Raw-starch-digesting and thermostable α-amylase from the yeast Cryptococcus sp. S-2: purification, characterization, cloning and sequencing

Abstract

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.

Amylase action pattern on starch polymers

“…For certain α-amylases, e.g. a raw starch degrading α-amylase from Cryptococcus sp., this domain seems also responsible for the thermostability of the amylase (Iefuji et al 1996). α-Amylases.…”

Microbial ?-amylases: physico-chemical properties, substrate specificity and domain structure

“…Продуценти α-амілаз виявлено і се-ред актиноміцетів (Nocardiopsis [29], Nocardia [1], Streptomyces [1], Thermomonospora [11,30], Thermobifida [31], Thermoactinomyces [11,30,32,33]) та дріжджів (Cryptococcus [34], Saccharomycopsis [35]). …”

Identification a Novel Raw-Starch-Degrading-α-Amylase from a Tropical Marine Bacterium

“…The latter enzymes possess the starch binding domain predominantly at their C-termini (for review see (Machovic and Janecek, 2006). α-Amylases degrading raw starch granules have been reported to occur in a wide range of organisms (Demirkan et al, 2005;Matsubara et al, 2004;Iefuji et al, 1996;Bandani, 2009a,2009b), however, few reports on α-amylases from marine sources are known (Vidilaseris et al, 2009). Here we report a hard coral associated bacterium producing rawstarchdegrading-α-amylase, Bacillus amyloliquifaciens ABBD.…”