Cloning and nucleotide sequence of a heat-stable amylase gene from an anaerobic thermophile, Dictyoglomus thermophilum.
A highly heat-stable amylase gene from an obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, was cloned and expressed in Escherichia coli. The nucleotide sequence of the amylase gene predicts a 686-amino-acid protein of relative molecular mass 81,200, which is consistent with that determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified enzyme. The NH2-terminal sequence determined using the enzyme purified from E. coli cells corresponds precisely to that predicted from the nucleotide sequence, except for the absence of the NH2-terminal methionine in the mature protein. When the amylase gene was expressed in E. coli cells, the enzyme was localized in the cytoplasmic fraction; this is probably explained by the absence of the signal sequence for secretion. By using the amylase purified from the E. coli transformant, some enzymatic properties, such as optimum pH, optimum temperature, pH-stability and heat-stability, were examined. The amylase was found to be a highly liquefying-type.
D000595:Amino Acid Sequence; D000681:Amylases / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids; D013696:Temperature
D000681:Amylases