Cloning, sequencing and expression of an alpha-amylase gene, amyA, from the thermophilic halophile Halothermothrix orenii and purification and biochemical characterization of the recombinant enzyme.
A recombinant clone expressing an amylase was identified from an Escherichia coli generated genomic library of the thermophilic, moderately halophilic, anaerobic bacterium Halothermothrix orenii by activity screening, and the gene encoding the enzyme was designated AmyA. The amyA gene was 1545 bp long, and encoded a 515 residue protein composed of a 25 amino acid putative signal peptide and a 490 amino acid mature protein. It possessed the five consensus regions characteristic of the alpha-amylase family and showed the greatest homology to the Bacillus megaterium group of alpha-amylases. The amyA gene was expressed in E. coli as a hexahistidine-tagged enzyme and purified. The purified recombinant enzyme was optimally active at 65 degrees C in 5% (w/v) NaCl at pH 7.5, with significant activity retained in the presence of up to 25% (w/v) NaCl. It had a specific activity of 22.32 U mg(-1) and required NaCl and CaCl(2) for optimum activity and thermostability. The relatively high proportion of acidic amino acids typically observed for many enzymes from halophiles was absent in H. orenii AmyA.
Mijts
Benjamin N
BN
Microbial Discovery Research Unit, School of Biomolecular and Biomedical Sciences, Faculty of Science, Griffith University, Brisbane, Queensland 4111, Australia1.
Patel
Bharat K C
BKC
Microbial Discovery Research Unit, School of Biomolecular and Biomedical Sciences, Faculty of Science, Griffith University, Brisbane, Queensland 4111, Australia1.
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D000516:alpha-Amylases