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Appl Environ Microbiol. Jan 1991; 57(1): 212–218.
PMCID: PMC182687

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

Abstract

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 degrees C. The alpha-amylase was stable at an acidic pH but very sensitive to thermal inactivation. It hydrolyzed soluble starch, with a Km of 3.6 g . liter-1 and a Kcat of 122 mol of reducing sugars . s-1 . mol-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.

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  • Allcock ER, Woods DR. Carboxymethyl cellulase and cellobiase production by Clostridium acetobutylicum in an industrial fermentation medium. Appl Environ Microbiol. 1981 Feb;41(2):539–541. [PMC free article] [PubMed]
  • Antranikian G, Herzberg C, Gottschalk G. Production of Thermostable alpha-Amylase, Pullulanase, and alpha-Glucosidase in Continuous Culture by a New Clostridium Isolate. Appl Environ Microbiol. 1987 Jul;53(7):1668–1673. [PMC free article] [PubMed]
  • Barton RR. A specific method for quantitative determination of glucose. Anal Biochem. 1966 Feb;14(2):258–260. [PubMed]
  • Buonocore V, Caporale C, De Rosa M, Gambacorta A. Stable, inducible thermoacidophilic alpha-amylase from Bacillus acidocaldarius. J Bacteriol. 1976 Nov;128(2):515–521. [PMC free article] [PubMed]
  • De Mot R, Verachtert H. Purification and Characterization of Extracellular Amylolytic Enzymes from the Yeast Filobasidium capsuligenum. Appl Environ Microbiol. 1985 Dec;50(6):1474–1482. [PMC free article] [PubMed]
  • De Mot R, Verachtert H. Purification and characterization of extracellular alpha-amylase and glucoamylase from the yeast Candida antarctica CBS 6678. Eur J Biochem. 1987 May 4;164(3):643–654. [PubMed]
  • Edman P, Begg G. A protein sequenator. Eur J Biochem. 1967 Mar;1(1):80–91. [PubMed]
  • ENGLARD S, SINGER TP. Physicochemical studies on beta-amylase. J Biol Chem. 1950 Nov;187(1):213–219. [PubMed]
  • FRENCH D, KNAPP DW. The maltase of Clostridium acetobutylicum; its specificity range and mode of action. J Biol Chem. 1950 Dec;187(2):463–471. [PubMed]
  • Hockenhull DJ, Herbert D. The amylase and maltase of Clostridium acetobutylicum. Biochem J. 1945;39(1):102–106. [PMC free article] [PubMed]
  • HSIU J, FISCHER EH, STEIN EA. ALPHA-AMYLASES AS CALCIUM-METALLOENZYMES. II. CALCIUM AND THE CATALYTIC ACTIVITY. Biochemistry. 1964 Jan;3:61–66. [PubMed]
  • Hyun HH, Zeikus JG. General Biochemical Characterization of Thermostable Extracellular beta-Amylase from Clostridium thermosulfurogenes. Appl Environ Microbiol. 1985 May;49(5):1162–1167. [PMC free article] [PubMed]
  • Kuhn H, Fietzek PP, Lampen JO. N-terminal amino acid sequence of Bacillus licheniformis alpha-amylase: comparison with Bacillus amyloliquefaciens and Bacillus subtilis Enzymes. J Bacteriol. 1982 Jan;149(1):372–373. [PMC free article] [PubMed]
  • Lee SF, Forsberg CW, Gibbins LN. Cellulolytic Activity of Clostridium acetobutylicum. Appl Environ Microbiol. 1985 Aug;50(2):220–228. [PMC free article] [PubMed]
  • Lee SF, Forsberg CW, Gibbins LN. Xylanolytic Activity of Clostridium acetobutylicum. Appl Environ Microbiol. 1985 Oct;50(4):1068–1076. [PMC free article] [PubMed]
  • Lee SF, Forsberg CW, Rattray JB. Purification and Characterization of Two Endoxylanases from Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol. 1987 Apr;53(4):644–650. [PMC free article] [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Madi E, Antranikian G, Ohmiya K, Gottschalk G. Thermostable amylolytic enzymes from a new clostridium isolate. Appl Environ Microbiol. 1987 Jul;53(7):1661–1667. [PMC free article] [PubMed]
  • Moseley MH, Keay L. Purification and characterization of the amylase of B. subtilis NRRL B3411. Biotechnol Bioeng. 1970 Mar;12(2):251–271. [PubMed]
  • Saito N. A thermophilic extracellular -amylase from Bacillus licheniformis. Arch Biochem Biophys. 1973 Apr;155(2):290–298. [PubMed]
  • SCOTT D, HEDRICK LR. The amylase of Clostridium acetobutylicum. J Bacteriol. 1952 Jun;63(6):795–803. [PMC free article] [PubMed]
  • SCOTT D, HEDRICK L. The amylase of Clostridium acetobutylicum. II. Adsorption. Appl Microbiol. 1959 May;7(3):135–138. [PMC free article] [PubMed]
  • Toda H, Kato I, Narita K. Correlation of the masked sulfhydryl group with the essential calcium in taka-amylase A. J Biochem. 1968 Mar;63(3):295–301. [PubMed]
  • Toda H, Narita K. Correlation of the sulfhydryl group with the essential calcium in Bacillus subtilis saccharifying alpha-amylase. J Biochem. 1968 Mar;63(3):302–307. [PubMed]
  • VALLEE BL, STEIN EA, SUMERWELL WN, FISCHER EH. Metal content of alpha-amylases of various origins. J Biol Chem. 1959 Nov;234:2901–2905. [PubMed]
  • Yang M, Galizzi A, Henner D. Nucleotide sequence of the amylase gene from Bacillus subtilis. Nucleic Acids Res. 1983 Jan 25;11(2):237–249. [PMC free article] [PubMed]
  • Yuuki T, Nomura T, Tezuka H, Tsuboi A, Yamagata H, Tsukagoshi N, Udaka S. Complete nucleotide sequence of a gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying alpha-amylases deduced from the DNA sequences. J Biochem. 1985 Nov;98(5):1147–1156. [PubMed]

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