• Journal of Life Science
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• v.20 no.5
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• pp.683-690
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• 2010

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 $45^{\circ}C$, respectively, whereas those for the extracellular glucoamylase were 5.0 and $50^{\circ}C$. The SGA were more sensitive to heat and SDS than the extracellular glucoamylase.

• KSBB Journal
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• v.11 no.3
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• pp.311-316
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• 1996

The effects of polyethylene glycol (PEG) on glucoamylase production in recombinant Saccharomyces cerevisiae were studied. By PEG addition, the cell growth was not affected. However, the glucoamylase production was increased in all range of PEG molecular weights and concentrations. The optimal molecular weight of PEG was 6000 and the optimal concentration was 1g/L. Using these optimals, the extracellular glucoamylase activity was 23% higher in PEG-containing medium than that in medium without PEG.

• Microbiology and Biotechnology Letters
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• v.24 no.6
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• pp.712-716
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• 1996

The effects of non-ionic surfactants (Triton X-100 and Tween 80) on cloned glucoamylase production and secretion in recombinant Saccharomyces cerevisiae culture were studied. Even though the extracellular glucoamylase activity was increased by addition of Tween 80 due to the increase of the cell mass, Tween 80 did not play a role in the increase of glucoamylase secretion. On the addition of Triton X-100 addition, the secretion efficiency was increased while the cell growth was inhibited. Triton X-100 was added to the culture broth after 24 hr of culture to minimize the inhibition of the cell growth, and consequently the glucoamylase activity in the culture broth was increased by 12%.

• Microbiology and Biotechnology Letters
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• v.28 no.4
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• pp.195-201
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• 2000

Glucoamylase of Saccharomyces diastaticus is produced as a large precursor composed of signal peptide (21 amino acid residues), Thr and Ser-rich region and functional glucoamylase. To evaluate the utility of the glucoamylase signal peptide (GSP) for the secretion of foreign proteins, four types of GSP mutants (ml : Pro-18 longrightarrowLeu-18, m2 : Tyr-13 longrightarrowLeu, m3 : Ser-9longrightarrowLeu-9, m4 : Asn-5 longrightarrowPro-5) were constructed and secretion efficiency of each mutant was compared with that of native GSP by the expression and secretion of Bacillus subtilis CMCase under the control of GAP in N-terminal domain and hydrophobic domain. n mutant 4, a polar amino acid was replaced by a helix - breaking Pro residue. CMCase activity assay and Western blot analysis revealed that CMCase secretion by GSP mutants replaced by Leu were increased compared with native GSP. In the case of m2 and m3, the substitution of Leu for Tyr-13 and Ser-9 in the hydrophobic region resulted in a twofold increase in the extracellular CMCase activity.

• Journal of the Korean Society of Food Science and Nutrition
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• v.16 no.2
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• pp.128-135
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• 1987

Extracellular ${\alpha}-amylase$, ${\beta}-amylase$ and glucoamylase produced by a thermophilic and cellulolytic bacterium, Herpetosiphon geysericola CUM 317, were partially purified by salting out with ammonium sulfate and by chromatography on a DEAE-cellulose column and on a CM-cellulose column. The Km values of ${\alpha}-amylase$, ${\beta}-amylase$ and glucoamylase for potato starch were $2.31mg/m{\ell}$, $7.69mg/m{\ell}$, and $8.33mg/m{\ell}$. The molecular weights of ${\alpha}-amylase$, ${\beta}-amylase$ and glucoamylase were calculated to be about 84000 dalton, 76000 dalton and 80000 dalton, respectively.

• Journal of Microbiology
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• v.37 no.1
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• pp.35-40
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• 1999

Sporulation-specific glucoamylase (SGA) gene was isolated from genomic library of Saccharomyces diastaticus 5114-9A by using glucoamylase non-producing mutant of S. diastaticus as a recipient. When the glucoamylase activities of culture supernatant, periplasmic, and intracellular fraction of cells transformed with hybrid plasmid containing SGA gene were measured, the highest activity was detected in culture supernatant. SGA produced by transformant and extracellular glucoamylase produced by S. diastaticus 5114-9A differed in enzyme characteristics such as optimum temperature, thermostability, and resistance to SDS and urea. But the characteristics of SGA produced by sporulating yeast cells and vegetatively growing transformants were identical.

• Korean Journal of Microbiology
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• v.30 no.5
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• pp.403-409
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• 1992

For the development of a glucanolytic yeast strain. the seceretion of endo-1.4-$\beta$-D-glucanase (CMCase) of Bacillus subtilis was performed in yeast using glucoamylase gene (STA1) of Saccharomyces diastaticus. A 1.7 kb-DNA fragment of STA1 gene containing authentic promoter, signal sequence, threonine serine-rich (TS) region and N-terminal region (98 amino acids) of mature glucoamylase was ligated to YEp 24. E. coli-yeast shuttle vector. And then. CMCase structural gene of B. subtilis was fused in frame with the 1.7 kb-DNA fragment of STA1 gene, resulting in recombinant plasmid pYES('24. Yeast transformant harboring pYESC24 had no CMCase activity. So. we deleted TS region and N-terminal region of mature glucoamylase existing between signal sequence and CMCase structural gene in pYESC24. consequently constructed recombinant plasmid pYESC11. The yeast transformed with the newly constructed recombinant plasmid pYESC11 efficiently secreted CMCase to extracellular medium. After 4 days culture. total CMCase activity of this transformant was 44.7 units/ml and over 93% of total CMCase activity was detected in culture supernatant.

• Microbiology and Biotechnology Letters
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• v.14 no.4
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• pp.311-318
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• 1986

Glucoamylase and ethanol productivities of HSDD-170 and HSDM-119 formed by S. cerevisiae and S. diastaticus protoplast fusion were investigated. For the production of the glucoamylase, soluble starch as carbon source, yeast extract and C. S. L as nitrogen source added into the basal medium were favorable. The production of the enzyme reached at maximum after cultivation of the fusant for 4 days at 3$0^{\circ}C$, aerobically. The properties of glucoamylase produced by fusants were very similar to those produced by S. diastaticus as based on optimum temperature, pH stability. In alcohol fermentation from starch, strain HSDD-170 fermented starch faster than either of its parental strains. The maximum of alcohol yield in 15% of liquefied potato starch was 7.5% (v/v).

• Journal of Microbiology and Biotechnology
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• v.18 no.3
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• pp.545-551
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• 2008

When cultivated aerobically, Aspergillus niger hyphae produced extracellular glucoamylase, which catalyzes the saccharification of unliquified potato starch into glucose, but not when grown under anaerobic conditions. The $K_m\;and\;V_{max}$ of the extracellular glucoamylase were 652.3 mg/l of starch and 253.3 mg/l/min of glucose, respectively. In mixed culture of A. niger and Saccharomyces cerevisiae, oxygen had a negative influence on the alcohol fermentation of yeast, but activated fungal growth. Therefore, oxygen is a critical factor for ethanol production in the mixed culture, and its generation through electrolysis of water in an electrochemical bioreactor needs to be optimized for ethanol production from starch by coculture of fungal hyphae and yeast cells. By applying pulsed electric fields (PEF) into the electrochemical bioreactor, ethanol production from starch improved significantly: Ethanol produced from 50 g/l potato starch by a mixed culture of A. niger and S. cerevisiae was about 5 g/l in a conventional bioreactor, but was 9 g/l in 5 volts of PEF and about 19 g/l in 4 volts of PEF for 5 days.

• Mycobiology
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• v.29 no.4
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• pp.183-189
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• 2001

Extracellular amylase properties were examined with the mycelium of Tricholoma matsutake isolated from ectomycorrhizal roots of Pinus densiflora. The molecular weights of $\alpha$-amylase and glucoamylase were estimated as 34.2 kD and 11.5 kD, respectively, after eluted through Superdex 75 column. The optimum pH of the purified enzyme was found in a range of pH $5.0{\sim}6.0$, with a peak at pH 5.0. The activities of these enzymes were stable from $4^{\circ}C\;to\;30^{\circ}C$. The $\alpha$-amylase of T. matsutake readily hydrolyzed soluble starch and amylose-B, while it weakly hydrolyzed glycogen, dextrin, amylose and amylose-A. The main products of hydrolysis were confirmed to be glucose, maltose and maltotriose on the basis of the similarities in the thin layer chromatographic mobility.