Haruyuki Iefuji1, Mariko Chino1, Miyoshi Kato1, Yuzuru Iimura1
1National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-hiroshima, 739 Japan
Tóm tắt
A starch-degrading enzyme produced by the yeast Cryptococcus sp. S-2 was purified in only one step by using an α-cyclodextrin–Sepharose 6B column, and was characterized as an α-amylase (EC 3.2.1.1). The molecular mass and isoelectric point of purified α-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 α-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.
