• Title/Summary/Keyword: Culture Temperatures

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Continuous Synthesis of Escherichia coli GroEL at a high Temperature

  • Kwak, Young-Hak;Lee, Kyong-Sun;Kim, Ji-Yeon;Lee, Dong-Seok;Kim, Han-Bok
    • Journal of Microbiology
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    • v.38 no.3
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    • pp.145-149
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    • 2000
  • GroEL is a typical molecular chaperone. GroEL synthesis patterns at various culture temperatures in Escherichia coli were investigated in this study. No significant differences in the amount of GroEL produced from the chromosome were found at 30 and 37$^{\circ}C$. However, GroEL production increased 3.4-fold at 42$^{\circ}C$. GroEL synthesis was not transient but continuous at 42$^{\circ}C$, although most heat shock gene expression is known to be transient. To understand the role of the groEL structural gene, a groE promoter-lacZ fusion was constructed. Interestingly , while transcriptional fusion is not thermally inducible, it is inducible by ethanol, suggesting that the secondary structure of the groEL transcript is involved in thermal regulation of the groEL gene. Secondary structures of groE mRNA at 37 and 42$^{\circ}C$ were compared using the computer program RNAdraw. Distinct structures at the two temperatures were found, and these structures may be related to a high level of GroEL expression at 42$^{\circ}C$.

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Screening and Characterization of Thermotolerant Alcohol-producing Yeast

  • Sohn, Ho-Yong
    • 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.

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Purification and Characterization of Two Thermostable Xylanases from Paenibacillus sp. DG-22

  • Lee, Yong-Eok;Lim, Pyung-Ok
    • Journal of Microbiology and Biotechnology
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    • v.14 no.5
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    • pp.1014-1021
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    • 2004
  • Two thermostable xylanases, designated XynA and XynB, were purified to homogeneity from the culture supernatant of Paenibacillus sp. DG-22 by ion-exchange and gel-filtration chromatography. The molecular masses of xylanases A and B were 20 and 30 kDa, respectively, as determined by SDS-PAGE, and their isoelectric points were 9.1 and 8.9, respectively. Both enzymes had similar pH and temperature optima (pH 5.0-6.5 and $70^{\circ}C$), but their stability at various temperatures differed. Xylanase B was comparatively more stable than xylanase A at higher temperatures. Xylanases A and B differed in their $K_m$ and $V_{max}$ values. XynA had a $K_m$ of 2.0 mg/ml and a $V_{max}$ of 2,553 U/mg, whereas XynB had a K_m$ of 1.2 mg/ml and a $V_{max}$, of 754 U/mg. Both enzymes were endo-acting, as revealed by their hydrolysis product profiles on birchwood xylan, but showed different modes of action. Xylotriose was the major product of XynA activity, whereas XynB produced mainly xylobiose. These enzymes utilized small oligosaccharides such as xylotriose and xylotetraose as substrates, but did not hydrolyzed xylobiose. The amino terminal sequences of XynA and XynB were determined. Xylanase A showed high similarity with low molecular mass xylanases of family 11.

A Laboratory-scale Recirculating Aquaculture System for Sea Cucumber Apostichopus japonicus (소형 순환여과양식시스템에서 어린 해삼(Apostichopus japonicus) 사육)

  • Jeong, U-Cheol;Jin, Feng;Choi, Jong-Kuk;Han, Jong-Cheol;Choi, Byong-Dae;Kang, Seok-Joong
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.49 no.3
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    • pp.343-350
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    • 2016
  • The sea cucumber Apostichopus japonicus is a commercially valuable aquaculture species in Korea. Aquaculture species require specific nursery culture conditions to increase survival and growth rates. Sea cucumbers hibernate during the high temperatures of summer and during the low temperatures of winter, and suboptimal temperature conditions decrease sea cucumber growth and survival rates. The natural South Korean environment is very unfavorable for culturing sea cucumber; therefore, developing a recirculating aquaculture system (RAS) capable of breeding and growing sea cucumber year-round is necessary. The aim of this study was to investigate growth performance of juvenile sea cucumber in a RAS. Growth and survival rates of juvenile sea cucumber were high during our 24-week experiment. Sea cucumber survival rates were 87.8-93.3%, and specific growth rates were 0.4689-0.7846.

Fasting and Postprandial Ammonia Nitrogen Excretion of Juvenile Oplegnathus fasciatus at Various Temperatures in a Recirculating System (순환여과식 내 수온에 따른 돌돔(Oplegnathus fasciatus) 치어의 절식 및 식후 암모니아 배설)

  • Oh, Sung-Yong;Choi, Hee-Jung
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.53 no.6
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    • pp.918-924
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    • 2020
  • This study investigated diurnal fasting and postprandial total ammonia nitrogen (TAN) excretion rates in juvenile Oplegnathus fasciatus (rock bream; mean body weight 45.8±1.1 g) at three temperatures (15, 20, and 25℃) in a recirculating system (three replicates). The fish were hand-fed twice daily (09:00 and 17:00 h) with commercial food (46.7% protein) until satiation. The results showed that the fasting and postprandial TAN excretion rates were temperature-dependent. The mean hourly rates were 7.7 (15℃), 10.2 (20℃), and 11.9 (25℃) mg kg fish-1 h-1 for fasting and 34.0 (15℃), 47.8 (20℃), and 60.2 (25℃) mg kg fish-1 h-1 for postprandial. At each temperature, two postprandial TAN excretion peaks were observed 2-4 h after feeding; the second peak was always higher. The TAN loss to nitrogen consumption ratio was 41.1-46.8 % and increased significantly with increasing temperature. Temperature affected fasting and postprandial TAN excretion in juvenile rock bream, providing insight for culture management.

Effect of Water Temperature and Photoperiod on the Oxygen Consumption Rate of Juvenile Pacific Cod Gadus macrocephalus (대구 Gadus macrocephalus 치어의 산소 소비율에 미치는 수온과 광주기의 영향)

  • Oh, Sung-Yong;Park, Heung-Sik;Kim, Chong-Kwan
    • Ocean and Polar Research
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    • v.32 no.3
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    • pp.229-236
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    • 2010
  • A study was conducted to investigate the effect of water temperature and photoperiod on the oxygen consumption of the fasting juvenile Pacific cod Gadus macrocephalus (mean body weight 79.9${\pm}$2.0 g) in order to quantify metabolic response of the species under given conditions. The oxygen consumption rate (OCR) of G. macrocephalus was measured under a combination of four different water temperatures (7, 10, 13 and $16^{\circ}C$) and three different photoperiods (24L:0D, 12L:12D and 0L:24D) with an interval of 5 minutes over a 24-hour period using a closed recirculating respirometer. Three replicates were set up in each treatment. OCRs increased with increased water temperatures under all photoperiod conditions (P<0.001). Mean OCRs at 7, 10, 13 and 16oC ranged from 793.7~1108.4, 1145.7~1570.3, 1352.8~1742.5 and 1458.2~1818.6 mg $O_2$ $kg^{-1}$ $h^{-1}$, respectively. Under all water temperature conditions except $7^{\circ}C$ (P<0.001), mean OCRs of G. macrocephalus were the highest in continuous light (24L:0D) followed by 12L:12D and 0L:24D photoperiods. Mean OCRs of fish exposed to the 12L:12D photoperiod were significantly higher during the light phase than during the dark phase under all temperature conditions (P<0.001). $Q_{10}$ values ranged from 3.19~5.13 between 7 and $10^{\circ}C$, 1.41~1.74 between 10 and $13^{\circ}C$ and 1.15~1.35 between 13 and $16^{\circ}C$, respectively. Based on overall results, water temperature, photoperiod and their combinations exerted a significant influence on the metabolic rate of juvenile cod. This study provides empirical data for estimating the amount of oxygen demand and managing the culture of cod under the given water temperatures and photoperiods.

Effects of Environmental Factors on the Zoospore Release and Germling Growth of the Green Macroalga Chaetomorpha linum (녹조류 실염주말의 유주자 방출과 배아생장에 미치는 환경요인의 영향)

  • Han, Su Jin;Na, Yeon Ju;Jeon, Da Vine;Kim, Young Sik;Choi, Han Gil;Nam, Ki Wan
    • Ocean and Polar Research
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    • v.38 no.1
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    • pp.47-57
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    • 2016
  • Effects of environmental factors on the zoospore release and germling growth of Chaetomorpha linum were examined. Zoospore release and germling growth experiments were carried out under a combination of temperatures ${\times}$ irradiances, and a single factor of irradiances, salinities, daylength and nutrients (nitrogen, phosphorus). Zoospore release was maximal under condition of $20^{\circ}C{\times}100{\mu}mol\;photons\;m^{-2}s^{-1}$ in a factorial experiment (temperatures ${\times}$ irradiances), at $100{\mu}mol\;photons\;m^{-2}s^{-1}$ in irradiance experiment and at 34 psu in salinity conditions. After 24 days in culture, germling growth was maximal at $25^{\circ}C{\times}100{\mu}mol\;photons\;m^{-2}s^{-1}$ in temperatures ${\times}$ irradiances and at normal seawater (34 psu) in salinity. Germling growth is faster at higher irradiance under same temperature condition and it increased with increasing daylengths (8-16 h). Chaetomorpha linum grew in wide range of N and P concentrations. The growth of germling was maximal at $50{\mu}M\;{PO_4}^{3-}$ and $40{\mu}M\;{NH_4}^+$. Germling growth was more effective under nitrogen addition than phosphorus addition in culture. In conclusion, optimal environmental conditions for zoospore release were $20^{\circ}C$, $100{\mu}mol\;photons\;m^{-2}s^{-1}$, and 34 psu. In addition, the optimal germling growth was observed at $25^{\circ}C$, $100{\mu}mol\;photons\;m^{-2}s^{-1}$, 34 psu, 16 h, $50{\mu}M\;({PO_4}^{3-})$ and $40{\mu}M\;{(NH_4}^+)$.

Studies on Potato Glycoalkaloid Determination by Acid-hydrolysis Method (산 가수분해 방법에 의한 감자 glycoalkaloid성분의 정량성 검토)

  • Yoon, Kyung-Soon;Byun, Gwang-In
    • Journal of the Korean Society of Food Culture
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    • v.24 no.1
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    • pp.84-89
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    • 2009
  • This paper was conducted to evaluate aglycones and carbohydrates produced by acid hydrolysis of three potato glycoalkaloids [(PGA); ${\alpha}$-chaconine, ${\alpha}$-solanine, and demissine] in potatoes. Standard solanidine and demissidine were dissolved in 1N HCl and then heated at $100^{\circ}C$ for 10-120 min. Solanidine was rapidly decomposed during acid hydrolysis and one peak that was identified as solantherene ($M^+$=379) by GC-MS was detected. The transformation solanidine to solanthrene was approximately 50% complete after 10 min, approximately 90% complete after 60 min and 100% complete after 120 min. Demissidine was hydrolyzed using the same method that was used to hydrolyze the solanidine. However, demissidine produced only one peak upon GC-MS ($M^+$=399) analysis and was found to be very stable at increased temperatures. Acidy hydrolysis of ${\alpha}$-chaconine, ${\alpha}$-solanine and demissine resulted in the decomposition of ${\alpha}$-chaconine and ${\alpha}$-solanine to solanidine and solanthrene, respectively. Therefore, this hydrolysis method should not be utilized to produce PGA combining with solanidine as aglycone. The individual carbohydrates produced by the two PGAs by hydrolysis were very stable at increased temperatures; therefore, it was possible to quantify these PGAs based on calculation of the individual carbohydrate content. Conversely, because demissidine produced by the hydrolysis of demissine was extremely stable at increased temperatures, it was possible to quantify the PGA based on the aglycone produced by hydrolysis.

Effect of Induction Temperature on the $P_L$ Promoter Controlled Production of Recombinant Human Interleukin-2 in Escherichia coli

  • Lee, In-Young;Kim, Myung-Kuk;Lee, Sun-Bok
    • Journal of Microbiology and Biotechnology
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    • v.2 no.1
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    • pp.26-34
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    • 1992
  • The effect of induction temperature on fermentation parameters has been investigated extensively using Escherichia coli M5248[pNKM21], a producer of recombinant human interleukin-2 (rhIL-2). In this recombinant microorganism, the gene expression of rhIL-2 is regulated by the cI857 repressor and $P_L$ promoter system. The recombinant fermentation parameters studied in this work include the cell growth, protein synthesis, cell viability, plasmid stability, $\beta$-lactamase activity, and rhIL-2 productivity. Interrelationships of such fermentation parameters have been analyzed through a quantitative assessment of the experimental data set obtained at eight different culture conditions. While the expression of rhIL-2 gene was repressed at culture temperatures below $34^\circ{C}$ with little effect on other fermentation parameters, under the conditions of rhIL-2 production $>(36~44^\circ{C})$ the cell growth, plasmid stability, and $\beta$-lactamase activity were, as induction temperature was increased, more profoundly reduced. Although the rhIL-2 content in the insoluble protein fraction was maximum at $40^\circ{C}$, total rhIL-2 production in the culture volume was found to be highest at the induction temperature of $36^\circ{C}$. This was in contrast to the previously known optimum induction temperature of the P$_{L}$ promoter system $>(40~42^\circ{C})$.Explanations for such a discrepancy have been proposed based on a product formation kinetics, and their implications have been discussed in detail.l.

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Lipase Diversity in Glacier Soil Based on Analysis of Metagenomic DNA Fragments and Cell Culture

  • Zhang, Yuhong;Shi, Pengjun;Liu, Wanli;Meng, Kun;Bai, Yingguo;Wang, Guozeng;Zhan, Zhichun;Yao, Bin
    • Journal of Microbiology and Biotechnology
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    • v.19 no.9
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    • pp.888-897
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    • 2009
  • Lipase diversity in glacier soil was assessed by culture-independent metagenomic DNA fragment screening and confirmed by cell culture experiments. A set of degenerate PCR primers specific for lipases of the hormone-sensitive lipase family was designed based on conserved motifs and used to directly PCR amplify metagenomic DNA from glacier soil. These products were used to construct a lipase fragment clone library. Among the 300 clones sequenced for the analysis, 201 clones encoding partiallipases shared 51-82% identity to known lipases in GenBank. Based on a phylogenetic analysis, five divergent clusters were established, one of which may represent a previously unidentified lipase subfamily. In the culture study, 11 lipase-producing bacteria were selectively isolated and characterized by 16S rDNA sequences. Using the above-mentioned degenerate primers, seven lipase gene fragments were cloned, but not all of them could be accounted for by the clones in the library. Two full-length lipase genes obtained by TAIL-PCR were expressed in Pichia pastoris and characterized. Both were authentic lipases with optimum temperatures of ${\le}40^{\circ}C$. Our study indicates the abundant lipase diversity in glacier soil as well as the feasibility of sequence-based screening in discovering new lipase genes from complex environmental samples.