• Journal of Microbiology and Biotechnology
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• 제26권12호
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• pp.2087-2097
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• 2016

Most cold-adapted enzymes possess higher $K_m$ and $k_{cat}$ values than those of their mesophilic counterparts to maximize the reaction rate. This characteristic is often ascribed to a high structural flexibility and improved dynamics in the active site. However, this may be less convincing to cold-adapted metabolic enzymes, which work at substrate concentrations near $K_m$. In this respect, cold adaptation of a shikimate kinase (SK) in the shikimate pathway from psychrophilic Colwellia psychrerythraea (CpSK) was characterized by comparing it with a mesophilic Escherichia coli homolog (EcSK). The optimum temperatures for CpSK and EcSK activity were approximately $30^{\circ}C$ and $40^{\circ}C$, respectively. The melting points were $33^{\circ}C$ and $45^{\circ}C$ for CpSK and EcSK, respectively. The ${\Delta}G_{H_2O}$ (denaturation in the absence of denaturing agent) values were 3.94 and 5.74 kcal/mol for CpSK and EcSK, respectively. These results indicated that CpSK was a cold-adapted enzyme. However, contrary to typical kinetic data, CpSK had a lower $K_m$ for its substrate shikimate than most mesophilic SKs, and the $k_{cat}$ was not increased. This observation suggested that CpSK may have evolved to exhibit increased substrate affinity at low intracellular concentrations of shikimate in the cold environment. Sequence analysis and homology modeling also showed that some important salt bridges were lost in CpSK, and higher Arg residues around critical Arg 140 seemed to increase flexibility for catalysis. Taken together, these data demonstrate that CpSK exhibits characteristics of cold adaptation with unusual kinetic parameters, which may provide important insights into the cold adaptation of metabolic enzymes.

• Journal of Microbiology and Biotechnology
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• 제29권2호
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• pp.244-255
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• 2019

Xylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (PbXI) from the psychrophilic soil microorganism, Paenibacillus sp. R4. Surprisingly, activity assay results showed that PbXI is not a cold-active enzyme, but displays optimal activity at $60^{\circ}C$. We solved the crystal structure of PbXI at $1.94-{\AA}$ resolution to investigate the origin of its thermostability. The PbXI structure shows a $({\beta}/{\alpha})_8$-barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the ${\beta}4-{\alpha}6$ loop region, was newly identified and is thought to be important for the stability of PbXI. Compared with previously determined thermostable and mesophilic XI structures, the ${\beta}1-{\alpha}2$ loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible ${\beta}1-{\alpha}2$ loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate low-temperature purpose-specific XI enzymes.

• Biodesign
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• 제6권4호
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• pp.84-91
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• 2018

Spo0F is a response regulator that modulates sporulation, undergoes phosphorylation for phosphorelay signal transduction, and interacts with various regulatory proteins; however, the mechanisms through which phosphorylation induces structural changes and regulates interactions with binding partners remain unclear. Here, we determined the unphosphorylated crystal structure of Spo0F from the psychrophilic bacterium Paenisporosarcina sp. TG-14 (PaSpo0F) and established a phosphorylation-state structural model. We found that PaSpo0F underwent structural changes (Lys54 and Lys102) by phosphorylation and generated new interactions (Lys102/Gln10 and Lys54/Glu84) to stabilize the ${\beta}4/{\alpha}4$ and ${\beta}1/{\alpha}1$ loop structures, which are important target-protein binding sites. Analysis of Bacillus subtilis Spo0 variants revealed movement by BsSpo0F Thr82 and Tyr84 residues following interaction with BsSpo0B, providing insight into the movement of corresponding residues in PaSpo0F (Thr80 and Tyr82), with further analysis of BsSpo0F/BsRapH interaction revealing alterations in the ${\beta}4/{\alpha}4$ loop region. These results suggest that phosphorylation-induced structural rearrangement might be essential for PaSpo0F activation and expand the understanding of Spo0F-specific activation mechanisms during sporulation.

• Journal of Microbiology and Biotechnology
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• 제28권3호
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• pp.448-453
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• 2018

In this study, a 107 kDa protease from psychrophilic Janthinobacterium lividum PAMC 26541 was purified by anion-exchange chromatography. The specific activity of the purified protease was 264 U/mg, and the overall yield was 12.5%. The J. lividum PAMC 25641 protease showed optimal activity at pH 7.0-7.5 and $40^{\circ}C$. Protease activity was inhibited by PMSF, but not by DTT. On the basis of the N-terminal sequence of the purified protease, the gene encoding the cold-adapted protease from J. lividum PAMC 25641 was cloned into the pET-28a(+) vector and heterologously expressed in Escherichia coli BL21(DE3) as an intracellular soluble protein.

• 한국미생물·생명공학회지
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• 제29권1호
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• pp.56-61
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• 2001

For the development of probiotics in aquaculture of marine organisms, three strains having psychrophilic and salt tolerant characteristics were isolated from Kimchi. Amng the isolated strains. MG19, MG89 and MG208 were identified as Lactobacillus brevis, Enterococcus faecium and Lactobacillus plantarum, respectively. The neutralized culture broth of isolated strains were tested in order to evaluate the antibactrial activity, 조초 showed high antibacterial activity against Edwardsiella tarda, Vibrio cholerae andPseudomonas fluorescens. In mixed culture of pathogens and isolated strain,pathogens were signifi-cantly inhibited after 2 days of cultivation but the isolated strains showed normal growth. When the Edwardsiella tarda was cultured with three isolated strains, its growth was completely inhibited after 254 hours of cultivation. The effect of isolated three strains as probiotics was investigated based on the changes in body weight of aquacultured flounder. After 50 days feeding trial, it was found that the mean body weight gain of the tested group fed freeze-dried isolated cells was significantly greater than that of the control group. These results suggest that these isolated strains can play an important role as probiotics in aquaculture.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.194-198
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• 2008

A newly designed Cytophaga-Flavobacteria-specific 16S rRNA gene primer pair was employed to investigate the CF community structure in the Bering Sea, revealing a previously unknown and unexpected high CF diversity in this high latitude cold sea. In total, 56 clones were sequenced and 50 unique CF 16 rRNA gene fragments were obtained, clustering into 16 CF subgroups, including nine cosmopolitan subgroups, five psychrophilic subgroups, and two putatively autochthonous subgroups. The majority of sequences (82%) were closely related to uncultured CF species and could not be classified into known CF genera, indicating the presence of a large number of so-far uncultivated CF species in the Bering Sea.

• 한국농공학회논문집
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• 제50권4호
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• pp.59-66
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• 2008

Anaerobic digestion system is getting more attractive in that it produces biogas in the process of organic waste stabilization. Net energy production is important when biogas production is concerned. In this study, net energy production was evaluated with respect to biogas production and heat losses in a hypothetical digester. Under the condition of digester operation with slurry inflow of 5% of TS, additional fuel is required to maintain digester temperature during the winder season. Substrate therefore, needs to have higher VS contents through co-digestion of silage or food waste that has greater values of methane production rate. Heating input slurry is important in cold season, which covers over 80% of heating requirement. Heat recovery from digestate is valuable to reduce the use of biogas for heating. It seems desirable to minimize slurry inflow when temperature is very low. Psychrophilic digestion may be a feasible option for reducing heating requirement.

• 한국미생물·생명공학회지
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• 제24권4호
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• pp.430-438
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• 1996

The eleven strains, which could be used lactic and acetic acids as carbon sources, were isolated from kimchi and identified; the strains were facultative microorganisms which could be grown at low temperature (10$\circ$C) and around pH 3.2. As results of morphological, biochemical and physiological tests, 5 species of 3 genera were identified as Debaryomyces coudertii, Pichia edia, Pichia chambardii, Pichia haplophilia and Saccharomyces fermentati. Each strain was grown in basal media. In acidic resistance and acid utilization test, Saccharomyces sp. YK- 17 and Saccharomyces fermentati YK-19 were grown well in basal and YM media containing 0.3% lactic acid. And two strains were grown in basal and YM media containing O.3% lactic acid and 0.6% acetic acid. Since strain YK-19 was grown better at 10$\circ$C than that in 25$\circ$C, strain YK-19 was known to be a psychrophilic strain.

• 농업과학연구
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• 제9권2호
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• pp.556-561
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• 1982

우리나라에서 처리(處理) 유통(流通)되고 있는 초고온멸균유(超高溫滅菌乳)의 선도(鮮度) 및 세균학적(細菌學的) 상태(狀態)를 확인(確認)함과 동시(同時)에 냉장(冷藏) 및 실온(室溫)에 있어서의 보존성(保存性)을 구명(究明)하기 위하여 3개지성(個地城)(천안(天安), 평택(平澤) 및 수원(水原))에서 처리(處理) 생산(生産)되고 있는 초고온멸균무균충전우유(超高溫滅菌無菌充塡牛乳)를 시료(試料)로 하여 신선도(新鮮度) 검사(檢査)와 세균학적(細菌學的) 상태(狀態)를 시험(試驗)한 결과(結果)는 다음과 같다. 1. 3개(個) 유처리장(乳處理場)에서 생산(生産)된 초고온멸균유(超高溫滅菌乳)를 $5^{\circ}C$에 보존(保存)할 때 6주(週)까지 0.16%의 산도(酸度)를 유지(維持)하고 10주(週)에 이르러서도 0.18% 이하(以下)의 산도(酸度)와 음성(陰性)의 주정검사(酒精檢査) 및 자비시험결과(煮沸試驗結果)를 나타내었다. 2. 초고온멸균유(超高溫滅菌乳)를 $25^{\circ}C$에 보존(保存)할 때에는 9 주(週)까지 0.18%이하(以下)의 산도(酸度)를 유지(維持)하고 주정검사(酒精檢査) 및 자비시험(煮沸試驗)에서도 전시료(全試料)가 음성(陰性)을 나타내었다. 3. 초고온멸균유(超高溫滅菌乳)는 $5^{\circ}C$ 보존(保存)의 경우 내열성균(耐熱性菌), 대장균(大腸菌)은 나타나지 않았으며 생균수(生菌數) 및 호냉성균(好冷性菌)은 5주(週), 6주(週) 및 10주등(週等)에 <10/ml의 균수(菌數)를 나타내었다. 4. 초고온멸균유(超高溫滅菌乳)를 $25^{\circ}C$에 보존(保存)할 때 내열성균(耐熱性菌) 및 대장균(大腸菌)은 나타나지 않았으며 생균수(生菌數) 및 호냉생균(好冷生菌) 3주(週)부터 10주(週)까지 사이에 간단적(間斷的)으로 <10/ml의 균수(菌數)를 표시(表示)하였다.

• 한국식품위생안전성학회지
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• 제32권5호
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• pp.363-370
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• 2017

본 실험에서는 냉동 새우(Litopenaeus vannamei)를 $4^{\circ}C$에서 12일동안 저온 저장하며, 키토산 코팅과 천연보존제의 병행처리(0.05% carvacrol, 0.05% thymol)가 미생물(중온균, 저온균, Pseudomonas spp., $H_2S$ 생성균)의 성장과 새우의 이화학적 특징(총 휘발성 질소화합물, pH) 및 관능적 품질(외관, 냄새, 전반적 수용도)에 미치는 영향을 살펴보았다. 키토산 코팅과 carvacrol 병행 처리, 키토산 코팅과 thymol 병행 처리는 모든 대상 미생물의 성장을 억제하였고 특히 중온균, 저온균, $H_2S$ 생성균의 경우 실험의 마지막 날인 12일째까지 유의적인 항균효과(p < 0.05)를 보였다. 이 처리구들은 총 휘발성 질소화합물과 관능적 특성(외관, 냄새, 전반적 수용도)에서도 12일째까지 유의적인 차이(p < 0.05)를 보였으나 pH에서는 유의적 차이가 없었다. 따라서 키토산 코팅과 carvacrol 병행 처리, 키토산 코팅과 thymol 병행 처리는 새우의 주요 부패 미생물의 성장을 효과적으로 억제하는 것으로 보이며 냉장 새우의 유통기간 연장에 활용할 수 있을 것으로 보여진다.