• Microbiology and Biotechnology Letters
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• v.29 no.4
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• pp.212-220
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• 2001

Rare mating was used to select a respiratory deficient mutant of Saccharomyces cerevisiae HDC52 strain. Glucoamylase gene of S. diastaticus K114 was developed into the RD mutant which could uptake maximum amount of non-fermentable sugars through the expression of glu- coamplyase gene and the fermentation characteristics of the developed strain HCS were investigated. The size of HCS yeast and HBD52 yeast strain were 13 $\mu\textrm{m}$ and 10$\mu\textrm{m}$ respectively. HCS strain which can uptake maximum amount of non-fermentable sugar through the expression of glucoamylase gene was developed. By karyotype anal- ysis. HCS stain but not RD mutant HBC52 showed a band of 1150 kb chromosome DNA This band should include glcoamylase gene from Saccharomyces diataticus K114 THis strain has glucoamylase which can degrade starch By transduction and contrnuance of glucoamylase gene HCS strain gegraded strach and formed halo. Also, HCS strain maintained the character after 50 generations. Glucoamylase activities of Saccharomyces diastaticus K114 and HCS yeast strains are 9.5 and 2.7~3.4(unit/ml) HCS and HBC52 strain showed similar sugar fermentation patterns and low flocculation In spore and film forming test, HCS and HBC52 strain formed neither spores nor films. In the limit fermentation test, HBC52 strain showed fermentation level of 68% and HCS strain showed 76~78% As the limit attenuation of HBC52 and HCS were ($2.00^{\circ}$P) and ($0.7~0.93^{\circ}$P) This study demon- strates and HCS strain may be used for low carbohydrate beer fermentation.

• 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.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.340-344
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• 2002

A gene, npl, encoding neopullulanase from Paenibacillus sp. KCTC 8848P was cloned and expressed in Escherichia coli. It consisted of an open reading frame of 1,530 bp for a protein that consisted of 510 amino acids with a molecular weight of 58,075 Da. The deduced amino acid sequence of the neopullulanase gene had $92\%$ identity with the neopullulanase of Bacillus polymyxa. The npl gene was also expressed in Saccharomyces cerevisiae secreting Schwanniomyces occidentalis glucoamylase (GAM1) under the control of the yeast actin gene (ACT1) promoter. Secretion of the neopullulanase was directed by the yeast mating pheromone ${\alpha}$ -factor ($MF{\alpha}1$) prepro region. Enzyme assays confirmed that co-expression of npl and GAM1 enhanced starch and pullulan degradation by S. cerevisiae.

• 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.

• 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.

• Korean Journal of Microbiology
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• v.52 no.2
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• pp.220-225
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• 2016

To improve antioxidant glutathione (GSH) content and saccharification ability in sake yeasts of Saccharomyces cerevisiae, the ${\gamma}$-glutamylcysteine synthetase gene (GSH1) from S. cerevisiae, glucoamylase gene (GAM1) and ${\alpha}$-amylase gene (AMY) from Debaryomyces occidentalis were co-expressed in sake yeasts for manufacturing a refreshing alcoholic beverage abundant in GSH from rice starch. The extracellular GSH content of the recombinant sake yeasts increased 1.5-fold relative to the parental wide-type strain. The saccharification ability by glucoamylase of the new yeast strain expressing both GAM1 and AMY genes was 2-fold higher than that of the yeast strain expressing only GAM1 gene when grown in the culture medium containing 2% (w/v) rice starch. It generated 11% (v/v) ethanol from 20% (w/v) rice starch and consumed up to 90% of the starch content after 7 days of fermentation.

• Preventive Nutrition and Food Science
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• v.6 no.4
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• pp.206-210
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• 2001

Starch is an abundant resource in plant biomass, and it should be hydrolyzed enzymatically into fermentable sugars for ethanol fermentation. A genetic recombinant yeast, Saccharomyces cerevisiae GA-7458, was constructed by integrating the structural gene of both $\alpha$-amylase from Bacillus stearothermophilus and the gene (STA1) encoding glucoamylase from S. diastaticus into the chromosome of S. cerevisiae SH7458. The recombinant yeast showed active enzymatic activities of $\alpha$-amylase and glucoamylase. The productivity of ethanol fermentation from the pH-controlled batch culture (pH 5.5) was 2.6 times greater than that of the pH-uncontrolled batch culture. Moreover, in a fed-batch culture, more ethanol was produced (13.2 g/L), and the production yield was 0.38 with 2% of corn starch. Importantly, the integrated plasmids were fully maintained during ethanol fermentation.

• Preventive Nutrition and Food Science
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• v.3 no.1
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• pp.98-104
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• 1998

As an efffort ot construct LAB (latice acid bacteria), capable of utilizing starch as fermentation substrate without the aid of externally supplied enzymes, plasmid vectors containing the amyL($\alpha$-amylase/pullulansase gene) from Clostridium thermophydrosulfuricum, and glucoamylase cDNA from Asperigillus shirousamii were constructed and introduced itno E. coli and L. lactis. For expression in procaryotes , 1.9kb glucoamylase cDNA encoding the mature form of enzyme was PCR amplified and translationaly fused to a PCR amplified 260 bp fragment containing the promotor and secretion signals of amyl in the same reading frame. The production of $\alpha$-amylase, Apu, and glucoamlase in E. coli and L. lactis was confirmed by enzyme assay and zymography . Enzymeswere detected in both cellpellets and supernatants, indicating theworking of scretion signals in heterologous hosts. The efficiencies of secretion were varibale depending on the gene and host. The highest $\alpha$- amylase acitivity observed was 1.1 units and most activiity was detected from thecell pellets. The degree of gene expression in both hosts and the effect on the growth of hosts were examined.

• Korean Journal of Microbiology
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• v.18 no.4
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• pp.161-171
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• 1980

A mutant strain having increased productivity of both enzymes, protease and amylase, was obtained from A. flavus KU 153, isolatd from South Korea for its high protease production by successive ultra-violet light irradiation, Two glucoamylases from the mutant strain selected were purified from wheat branculture by successive salting out, followed by dialysis and column chromatography, and their characteristics were compared with those of the wild strain. Glucoamylase production of the mutant selected was increased about 3.3 times compared with the wild strain, and 2.1 times compared with the parental strain, ${\alpha}-amylase$ activity of the mutant selected was about 2 times hugher than that of the wild strain or the parental strain. Protease and cellulase productivities of the muant selected were all alike compared with those of the highly proteolytic mutant, the parental strain. Therefore, it was considered that the back mutation on the protease production did not occurred in the formation process of the glucoamylase producing mutant. Total activities of glucoamylase I and II from the mutant selected were 2.86 and 3.65 times higher compared with those from the wild strain, respectively. Considering the optimal pH-thermal stability and Km-Vmax value of glucoamylase I and II from both strains, wild and mutant, it was deduced that the characteristics of glucoamylase I and II from the wild strain did not altered during the mutation process. Therefore, it was concluded that the selected mutant did not induce the formation of another glucoamylase isozyme, or the changes in the characteristics of the glucoamylase, but induce the productivity of the same glucoamylase I and II by the action of regulatory gene.

• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.767-774
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• 2010

In this study, the problems of high caloric content, increased maturation time, and off-flavors in commercial beer manufacture arising from residual sugar, diacetyl, and acetaldehyde levels were addressed. A recombinant industrial brewing yeast strain (TQ1) was generated from T1 [Lipomyces starkeyi dextranase gene (LSD1) introduced, ${\alpha}$-acetohydroxyacid synthase gene (ILV2) disrupted] by introducing Saccharomyces cerevisiae glucoamylase (SGA1) and a strong promoter (PGK1), while disrupting the gene coding alcohol dehydrogenase (ADH2). The highest glucoamylase activity for TQ1 was 93.26 U/ml compared with host strain T1 (12.36 U/ml) and wild-type industrial yeast strain YSF5 (10.39 U/ml), respectively. European Brewery Convention (EBC) tube fermentation tests comparing the fermentation broths of TQ1 with T1 and YSF5 showed that the real extracts were reduced by 15.79% and 22.47%; the main residual maltotriose concentrations were reduced by 13.75% and 18.82%; the caloric contents were reduced by 27.18 and 35.39 calories per 12 oz. Owing to the disruption of the ADH2 gene in TQ1, the off-flavor acetaldehyde concentrations in the fermentation broth were 9.43% and 13.28%, respectively, lower than that of T1 and YSF5. No heterologous DNA sequences or drug resistance genes were introduced into TQ1. Hence, the gene manipulations in this work properly solved the addressed problems in commercial beer manufacture.