• Korean Journal of Microbiology
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• v.42 no.3
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• pp.195-198
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• 2006

Lethal toxin is a critical virulence factor of anthrax. It is composed two protein: protective antigen (PA) and lethal factor (LF). PA binds to specific cell surface receptors and, forms a membrane channel that mediates entry of LF into the cell. LF is a zinc-dependent metalloprotease, which cleaves MKKs [MAPK (mitogen-activated protein kinase) kinases] at peptide bonds very close to their N-termini. In this study, we suggest application of cell-based assays in the early phase of drug discovery, with a particular focus on the use of yeast cells. We constructed MEK1 expression system in yeast to determine LF activity and approached cell-based assay system to screen inhibitors, in which the results covering the construction of LF-substrate in yeast expression vector, expression, and LF-mediated proteolysis of substrate were described. These results could provided the basic steps in design of cell-based assay system with the high efficiency, rapidly and easy way to screening of inhibitors.

• Journal of Microbiology and Biotechnology
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• v.16 no.3
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• pp.360-367
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• 2006

The endoinulinase gene (inu1) from Pseudomonas mucidolens was expressed on the cell surface of Saccharomyces cerevisiae by fusing with Aga2p linked to the membrane anchored protein, Aga1p. The inu1 gene of P. mucidolens was subcloned into the surface display vector, pCTcon (GAL1 promoter). The constructed plasmid, pCTENIU (8.5kb), was then introduced to S. cerevisiae EBY100 cells and the yeast transformants selected on synthetic defined media lacking uracil and inulin-containing media. The inu1 gene under the control of the GAL1 promoter was successfully expressed in the yeast transformants, and the surface display of endoinulinase confirmed by immunofluorescence microscopy, along with its enzymatic ability to form inulooligosaccharides (IOSs) from inulin. The total endoinulinase activity reached about 2.31 units/ml when the yeast transform ants were cultivated on a YPDG medium. To efficiently hydrolyze the inulin, various reaction conditions were examined, including the pH, temperature, and inulin source. The optimized conditions were then determined as follows: pH, 7.0; temperature, $50^{\circ}C$; inulin source, Jerusalem artichoke. Under the optimized condition and 46 units of endoinulinase per g of inulin, IOSs started to be produced after 10 min of enzymatic reaction. The highest yield, 71.2% of IOSs, was achieved after 30 h of reaction without any significant loss of the initial enzyme activity. As a result of the reaction with inulin, IOSs consisting of inulobiose (F2), inulotriose (F3), inulotetraose (F4), and inulopentaose (F5) were produced, and F4 was the major product.

• Korean Journal of Food Science and Technology
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• v.17 no.3
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• pp.203-207
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• 1985

Relationship between cell surface hydrophobicity and pellicle formation was studied in a film strain isolated from stored apple wine and identified as Hansenula beijerinckii FY-5. In the media containing non-ionic surface-active agents the pellicle formation of strain FY-5 was efficiently repressed, whereas growth of the yeast was possible, and also cell surface hydrophobicity was greatly decreased by the addition of these agents. These results indicate that a pellicle formation factor, which keeps yeast cells floating on the medium surface, is necessary for the pellicle formation, and surely this factor is the hydrophobicity of the cell surface. The pellicle formation in the film strains was abundant with the increase of the cell surface hydrophobicity, whereas the non-film strains had less hydrophobicity as compared with the film strains. Ethanol, as a sole carbon source, efficiently increased hydrophobicity more than glucose, and the hydrophobicity was lowered with the rise of pH. In the experiments of time course, the hydrophobicity was increased in proportion to cell growth, and was maximum during the stationary phase.

• Molecules and Cells
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• v.41 no.1
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• pp.35-44
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• 2018

Mitochondria are responsible for supplying of most of the cell's energy via oxidative phosphorylation. However, mitochondria also can be deleterious for a cell because they are the primary source of reactive oxygen species, which are generated as a byproduct of respiration. Accumulation of mitochondrial and cellular oxidative damage leads to diverse pathologies. Thus, it is important to maintain a population of healthy and functional mitochondria for normal cellular metabolism. Eukaryotes have developed defense mechanisms to cope with aberrant mitochondria. Mitochondria autophagy (known as mitophagy) is thought to be one such process that selectively sequesters dysfunctional or excess mitochondria within double-membrane autophagosomes and carries them into lysosomes/vacuoles for degradation. The power of genetics and conservation of fundamental cellular processes among eukaryotes make yeast an excellent model for understanding the general mechanisms, regulation, and function of mitophagy. In budding yeast, a mitochondrial surface protein, Atg32, serves as a mitochondrial receptor for selective autophagy that interacts with Atg11, an adaptor protein for selective types of autophagy, and Atg8, a ubiquitin-like protein localized to the isolation membrane. Atg32 is regulated transcriptionally and post-translationally to control mitophagy. Moreover, because Atg32 is a mitophagy-specific protein, analysis of its deficient mutant enables investigation of the physiological roles of mitophagy. Here, we review recent progress in the understanding of the molecular mechanisms and functional importance of mitophagy in yeast at multiple levels.

• Journal of the Korean Society of Food Science and Nutrition
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• v.14 no.4
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• pp.401-408
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• 1985

The yeast cell wall lytic action of the alkaline AL-protease, which was found out of the crude Zymolyase that a kind of yeast cell wall lytic $endo-{\beta}-1$, 3-glucanase produced from Arthrobacter luteus, was investigated with the viable cells of S. sake and it's cell wall preparation. AL-protease on the lysis of the viable yeast cells showed very low activities with the alone, but the lytic activities were highly increased with the combination of AL-protease and Zymolyase. On the stepwise treatment of the viable yeast cells with AL-protease and Zymolyase, the cells were lysed highly only by the course having a treatment with Zymolyase after pretreatment with AL-protease. Thus synergistic action of AL-protease was not observed with any some commercial enzymes, known as a type of alkaline and serine protease such as AL-protease, and was also found to be affected greatly by the culture conditions and species of the yeast tested. AL-protease caused the release of some peptide and a lot of sugar from the cell wall preparation, but could not lysed the cell wall more than 66%. Whereas Zymolyase could lysed the cell walls almost completely with alone. On the basis of these results, the synergistic action of AL-protease on the lysis of S. sake cells is hypothesized that at first AL-protease bind to the yeast cell surface layer consisting of mannan and protein, and then changes their conformation to facilitate the penetration of Zymolyase from the outside to the inside framework layer constituted of alkali insoluble ${\beta}-1,\;3-glucan$.

• Journal of Microbiology
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• v.37 no.3
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• pp.128-135
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• 1999

Among oil-degrading microorganisms isolated from oil-polluted industrial areas, one yeast strain showed high degradation activity of aliphatic hydrocarbons. From the analyses of 18S rRNA sequences, fatty acid, coenzyme Q system, G+C content of DNA, and biochemical characteristics, the strain was identified as Yarrowia lipolytica 180. Y. lipolytica 180 degraded 94% of aliphatic hydrocarbons in minimal salts medium containing 0.2% (v/v) of Arabian light crude oil within 3 days at 25$^{\circ}C$. Optimal growth conditions for temperature, pH, NaCl concentration, and crude oil concentration were 30$^{\circ}C$, pH 5-7, 1%, and 2% (v/v), respectively. Y. lipolytica 180 reduced surface tension when cultured on hydrocarbon substrates (1%, v/v), and the measured values of the surface tension were in the range of 51 to 57 dynes/cm. Both the cell free culture broth and cell debris of Y. lipolytica 180 were capable of emulsifying 2% (v/v) crude oil by itself. They were also capable of degrading crude oil (2%). The strain showed a cell surface hydrophobicity higher than 90%, which did not require hydrocarbon substrates for its induction. These results suggest that Y. lipolytica has high oil-degrading activity through its high emulsifying activity and cell hydrophobicity, and further indicate that the cell surface is responsible for the metabolism of aliphatic hydrocarbons.

• BMB Reports
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• v.30 no.3
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• pp.167-172
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• 1997

The yeast Saccharomyces cerevisiae, mutant CH117, shows a drug-hypersensitivity (dhs) to cycloheximide, bleomycin, actinomycin D, 5-fluorouracil. nystatin, nigericin and several other antibiotics. CH 117 was also temperature-sensitive (ts). being unable to grow at $37^{\circ}C$ and secreted more invertase and acid phosphatase into the medium than the parent yeast. CH117 grows very slowly and the cell shape is somewhat larger and more sensitive to zymolyase than the wild type cells. Light microscopic and electron microscopic observation also revealed abnormality of the mutant cell wall. These characteristics indicate that CH117 has a defect in an essential component of the cell surface and that the cell wall which performs barrier functions has become leaky in the mutant. We screened a genomic library of wild type yeast for clones that can complement the mutation of CH117. A plasmid, pCHX1, with an insert of 3.6 kilobases (kbs) could complement the dhs and ts of CH117. Deletion and subcloning of the 3.6 kb insert showed that a gene for the complementation of mutant phenotypes was located in 1.9 kbs Puvll-Hindlll fragment.

• Microbiology and Biotechnology Letters
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• v.14 no.4
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• pp.299-304
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• 1986

The cell volume, cell surface, cell concentration, dry cell weight, frequence of respiratory deficient mutation, resistance against ultraviolet irradiation, fermentation power, DNA contents of haploid diploid, triploid and tetraploid of Saccharomyces cerevisiae strain were investigated. Respiratory deficient mutants by spontaneous mutation were absolved more frequently in the haploid than in the diploid, triploid and tetraploid. And cell volume, cell surface, cell concentration, dry cell weight, resistance against ultraviolet irradiation, fermentation power, and DNA contents were significantly increased as the ploidy increased.

• Journal of Microbiology and Biotechnology
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• v.25 no.11
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• pp.1856-1862
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• 2015

In order to improve the stability of endoglucanase under thermal and acidic conditions, the endoglucanase gene was fused to the N-terminus of the Saccharomyces cerevisiae pir gene, encoding the cell wall protein PIR. The fusion gene was transformed into Pichia pastoris GS115 for expression. A resulting strain with high expression and high activity was identified by examining resistance to Geneticin 418, Congo red staining, and quantitative analysis of enzyme activity. SDS-PAGE analysis revealed that the endoglucanase was successfully displayed on the yeast cell surface. The displayed endoglucanase (DEG) showed maximum activity towards sodium carboxyl methyl cellulose at approximately 275 IU/g cell dry weight. DEG exhibited greater than 60% residual activity in the pH range 2.5-8.5, higher than free endoglucanase (FEG), which had 40% residual activity at the same pH range. The highest tolerated temperature for DEG was 70℃, much higher than that of FEG, which was approximately 50℃. Moreover, DEG showed 91.1% activity at 65℃ for 120 min, while FEG only kept 77.8% residual activity over the same period. The half-life of DEG was 270 min at 65℃, compared with only 150 min for FEG. DEG could be used repeatedly at least three times. These results suggest that the DEG has broad applications as a yeast whole-cell biocatalyst, due to its novel properties of high catalytic efficiency, acid-thermal stabilities, and reusability.

• Journal of Microbiology and Biotechnology
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• v.9 no.1
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• pp.70-77
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• 1999

An extremely cadmium-tolerant budding yeast, Hansenula anomala B-7 underwent a morphological switch in response to either heat shock treatment or cadmium stress, respectively. It exhibited a morphological transition from a unicellular yeast form to a pseudohyphae-like coagulation when subjected to prolonged heat shock treatment. In contrast, the yeast cells showed an irregularity in surface morphology when given thermal stress for a short time. Patterns of proteins expressed in the pseudohyphae-like cells demonstrated that several proteins were overexpressed while others were underexpressed in comparison with those prepared from the cells in the yeast form. It was a striking feature, however, that nearly 40％ of the proteins extracted from the cells in the pseudohyphae form appeared to be composed of a single polypeptide. This polypeptide was apparently overexpressed during the pseudohyphae phase and its molecular weight was estimated to be 58 kDa according to SDS-PAGE analysis. However, a significant level of the protein was not observed in the cells before transition to pseudohyphae. The architecture of the cell shape was also damaged when incubated in a medium containing more than 1,000 ppm (8.9mM) of cadmium ions, although able to proliferate at a slow rate. However, the irregularity in the cell morphology exerted either by the brief heat shock treatment or by the cadmium stress with the high concentrations of the metal ions was not repaired, even though the damaged cells were allowed to grow for sufficient time in fresh, cadmium-free medium.