• Applied Biological Chemistry
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• v.31 no.3
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• pp.267-273
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• 1988

Transformed, hybrid strains of the yeast Saccharomyces capable of simultaneous secretion of both glucoamylase and ${\alpha}-amylase$ have been produced. These strains can carry out direct, one-step assimilation of starch with conversion efficiency greater than 93% during a 5 day growth period. One of the transformants converts 92.8% of available starch into reducing sugars in only 2 days. Glucoamylase secretion by these strains results from expression of one or more chromosomal STA genes derived from Saccharomyces diastaticus. The strains were transformed by a plasmid(pMS12) containing mouse salivary ${\alpha}-amylase$ cDNA in an expression vector containing yeast alcohol dehydrogenase promoter and a segment of yeast $2{\mu}$ plasmid. The major starch hydrolysis product produced by crude amylases found in culture broths is glucose, indicating that ${\alpha}-amylase$ and glucoamylase act cooperatively.

• 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.

• Microbiology and Biotechnology Letters
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• v.14 no.5
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• pp.359-363
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• 1986

S. diastaticus hydrolysised $\alpha$-1.4 linkage of the starch and was known fermenting yeast strain, but poorly hydrolized $\alpha$-1.6 linkage of the starch. To improve the starch fermentation ability of yeast, we tried that protoplast fusion between S. diastaticus and C. tropicalis and finally two starins of fusant (FPDC42, FPDC43) were obtained. C. tropicalis well hydrolysis both $\alpha$-1.4 and $\alpha$-1.6 linkanges in the starch. The protoplast of parental auxotrophic cells were fused in the presence of 10mM CaCl$_2$ and 35% of polyethylene glycol (M. W. 4,000). The fusion frequency was 10$^{-5}$ to 10$^{-6}$. Properties of the fusants(genetic stability, assimilation of carbon sources, random spore formation, copper resistance, NaCl tolerance, DNA content, cell size and growth rate) were investigated.

• Microbiology and Biotechnology Letters
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• v.14 no.5
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• pp.365-369
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• 1986

The activity of glucoamylase and pullulanase, properties of glucoamylase and ethanol productivities of fusants were studied. Glucoamylase and pullulanase activity of fusants were higher than parents. The optimal pH and temperature of glucoamylase of fusants were very similar to the those produced by S. diastaticus. In alcohol fermentation. fermenting ability and fermentation rate of fusants were higher and faster than either of its parental strain. The maximum of alcohol yield in 15% of liquefied potato starch was 7.8% (v/v)

• Microbiology and Biotechnology Letters
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• v.22 no.6
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• pp.584-592
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• 1994

Several mutation procedures have been compared to obtain an ethanol-tolerant Saccha- romyces diastaticus strain secreting glucoamylase. These procedures include spontaneous mutation, EMS treatment, UV irradiation, and combination of EMS treatment and UV irradiation. All these methods were followed by adaptation of the yeast cells to gradually higher ethanol concentration. Among these procedures, the combined method of EMS treatment and UV irradiation gave the promising result, i.e. the ethanol tolerance of the yeast increased from 11.5%(v/v) to 14.0%(v/v). Respiratory deficient petite mutants of industrial and ethanol-tolerant yeast strains have been isola- ted and hybridized with haploid S. diastaticus strains. The resulting hybrids showed increased ethanol tolerance and starch-fermentability.

• Microbiology and Biotechnology Letters
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• v.24 no.5
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• pp.604-612
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• 1996

To improve the fermentation characteristics of the haploid starch-fermenting recombinant yeast strain K114/YIpMS$\Delta$R(LEU2/URA3) secreting both $\alpha$-amylase and glucoamylase was rare-mated with polyploid industrial yeast Saccharomyces sp. K35. The K35 strain had good fermentation-characteristics such as ethanol-tolerance, high temperature and sugar-tolerance, and high fermentation rate. Among the resulting 66 hybrids, the best strain RH51 was selected. The RH51 exhibited amylolytic activity of K114/YIpMS$\Delta$R(LEU2/URA3) as well as ethanol and sugar tolerance of K35. The optimum temperature of hybrid RH51 for starch fermentation was 34$\circ$C which was same as that of K35 but different from that (30$\circ$C) of K114/YIpMS$\Delta$R(LEU2/URA3). The optimum pH was 5.0. The optimum size of inoculum was 2% as the pellet (w/v) of yeast cells. The hybrid strain RH51 produced 7.0% ethanol (w/v) from 20% (w/v) soluble starch while K35 formed almost no ethanol, 0.3% (w/v). RH51 strain produced 7.5% (w/v) ethanol after 8 days in a 2.5 l fermenter containing 800 ml of 20% (w/v) soluble starch. The residual starch content in the fermentation medium was 1.68% (w/v), and therefore almost all the starch was fermented completely.

• 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).

• Korean Journal of Microbiology
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• v.32 no.4
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• pp.271-279
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• 1994

To develop a yeast strain which stably secretes both $\alpha$-amylase and glucoamylase and therefore is able to convert starch directly to ethanol, a mouse salivary $\alpha$-amylase cDNA gene with a yeast alcohol dehydrogenase I promoter has been introduced into the cell of a Saccharomyces diactaticus hybrid strain secreting only glucoamylase. To secrete both enzymes more stably without loss of the $\alpha$-amylase gene during a cell-multiplication, an integrating plasmid vector containing $\alpha$-amylase gene was constructed and introduced into the yeast cell. The results showed that the linearized form of the integrating vector was superior in the transformation efficiency and the rate of the expression of the $\alpha$-amylase gene than the circular type of the vector. The yeast transformant having a linearized plasmid vector exhibited higher mitotic stability than the yeast transformant habouring episomat plasmid vector. The transformant containing the linearized vector producing both $\alpha$-amylase and glucoamylase exhibited 2-3 times more amylolytic activity than the original untransformed strain secreting only glucoamylase.

• Mycobiology
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• v.39 no.1
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• pp.33-39
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• 2011

Wild yeasts on the surface of various fruits including grapes were surveyed to obtain yeast strains suitable for fermenting a novel wine with higher alcohol content and supplemented with rice starch. We considered selected characteristics, such as tolerance to alcohol and osmotic pressure, capability of utilizing maltose, and starch hydrolysis. Among 637 putative yeast isolates, 115 strains exhibiting better growth in yeast-peptone-dextrose broth containing 30% dextrose, 7% alcohol, or 2% maltose were selected, as well as five ${\alpha}$-amylase producers. Nucleotide sequence analysis of the 26S rDNA gene classified the strains into 13 species belonging to five genera; Pichia anomala was the most prevalent (41.7%), followed by Wickerhamomyces anomalus (19.2%), P. guilliermondii (15%), Candida spp. (5.8%), Kodamaea ohmeri (2.5%), and Metschnikowia spp. (2.5%). All of the ${\alpha}$-amylase producers were Aureobasidium pullulans. Only one isolate (NK28) was identified as Saccharomyces cerevisiae. NK28 had all of the desired properties for the purpose of this study, except ${\alpha}$-amylase production, and fermented alcohol better than commercial wine yeasts.

• Microbiology and Biotechnology Letters
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• v.38 no.4
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• pp.385-390
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• 2010

Bacillus licheniformis fermenting soybean product with highest score in consumer acceptance had been isolated from homemade Cheongkookjang. In order to develop the medium composition, effects of ingredients including nitrogen sources, carbon sources, metal ions and phosphate were examined for protease production of the isolate. Potato starch increased the protease productivity, while glucose repressed it. Yeast extract was the most effective nitrogen source for enzyme production. The calcium was found to increase protease activity slightly while cell growth and enzyme production was completely inhibited by divalent ions such as $Zn^{2+}$, $Cu^{2+}$ and $Co^{2+}$. The maximum protease productivity was reached approximately 800 unit/mL in the optimized medium consisting of potato starch (1.5%), yeast extract (1.5%), $CaCl_2$(0.7%), $K_2HPO_4$(0.03%) and $KH_2PO_4$(0.03%). The protease activity of culture filtrate was gradually decreased after incubation for 28 h.