Cloning, Sequencing, Characterization, and Expression of an Extracellular α-Amylase from the Hyperthermophilic ArchaeonPyrococcus furiosus in Escherichia coli andBacillus subtilis

• Published in: The Journal of biological chemistry Vol. 272; no. 26; p. 16335
• Main Authors: Steen Jørgensen, Constantin E. Vorgias, Garabed Antranikian
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 27.06.1997
• DOI: 10.1074/jbc.272.26.16335
• ISSN: 0021-9258

A gene encoding a highly thermostable extracellular α-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 α-amylase was biochemically characterized and shows an activity optimum at pH 4.5, whereas the optimun temperature for enzymatic activity is close to 100 °C. α-Amylase shows sequence homology to the other known α-amylases and belongs to family 13 of glycosyl hydrolases. This extracellular α-amylase is not homologous to the subcellular α-amylase previously isolated from the same organism.