Raw-starch-digesting and thermostable alpha-amylase from the yeast Cryptococcus sp. S-2: purification, characterization, cloning and sequencing.
A starch-degrading enzyme produced by the yeast Cryptococcus sp. S-2 was purified in only one step by using an alpha-cyclodextrin-Sepharose 6B column, and was characterized as an alpha-amylase (EC 3.2.1.1). The molecular mass and isoelectric point of purified alpha-amylase (AMY-CS2) were estimated to be 66 kDa and 4.2 respectively. AMY-CS2 has raw-starch-digesting and raw-starch-absorbing activities. Furthermore it was shown to be thermostable. An open reading frame of the cDNA specified 611 amino acids, including a putative signal peptide of 20 amino acids. The N-terminal region of AMY-CS2 (from the N-terminus to position 496) had 49.7% similarity with the whole region of alpha-amylase from Aspergillus oryzae (Taka-amylase), whereas the C-terminal region had a sequence that was similar to the C-terminal region of glucoamylase G1 from A. niger. In addition, putative raw-starch-binding motifs exist in some amylolytic enzymes. A mutant AMY-CS2 that lacks the C-terminal domain lost not only its ability to bind or digest raw starch, but also its thermostability. Consequently it is possible that the putative raw-starch-binding domain of AMY-CS2 plays a role not only in the molecule's raw-starch-digesting ability but also in its thermostability.
Iefuji
H
H
National Research Institute of Brewing, Higashi-Hiroshima, Japan.
Chino
M
M
Kato
M
M
Iimura
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003454:Cryptococcus / Q000201:enzymology* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013213:Starch / Q000378:metabolism*; D013696:Temperature; D000516:alpha-Amylases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D018076:DNA, Complementary; D004271:DNA, Fungal; D008670:Metals; D013213:Starch; D000516:alpha-Amylases