Gene sequence, bioinformatics and enzymatic characterization of alpha-amylase from Saccharomycopsis fibuligera KZ

A fragment coding for a putative extracellular alpha-amylase, from the genomic library of the yeast Saccharomycopsis fibuligera KZ, has been subcloned into yeast expression vector pVT100L and sequenced. The nucleotide sequence revealed an ORF of 1,485 bp coding for a 494 amino acid residues long protein with 99% identity to the alpha-amylase Sfamy from S. fibuligera HUT 7212. The S. fibuligera KZ alpha-amylase (Sfamy KZ) belongs to typical extracellular fungal alpha-amylases classified in the glycoside hydrolase family 13, subfamily 1, as supported also by clustering observed in the evolutionary tree. Sfamy KZ, in addition to the essential GH13 alpha-amylase three-domain arrangement (catalytic TIM barrel plus domains B and C), does not contain any distinct starch-binding domain. Sfamy KZ was expressed as a recombinant protein in Saccharomyces cerevisiae and purified to electrophoretic homogeneity. The enzyme had a molecular mass 53 kDa and contained about 2.5% of carbohydrate. The enzyme exhibited pH and temperature optima in the range of 5-6 and 40-50 degrees C, respectively. Stable adsorption of the enzyme to starch granules was not detected but a low degradation of raw starch in a concentration-dependent manner was observed.