Haloalkaliphilic maltotriose-forming alpha-amylase from the archaebacterium Natronococcus sp. strain Ah-36.
A haloalkaliphilic archaebacterium, Natronococcus sp. strain Ah-36, produced extracellularly a maltotriose-forming amylase. The amylase was purified to homogeneity by ethanol precipitation, hydroxylapatite chromatography, hydrophobic chromatography, and gel filtration. The molecular weight of the enzyme was estimated to be 74,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 8.7 and 55 degrees C in the presence of 2.5 M NaCl. The activity was irreversibly lost at low ionic strength. KCl, RbCl, and CsCl could partially substitute for NaCl at higher concentrations. The amylase was stable in the range of pH 6.0 to 8.6 and up to 50 degrees C in the presence of 2.5 M NaCl. Stabilization of the enzyme by soluble starch was observed in all cases. The enzyme activity was inhibited by the addition of 1 mM ZnCl2 or 1 mM N-bromosuccinimide. The amylase hydrolyzed soluble starch, amylose, amylopectin, and, more slowly, glycogen to produce maltotriose with small amounts of maltose and glucose of an alpha-configuration. Malto-oligosaccharides ranging from maltotetraose to maltoheptaose were also hydrolyzed; however, maltotriose and maltose were not hydrolyzed even with a prolonged reaction time. Transferase activity was detected by using maltotetraose or maltopentaose as a substrate. The amylase hydrolyzed gamma-cyclodextrin. alpha-Cyclodextrin and beta-cyclodextrin, however, were not hydrolyzed, although these compounds acted as competitive inhibitors to the amylase activity. Amino acid analysis showed that the amylase was characteristically enriched in glutamic acid or glutamine and in glycine.
Kobayashi
T
T
Laboratory of Microbiology, RIKEN Institute, Saitama, Japan.
Kanai
H
H
Hayashi
T
T
Akiba
T
T
Akaboshi
R
R
Horikoshi
K
K
D000596:Amino Acids / Q000032:analysis; D000693:Anaerobiosis; D001105:Archaea / Q000145:classification / Q000201:enzymology*; D003505:Cyclodextrins / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D011489:Protein Denaturation; D012492:Salts / Q000494:pharmacology; D014312:Trisaccharides / Q000096:biosynthesis*; D000516:alpha-Amylases / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification*
D000596:Amino Acids; D003505:Cyclodextrins; D008670:Metals; D012492:Salts; D014312:Trisaccharides; C008317:maltotriose; D000516:alpha-Amylases