[1]

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1. Centre National de la Recherche Scientifique Unite Associee, Toulouse (France)

The extracellular {alpha}-amylase (1,4-{alpha}-D-glucanglucanohydrolase; EC 3.2.1.1) from Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography (Mono Q) and gel filtration (Superose 12). The enzyme had an isoelectric point of 4.7 and a molecular weight of 84,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a monomeric protein, the 19-amino-acid N terminus of which displayed 42% homology with the Bacillus subtilis saccharifying {alpha}-amylase. The amino acid composition of the enzyme showed a high number of acidic and hydrophobic residues and only one cysteine residue per mole. The activity of the {alpha}-amylase was not stimulated by calcium ions (or other metal ions) or inhibited by EDTA, although the enzyme contained seven calcium atoms per molecule. {alpha}-Amylase activity on soluble starch was optimal at pH 5.6 and 45{degree}C. The {alpha}-amylase was stable at an acidic pH but very sensitive to thermal inactivation. It hydrolyzed soluble starch, with a K{sub m} of 3.6 g {center dot} liter{sup {minus}1} and a K{sub cat} of 122 mol of reducing sugars {center dot} s{sup {minus}1} {center dot} mol{sup {minus}1}. The {alpha}-amylase showed greater activity with high-molecular-weight substrates than with low-molecular-weight maltooligosaccharides, hydrolyzed glycogen and pullulan slowly, but did not hydrolyze dextran or cyclodextrins. The major end products of maltohexaose degradation were glucose, maltose, and maltotriose; maltotetraose and maltopentaose were formed as intermediate products. Twenty seven percent of the glucoamylase activity generally detected in the culture supernatant of C. acetobutylicum can be attributed to the {alpha}-amylase.

• OSTI ID: 5638300
• Journal Information: Applied and Environmental Microbiology; (USA), Vol. 57:1; ISSN 0099-2240
• Country of Publication: United States
• Language: English

Related Subjects

09 BIOMASS FUELS
59 BASIC BIOLOGICAL SCIENCES
AMYLASE
FRACTIONATION
CLOSTRIDIUM ACETOBUTYLICUM
BIOCHEMICAL REACTION KINETICS
ENZYME ACTIVITY
AMINO ACID SEQUENCE
BACILLUS
EDTA
ELECTROPHORESIS
ION EXCHANGE CHROMATOGRAPHY
OLIGOSACCHARIDES
AMINO ACIDS
BACTERIA
CARBOHYDRATES
CARBOXYLIC ACIDS
CHELATING AGENTS
CHROMATOGRAPHY
CLOSTRIDIUM
ENZYMES
GLYCOSYL HYDROLASES
HYDROLASES
KINETICS
METHANOGENIC BACTERIA
MICROORGANISMS
MOLECULAR STRUCTURE
O-GLYCOSYL HYDROLASES
ORGANIC ACIDS
ORGANIC COMPOUNDS
REACTION KINETICS
SACCHARIDES
SEPARATION PROCESSES
090900* - Biomass Fuels- Processing- (1990-)
550200 - Biochemistry