Proteolytic Processing of Glucoamylase in the Yeast Saccharomyces diastaticus

概要

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Glucoamylase was purified from the culture fluid of the yeast Saccharomyces diastaticus carrying STA1. The molecular weight of the protein was about 250K by both gel-filtration and acrylamide gel electrophoresis. The protein was glycosylated with asparagine-linked glycosides whose molecular weight was 70K. The amino-terminal sequence of the protein began from the 33rd amino acid residue from the first mcthionine of the putative precursor deduced from the DNA sequence of STA1. The amino acid composition of the purified protein matched the predicted amino acid composition. These results confirmed that STA1 codes for a structural gene of glucoamylase. Yeast cells secreted enzymatically active β-lactamase into the culture medium after transformation with hybrid plasmids containing the glucoamylase promoter and the DNA sequence encoding the extended peptide of 32 amino acids fused to a structural gene for Escherichia coli β-lactamase. The result suggested that the amino-terminal peptide of the putative glucoamylase precursor functions as a signal sequence for protein secretion. Taking into consideration the subunit structure of the previously purified glucoamylase [Yamashita et al., Agric. Biol. Chem. 48, 1611 (1984)], we present the molecular structure of the glucoamylase precursor and also a model for its proteolytic processing.
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