• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 1996년도 정기총회 및 학술발표회
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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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• 제31권4호
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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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• 제28권2호
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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.

• 생명과학회지
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• 제21권1호
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• pp.68-73
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• 2011

갈조류 유래의 다당류인 laminarin을 기질로써 호열성 미생물인 Pyrococcus furiosus의 $\beta$-1,3-glucanase와 반응 시킨 뒤, 분해산물을 yeast를 이용한 알코올 발효과정을 통하여 에탄올을 생산하고자 하는 연구를 수행하였다. 33 kDa (297 a.a, 894 bp)의 재조합 $\beta$-1,3-glucanase를 대장균에게 발현 후 순수하게 정제 하였으며, 정제한 $\beta$-1,3-glucanase와 laminarin을 반응시킨 결과 단당을 포함하여 oligo당 형태로 분해됨을 TLC와 HPLC로써 확인하였다. 그리고 이러한 분해산물을 에탄올 생산 배지의 유일한 탄소원으로써 첨가하여 yeast를 배양한 결과 48시간뒤에는 세포 외로 최소 0.3%의 알코올을 생산함을 gas chromatography로써 확인하였다. 따라서 $\beta$-1,3-glucanase와 laminarin의 최적 분해반응 및 yeast의 최적 알코올 발효 조건을 확립한다면 본 연구의 방법을 이용한 해조류로부터의 bio-ethanol의 생산을 성공적으로 수행 할 수 있으리라고 판단된다.

• Journal of Microbiology and Biotechnology
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• 제20권5호
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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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• 제12권5호
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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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• 제39권4호
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• pp.245-249
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• 2001

• Journal of Microbiology
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• 제39권3호
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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.

• 미생물학회지
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• 제51권1호
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• pp.68-74
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• 2015

Thermococcus pacificus P-4의 유전체 서열 분석을 통하여 예측되는 GH1 ${\beta}$-glucosidase를 암호화하는 유전자를 동정하였다. 그 유전자는 487 아미노산들을 암호화하는 1,464 bp 나타내었으며, 그 아미노산 서열은 Pyrococcus furiosus ${\beta}$-glucosidase와 77% 상동성을 나타내었다. 그 유전자는 Escherichia coli 시스템 내에서 복제 및 발현하였다. 재조합 된 단백질은 금속 친화크로마토그래피를 통하여 정제하고 특성을 분석하였다. 정제된 단백질(Tpa-Glu)은 pH 7.5와 $75^{\circ}C$에서 최적활성을 나타내었으며, 열안정성은 $90^{\circ}C$에서 약 6시간의 반감기를 보였다. Tpa-Glu는 pNP-${\beta}$-glucopyranoside, pNP-${\beta}$-galactopyranoside, pNP-${\beta}$-mannopyranoside, 그리고 pNP-${\beta}$-xylopyranoside 순으로 우수한 $k_{cat}/K_m$ 값을 나타내었다. 또한, Tpa-Glu는 ${\beta}$-1,3-linked polysaccharide (laminarin) 그리고 ${\beta}$-1,3-와 ${\beta}$-1,4-linked oligosaccharides에 대하여 exo-hydrolyzing 활성을 보였다. 본 연구는 초고온 고세균으로터 ${\beta}$-glucosidase가 exohydrolyzing 활성을 처음 확인한 것으로 이 효소는 laminarin의 당화공정에 ${\beta}$-1,3-endoglucanase와 함께 적용할 수 있을 것으로 기대된다.

• 한국신재생에너지학회:학술대회논문집
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• 한국신재생에너지학회 2010년도 춘계학술대회 초록집
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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.