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 w as 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 a-amylase (AMY-CS2) were estimated to be 66 kDa and 4.2 respectively. AMY-CS2 has raw-star ch-digesting and raw-starch-absorbing activities. Furthermore it was s hown to be thermostable. An open reading frame of the cDNA specified 6 11 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 a-amylase from Aspergi llus oryzae (Taka-amylase), whereas the C-terminal region had a sequen ce 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-sta rch-binding domain of AMY-CS2 plays a role not only in the molecule's raw-starch-digesting ability but also in its thermostability.