Abstract
A gene encoding a highly thermostable alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus was cloned and expressed in Escherichia coli. The nucleotide sequence of the gene predicts a 649-amino acid protein with a calculated molecular mass of 76.3 kDa, which corresponds well with the value obtained from purified enzyme using denaturing polyacrylamide gel electrophoresis. The NH2 terminus of the deduced amino acid sequence corresponds precisely to that obtained from the purified enzyme, excluding the NH2-terminal methionine. The amylase expressed in E. coli exhibits temperature-dependent activation characteristic of of the original enzyme from P. furiosus, but has a higher apparent molecular weight which is attributed to the improper formation of the native quaternary structure. No homology was found with previously characterized promotor or termination sequences. The deduced amino acid sequence displayed strong homology to the alpha-amylase A of Dictyoglomus thermophilum, an obligately anaerobic, extremely thermophilic bacterium. Evolutionary implications of this homology are discussed.
Highlights
From the $Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218 and STakara Shuzo Co., Seta 3-4-1, Otsu, Shiga 520-21,Japan
It is possible that this represents a tendency in the usage of this initiation codon in hyperthermophilic archaebacteria
It isknown thatthe production of a-amylase from P. furiosus is increased by the presence of starch(datanot shown) indicating that the gene possesses an inducible promotor, a feature previously uncharacterized in hyperthermophilic archaebacteria
Summary
A gene encoding a highly thermostable a-amylase from the hyperthermophilic archaebacterium P y r o coccus furiosus was cloned and expressed in Escherichia coli. The nucleotide sequence of the gene predicts a 649-amino acid protein with a calculated molecular mass of 76.3 kDa, whichcorrespondswell with the value obtained from purified enzyme using denaturing polyacrylamide gel electrophoresis. The NH2 terminus of the deduced amino acid sequence corresponds precisely to that obtained from the purified enzyme, excluding the NH2-terminal methionine. The deduced amino acid sequence displayed strong homology to the a-amylase A of Dictyoglomus thermophihm, an obligately anaerobic, extremely thermophilic bacterium. Evolutionary implications of this homology are discussed. In addition the 3’- and 5”noncoding regions were analyzed in an attempt toidentify sequences involved in transcriptional regulation, and a search for homology between the deduced amino acid sequence and other a-amylases was completed
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