• Archives of Pharmacal Research
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• v.21 no.1
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• pp.46-53
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• 1998

When NIH3T3 cells were exposed to mild heat and recovered at $37^{\circ}C$ for various time intervals, they were thermotolerant and resistant to subsequent stresses including heat, oxidative stresses, and antitumor drug methotrexate which are apoptotic inducers. The induction kinetics of apoptosis by stresses were determined by DNA fragmentation and protein synthesis using $[35^S]$methionine pulse labeling. We investigated the hypothesis that thermotolerant cells were resistant to apoptotic cell death compared to control cells when both cells were exposed to various stresses inducing apoptosis. The cellular changes in thermotolerant cells were examined to determine which components are involved in this resistance. At first, the degree of resistance correlates with the extent of heat shock protein synthesis which were varied depending on the heating times at $45^{\circ}C$ and recovery times at $37^{\circ}C$after heat shock. Secondly, membrane permeability change was observed in thermotolerant cells. When cells prelabeled with $[^{3}H]$thymidine were exposed to various amounts of heat and recovered at $37^{\circ}C$ for 1/2 to 24 h, the permeability of cytosolic $[^{3}H]$thymidine in thermotolerant cells was 4 fold higher than that in control cells. Thirdly, the protein synthesis rates in thermotolerant and control cells were measured after exposing the cells to the same extent of stress. It turned out that thermotolerant cells were less damaged to same amount of stress than control cells, although the recovery rates are very similar to each other. These results demonstrate that an increase of heat shock proteins and membrane changes in thermotolerant cells may protect the cells from the stresses and increase the resistance to apoptotic cell death, even though the exact mechanism should be further studied.

• KSBB Journal
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• v.30 no.3
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• pp.125-131
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• 2015

Kluyveromyces marxianus is a well-known thermotolerant yeast. Although Saccharomyces cerevisiae is the most commonly used yeast species for ethanol production, the thermotolerant K. marxianus is more suitable for simultaneous saccharification and fermentation (SSF) processes. This is because enzymatic saccharification usually requires a higher temperature than that needed for the optimum growth of S. cerevisiae. In this study, we compared the fermentation patterns of S. cerevisiae and K. marxianus under various temperatures of fermentation. The results show that at a fermentation temperature of $45^{\circ}C$, K. marxianus exhibited more than two fold higher growth rate and ethanol production rate in comparison to S. cerevisiae. For SSF using starch or corn stover as the sole carbon source by K. marxianus, the high temperature ($45^{\circ}C$) fermentations showed higher enzymatic activities and ethanol production compared to SSF at $30^{\circ}C$. These results demonstrate the potential of the thermotolerant yeast K. marxianus for SSF in the industrial production of biofuels.

• Journal of Microbiology and Biotechnology
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• v.4 no.3
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• pp.215-221
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• 1994

Two strains of yeast (RA-74-2 and RA-912) showing superior fermenting ability at a high temperature were isolated from soils and wastewaters by an enrichment culture method. Based on the morphological and physiological charateristics, the two strains were identified as Saccharomyces cerevisiae and Kluyveromyces marxianus, respectively. RA-74-2 was able to grow upto $43^{\circ}C$ and sustain similar fermenting ability in the temperatures range from 30 to $40^{\circ}C$. In addition, the sugar- and ethanol-tolerance of RA-74-2 were 30% (w/v) glucose and 10% (v/v) ethanol, which appeared to be higher than those of nine other industrial yeast strains currently being used in the alcohol factories. The thermotolerant ethanol fermenting yeast RA-912 showed identical growth in the temperatures range from 35 to $45^{\circ}C$ and was resistant to various heavy metals. The quality and quantity of byproducts of the isolated yeast strains in fermentation broth after fermentation at $40^{\circ}C$ and $45^{\circ}C$ were similiar with those obtained at $30^{\circ}C$. These results show that RA-74-2 can be adopted for the ethanol fermentation process where the expenses for cooling system is significant, and suggest that RA-912 may be applied in either SSF(simultaneous saccharification and fermentation) or Flash-fermentation process and RA-912 may be used as a gene donor for the development of thermotolerant ethanol-fermenting yeasts.

• Journal of Microbiology and Biotechnology
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• v.7 no.1
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• pp.62-67
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• 1997

To enhance the hyperproductive and low energy-consuming ethanol fermentation rate, the thermotolerant yeast S. cerevisiae RA-74-2 cells were immobilized. An efficient immobilization condition was proved to be $1.5{\%}$ (w/v) alginate solution, neutral pH and 20 h activation of beads. The fermentation characteristics and stability at various temperatures were examined as compared with free S. cerevisiae RA-74-2 cells. The immobilized cells had excellent fermentation rate at the range of pH 3-7 at 30-$42^{\circ}C$ in 15-$20{\%}$ glucose media. When the seed volume was adjusted to 0.12 (v/v) (6ml bead/50 ml medium), $11{\%}$ (w/v) ethanol was produced during the first 34 hand $12.15{\%}$ (w/v) ethanol [$95{\%}$ (w/v) of theoretical yield] during the first 60 h in $25{\%}$ glucose medium. In repetitive fermentation using a 2 litre fermentor, 5.79-$7.27{\%}$ (w/v) ethanol [76-$95{\%}$ (w/v) of theoretical yield] was produced during the 40-55 h in $15{\%}$ glucose media. These data suggested the fact that alginate beads of thermotolerant S. cerevisiae RA-74-2 cells would contribute to economic and hyperproductive ethanol fermentation at high temperature.

• Microbiology and Biotechnology Letters
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• v.23 no.5
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• pp.617-623
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• 1995

A new thermotolerant yeast strain was siolated, and its characteristics have been studied. The strain was identified and named Saccharomyces cerevisiae F38-1. This strain could grow not only at high temperature, but also in high concentrations of sugar and ethanol. S. cerevisiae F38-1 could grow in a medium containing 50% glucose. The isolate produced ethanol at 43$\circ$C, but didn't grow at 40$\circ$C in the presence of 8% ethanol. Fermentation studies showed that the isolate ferments 20% glucose to 9.8% (V/V) ethanol at 40$\circ$C in the presence of 0.2%, yeast extract.

• The Korean Journal of Food And Nutrition
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• v.12 no.5
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• pp.490-495
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• 1999

A thermotolerant yeast Saccharomyces cerevisiae OE-16 was isolated from traditional Meju was investigated on their physiological characteristics and ethanol fermentation ability. Saccharomyces cerevisiae OE-16 were able to grow up to 45$^{\circ}C$ and 40% of glucose. Saccharomyces cerevisiae OE-16 was also resistant to 15% of KCl 1,200ppm of Pb2+, Hg2+ and 500ppm of potassium sorbate. From 20% glucose media Saccharomyces cerevisiae OE-16 produced 83.4g per liter of ethanol at 3$0^{\circ}C$ and 9.5g per liter of ethanol at 4$0^{\circ}C$ for 72 hours.

• Journal of Microbiology and Biotechnology
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• v.33 no.7
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• pp.875-885
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• 2023

Volatile organic compounds such as benzene, toluene, ethylbenzene, and isomers of xylenes (BTEX) constitute a group of monoaromatic compounds that are found in petroleum and have been classified as priority pollutants. In this study, based on its newly sequenced genome, we reclassified the previously identified BTEX-degrading thermotolerant strain Ralstonia sp. PHS1 as Cupriavidus cauae PHS1. Also presented are the complete genome sequence of C. cauae PHS1, its annotation, species delineation, and a comparative analysis of the BTEX-degrading gene cluster. Moreover, we cloned and characterized the BTEX-degrading pathway genes in C. cauae PHS1, the BTEX-degrading gene cluster of which consists of two monooxygenases and meta-cleavage genes. A genome-wide investigation of the PHS1 coding sequence and the experimentally confirmed regioselectivity of the toluene monooxygenases and catechol 2,3-dioxygenase allowed us to reconstruct the BTEX degradation pathway. The degradation of BTEX begins with aromatic ring hydroxylation, followed by ring cleavage, and eventually enters the core carbon metabolism. The information provided here on the genome and BTEX-degrading pathway of the thermotolerant strain C. cauae PHS1 could be useful in constructing an efficient production host.

• Microbiology and Biotechnology Letters
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• v.23 no.5
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• pp.624-631
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• 1995

The fermentation characteristics of Saccharomyces cerevisiae F38-1, a newly isolated thermotolerant yeast strain from a high temperature environment have been studied using a fermentation medium containing 20% glucose, 0.2% yeast extract, 0.2% polypeptone, 0.3% (NH$_{4}$)$_{2}$SO$_{4}$, 0.1% KH$_{2}$PO$_{4}$, and 0.2% MgSO$_{4}$ without shaking at 30$\circ$C to 43$\circ$C for 5 days. The fermentability was over 90% at 30$\circ$C, 88% at 37$\circ$C, 77% at 40$\circ$C and 30% at 43$\circ$C. A similar fermentation result was obtained at pH between 4 and 6 at 30$\circ$C and 40$\circ$C. Aeration stimulated the growth of the strain at the beginning of the fermentation, but it reduced alcohol production at the end of alcohol fermentation. Optimal glucose concentration was determined to be between 18 and 22% at 40$\circ$C as well as 30$\circ$C, but the growth was inhibited at the glucose concentration of over 30%. A fermentability of over 90% was observed at 40$\circ$C in 2 days when the medium was supplemented by 2% yeast extract. A higher inoculum size increased the initial fermentation rate, but not the fermentation. A fermentability of over 90% was achieved in 2 days at 40$\circ$C in a fermentor experiment using an optimized medium containing 20% glucose and 1% yeast extract.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.265-269
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• 1988

For the purpose of developing new thermotolerant yeast strains for ethanol fermentation, yeasts were isolated from molasses and screened for their fermentation ability at elevated temperatures. Three candidate strains were screened. These strains preferred pH 5.0 and 34$^{\circ}C$ for their ethanol production. Under such conditions the three strains showed average ethanol productivity of 75g ethanol per liter of fermentation broth in n synthetic medium containing glucose as substrate. These strains were identified as Saccharomyces cerevisiae and Kluveromyces marxianus.

• The Korean Journal of Pharmacology
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• v.32 no.3
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• pp.433-444
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• 1996

We describe a novel approach to evaluate quantitatively the amounts of denatured proteins in cells upon heat exposure. A thiol compound, diamide [azodicarboxylic acid bis (dimethylamide)] causes protein cross-linking with exposed sulfyhydryl residues of denatured proteins. Since denatured proteins expose normally well-hidden sulfhydryl groups, these will be preferentially cross-linked by diamide. Thus diamide acts to 'trap' denatured proteins. We observed that protein aggregates (high molecular weight protein aggregates, HMA) appeared on SDS-polyacrylamide gels run under non-reducing conditions and that the amount of HMA can be quantified by scanning the gels using a gas flow counter. Heating cells followed by a fixed dose of diamide exposure resulted in HMA increases in a heat-dose dependent manner, demonstrating that the quantitation of HMA could serve as a measure of heat-denatured proteins. We compared thermotolerant and nontolerant cells and found decreased HMA in tolerant cells upon heat treatment. As an attempt to examine the kinetics of protein renaturation (or 'repair'), we measured the amounts of aggregates formed by the addition of diamide at various times after heat shock. Such experiments demonstrate an equally rapid disappearance of HMA in previously unheated and in thermotolerant cells. Levels of HMA in tolerant cells increased significantly after electroporation of HSP70 specific mAbs, suggesting an involvement of HSP70 in reducing HMA levels in thermotolerant cells upon heat exposure. Immunoprecipitation studies using anti-HSP70 antibody indicated an association of HSP70 with heat-denatured proteins. Our results suggest that heat induces protein denaturation, and that elevated level of HSP70 present in thermotolerant cells protects them by reducing the level of protein denaturation rather than by facilitating the 'repair' (or degradation) process.