• Microbiology and Biotechnology Letters
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• v.14 no.5
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• pp.385-389
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• 1986

Both hydrogenase and nitrogenase were found to be involved in hydrogen evolution independently in Rhodopseudomonas sp. KCTC 1437. The hydrogen formation in this bacterium was independent on light illumination and presence of N $H_4^{+}$ After establishment of conditions to measure the amount of hydrogen evolved by each of the enzymes in vivo, the several factors affecting on the hydrogen evolution, e.g. presence of gases ( $C_2$ $H_2$, $H_2$, $O_2$ or $N_2$), C/N ratio, were investigated, Hydrogenase was less inhibited than nitrogenase under $O_2$ and was active independent on the presence of $N_2$ or $C_2$ $H_2$ which were the strong inhibitor of nitrogenase. Besides, the hydrogenase activity was increased after incubation with $H_2$. And it was verified that this bacterium consume hydrogen and photoreduce $CO_2$ by hydrogenase. From above results, it is concluded that hydrogenase in Rhodopseudomonas sp. KCTC 1437 can produce hydrogen under more favorable condition that nitrogenase.e.

• 한국신재생에너지학회:학술대회논문집
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• 2005.11a
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• pp.631-633
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• 2005

For biohydrogen production, hydrogenase is a key enzyme. In the present work we performed search of [NiFe]-hydrogenases from hydrogen producing microorganisms using degenerate polymerase chain reaction (PCR) strategy. Degenerate primers were designed from the conserved region of [NiFe]-hydrogenase group I especially on structural genes encoding for catalytic subunit of [NiFe]-hydrogenase from bacteria producing hydrogen. Most of [NiFe]-hydrogenase (group I) are expressed via complex mechanism with aid of auxiliary protein and localized through twin-arginine translocation pathway. [NiFe]-hydrogenase is composed of large and small subunits for catalytic activity. It is known that only small subunit has signal peptide for periplasmic localization and large & small subunitscome together before localization. During this process, large subunit is treated by endopeptidase for maturation. Based on these information we used signal peptide sequence and C-terminal of large subunit by recognized by endopeptidase as templates for degenerate primers. About 2,900 bp of PCR products were successfully amplified using the designed degenerate primers from genomic DNAs of several microorganisms. The amplified PCR products were inserted into T-vector and then sequenced to confirm.

• Transactions of the Korean hydrogen and new energy society
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• v.19 no.2
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• pp.124-131
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• 2008

Crude cytoplasmic fraction of phototrophic purple sulfur bacterium, Thiocapsa roseopersicina NCIB 8347, were initially prepared and purified by sonication, ultracentrifugation, ammonium sulfate fractionation and heat-treatment and it has been previously reported. Using various applications of chromatography far the purification of membrane-bound and soluble hydrogenases from heat-treated enzyme fraction were studied at present report. When the heat-treated enzyme preparation was applied to the anion column chromatography using Q-sepharose, Fraction I and II, which were extracted with the KCl 0-0.5 M gradient, showed the specific evolution hydrogenase activity 3.86 and 2.27 U/mg-protein respectively. Specific hydrogenase activitys of Fraction I and II were further increased to 4.35 and 7.46 U/mg-protein for Fraction I and to 2.49 and 4.41 U/mg-protein fur Fraction II respectively, when hydrophobic interaction column, Phenyl superose, and anion exchange column, Mono-Q, were applied. Size exclusion chromatography using superdex 200 concentrated the hydrogenase Fraction I and II to 9.19 and 7.84 U/mg-protein respectively at the final step of purification.

• Transactions of the Korean hydrogen and new energy society
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• v.17 no.4
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• pp.371-378
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• 2006

Effect of $(NH_4)_2SO_4$ precipitation and heat-treatment on hydrogenase which was extracted from the cytoplasmic fraction of the phototrophic purple sulfur bacterium Thiocapsa roseopersicina NCIB 8347 was studied. Crude enzyme extract was prepared by centrifugation($28,000{\times}g$, $400,000{\times}g$) after sonication of cells grown under photosynthetic condition for 96 hrs. Various conditions of $(NH_4)_2SO_4$ precipitation and heat-treatment were examined and the effect of protein concentration was analyzed by SDS-electrophoresis between the treatments. Optimum conditions for $(NH_4)_2SO_4$ precipitation and heat-treatment for evolution hydrogenase activity were 40-60% saturation and $60^{\circ}C$ for 20 min, respectively, which exhibited the specific hydrogenase activity of 0.78 U/mg-protein. Specific hydrogenase activity was decreased to 31.6% when the heat-treatment at $60^{\circ}C$ increased from 20 min to 5 hrs.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.717-724
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• 2003

A newly isolated Citrobacter sp. Y19 catalyzes the CO-dependent $H_2$ production (biological water-gas shift reaction) by the actions of CO dehydrogenase (CODH) and hydrogenase. Y 19 requires $O_2$ for fast growth, but its $H_2$ production activity is significantly inhibited by $O_2$. In the present study, the effect of $O_2$ on the activities of CODH ard hydrogenase was investigated quantitatively in both whole cells and broken cells, based on CO-dependent or methyl viologen (MV)-dependent $H_2$ production in addition to CO-dependent MV reduction. In crude cell extracts, CODH activity was mostly found in the soluble fraction. Inactivation of CODH and hydrogenase activities by $O_2$ followed the first-order decay kinetics, and the dependence of the rate constants on $O_2$ partial pressure could be expressed by the Michaelis-Menten equation. In whole cells, the maximum deactivation rate constants ($k_{d,max}$ of hydrogenase and CODH were quite similar: $0.07{\pm}0.03 min^{-1}\;and\;0.10{\pm}0.04 min^{-1}$, respectively. However, the first-order rate constant ($k_{d,max}/K_s$) of CODH ($0.25\;min^{-1}\;atm^{-1}$) at low $O_2$ partial pressures was about 3-fold higher than that of the hydrogenase, since the half-saturation constant ($K_s$) of CODH was about half of that of hydrogenase. In broken cells, both enzymes became significantly more sensitive to $O_2$ compared to the unbroken cells, while $k_{d,max}/K_s$ increased 37-fold for hydrogenase and 6.7-fold for CODH. When whole cells were incubated under anaerobic conditions after being exposed to air for 1 h, hydrogenase activity was recovered more than 90% in 2 h suggesting that the deactivation of hydrogenase by $O_2$ was reversible. On the contrary, CODH activity was not recovered once deactivated by $O_2$ and the only way to recover the activity was to synthesize new CODH. This study indicates that $O_2$ sensitivity of $H_2$ production activity of Citrobacter sp. Y19 is an important drawback as in other $H_2-producing$ bactria.

• Transactions of the Korean hydrogen and new energy society
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• v.33 no.5
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• pp.507-514
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• 2022

There is growing interest in sustainable energy sources that can reduce fossil fuel dependence and environmental pollution while meeting rapidly growing energy demands. Hydrogen have been investigated as one of the ideal alternative energies because it has relatively high efficiency without emitting pollutants. The light-sensitized enzymatic (LSE) system, which uses hydrogenase-enzymes, is one of the methods towards economically feasible system configurations that enhance the rate of hydrogen generation. Hydrogenase is an enzyme that catalyzes a reversible reaction that oxidizes molecular hydrogen or produces molecular hydrogen from protons and electrons. In this paper, utilization of [NiFe]-hydrogenase (from Pyrococcus furiosus) in photoelectrochemical hydrogen production system such as handling, immobilization, physicochemical and electrochemical analysis, process parameters, etc. was introduced.

• 한국신재생에너지학회:학술대회논문집
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• 2009.06a
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• pp.782-786
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• 2009

Thiocapsa roseopersicina NCIB 8347은 purple sulfur bacteria이며 광합성종속영양 조건에서는 nitrogenase 효소계가 유도되어 질소를 고정하며, 수소를 발생한다. 또한 광합성독립영양 조건에서는 hydrogenase 효소계가 유도되어 3~4개 종류의 특성이 다른 hydrogenase가 membrane에 결합되어 있거나, cytoplasma에 존재하며, 이 중의 일부는 산소농도와 온도의 상승에도 비교적 안정하다. 본 연구에서는 T. roseopersicina NCIB 8347이 광합성종속영양 조건에서 수소를 생산할 수 있는 제반 배양조건을 최적화하고, nitrogenase와 일부 hydrogenase역가를 측정하여 purple non-sulfur bacteria, Rhodobacter sphaeroides KD131의 nitrogenase와 비교하여 수소생산을 최적화하였다. 할로겐램프를 8-9 $Klux/m^2$로 조사할 때와 배양온도 $26{\sim}30^{\circ}C$, 배양시간 72시간에서 균체 성장과 수소생산이 가장 높았다. T. roseopersicina NCIB 8347는 광합성 독립영양, 종속영양 조건에서 모두 성장 할 수 있었다.

• Rapid Communication in Photoscience
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• v.1 no.1
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• pp.21-24
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• 2012

Influence of gamma rays on the cyanobacterium Synechocystis sp. PCC 6803 cells was investigated in terms of a bidirectional hydrogenase, which is encoded by hoxEFUYH genes and responsible for biohydrogen production. Irradiated cells revealed a substantial change in stoichiometry of photosystems at one day after gamma irradiation at different doses. However, as evaluated by the maximal rate of photosynthetic oxygen evolution, maximal photochemical efficiency of photosystem II, and chlorophyll content, net photosynthesis or photosynthetic capacity was not significantly different between the control and irradiated cells. Instead, transcription of hoxE, hoxH, or lexA, which encodes a subunit of bidirectional hydrogenase or the only transcriptional activator, LexA, for hox genes, was commonly enhanced in the irradiated cells. This transcriptional enhancement was more conspicuously observed immediately after gamma irradiation. In contrast, hydrogenase activities were found to somewhat lower in the irradiated cells. Therefore, we propose that transcription of hox genes should be enhanced by gamma irradiation in a LexA-mediated and possibly photosynthesis-independent manner and that this enhancement might not induce a subsequent increase in hydrogenase activities, probably due to the presence of post-transcriptional and/or post-translational regulatory mechanisms.

• Journal of Microbiology and Biotechnology
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• v.2 no.4
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• pp.248-254
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• 1992

The initial growth of Clostridium acetobutylicum was not inhibited by 1 atm of H$_2$ while H$_2$ reduced glucose consumption in a solventogenic culture of a phosphate limited 2-stage chemostat. Under 1 atm of H$_2$, a solventogenic culture consumed hydrogen, but an acidogenic culture produced hydrogen. H$_2$ consumption by the solventogenic culture was enhanced by the addition of 5 mM neutral red, an artificial electron carrier with a redox potential of -325 mV. Hydrogenase activity, measured in both directions of production and consumption, showed that activity coupled with methyl viologen is higher in an acidogenic culture than in a solventogenic culture, and that the two cultures have similar activities for methylene blue reduction. The solventogenic culture showed a higher activity coupled with neutral red than the acidogenic culture. From these results, it is hypothesized that hydrogen producing hydrogenase activity is high during the acidogenic phase, and decreases as solventogenesis starts, and that the solventogenic culture produces a second hydrogenase which uses an electron carrier other than ferredoxin. This hypothesis was supported by the fact that enzyme activities involved in electron flow can be coupled to neutral red, indepedent of ferredoxin, and that neutral red addition to the fermentation system increased butanol yield, with a decrease in production of less reduced fermentation products, and $H^2$.

• Korean Journal of Microbiology
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• v.29 no.5
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• pp.296-300
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• 1991

The membrane-bound Escherichia coli hydrogenases were purified partially by the solubilization with detergents. the E. coli crude extract was solubilized with sodium deoxycholate and dialyzed against the buffer containing Triton X-100. Two different hydrogenases were obtained by the DEAE-cellulose, hydroxyapatite and Sephedex G-200 column chromatography. The one was unstable during purification and contained 70- and 47-kDa polypeptides as major proteins. The other showed high H2-evolving activity and had major polypeptides of Mr 31 and 27. Those polypeptides were detected by the two-dimensional electrophoresis.