Cloning, sequencing, characterization, and expression of an extracellular alpha-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis.
A gene encoding a highly thermostable extracellular alpha-amylase from the hyperthermophilic archaeon Pyrococcus furiosus was identified. The gene was cloned, sequenced, and expressed in Escherichia coli and Bacillus subtilis. The gene is 1383 base pairs long and encodes a protein of 461 amino acids. The open reading frame of the gene was verified by microsequencing of the recombinant purified enzyme. The deduced amino acid sequence is 25 amino acids longer at the N terminus than that determined by sequencing of the purified protein, suggesting that a leader sequence is removed during transport of the enzyme across the membrane. The recombinant alpha-amylase was biochemically characterized and shows an activity optimum at pH 4.5, whereas the optimun temperature for enzymatic activity is close to 100 degrees C. alpha-Amylase shows sequence homology to the other known alpha-amylases and belongs to family 13 of glycosyl hydrolases. This extracellular alpha-amylase is not homologous to the subcellular alpha-amylase previously isolated from the same organism.
Jorgensen
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S
Novo Nordisk A/S, Enzyme Research, Bacterial Gene Technology, Novo Allé, DK 2880 Bagsvaerd, Denmark.
Vorgias
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CE
Antranikian
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G
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology*; D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics*
D000516:alpha-Amylases