研究者詳細 - 今中　洋行

論文 - 今中　洋行

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温度走査フーリエ変換赤外分光分析による糖-高分子複合アモルファスマトリクスにおける分子間相互作用の解析

大山健一, 谷加寿子, 今中洋行, 今村維克, 中西一弘

化学工学会研究発表講演要旨集 2005 293 - 293 2005年

膜面液体培養法を用いた遺伝子組換え麹カビによるNuclease S1生産

森田正和, 安達昇, 奥村敦, 今中洋行, 今村維克, 中西一弘

化学工学会研究発表講演要旨集 2005 242 - 242 2005年

放線菌 Streptomyces mobaraensis 由来新規アシラーゼの精製・特性解析および合成反応

是石真友子, 川崎涼子, 今中洋行, 今村維克, 中西一弘, 丹尾式希

化学工学会研究発表講演要旨集 2005 270 - 270 2005年

異なる金属表面への種々の有機物質の吸着挙動の比較

長安武司, 今中洋行, 今村維克, 中西一弘

化学工学会研究発表講演要旨集 2005 292 - 292 2005年

ポリスチレン親和性ペプチド融合タンパク質の吸着特性とその応用

徳永安秀, 熊田陽一, 今石大輔, 今中洋行, 今村維克, 崎山高明, 中西一弘

化学工学会研究発表講演要旨集 2005f 311 - 311 2005年

Genetic evidence identifying the true gluconeogenic fructose-1,6-bisphosphatase in Thermococcus kodakaraensis and other hyerthermophiles. 査読

T Sato, H Imanaka, N Rashid, T Fukui, H Atomi, T Imanaka

JOURNAL OF BACTERIOLOGY 186 ( 17 ) 5799 - 5807 2004年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC MICROBIOLOGY

Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes in gluconeogenesis. Although FBPase activity has been detected in several hyperthermophiles, no orthologs corresponding, to the classical FBPases from bacteria and eukaryotes have been identified in their genomes. An inositol monophosphatase (IMPase) from Methanococcus jannaschii which displayed both FBPase and IMPase activities and a structurally novel FBPase (Fbp(Tk)) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 have been proposed as the "missing" FBPase. For this study, using T. kodakaraensis, we took a genetic approach to elucidate which candidate is the major gluconeogenic enzyme in vivo. The IMPase/FBPase ortholog in T. kodakaraensis, Imp(Tk), was confirmed to possess high FBPase activity along with IMPase activity, as in the case of other orthologs. We therefore constructed Deltafbp and Deltaimp strains by applying a gene disruption system recently developed for T. kodakaraensis and investigated their phenotypes. The Deltafbp strain could not grow under gluconeogenic conditions while glycolytic growth was unimpaired, and the disruption resulted in the complete abolishment of intracellular FBPase activity. Evidently, fbp(Tk) is an indispensable gene for gluconeolgenesis and is responsible for almost all intracellular FBPase activity. In contrast, the endogenous imp(Tk) gene could not complement the defect of the fbp deletion, and its disruption did not lead to any detectable phenotypic changes under the conditions examined. These facts indicated that imp(Tk) is irrelevant to gluconeogenesis, despite the high FBPase activity of its protein product, probably due to insufficient transcription. Our results provide strong evidence that the true FBPase for gluconeogenesis in T. kodakaraensis is the FbpTk ortholog, not the IMPase/FBPase ortholog.

DOI： 10.1128/JB.186.17.5799-5807.2004

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Presence of a novel phosphopentomutase and a 2-deoxyribose 5-phosphate aldolase reveals a metabolic link between pentoses and central carbon metabolism in the hyperthermophilic archaeon Thermococcus kodakaraensis 査読

N Rashid, H Imanaka, T Fukui, H Atomi, T Imanaka

JOURNAL OF BACTERIOLOGY 186 ( 13 ) 4185 - 4191 2004年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC MICROBIOLOGY

Numerous bacteria and mammalian cells harbor two enzymes, phosphopentomutase (PPM) and 2-deoxyribose 5-phosphate aldolase (DERA), involved in the interconversion between nucleosides and central carbon metabolism. In this study, we have examined the presence of this metabolic link in the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. A search of the genome sequence of this strain revealed the presence of a closely related orthologue (TK2104) of bacterial DERA genes while no orthologue related to previously characterized PPM genes could be detected. Expression, purification, and characterization of the TK2104 protein product revealed that this gene actually encoded a DERA, catalyzing the reaction through a class I aldolase mechanism. As PPM activity was detected in T. kodakaraensis cells, we partially purified the protein to examine its N-terminal amino acid sequence. The sequence corresponded to a gene (TK1777) similar to phosphomannomutases within COG1109 but not COG1015, which includes all previously identified PPMs. Heterologous gene expression of TK1777 and characterization of the purified recombinant protein clearly revealed that the gene indeed encoded a PPM. Both enzyme activities could be observed in T. kodakaraensis cells under glycolytic and gluconeogenic growth conditions, whereas the addition of ribose, 2-deoxyribose, and 2'-deoxynucleosides in the medium did not lead to a significant induction of these activities. Our results clearly indicate the presence of a metabolic link between pentoses and central carbon metabolism in T. kodakaraensis, providing an alternative route for pentose biosynthesis through the functions of DERA and a structurally novel PPM.

DOI： 10.1128/JB.186.13.4185-4191.2004

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Gene cloning and characterization of fructose-1,6-bisphosphate aldolase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 査読

H Imanaka, T Fukui, H Atomi, T Imanaka

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 94 ( 3 ) 237 - 243 2002年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN

The fructose-1,6-bisphosphate (FBP) aldolase gene from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 was cloned. The gene encoding FBP aldolase (Tk-fba) was expressed in Escherichia coli and the purified recombinant protein was characterized at high temperature. Tk-Fba is a homodecamer with a subunit molecular mass of 31,283 Da. The amino acid sequence, decameric conformation, formation of a Schiff-base intermediate, and stimulation (286%) of FBP cleavage activity by citrate suggested that Tk-Fba belonged to Class IA, a subtype of the classical Class I aldolases. The specific activity for the FBP cleavage reaction was 18.9 U/mg, which was much higher than those of other Class IA type FBP aldolases. Tk-Fba was extremely thermostable since the optimum temperature seemed to be above 100degreesC. The optimum pH for Tk-Fba was determined to be 5.0 in the absence of citrate, while it shifted to around 7.0 in the presence of citrate. Tk-Fba accepted FBP and fructose-1-phosphate as substrates and K-m values were determined to be 0.063 mM and 4.37 mM, respectively. In addition to citrate, phosphoenolpyruvate and pyrophosphate were also found to be potent activators of Tk-Fba, enhancing activities up to 346% and 201%, respectively. Erythrose-4-phosphate acted as an inhibitor and caused a decrease in the activity to 49%. Tk-Fba also catalyzed the condensation reaction with a similar activity level (14.9 U/mg) to that for FBP cleavage. However, none of the above compounds seemed to have a significant effect on the condensation reaction by Tk-Fba. These results suggest a regulatory function of Tk-Fba toward the catabolic direction of sugar metabolism in T. kodakaraensis KOD1.

DOI： 10.1263/jbb.94.237

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A novel candidate for the true fructose-1,6-bisphosphatase in Archaea 査読

N Rashid, H Imanaka, T Kanai, T Fukui, H Atomi, T Imanaka

JOURNAL OF BIOLOGICAL CHEMISTRY 277 ( 34 ) 30649 - 30655 2002年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes of the gluconeogenic pathway. Although enzyme activity had been detected in Archaea, the corresponding gene had not been identified until a presumable inositol monophosphatase gene from Methanococcus jannaschii was found to encode a protein with both inositol monophosphatase and FBPase activities. Here we display that a gene from the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, which does not correspond to the inositol monophosphatase gene from M. jannaschii, displays high FBPase activity. The FBPase from strain KOD1 was partially purified, its N-terminal amino acid sequence was determined, and the gene (Tk-fbp) was cloned. Tk-fbp encoded a protein of 375 amino acid residues with a molecular mass of 41,658 Da. The recombinant Tk-Fbp was purified and characterized. Tk-Fbp catalyzed the conversion of fructose 1,6-bisphosphate to fructose 6-phosphate following Michaelis-Menten kinetics with a K-m value of 100 mum toward fructose 1,6-bisphosphate, and a k(cat) value of 17 s(-1) subunit(-1) at 95degreesC. Unlike the inositol monophosphatase from M. jannaschii, Tk-Fbp displayed strict substrate specificity for fructose 1,6-bisphosphate. Activity was enhanced by Mg2+ and dithioerythritol, and was slightly inhibited by fructose 2,6-bisphosphate. AMP did not inhibit the enzyme activity. We examined whether expression of Tk-fbp was regulated at the transcription level. High levels of Tk-fbp transcripts were detected in cells grown on pyruvate or amino acids, whereas no transcription was detected when starch was present in the medium. Orthologue genes corresponding to Tk-fbp with high similarity are present in all the complete genome sequences of thermophilic Archaea, including M. jannaschii, Pyrococcus furiosus, Sulfolobus solfataricus, and Archaeoglobus fulgidus, but are yet to be assigned any function. Taking into account the high FBPase activity of the protein, the strict substrate specificity, and its sugar-repressed gene expression, we propose that Tk-Fbp may represent the bona fide FBPase in Archaea.

DOI： 10.1074/jbc.M202868200

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Removal of aromatic compounds in gas by electron attachment 査読

H Tamon, H Imanaka, N Sano, M Okazaki, W Tanthapanichakoon

INDUSTRIAL & ENGINEERING CHEMISTRY RESEARCH 37 ( 7 ) 2770 - 2774 1998年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER CHEMICAL SOC

A corona-discharge reactor was applied to remove benzene and p-dichlorobenzene from nitrogen and a nitrogen-oxygen mixture. Although benzene was not effectively removed from nitrogen by electron attachment, the removal efficiency was improved greatly by mixing oxygen. On the other hand, the high removal efficiencies of p-dichlorobenzene were obtained in nitrogen or a nitrogen-oxygen mixture. The removal mechanism was studied based on the contribution of the ozone reaction and the analysis of the deposit on the anode of the reactor. As a result, the ozone reaction does not contribute to the removals. An FT-IR measurement and thermogravimetry suggest that benzene or p-dichlorobenzene is decomposed by dissociative electron attachment and deposits as polycyclic aromatic compounds of a high boiling point on the anode surface.

DOI： 10.1021/ie980071v

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