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KMID : 0613820100200050683
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Journal of Life Science
2010 Volume.20 No. 5 p.683 ~ p.690
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Characterization of Sporulation-Specific Glucoamylase of Saccharomyces diastaticu
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Kim Eun-Ju
Ahn Jong-Seog
Kang Dae-Ook
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Abstract
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The yeast strains of Saccharomyces diastaticus produce one of three isozymes of an extracellular glucoamylase Ⅰ, Ⅱ or Ⅲ, a type of exo-enzyme which can hydrolyse starch to generate glucose molecules from non-reducing ends. These enzymes are encoded by the STA1, STA2 and STA3 genes. Another gene, sporulation-specific glucoamylase (SGA), also exists in the genus Saccharomyces which is very homologous to the STA genes. The SGA has been known to be produced in the cytosol during sporulation. However, we hypothesized that the SGA is capable of being secreted to the extracellular region because of about 20 hydrophobic amino acid residues at the N-terminus which can function as a signal peptide. We expressed the cloned SGA gene in S. diastaticus YIY345. In order to compare the biochemical properties of the extracellular glucoamylase and the SGA, the SGA was purified from the culture supernatant through ammonium sulfate precipitation, DEAE-Sephadex A-50, CM-Sephadex C-50 and Sephadex G-200 chromatography. The molecular weight of the intact SGA was estimated to be about 130 kDa by gel filtration chromatography with high performance liquid chromatography (HPLC) column. Sodium dedecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed it was composed of two heterogeneous subunits, 63 kDa and 68 kDa. The deglycosylation of the SGA generated a new 59 kDa band on the SDS-PAGE analysis, indicating that two subunits are glycosylated but the extent of glycosylation is different between them. The optimum pH and temperature of the SGA were 5.5 and 45℃, respectively, whereas those for the extracellular glucoamylase were 5.0 and 50℃. The SGA were more sensitive to heat and SDS than the extracellular glucoamylase.
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KEYWORD
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Extracellular glucoamylase, purification, Saccharomyces diastaticus, secretion, sporulationspecific glucoamylase
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