• Applied Microscopy
• /
• 제30권4호
• /
• pp.413-421
• /
• 2000

초고온 archaeon인 Thermococcus profundus에서 실린드형태의 단백질 복합체를 분리, 생화학적 특성과 구조를 규명하였다. 전자현미경과 영상처리 (image processing)를 이용한 구조 분석으로 8개의 subunit으로 이루어진 링 (ring) 형태가 두 개 겹쳐져 가운데 통로를 가지는 실린드형태로 이루어진 복합체임을 알 수 있었다. 또한 60kDa단백질(P60 complex)로 구성된 homomultimer임을 보여 주고 있다. 이 복합체는 $80^{\circ}C$ 이상에서 도 강한 ATPase의 활성을 보여주는 강한 내열성 단백질임을 알 수 있다. P60 complex는 초고온 archaeon이 높은 온도에서 적응하며 생존하는데 매우 중요한 역할을 할 것으로 추정할 수 있다.

• 한국생명과학회:학술대회논문집
• /
• 한국생명과학회 2006년도 제47회 학술심포지움 및 추계국제학술대회
• /
• pp.91.2-91.2
• /
• 2006

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제4권1호
• /
• pp.21-25
• /
• 1999

Two low-salt complex media, bactopeptone and desalted yeast extract, were used for high density cultivation of the hyperthermophilic archaeon Sulfolobus solfataricus (DSM 1617). Bactopeptone, which has low mineral ion content among various complex media, was good for cell growth in batch cultures; the maximal cell density in bactopeptone was comparable to that in yeast extract. However, cell growth was rather poor when bactopeptone was added by the fed-batch procedure. Since several vitamins are deficient in abctopeptone, the effect of vitamins on cell growth was examined. Among the vitamins tested, pyridoxine was found to improve the growth rate of S. solfataricus. To reduce the growth inhibition caused by mineral ions, yeast extract was dialyzed against distilled water and then fed-batch cultures were carried out using a fed medium containing desalted yeast extract. Although the concentrations of mineral ions in yeast extract were significantly lowered by the dialysis whether low molecular weight solutes in yest extract are crucial for cell growth, we investigated the effect of trehalose, a most abundant compatible solute in yeast extract, on the growth pattern. Cell densities were increased and the length of the lag phase was markedly shortened by the presence of trehalose, indicating that trehalose plays an important role in the growth of S. solfataricus.

• Journal of Microbiology and Biotechnology
• /
• 제16권7호
• /
• pp.1144-1148
• /
• 2006

DNA can oxidatively be deaminated by ROS, which converts DNA base amino groups to keto groups and can trigger abnormal mutations, resulting in mutagenesis in organisms. In this study, a noncanonical purine dNTP pyrophosphatase (AfPPase) from a hyperthermophilic archaeon Archaeoglobus fulgidus, which hydrolyzes aberrant nucleoside triphosphates, was overexpressed in E. coli, purified, and characterized. The purified AfPPase showed remarkably high activity for XTP and dITP, suggesting that the 6-keto group of these nucleotides is critical for the reactivity. Under optimal reaction conditions, the reaction rate for these substrates was about 120 times that with dGTP. Therefore, AfPPase may play a significant role in DNA repair by hydrolysis of noncanonical nucleotides before they are misincorporated into DNA.

• Journal of Microbiology and Biotechnology
• /
• 제17권8호
• /
• pp.1242-1248
• /
• 2007

Genomic analysis of a hyperthermophilic archaeon, Thermococcus onnurineus NA1 [1], revealed the presence of an open reading frame consisting of 1,377 bp similar to ${\alpha}$-amylases from Thermococcales, encoding a 458-residue polypeptide containing a putative 25-residue signal peptide. The mature form of the ${\alpha}$-amylase was cloned and the recombinant enzyme was characterized. The optimum activity of the enzyme occurred at $80^{\circ}C$ and pH 5.5. The enzyme showed a liquefying activity, hydrolyzing maltooligosaccharides, amylopectin, and starch to produce mainly maltose (G2) to maltoheptaose (G7), but not pullulan and cyclodextrin. Surprisingly, the enzyme was not highly thermostable, with half-life ($t_{1/2}$) values of 10 min at $90^{\circ}C$, despite the high similarity to ${\alpha}$-amylases from Pyrococcus. Factors affecting the thermostability were considered to enhance the thermo stability. The presence of $Ca^{2+}$ seemed to be critical, significantly changing $t_{1/2}$ at $90^{\circ}C$ to 153 min by the addition of 0.5 mM $Ca^{2+}$. On the other hand, the thermostability was not enhanced by the addition of $Zn^{2+}$ or other divalent metals, irrespective of the concentration. The mutagenetic study showed that the recovery of zinc-binding residues (His175 and Cys189) enhanced the thermo stability, indicating that the residues involved in metal binding is very critical for the thermostability.

• Journal of Microbiology and Biotechnology
• /
• 제16권11호
• /
• pp.1826-1831
• /
• 2006

A novel hyperthermophilic, anaerobic, heterotrophic archaeon, designated strain $NA1^T$, was isolated from a deep-sea hydrothermal vent area (depth, 1,650 m) within the Papua New Guinea-Australia-Canada-Manus (PACMANUS) field. Cells of this strain were motile by means of polar flagella, coccoid-shaped with a diameter of approximately $0.5-1.0{\mu}m$, and occurred as single cells. Optimal temperature, pH, and NaCl concentration for growth were $80^{\circ}C$, 8.5, and 3.5%, respectively. The new isolate was an obligate heterotroph that utilized yeast extract, beef extract, tryptone, peptone, casein, and starch as carbon and energy sources. Elemental sulfur was required for growth and was reduced to hydrogen sulfide. The G+C content of the genomic DNA was 52.0 mol%. Phylogenetic analysis of the 16S rRNA gene indicated that strain $NA1^T$ belongs to the genus Thermococcus, and the organism is most closely related to T. gorgonarius, T. peptonophilus, and T. celer; however, no significant homology was observed among species by DNA-DNA hybridization. Strain $NA1^T$ therefore represents a novel species for which the name Thermococcus onnurineus sp. novo is proposed. The type strain is $NA1^T$ (=KCTC 10859, =JCM 13517).

• 한국자기공명학회논문지
• /
• 제8권1호
• /
• pp.1-18
• /
• 2004

A thioredoxin from hyperthermophile, Methanococcus jannashii (MjTRX) was characterized by use of the differential scanning calorimetry to understand the mechanisms of thermodynamic stability. MjTRX has an unfolding transition temperature of 116.5$^{\circ}C$, although the maximum free energy of the unfolding (9.9 Kcal/mol) is similar to that of E. coli thioredoxin (ETRX, 9.0 Kcal/mol). However, the temperature of maximum stability is higher than ETRX by 20$^{\circ}C$, indicating that the unfolding transition temperature increased by shifting the temperature of maximum stability. MjTRX has lower enthalpy and entropy of the unfolding compared to ETRX maintaining a similar free energy of the unfolding. From the structure and the thermodynamic parameters of MjTRX, we showed that the unfolding transition temperature of MjTRX is increased due to the decreased entropy of the unfolding. Decreasing the unfolded state entropy and increasing the folded state entropy can decrease the entropy of the unfolding. In the case of MjTRX, the increased number of proline residues decreased the unfolded state entropy and the increased enthalpy in the folded state increased the folded state entropy.

• Journal of Microbiology and Biotechnology
• /
• 제17권7호
• /
• pp.1090-1097
• /
• 2007

Genomic analysis of Thermococcus sp. NA1 revealed the presence of a 3,927-base-pair (bp) family B-type DNA polymerase gene, TNA1_pol. TNA1_pol, without its intein, was overexpressed in Escherichia coli, purified using metal affinity chromatography, and characterized. TNA1_pol activity was optimal at pH 7.5 and $75^{\circ}C$. TNA1_pol was highly thermostable, with a half-life of 3.5h at $100^{\circ}C$ and 12.5h at $95^{\circ}C$. Polymerase chain reaction parameters of TNA1_pol such as error-rate, processivity, and extension rate were measured in comparison with rTaq, Pfu, and KOD DNA polymerases. TNA1_pol averaged one incorrect bp every 4.45 kilobases (kb), and had a processivity of 150 nucleotides (nt) and an extension rate of 60 bases/s. Thus, TNA1_pol has a much faster elongation rate than Pfu DNA polymerase with 7-fold higher fidelity than that of rTaq.