The complete nucleotide sequence of the glucoamylase gene GLUI from the yeast Saccharomycopsisfibuligera has been determined. The GLUI DNA hybridized to a polyadenylated RNA of 2.1 kilobases. A single open reading frame codes for a 519-amino-acid protein which contains four potential N-glycosylation sites. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. Glucoamylase was purified from a culture fluid of the yeast Saccharomyces cerevisiae which had been transformed with a plasmid carrying GLUI. The molecular weight of the protein was 57,000 by both gel filtration and acrylamide gel electrophoresis. The protein was glycosylated with asparagine-linked glycosides whose molecular weight was 2,000. The amino-terminal sequence of the protein began from the 28th amino acid residue from the first methionine of the putative precursor. The amino acid composition of the purified protein matched the predicted amino acid composition. These results confirmed that GLUI encodes glucoamylase. A comparison of the amino acid sequence of glucoamylases from several fungi and yeast shows five highly conserved regions. One homology region is absent from the yeast enzyme and so may not be essential to glucoamylase function.
The complete nucleotide sequence of the secretable a-amylase gene ALP1 from the yeast Saccharomycopsis jbuiigera has been determined. The ALP1 DNA hybridized to a polyadenylated RNA of 2.0 kilobases. A single open reading frame encodes a 494-amino acid protein which is highly homologous with a-amylase (Taka-amylase) of a fungus Aspergillus oryzae.
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