• Proceedings of the Korean Biophysical Society Conference
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• 1996.07a
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• pp.21-21
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• 1996

The hyperthermostable protein, rubredoxin from Pyrococcus furiosus is 53-residue protein with a three-stranded anti-parallel $\beta$-sheet and several loops. To investigate the effect of changes of electrostatic and hydrophobic interactions on the structure and dynamic property of P. furiosus rubredoxin, molecular dynamics simulations in water were performed on three mesophilic rubredoxins, P, furiosus rubresoxin, and 5 mutants of P. furiosus rubredoxin. (omitted)

• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.570-583
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• 2021

Pyrococcus furiosus α-amylase can hydrolyze α-1,4 linkages in starch and related carbohydrates under hyperthermophilic condition (~ 100℃), showing great potential in a wide range of industrial applications, while its relatively low productivity from heterologous hosts has limited the industrial applications. Bacillus subtilis, a gram-positive bacterium, has been widely used in industrial production for its non-pathogenic and powerful secretory characteristics. This study was conducted to increase production of P. furiosus α-amylase in B. subtilis through three strategies. Initial experiments showed that co-expression of P. furiosus molecular chaperone peptidyl-prolyl cis-trans isomerase through genomic integration mode, using a CRISPR/Cas9 system, increased soluble amylase production. Therefore, considering that native P. furiosus α-amylase is produced within a hyperthermophilic environment and is highly thermostable, heat treatment of intact culture at 90℃ for 15 min was performed, thereby greatly increasing soluble amylase production. After optimization of the culture conditions (nitrogen source, carbon source, metal ion, temperature and pH), experiments in a 3-L fermenter yielded a soluble activity of 3,806.7 U/ml, which was 3.3- and 28.2-fold those of a control without heat treatment (1,155.1 U/ml) and an empty expression vector control (135.1 U/ml), respectively. This represents the highest P. furiosus α-amylase production reported to date and should promote innovation in the starch liquefaction process and related industrial productions. Meanwhile, heat treatment, which may promote folding of aggregated P. furiosus α-amylase into a soluble, active form through the transfer of kinetic energy, may be of general benefit when producing proteins from thermophilic archaea.

• Journal of Microbiology and Biotechnology
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• v.28 no.2
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• pp.255-261
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• 2018

Aglycon protopanaxatriol (APPT) has valuable pharmacological effects such as memory enhancement and tumor inhibition. ${\beta}$-Glycosidase from the hyperthermophilic bacterium Dictyoglomus turgidum (DT-bgl) hydrolyzes the glucose residues linked to APPT, but not other glycoside residues. ${\beta}$-Glycosidase from the hyperthermophilic bacterium Pyrococcus furiosus (PF-bgl) hydrolyzes the outer sugar at C-6 but not the inner glucose at C-6 or the glucose at C-20. Thus, the combined use of DT-bgl and PF-bgl is expected to increase the biotransformation of PPT-type ginsenosides to APPT. We optimized the ratio of PF-bgl to DT-bgl, the concentrations of substrate and enzyme, and the reaction time to increase the biotransformation of ginsenoside Re and PPT-type ginsenosides in Panax ginseng leaf extract to APPT. DT-bgl combined with PF-bgl converted 1.0 mg/ml PPT-type ginsenosides in ginseng leaf extract to 0.58 mg/ml APPT without other ginsenosides, with a molar conversion of 100%. We achieved the complete biotransformation of ginsenoside Re and PPT-type ginsenosides in ginseng leaf extract to APPT by the combined use of two ${\beta}$-glycosidases, suggesting that discarded ginseng leaves can be used as a source of the valuable ginsenoside APPT. To the best of our knowledge, this is the first quantitative production of APPT using ginsenoside Re, and we report the highest concentration and productivity of APPT from ginseng extract to date.

• Journal of Life Science
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• v.21 no.1
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• pp.68-73
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• 2011

$\beta$-1,3-glucanase from Pyrococcus furiosus was applied for the saccharification of laminarin, which is a major oligo-saccharide component of brown algae, and the reaction mixture produced from laminarin was utilized as a substrate for alcohol fermentation using yeast. To prepare the recombinant $\beta$-1,3-glucanase, a $\beta$-1,3-glucanase gene was overexpressed in Escherichia coli and purified. Laminarin was degraded to an oligo- and mono-saccharide, such as glucose, after reaction with the purified recombinant $\beta$-1,3-glucanase, and the products after enzymatic treatment were confirmed by TLC and HPLC analysis. Decomposed laminarin after enzyme reaction was only added to the medium as a C-source for yeast alcohol production reaction. 0.3% alcohol production was detected from the cultured broth by gas chromatography after 48 hr of incubation. Further evaluation for optimal conditions of saccharification and alcohol fermentation can be suggested, as well as the possibility of using this enzymatic method to produce ethanol using laminarin.

• 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.

• Journal of Microbiology and Biotechnology
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• v.12 no.5
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• pp.841-843
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• 2002

Pfu DNA polymerase from Pyrococcus furiosus was expressed in the E. coli periplasm, and the fully active polymerase was partially purified by applying osmotic shock, ammonium sulfate precipitation, and heat treatment. This method represents a new way of expressing and purifying functional Pfu DNA polymerase without the use of chromatography.

• Journal of Microbiology
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• v.39 no.4
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• pp.245-249
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• 2001

• Journal of Microbiology
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• v.39 no.3
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• pp.149-153
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• 2001

The electron transfer reaction is one of the most essential processes of life. Not only does it provide the means of transforming solar and chemical energy into a utilizable form for all living organisms, it also extends into a range of metabolic processes that support the life of a cell. Thus, it is of great interest to understand the physical basis of the rates and reduction potentials of these reactions. To identify the major determinants of reduction potentials in redox proteins, we have chosen the simplest electron transfer protein, rubredoxin, a small (52-54 residue) iron-sulfur protein family, widely distributed in bacteria and archaea. Rubredoxins can be grouped into two classes based on the correlation of their reduction potentials with the identity of residue 44; those with Ala44 (ex: Pyrococcus furiosus) have reduction potentials that are ∼50 mV higher than those with Va144 (ex: Clostridium pasteurianum). Based on the crystal structures of rubredoxins from C. pasteurianum and P. furiosus, we propose the identity of residue 44 alone determines the reduction potential by the orientation of the electric dipole moment of the peptide bond between 43 and 44. Based on 1.5 $\AA$ resolution crystal structures and molecular dynamics simulations of oxidized and reduced rubredoxins from C. pasteurianum, the structural rearrangements upon reduction suggest specific mechanisms by which electron transfer reactions of rubredoxin should be facilitated.

• Korean Journal of Microbiology
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• v.51 no.1
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• pp.68-74
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• 2015

Based on the genomic analysis of Thermococcus pacificus P-4, we identified a putative GH1 ${\beta}$-glucosidase-encoding gene (Tpa-glu). The gene revealed a 1,464 bp encoding 487 amino acid residues, and the deduced amino acid residues exhibited 77% identity with Pyrococcus furiosus ${\beta}$-glucosidase (accession no. NP_577802). The gene was cloned and expressed in Escherichia coli system. The recombinant protein was purified by metal affinity chromatography and characterized. Tpa-Glu showed optimum activity at pH 7.5 and $75^{\circ}C$, and thermostability with a half life of 6 h at $90^{\circ}C$. Tpa-Glu exhibited hydrolyzing activity against various pNP-glycopyranosides, with kcat/Km values in the order of pNP-${\beta}$-glucopyranoside, pNP-${\beta}$-galactopyranoside, pNP-${\beta}$-mannopyranoside, and pNP-${\beta}$-xylopyranoside. In addition, the enzyme exhibited exo-hydrolyzing activity toward ${\beta}$-1,3-linked polysaccharide (laminarin) and ${\beta}$-1,3- and ${\beta}$-1,4-linked oligosaccharides. This is the first description of an enzyme from hyperthermophilic archaea that displays exo-hydrolyzing activity toward ${\beta}$-1,3-linked polysaccharides and could be applied in combination with ${\beta}$-1,3-endoglucanase for saccharification of laminarin.

• 한국신재생에너지학회:학술대회논문집
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• 2010.06a
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• pp.227.2-227.2
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• 2010

본 연구에서는 양극산화된 $TiO_2$ 전극(anodized tubular $TiO_2$ electrode, ATTE)을 수소제조용 PEC(Photoelectrochemical)시스템에서 광어노드와 기존의 백금전극을 대체하고 $H^+$ 환원능을 향상시키기 위하여 엔자임(Pyrococcus furiosus, Pfu)을 고정화한 후 캐소드로 동시에 활용하였으며, 엔자임 고정을 위한 crosslinker 종류 및 금속담지 여부, ATTE 길이를 통한 수소발생양에 미치는 영향을 연구하였다. ATTE 표면과 엔자임의 amine group의 연결을 위하여 heterobifunctional crosslinker로써 사슬 길이가 상대적으로 짧은 Sulfo-SDA가 유리하였으며, 금속담지의 경우 짧은 튜브의 경우 1% 내에서 효과가 증진되었으나 긴 튜브의 경우는 오히려 광전류 및 궁극적으로 수소발생속도에 불리하게 작용하였다. 또한, 튜브 길이가 긴 ATTE가 짧은 ATTE 보다 수소발생양에서 더욱 효율적임을 알 수 있었다. 텅스텐산화물 담지의 가시광감응에의 담지 효과는 예비 실험 결과로 나타나지 않아, 추가적인 연구가 필요한 것으로 판단된다.