ALPHA-AMYLASE FROM THE HYPERTHERMOPHILIC ARCHAEBACTERIUM PYROCOCCUS-FURIOSUS - CLONING AND SEQUENCING OF THE GENE AND EXPRESSION IN ESCHERICHIA-COLI

A gene encoding a highly thermostable alpha-amylase from the hyperther mophilic archaebacterium Pyrococcus furiosus was cloned and expressed in Escherichia coli. The nucleotide sequence of the gene predicts a 64 9-amino acid protein with a calculated molecular mass of 76.3 kDa, whi ch corresponds well with the value obtained from purified enzyme using denaturing polyacrylamide gel electrophoresis. The NH2 terminus of th e deduced amino acid sequence corresponds precisely to that obtained f rom the purified enzyme, excluding the NH2-terminal methionine. The am ylase expressed in E. coli exhibits temperature-dependent activation c haracteristic of the original enzyme from P. furiosus, but has a highe r apparent molecular weight which is attributed to the improper format ion of the native quaternary structure. No homology was found with pre viously characterized promotor or termination sequences. The deduced a mino acid sequence displayed strong homology to the alpha-amylase A of Dictyoglomus thermophilum, an obligately anaerobic, extremely thermop hilic bacterium. Evolutionary implications of this homology are discus sed.