• Ocean and Polar Research
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• 제33권3호
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• pp.303-308
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• 2011

Polar psychrophiles which thrive under extreme conditions such as cold temperature, high salinity, and high dose ultraviolet light, emerge as novel targets for biotechnology. To prevent genetic drift and the possibility of contamination by subculturing, cryopreservation was employed for two psychrophilic microalgae, Porosira sp. (KOPRI AnM0008) and Pyramimonas sp. (KOPRI AnM0046), which have anti-freeze activities. Five cryoprotectants (dimethyl sulphoxide, ethylene glycol, glycerol, methanol and propylene glycol) showed toxicity at 20-30% (v/v). The optimal cryoprotectant concentration and equilibration time were less than 20% and 10 min, respectively. Cryopreservation was carried out in the presence of cryoprotectants either by direct freezing in liquid nitrogen ($LN_2$) or controlled freezing using a controlled rate freezer followed by storage in the $LN_2$ tank. As a result, Pyramimonas sp. (KOPRI AnM0046), a psychrophilic chlorophyta was revived. Cryopreserved Porosira sp. was not revived from either freezing protocols probably due to the silicic cell wall and its relatively large cell size. In the case of Pyramimonas sp. (KOPRI AnM0046), the controlled freezing method showed higher revival yield than the direct freezing method.

• Journal of Microbiology and Biotechnology
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• 제27권12호
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• pp.2180-2189
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• 2017

Psychrophilic phytases suitable for aquaculture are rare. In this study, a phytase of the histidine acid phosphatase (HAP) family was identified in Morchella importuna, a psychrophilic mushroom. The phytase showed 38% identity with Aspergillus niger PhyB, which was the closest hit. The M. importuna phytase was overexpressed in Pichia pastoris, purified, and characterized. The phytase had an optimum temperature at $25^{\circ}C$, which is the lowest among all the known phytases to our best knowledge. The optimum pH (6.5) is higher than most of the known HAP phytases, which is fit for the weak acidic condition in fish gut. At the optimum pH and temperature, MiPhyA showed the maximum activity level ($2,384.6{\pm}90.4{\mu}mol{\cdot}min^{-1}{\cdot}mg^{-1}$, suggesting that the enzyme possesses a higher activity level over many known phytases at low temperatures. The phytate-degrading efficacy was tested on three common feed materials (soybean meal/rapeseed meal/corn meal) and was compared with the well-known phytases of Escherichia coli and A. niger. When using the same amount of activity units, MiPhyA could yield at least $3{\times}$ more inorganic phosphate than the two reference phytases. When using the same weight of protein, MiPhyA could yield at least $5{\times}$ more inorganic phosphate than the other two. Since it could degrade phytate in feed materials efficiently under low temperature and weak acidic conditions, which are common for aquacultural application, MiPhyA might be a promising candidate as a feed additive enzyme.

• Journal of Dairy Science and Biotechnology
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• 제35권1호
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• pp.55-72
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• 2017

The second article of 'Effects of heat treatment on the nutritional quality of milk,' titled 'Destruction of microorganisms in milk by heat treatment' and authored by Dr. Seong Kwan Cha, who worked at the Korea Food Research Institute, covers the heat-stable microorganisms that exist in milk after pasteurization. The article focusses on the microbiological quality of raw milk and market milk following heat treatment, and is divided into four sub-topics: microbiological quality of raw milk, survey and measurement of microorganisms killed in raw milk, effect on psychrophilic and mesophilic microorganisms, and effect of heat treatment methods on thermoduric microorganisms. Bacillus spp. and Clostridium spp. are sporeforming gram-positive organisms commonly found in soil, vegetables, grains, and raw and pasteurized milk that can survive most food processing methods. Since spores cannot be inactivated by LTLT (low temperature long time) or HTST (high temperature short time) milk pasteurization methods, they are often responsible for food poisoning. However, UHT (ultra high temperature) processing completely kills the spores in raw milk by heating it to temperatures above $130^{\circ}C$ for a few seconds, and thus, the UHT method is popularly used for milk processing worldwide.

• 한국가정과학회지
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• 제5권2호
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• pp.45-54
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• 2002

Packaged Backsulgi cooked by steam/convection oven and then rapidly chilled was examined by research of microbiological test and sensory evaluation while storing them at the temperatures of 3$^{\circ}C$ and 3$0^{\circ}C$ for 0, 2, 4, 6, 10 days . The pH and reducing sugar content were seemed to change little at 3$^{\circ}C$. However the pH was rapidly reduced until 4 days and then decreased a little at 3$0^{\circ}C$, the reducing sugar content was inclosed little by little. In the microbiological test, any microbial growth in total aerobic, psychrophilic, anaerobic, spore forming bacteria, yeast and molds was not observed until 10 days at 3$^{\circ}C$, but microbial changes of aerobic, psychrophilic and anaerobic bacteria increased to 6 logCFU/g until 10 days at 3$0^{\circ}C$. However microbial changes of them decreased from 6 logCFU/g to 5 logCFU/g. As a result of the sensory evaluation, appearance, taste, color, softness, chewiness and overall Quality were significantly decreased during storage times(p<0.05), but scores of taste and overall quality on 6th days were 7.38${\pm}$1.06, 7.00${\pm}$0.93. Therefore we concluded that there was no problem about stability of storage 6 days at 3$^{\circ}C$.

• Bulletin of the Korean Chemical Society
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• 제31권4호
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• pp.887-890
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• 2010

The family of ClpB protein is a molecular chaperone which protects cellular proteins from being aggregated upon exposure to severe environmental stresses in association with DnaK/DanJ/GrpE in the ATP-dependent manner. In a psychrophilic bacterium which survives at a subzero temperature, any functional role of cold-active ClpB protein can be rather crucial. In order to identify a ClpB encoding gene from a cold-adapted bacterium whose genome sequence has not been fully discovered, we have employed a series of PCR technologies, including a gradient PCR with homologous primers, an inverse PCR and a cassette PCR. The full sequence of PaclpB gene was successfully identified and compared with those of other psychrophilic species. We have further cloned the gene in E.coli expression systems and were able to induce PaClpB protein expression by IPTG, which help us understand a molecular mechanism for survival against extremely cold environments.

• 청정기술
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• 제22권3호
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• pp.175-180
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• 2016

본 연구에서는, 저온 및 약산성 조건에서 높은 활성을 보이는 티로시나아제인 tyrosinase-CNK의 반응 안정성을 조사하였다. Tyrosinase-CNK는 지금까지 알려진 중온성(mesophilic) 및 호열성(thermophilic) 환경 유래의 티로시나아제 보다는 열 안정성이 낮았으나 0 ℃에서 효소 안정성이 매우 뛰어났고, 반복적인 동결-해동(freeze-thaw) 과정에서도 효소 활성을 안정적으로 유지하였다. 또한, 물과 ethanol 및 acetonitrile이 혼합된 유기용매 환경에서 초기 상대 활성 값의 변화가 관찰되었으나 유기용매 농도의 증가로 인한 추가적인 활성 저하를 유발하지 않고 활성을 일정하게 유지하였다. 한편, 효소 반응의 염(salt)에 대한 저해는 chaotropic 특성이 많이 나타나는 염일수록 적게 나타났다. 결과적으로, tyrosinase-CNK는 통상의 티로시나아제를 이용하여 반응을 수행하기 어려운 환경에서도 원하지 않는 반응을 억제하고 효소 불활성화를 최소화면서 반응을 촉매할 것으로 기대된다.

• 한국수산과학회지
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• 제28권4호
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• pp.401-411
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• 1995

일본의 Sagami bay와 Suruga bay의 810-4,000m되는 심해에서 채취한 시료에서 Flavvobacterium sp., Pseudomonas sp., Vibrio sp. 균종을 분리하였다. 이들 중 barophilic과 psychrophilic bacteria인 sp.-86과 sp.-87를 분리동정하고 압력단계별 성장속도를 조사한 결과 400기압에서도 잘 성장하였고 특히 100기압에서 가장 성장속도가 좋았다. 균의 형태는 1기압의 균 형태에 비하여 400기압에서 성장한 간균이 30-50배 이상 긴 간균 형태였으나(Fig. 3), 다시 1기압에 방치하면 단간균으로 균괴(flock)를 형성한다(Fig. 4) 이들 균의 발육온도 범위는 $5-37^{\circ}C$까지 발육하며, NaCl 농도의 범위는 sp.-86은 $0.5-9\%$이고 최적성장 농도가 $3\%$, sp.-87 균주의 지방산 조성은 $C_{20-22}\;C_{16:1},\;C_{16:0}$ 순으로 높은 함량이었고 400기압에서는 $C_{18:1}\;C_{18:0},\;C_{20-22:0}$ 의 함량순을 보였다. sp.-87균주는 $C_{16:1}\;C_{14:0},\;C_{20-22:0}$의 순이었고 400기압에서는 $C_{14:1}\;C_{12:0},\;C_{16:1}$의의 순으로 함량조성이 변하였다. 아미노산 조성은 sp.-86의 깅우 aspartic acid, methionine, glutamic acid 함량순이었고 400기압에서는methionine, glutamic acid, aspartic acid 함량순이었다. sp.-87의 균주는 methionine, glutamic acid, aspratic acid 순이었고 400기압에서는 methionine이 가장 높은 함량이고 glutamic acid, isoleucine 순으로 높은 함량이었다.

• 한국지하수토양환경학회지:지하수토양환경
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• 제14권5호
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• pp.10-17
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• 2009

생물학적 정화는 TPH로 오염된 지역을 정화하는 효과적인 방법으로 적용되고 있다. 하지만 미생물의 분해 활성이 적정온도 이하, 이상의 온도에서는 감소하기 때문에, 생분해 효율이 온도의 변화에 많은 영향을 받는 것으로 알려져 있다. 따라서 이번 연구의 목적은 유류 분해 효율이 우수한 중저온성 미생물을 분리하여 TPH로 오염된 지역에 적용할 때의 정화효율을 평가해 보는 것이다. 먼저 탄화수소 분해효율이 뛰어난 중온성($30^{\circ}C$)미생물 5종과 저온성($80^{\circ}C$) 미생물 3종의 consortia를 분리하였으며, 이들 미생물 consortia를 실험실내에서 유류로 오염된 토양에 적용해 본 결과, 중온성 미생물의 경우 초기 TPH 4,044 mg/kg이 10일 경과 후 1,084 mg/kg으로 73.2%, 저온성 미생물은 TPH 5,427 mg/kg이 50일 경과 후 1,756 mg/kg으로 67.6%의 처리효율을 보였다. 이 분해율은 휘발이나 희석에 의한 물리적 저감을 포함한다. 이후 분리된 미생물들을 토양 경작 현장에 적용해 본 결과, TPH 2,560 mg/kg의 오염이 56일 경과 후 87.1%의 제거율을 보였으며, 이때의 생분해 반응 속도상수는 $0.0374\;day^{-1}$이었다. 본 연구 결과는 저온, 중온 상태에서 미생물을 이용한 생물학적 정화가 더 다양하게 이용될 수 있는 가능성을 보여준 것으로 판단된다.

• Bulletin of the Korean Chemical Society
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• 제32권6호
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• pp.1925-1930
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• 2011

Psychrophilic bacteria have acquired cold-resistance in order to protect themselves against freezing temperatures, which would otherwise be lethal. DnaK/DnaJ/GrpE systems are molecular chaperones which facilitate proper folding of newly synthesized proteins. Efficient folding processes are of great importance especially in a cold environment, such as the Arctic. In order to understand the protection mechanisms of psychrophilic bacteria against cold temperatures, we have explored a genome of KOPRI22215, tentatively identified as Psychromonas arctica, whose genome sequence has not yet been discovered. With an aim of searching for a coding gene of DnaK from KOPRI22215, we have applied a series of polymerase chain reactions (PCR) with homologous primers designed from other Psychromonas species and LA PCR in vitro cloning. 1917 bp complete coding sequence of dnaK from KOPRI22215 was identified including upstream promoter sites. Recombinant plasmids to overexpress PaDnaK along with EcDnaK (DnaK of E. coli) were then constructed in pAED4 vector and the pET-based system to induce PaDnaK expression by IPTG. Characterization assays of expressed PaDnaK were carried out by measuring survival rates upon 4 day incubation at 4 $^{\circ}C$: a refolding assay as molecular chaperone, and ATPase assay for functional activity. Taking account of all the data together, we conclude that PaDnaK was identified, successfully expressed, and found to be more efficient in providing cold-resistance for bacterial cells.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2007년도 International Meeting of the Microbiological Society of Korea
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• pp.80-82
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• 2007

We have isolated numerous cold deep-sea adapted microorganisms (piezophilic, formerly referred to as "barophilic" bacteria) using deep-sea research submersibles. Many of the isolates are novel psychrophilic bacteria, and we have identified several new piezophilic species, i.e., Photobacterium profundum, Shewanella violacea, Moritella japonica, Moritella yayanosii, Psychromonas kaikoi, and Colwellia piezophila. These piezophiles are involving to five genera in gamma-Proteobacteria subgroup and produce significant amounts of unsaturated fatty acids in their cell membrane fractions to maintain the membrane fluidity in cold and high-pressure environments. Piezophilic microorganisms have been identified in many deep-sea bottoms of many of the world oceans. Therefore, these microbes are well distributed on our planet. One of the isolated deep-sea piezophiles, Shewanella violacea strain DSS12 is a psychrophilic, moderately piezophilic bacterium from a sediment sample collected at the Ryukyu Trench (depth: 5,110 m), which grows optimally at 30 MPa and $8^{\circ}C$ but also grows at atmospheric pressure (0.1 MPa) and $8^{\circ}C$. We have examined this strain to elucidate the molecular basis for gene regulation at different pressure conditions because this strain is useful as a model bacterium for comparing the various features of bacterial physiology under pressure conditions. In addition, we completed the sequencing of the entire genome of this piezophilic bacterium and we expect that many biotechnologically useful genes will be identified from the genome information.