• Journal of Microbiology and Biotechnology
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• v.20 no.5
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• pp.889-892
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• 2010

We investigated a potential for glucose production from cellulose material using two kinds of hyperthermophilic enzymes, endocellulase (EG) and beta-glucosidase (BGL). Two BGLs, from hyperthermophile Pyrococcus furiosus and mesophile Aspergillus aculeatus, were compared with P. horikoshii endocellulase (EGPh) for complete hydrolysis of cellulose. The combination reactions by each BGL enzyme and EGPh could produce only glucose without the other oligosaccharides from phosphoric acid swollen Avicel (PSA). The combination of both the hyperthermophilic cellulases, BGLPf and EGPh, will be adaptable to a high efficiency system to produce glucose at high temperature.

• Biotechnology and Bioprocess Engineering:BBE
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• v.4 no.1
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• pp.21-25
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• 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
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• v.14 no.5
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• pp.1022-1030
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• 2004

The gene encoding Aquifex pyrophilus (Apy) DNA polymerase was cloned and sequenced. The Apy DNA polymerase gene consists of 1,725 bp coding for a protein with 574 amino acid residues. The deduced amino acid sequence of Apy DNA. polymerase showed a high sequence homology to Escherichia coli DNA polymerase I-like DNA polymerases. It was deduced by amino acid sequence alignment that Apy DNA polymerase, like the Klenow fragment, has only the two domains, the $3'{\rightarrow}5'$ exonuclease domain and the $5'{\rightarrow}3'$ polymerase domain, containing the characteristic motifs. The Apy DNA polymerase gene was expressed under the control of T7lac promoter on the expression vector pET-22b(+) in E. coli. The expressed enzyme was purified by heat treatment, and Cibacron blue 3GA and $UNO^{TM}$ Q column chromatographies. The optimum pH of the purified enzyme was 7.5, and the optimal concentrations of KCl and $Mg^{2+}$ were 20 mM and 3 mM, respectively. Apy DNA polymerase contained a double strand-dependent $3'{\rightarrow}5'$ proofreading exonuclease activity, but lacked any detectable $5'{\rightarrow}3'$ exonuclease activity, which is consistent with its amino acid sequence. The somewhat lower thermostability of Apy DNA polymerase than the growth temperature of A. pyrophilus was analyzed by the comparison of amino acid composition and pressure effect.

• BMB Reports
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• v.31 no.2
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• pp.130-134
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• 1998

An aldolase gene has been cloned from Methanococcus jannaschii. The coding region of the gene has been expressed in E. coli using a pET system to a level of 30% of total cellular proteins. The protein was purified to more than 95 % homogeneity by heat treatment and ion exchange chromatography. The protein performed an aldol condensation reaction with glyceraldehyde as substrate and dihydroxyacetone phosphate as a carboxyl donor. The protein was determined to be a type II aldolase which requires the $Zn^{2+}$ ion as a metal cofactor. This enzyme has a broad range of optimum pH (7-9) and temperature ($50-80^{\circ}C$). It shows strong stability against heat, chemical denaturants, as well as a high percentage' of organic solvents. The half-life of this enzyme at $85^{\circ}C$ is more than 24 h and it maintains more than 90% of aldolase activity in the presence of 6 M urea, 50% acetonitrile, or 15% isopropyl alcohol.

• Journal of Microbiology and Biotechnology
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• v.9 no.4
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• pp.404-413
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• 1999

Methanopyrus kandleri is a hyperthermophilic methanogen that represents one of the most heat-resistant organisms: the maximum growth temperature of M. kandleri is $110^{\circ}C$. A random sequence analysis of the genomic DNA of M. kandleri has been performed to obtain genomic information. More than 200 unique sequence tags were obtained and compared with the sequences in the GenBank and PIR databases. About 30% of the analyzed tags showed strong sequence similarity to previously identified genes involved in various cellular processes such as biosynthesis, transport, methanogenesis, or metabolism. When statistics relating to the frequency of codons were examined, the sequenced open reading frames showed highly biased codon usage and a high content of charged amino acids. Among the identified genes, a homologue of the catalytic subunit of carbon monoxide dehydrogenase (CODH) that reduces $CO_2$ to CO was cloned and sequenced in order to examine its detailed gene structure. The cloned gene includes consensus promoters. The amino acid sequence of the cloned gene shows a strong homology with the CODH genes from methanogenic Archaea, especially in the presumed binding sites for Fe-S centers.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.838-844
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• 2001

The enzyme Fuc aldolase from Methanococcus jannaschii that catalyzes the aldol condensation of DHAP and L-lactaldehyde to give fuculose-1-phosphate was inactivated by DEP. The inactivation was pseudo first-order in the enzyme and DEP, which was biphasic. A pseudo second-order rate constant of 120$M^{-1}min^{-1}$ was obtained at pH 6.0 and $25{\circ}C$. Quantifying the increase in absorbance at 240nm showed that four histidine residues per subunit were modified during the nearly complete inactivation. The statistical analysis and the time course of the modification suggested that two or three histidine residues were essential for activity. The rate of inactivation was dependent on the pH, and the pH inactivation data implied the involvement of the amino acid residue with a $pK_a$ value of 5.7. Fuc aldolase was protected against DEP inactivation by DHAP, indicating that the histidine residues were located at the active site of Fuc aldolase. DL-Glyceraldehyde, as an alternative substrate to L-lactaldehyde, showed no specific protection for the Fuc aldolase.

• Journal of the Korean Magnetic Resonance Society
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• v.8 no.1
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• pp.1-18
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• 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
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• v.17 no.8
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• pp.1242-1248
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• 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
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• v.16 no.11
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• pp.1826-1831
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• 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).

• Journal of Microbiology and Biotechnology
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• v.17 no.7
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• pp.1090-1097
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• 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.