• 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.

• Korean Journal of Agricultural Science
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• v.23 no.1
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• pp.80-89
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• 1996

The expression and secretion of human lactoferrin (hLf) in Sacclnromyces diastaticus were performed. 1. For the secretion of hLf in yeast, recombinant plasmid pYEGLf was constructed using promoter, secretion signal sequence of glucoamylase I gene (STA1) and transcriptional terminator of GAL7 gene. 2. Each correct recombinant plasmid was selected by mini-preparation of plasmid DNA from E coli transformant and restriction enzyme digestion analysis. The selected plasmids, pYEGLf, were transformed into S. diastaticus YIY345 as a expression host, respectively. 3. Western blot analysis using rabbit anti-hLf was carried out to identify expressed hLf. Positive signals were shown in culture supernatant of pYEGLf transformant. 4. About $100{\mu}g-1mg$ of concentrated culture supernatant of positive clone were loaded on paper disc and tested for the antimicrobial activity against E coli. However, no activity was observed. We concluded that this fact results from low concentration of hLf secreted from yeast, compared with the fact that MIC of hLf is as high as $3mg/m{\ell}$. Therefore, the purification of secreted hLf may be require to investigate the antimicrobial activity. From this study, the feasibility of low-cost production of sufficient quantities of human lactofferin for nutritional and therapeutical applications were suggested.

• Microbiology and Biotechnology Letters
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• v.36 no.2
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• pp.158-164
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• 2008

The fusants which contain starch utilizing ability and alcohol fermenting ability were developed by protoplast fusion of Saccharomyces cerevisiae KOY-1 and Saccharomyces diastaticus KCTC 1804. Sacharomyces cerevisiae KH-12 was obtained by haploid induction from Saccharomyces cerevisiae KOY-1. The auxotropic mutants of yeast were obtained by using an ethylmethane sulfonate (EMS). The frequency of protoplast formation in Saccharomyces cerevisiae KOY-1 $(Met^-)$ and Saccharomyces diastaticus KCTC 1804 $(Trp^-)$ were 90.5% and 97.7%, respectively. The frequency of fusant formation was $1.79{\times}10^{-4 }$ for the regenerated protoplast and the 1,000 fusants were obtained. Fusant FA 776 was selected as a potential yeast which contain an alcohol fermenting ability in the starch medium. The genetic stability was 4.64% for 10 passages of generation. Fusant FA 776 produced 13mg/ml of alcohol in 24% starch medium and showed 1.86-fold higher alcohol fermenting ability than Saccharomyces diastaticus KCTC 1804.

• Journal of Life Science
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• v.22 no.2
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• pp.142-147
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• 2012

The sporulation-specific glucoamylase (SGA) of Saccharomyces diastaticus is known to be produced in the cytoplasm during sporulation. For the purpose of proving that SGA has secretory potential, we constructed a hybrid plasmid, pYESC25, containing the promoter and the putative signal sequence of the SGA fused in frame to the endo-1,4-${\beta}$-D-glucanase (CMCase) gene of Bacillus subtilis without its own signal sequence. The recipient yeast strain of S. diastaticus YIY345 was transformed with the hybrid plasmid. CMCase secretion from S. diastaticus harboring pYESC25 into culture medium was confirmed by the formation of yellowish halos around transformants after staining with Congo red on a CMC agar plate. The transformant culture was fractionated to the extracellular, periplasmic, and intracellular fraction, followed by the measurement of CMCase activity. About 63% and 13% enzyme activity were detected in the culture supernatant (extracellular fraction) and periplasmic fraction, respectively. Furthermore, ConA-Sepharose chromatography, native gel electrophoresis, and activity staining revealed that CMCase produced in yeast was glycosylated and its molecular weight was larger than that of the unglycosylated form from B. subtilis. Taking these findings together, SGA has the potential of secretion to culture medium, and the putative signal sequence of SGA can efficiently direct bacterial CMCase to the yeast secretion pathway.

• The Korean Journal of Mycology
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• v.27 no.6 s.93
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• pp.386-393
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• 1999

Ethanol is considered as one of the most suitable substitutes for the petroleum, since it offers attractive functional features at an economical cost. Glucoamylase producing yeasts were isolated and characterized. Based on the morphological character, carbon fermentations, assimilation of carbon and nitrate, growth on vitamine-free medicine, and urease activity, five isolates of Saccharomyces diastaticus, two isolates of Saccharomycopsis fibuligera, and two of Schwanniomyces occidentalis, and each isolate of Ambrosiozyma monospora and Lipomyces kononenkoae were identified. Among 12 isolates, one of the S. diastaticus, E3 showed the highest activity of glucoamylase and identified as Saccharomyces diastaticus. The hydrolysis of starch by the E3 strain showed the release of considerable amount of reducing sugar, along with the reduction in iodine staining capacity. The product of action of glucoamylase, glucose was determined by thin-layer chromatography. The enzyme activity was found to be stable in broad pH range of $5.0{\sim}7.0$ with optimal activity at pH $5.0{\sim}6.0$. The enzyme showed optimal antivity at $50^{\circ}C{\sim}60^{\circ}C$. Soluble starch and glucose were better carbon sources for the enzyme production than xylose and glycerol. $Na^+\;and\;Mg^{2+}$ increased the glucoamylase activity, however $Hg^{2+}\;and\;Ag^{2+}$ inhibited the activity. Soluble starch was the best substrate for the enzyme activity.

• Microbiology and Biotechnology Letters
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• v.24 no.6
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• pp.726-732
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• 1996

To improve the fermentation characteristics(such as starch-degradability, ethanol tolerance, sugar and high-temperature tolerance) of recombinant haploid yeast Saccharomyces diastaticus K114, hybridization technique was used. The hybridization partner was S. diastaticus 1177 which had good glucoamylase activity and fermentabi- lity. The best hybrid HH64 showed improved ethanol tolerance, sugar and high-temperature tolerance. Especia- lly, the starch-fermentability was significantly improved, since the hybrid produced 1.60% (w/v) ethanol from 4% (w/v) starch, while the recombinant haploid K114 produced 1.30% (w/v) ethanol. The optimum temperature and pH for the starch-fermentation by the hybrid HH64 was 30$\circ$C and 5, respectively. The hybrid yeast HH64 produced 7.5% (w/v) ethanol directly from 20% (w/v) starch.

• Korean Journal of Agricultural Science
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• v.16 no.1
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• pp.44-55
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• 1989

Out of 826 isolates of Streptomyces isolated from different soils, 65 isolates were selected and identified according to their morphological and physiological characters. A total of 46 species of Streptomyces were identified, and the following 38 species were newly recorded in Korea. 1) Melanoid pigments producing group; S. lavendulae, S. purpurascens, S. violarus, S. lateritius, S. olivochromogenes, S. purpeofuscus, S. fulvoviolaceus, S. gallilaeus, S. naganishii, S. hygroscopicus subsp. ossamyceticus, S. nevagawaensis, S. anandii, S. phaeopurpureus, S. mirabilis, S. arenae, S. massasporeus, S. eurythermus, S. tuirus. 2) Melanoid pigments non-producing group; S. fellus, S. flavidovirens, S. exfoliatus, S. fumanus, S. termitum, S. glomeroaurantiacus, S. luteofluorescens, S. prunicolor, S. fradiae, S. tauricus, S. griseolus, S. misakiensis, S. poonensis, S. antimycoticus, S. diastaticus subsp. ardesiacus, S. nigrifaciens, S. tendae, S. narbonensis subsp. josamvceticus, S. atroolivaceus, S. chrysomallus subsp. fumigatus.

• Journal of Food Hygiene and Safety
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• v.10 no.1
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• pp.23-28
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• 1995

The effect of the ethanol extract from salviae miltiorrhizae radix (Salvia miltiorrhiza) on the microbial growht and the stability of the extracted antimicrobial material were investigated. The ethanol extract had strong growth inhibition activity (MIC, 3.13~50.0 $\mu\textrm{g}$/ml) against Gram-positive bacteria such as B. subtilis, L. monocytogenes and S. aureus. Among Gram-positive bacteria tested, B. subtilis was the most susceptible to the extracted substance. While the antimicrobial activity of the ethanol extract was weak (MIC, 400~800 $\mu\textrm{g}$/ml) to E. coli and yeasts (C. albicans, Sacch. diastaticus). The ethanol extract had bactericidal action at higher concentration than MIC against B. subtilis, while the extract had only bacteriostatic action against S. aureus. The extracted antimicrobial substance was stable in the pH range of 4.0 to 10.0, heat treatment at 121$^{\circ}C$ for 15 min, and freezing and thawing

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.546-552
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• 1992

Ethanol-tolerant strain, S. eerevisiae BUI a26 ($alc^r thr^-$) and gJucoamylase-producing strain, S diastatieus AI5a6 (STA+ hom-) were prepared by means of genetic manipulation, Protoplast fusion was carried out to introduce STA gene from AI5a6 strain to BUla26 strain, Protoplast formation was shown at 0,8 M sorbitol and 200 Jig/ml to 400 Jig/ml zymolyase treatment for 2 hours incubation, Fusion frequency was $ 3.25 {\times} 10^{-3}$ to the regenerated protoplast number using PEG 6000 for 90 min incubation. The excellent fusants with genotype of STA- $alc^r thr^-$ hom+/STA+ ($alc^s thr^+$ hom- (2n), F7 and FIO, were selected by ethanol-tolerant, ethanol fermentation, and glucoamylase production tests, Glucoamylase production of AI5a6 showed 2,7 units, but 4.2 or 8.4 units for F7 or FIO fusant at $30^{\circ}C$, Ethanol fermentation from 32% glucose by BUla26 was 14,0%(v/v) in fermentaion medium for 5 days incubation, but 14.5% or 15,0% for F7 or FIO strain, respectively. Ethanol fermentation from 5% starch was 2,0% by F7, or 1.8% by FIO strain in fermentation medium for 5 days fermentation.

• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.494-498
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• 1988

The optimum conditions for glucoamylase production, and ethanol productivity of the transformant TSD-14 were investigated as compared with the parental strains. The properties of TSD-14 were comparatively similar to the donor S. diastaticus IFO 1046 as regards the conditions of glucoamylase production and ethanol productivity. The soluble starch was the most effective carbon source for the glucoamylase production. While inorganic nitrogen sources did not prompt cell growth and enzyme production, the organic nitrogen sources generally enhanced both cell growth and glucoamylase production. The metal salts such as FeSO$_4$, MgSO$_4$, MnCl$_2$, and NiSO$_4$were favorable to the enzyme production. And the optium temperature and initial pH for glucoamylase production were 3$0^{\circ}C$ and 5. The transformant TSD-14 produced 8.3%(v/v) ethanol from 15% sucrose medium, 4.8%(v/v) ethanol from 15% soluble starch medium, and 7.5%(v/v) ethanol from 15% liquefied potato starch medium. The corresponding fermentation efficiency were 84% , 45% and 70%, respectively.