掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.jbiosc.2021.11.008

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

The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 catalyzes the oxidative deamination of l-glutamate to produce 2-ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l-glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l-glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l-arginine. The oxidative deamination activity of LGOX to l-arginine is higher than that of l-arginine oxidase form from Pseudomonas sp. TPU 7192. X-ray crystal structure analysis revealed that the guanidino group of l-arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild-type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l-arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l-arginine by LGOX R305E, is proportional to the concentration of l-arginine in a range from 0 to 100 mu M. The linear relationship is maintained around 1 mu M of l-arginine. Thus, LGOX R305E is suitable for the determination of l-arginine.

DOI： 10.1002/pro.4070

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

L-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 is a vitamin B-6-dependent enzyme and catalyzes the non-oxidative decarboxylation of L-methionine to produce 3-methylthiopropylamine and carbon dioxide. We present here the crystal structures of the ligand-free form of MetDC and of several enzymatic reaction intermediates. Group II amino acid decarboxylases have many residues in common around the active site but the residues surrounding the side chain of the substrate differ. Based on information obtained from the crystal structure, and mutational and biochemical experiments, we propose a key role for Gln64 in determining the substrate specificity of MetDC, and for Tyr421 as the acid catalyst that participates in protonation after the decarboxylation reaction.

DOI： 10.1002/pro.4027

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記述言語：英語  

DOI： 10.1007/s10126-020-09999-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) β-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory-based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA.

DOI： 10.1093/bbb/zbab011

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.yjsbx.2021.100044

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掲載種別：研究論文（学術雑誌）   出版者・発行元：Wiley  

DOI： 10.1002/pro.3946

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その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/pro.3946

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Silica cell walls of diatoms have attracted attention as a source of nanostructured functional materials and have immense potential for a variety of applications. Previous studies of silica cell wall formation have identified numerous involved proteins, but most of these proteins are species-specific and are not conserved among diatoms. However, because the basic process of diatom cell wall formation is common to all diatom species, ubiquitous proteins and molecules will reveal the mechanisms of cell wall formation. In this study, we assembled de novo transcriptomes of three diatom species, Nitzschia palea, Achnanthes kuwaitensis, and Pseudoleyanella lunata, and compared protein-coding genes of five genome-sequenced diatom species. These analyses revealed a number of diatom-specific genes that encode putative endoplasmic reticulum-targeting proteins. Significant numbers of these proteins showed homology to silicanin-1, which is a conserved diatom protein that reportedly contributes to cell wall formation. These proteins also included a previously unrecognized SET domain protein methyltransferase family that may regulate functions of cell wall formation-related proteins and long-chain polyamines. Proteomic analysis of cell wall-associated proteins in N. palea identified a protein that is also encoded by one of the diatom-specific genes. Expression analysis showed that candidate genes were upregulated in response to silicon, suggesting that these genes play roles in silica cell wall formation. These candidate genes can facilitate further investigations of silica cell wall formation in diatoms.

DOI： 10.1007/s10126-020-09976-1

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1080/09168451.2020.1715781

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Springer Science and Business Media {LLC}  

Feline herpesvirus type 1 (FHV-1) causes a potentially fatal disease in cats. Through the use of virus inhibition and cytotoxicity assays, sinefungin, a nucleoside antibiotic, was assessed for its potential to inhibit the growth of FHV-1. Sinefungin inhibited in vitro growth of FHV-1 most significantly over other animal viruses, such as feline infectious peritonitis virus, equine herpesvirus, pseudorabies virus and feline calicivirus. Our results revealed that sinefungin specifically suppressed the replication of FHV-1 after its adsorption to the host feline kidney cells in a dose-dependent manner without obvious cytotoxicity to the host cells. This antibiotic can potentially offer a highly effective treatment for animals infected with FHV-1, providing alternative medication to currently available antiviral therapies.

DOI： 10.1038/s41429-019-0234-4

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記述言語：日本語   掲載種別：研究論文（その他学術会議資料等）  

添付ファイル： 生化学PLP.pdf

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Oxidative folding of extracellular proteins is pivotal for the biogenesis of bacterial virulence factors. Escherichia coli DsbA catalyzes disulfide bond formation in extracellular proteins and in multicomponent architectures on the cell surface. The present study assessed the significance of the redox properties of DsbA by exploiting the plaque-forming ability of bacteriophage M13, which specifically recognizes F-pili during infection of the host cell. A library of mutant dsbA genes was constructed by randomizing the dipeptide XX sequence in the active-site redox motif CXXC and then screened for mutants that altered plaque yield and appearance. In total, 24 dsbA mutant alleles produced substantially different degrees of complementation, and one mutant dsbA gene that encodes a CDIC sequence produced over 40-fold more clear plaques than wild type dsbA. The redox potential of purified DsbA [CDIC] was -172 mV, representing a less-oxidizing catalysis than the wild type DsbA (-122 mV), but one that is closer to yeast protein disulfide isomerase (-175 mV). DsbA [CDIC] exhibited a greater ability to refold fully denatured glutathionylated ribonuclease A than the wild type enzyme and a DsbA [CRIC] mutant, which has the same redox potential of -172 mV. Homology modeling and molecular dynamics simulation suggest that the CDIC mutant may have an enlarged substrate-binding cleft near the redox center, which confers kinetic advantages when acting on protein substrates.

DOI： 10.1016/j.bbapap.2018.12.005

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Many species of chiton are known to deposit magnetite (Fe3O4) within the cusps of their heavily mineralized and ultrahard radular teeth. Recently, much attention has been paid to the ultrastructural design and superior mechanical properties of these radular teeth, providing a promising model for the development of novel abrasion resistant materials. Here, we constructed de novo assembled transcripts from the radular tissue of C. stelleri that were used for transcriptome and proteome analysis. Transcriptomic analysis revealed that the top 20 most highly expressed transcripts in the non-mineralized teeth region include the transcripts encoding ferritin, while those in the mineralized teeth region contain a high proportion of mitochondrial respiratory chain proteins. Proteomic analysis identified 22 proteins that were specifically expressed in the mineralized cusp. These specific proteins include a novel protein that we term radular teeth matrix protein1 (RTMP1), globins, peroxidasins, antioxidant enzymes and a ferroxidase protein. This study reports the first de novo transcriptome assembly from C. stelleri, providing a broad overview of radular teeth mineralization. This new transcriptomic resource and the proteomic profiles of mineralized cusp are valuable for further investigation of the molecular mechanisms of radular teeth mineralization in chitons.

DOI： 10.1038/s41598-018-37839-2

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記述言語：英語   出版者・発行元：ELSEVIER SCIENCE INC  

DOI： 10.1016/j.freeradbiomed.2017.10.092

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni-Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulphate-reducing delta-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homology modelling and molecular dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under absolute anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions.

DOI： 10.1038/srep19742

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jbiosc.2015.02.019

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

DOI： 10.1080/09168451.2015.1012148

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

L-Lysine alpha-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L-lysine to form alpha-keto-epsilon-aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 <remove> resolution revealed that the overall structure is similar to that of snake venom L-amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX.

DOI： 10.1093/jb/mvv012

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その他リンク： http://orcid.org/0000-0003-1342-8885

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：American Society for Microbiology  

A draft genome sequence of Streptomyces incarnatus NRRL8089, which produces the nucleoside antibiotic sinefungin, is described here. The genome contains 8,897,465 bp in 76 contigs and 8,266 predicted genes. Interestingly, the genome encodes an open reading frame for selenocysteine-containing formate dehydrogenase-O and the selenoprotein biosynthetic gene cluster selABCD.

添付ファイル： Microbiology Resource Announcements-2015-Oshima-e00715-15.full.pdf

DOI： 10.1128/genomeA.00715-15

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.89.2_80

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.89.8_409

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記述言語：日本語   出版者・発行元：公益社団法人日本生物工学会  

グリセロールキナーゼは,中性脂肪測定用臨床検査薬用原料として利用されている.臨床検査薬は,近年溶液状態の検査薬が主流になり,長期間の保存を可能にするため,防腐剤を添加する事が一般的である.しかし,これらの防腐剤は酵素の安定性へ悪影響を及ぼす事もあり,臨床検査薬用原料酵素には溶液中の安定性に加え,これらの防腐剤への耐性も求められるようになっている.我々はCellulomonas sp. NT3060由来グリセロールキナーゼがN-methylisothiazoloneやimidazolidinylureaのような防腐剤に対し優れた安定性を示す事を見いだし,その酵素学的性質を明らかにした.Cellulomonas sp. NT3060由来のグリセロールキナーゼは,1,518塩基にコードされ,505アミノ酸より構成されていた.さらに,本遺伝子を導入した大腸菌により発現されたCellulomonas sp. NT3060由来グリセロールキナーゼを各種カラムクロマトグラフィーにより均一に精製し,その酵素学的特性を明らかにした.本グリセロールキナーゼの安定性は好熱性細菌由来のグリセロールキナーゼと比較すると不安定であるが,37℃付近の実用温度では十分安定であり,弱酸性領域から弱アルカリ領域まで幅広いpH領域で安定であった.また,基質であるグリセロールに対してKm値は6.9μMと高い親和性を示した.さらに,他起源のグリセロールキナーゼと比較して複数の防腐剤に対して高い耐性を示した.以上の性質より,本グリセロールキナーゼは臨床診断薬用原料として総合的に優れた性質を有した酵素である事が示唆された.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1271/bbb.110906

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Recently, we have solved the crystal structure of L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 (PDB code: 2E1M), the substrate specificity of which is strict toward L-glutamate. By a docking simulation using.-glutamate and structure of LGOX, we selected three residues, Arg305, His312, and Trp564 as candidates of the residues associating with recognition of L-glutamate. The activity of LGOX toward L-glutamate was significantly reduced by substitution of selected residues with Ala. However, the enzyme, Arg305 of which was substituted with Ala, exhibited catalytic activity toward various L-amino acids. To investigate the role of Arg305 in substrate specificity, we constructed Arg305 variants of LGOX. In all mutants, the substrate specificity of LGOX was markedly changed by the mutation. The results of kinetics and pH dependence on activity indicate that Arg305 of LGOX is associated with the interaction of enzyme and side chain of substrate. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2011.12.033

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記述言語：英語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.52.S123_1

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.86.7_412

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その他リンク： http://search.jamas.or.jp/link/ui/2013003170

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

Selenite (SeO32-) assimilation into a bacterial selenoprotein depends on thioredoxin (trx) reductase in Esherichia coli, but the molecular mechanism has not been elucidated. The mineral-oil overlay method made it possible to carry out anaerobic enzyme assay, which demonstrated an initial lag-phase followed by time-dependent steady NADPH consumption with a positive cooperativity toward selenite and trx. SDS-PAGE/auto-radiography using Se-75-labeled selenite as substrate revealed the formation of trx-bound selenium in the reaction mixture. The protein-bound selenium has metabolic significance in being stabilized in the divalent state, and it also produced the selenopersulfide (-S-SeH) form by the catalysis of E. coli trx reductase (TrxB).

DOI： 10.1271/bbb.100847

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

Mammalian thioredoxin reductases (TrxRs) contain selenium as selenocysteine (Sec) in the C-terminal redox center -Gly-Cys-Sec-Gly-OH to reduce Trx and other substrates; a Sec-to-Cys substitution in mammalian TrxR yields an almost inactive enzyme. The corresponding tetrapeptide sequence in Drosophila melanogaster TrxR (Dm-TrxR), -Ser-Cys-Cys-Ser-OH, endows the orthologous enzyme with a catalytic competence similar to mammalian selenoenzymes, but implementation of the Ser-containing tetrapeptide sequence SCCS into the mammalian enzyme does not restore the activity of the Sec-to-Cys mutant form (turnover number < 2/min). MOPAC calculation suggested that the C-terminal hexapeptide Pro-Ala-Ser-Cys-Cys-Ser-OH functions as a redox center that alleviates the necessity for selenium in Dm-TrxR, and a mutant form of human lung TrxR that mimics this hexapeptide sequence showed improved catalytic turnover (17.4/min for DTNB and 13.2/min for E. coli trx) compared to the Sec-to-Cys mutant. MOPAC calculation also suggested that the dominant form of the Pro-containing hexapeptide is a C+ conformation, which perhaps has a catalytic advantage in facile reduction of the intramolecular disulfide bond between Cys497 and Cys498 by the N-terminal redox center in the neighboring subunit.

DOI： 10.1271/bbb.100740

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.85.7_346

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その他リンク： http://search.jamas.or.jp/link/ui/2011324915

記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.85.1_27

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.84.2_77

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その他リンク： http://search.jamas.or.jp/link/ui/2010133267

記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.84.5-6_274_2

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記述言語：英語   出版者・発行元：日本放線菌学会  

DOI： 10.3209/saj.SAJ240204

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記述言語：英語   出版者・発行元：日本微量元素学会  

DOI： 10.11299/brte.19.84

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その他リンク： http://search.jamas.or.jp/link/ui/2008358486

記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.82.1_61_1

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.82.11_617_1

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.81.5-6_258

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記述言語：日本語   出版者・発行元：一般社団法人 日本生物物理学会  

DOI： 10.2142/biophys.45.S40_4

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.79.1_43_2

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.79.3_194_1

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER-VERLAG TOKYO  

Sixty strains of thermoacidophilic bacteria have been isolated from soil and water samples obtained from various acidic environments in Japan. An initial comparative sequence analysis of the hypervariable regions of the 16S rDNA revealed that all strains could be assigned to the Alicyclobacillus acidocaldarius-Alicyclobacillus genomic species 1 group, which could be further subdivided into three clusters (Clusters I-III). On the basis of phenotypic characteristics, chemotaxonomic profiles, and phylogenetic data of six selected strains, five strains were identified as either A. acidocaldarius or Alicyclobacillus genomic species 1; however, one strain (MIH 332) could not be determined to belong to either of these species. 16S rDNA sequence homology values between strain MIH 332 and the reference strains of A. acidocaldarius (ATCC 27009(T)) and Alicyclobacillus genomic species 1 (DSM 11984) were 98.8% and 99.1%, respectively, which were higher than the corresponding similarity between the reference strains (98.4%). On the other hand, DNA-DNA hybridization levels between strain MIH 332 and the reference strains were 39% and 44%, respectively, which were lower than the value between the reference strains (59% or 65%). However, the phenotype of strain MIH 332 was also similar to those of the reference strains, and a typical phenotype could not be found for the strain, thus indicating that the strain may be a new genomic species of A. acidocaldarius, for which the name Alicyclobacillus genomic species 2 is tentatively proposed. The results of this study suggest that A. acidocaldarius and its related species are widely distributed in acidic environments in Japan, with slight regional variations in morphological and genotypic characteristics.

DOI： 10.1007/s00792-001-0262-3

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1271/bbb.64.2336

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/oxfordjournals.jbchem.a022760

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC  

L-Lactate and L-beta-phenyllactate have been identified in the culture broth of Streptomyces sp. KY-11 as reversible noncompetitive inhibitors of Aspergillus oryzae aminoacylase-l and porcine kidney aminoacylase I. A series of alpha-hydroxyl acids (DL-R-CH(OH)-COOH, R = Et, n-pro, n-butyl, n-pentyl, n-hexyl) also inhibited the two enzymes in reversible noncompetitive kinetics, and the inhibition potency (-log K-i) increased with the increased hydrophobicity of the R group. The two eukaryotic enzymes showed distinct preferences to the ligand alpha-alkyl group, and the fungus enzyme was inhibited by L-beta-phenyllactate (R = benzyl) 10(3)-fold more potently than the mammalian enzyme. L-alpha-Fluoro-beta-phenyl-propionate and its D-isomer were used to show that the L-configuration of the alpha-substituent was important for potent inhibition of both the enzymes. The fungus aminoacylase-1 steeply decreased the affinity to alpha-fluoro-and alpha-hydroxy-n-caproate as pH was raised from 7 to 11, whereas the mammalian enzyme retained the affinity to these ligands under alkaline conditions. These results suggest that A. oryzae aminoacylase-1 has an acidic residue that interacts with OH or -F, while the mammalian enzyme would have a basic residue that recognizes the alpha-substituents. (C) 2000 Academic Press.

DOI： 10.1006/abbi.2000.1869

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DOI： 10.11465/milk.49.81

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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  

A 15-kb region of Pseudomonas putida chromosomal DNA containing the mde operon and an upstream regulatory gene (mdeR) has been cloned and sequenced. The mde operon contains two structural genes involved in L-methionine degradative metabolism: the already-identified mdeA, which encodes L-methionine gamma-lyase (H. Inoue, K. Inagaki, M. Sugimoto, N. Esaki, K. Soda, and fi, Tanaka, J. Biochem, (Tokyo) 117:1120-1125, 1995), and mdeB, which encodes a homologous protein to the homodimeric-type E1 component of pyruvate dehydrogenase complex, A rho-independent terminator was present just downstream of mdeB, and open reading frames corresponding to other components of alpha-keto acid dehydrogenase complex were not found, When MdeB was overproduced in Escherichia coli, the cell extract showed the E1 activity with high specificity for alpha-ketobutyrate rather than pyruvate. These results suggest that MdeB plays an important role in the metabolism of alpha-ketobutyrate produced by MdeA from L-methionine, Accordingly, MdeB encodes a novel E1 component, alpha-ketobutyrate dehydrogenase E1 component, of an unknown alpha-keto acid dehydrogenase complex in P. putida, In addition, we found that the mdeR gene was located on the opposite strand and began at 127 bp from the translational start site of mdeA. The mdeR gene product has been identified as a member of the leucine-responsive regulatory protein (Lrp) family and revealed to act as an essential positive regulator allowing the expression of the mdeAB operon.

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記述言語：日本語   出版者・発行元：(公社)日本ビタミン学会  

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記述言語：英語   出版者・発行元：INT UNION CRYSTALLOGRAPHY  

Cystathionine gamma-synthase (CGS; EC 2.5.1.48), a pyridoxal 5'-phosphate (PLP)dependent enzyme, catalyzes the formation of cystathionine from an l-homoserine derivative and l-cysteine in the first step of the transsulfuration pathway. Recombinant CGS from the thermoacidophilic archaeon Sulfolobus tokodaii (StCGS) was overexpressed in Escherichia coli and purified to homogeneity by heat treatment followed by hydroxyapatite and gel-filtration column chromatography. The purified enzyme shows higher enzymatic activity at 353 K under basic pH conditions compared with that at 293 K. Crystallization trials yielded three crystal forms from different temperature and pH conditions. Form I crystals (space group P2(1); unit-cell parameters a = 58.4, b = 149.3, c = 90.2 angstrom , beta = 108.9 degrees) were obtained at 293 K under acidic pH conditions using 2-methyl-2,4-pentanediol as a precipitant, whereas under basic pH conditions the enzyme crystallized in form II at 293 K (space group C222(1); unit-cell parameters a = 117.7, b = 117.8, c = 251.3 angstrom ) and in form II' at 313 K (space group C2221; unit-cell parameters a = 107.5, b = 127.7, c = 251.1 angstrom ) using polyethylene glycol 3350 as a precipitant. X-ray diffraction data were collected to 2.2, 2.9 and 2.7 angstrom resolution for forms I, II and III', respectively. Structural analysis of these crystal forms shows that the orientation of the bound PLP in form II is significantly different from that in form II', suggesting that the change in orientation of PLP with temperature plays a role in the thermophilic enzymatic activity of StCGS.

DOI： 10.1107/S2053230X17002011

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：英語   出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures (T-m), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60 degrees C (t(1/2)). Three mutants showed increased stabilities compared to the wild type protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2015.09.011

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記述言語：英語   出版者・発行元：TAYLOR & FRANCIS LTD  

DL-Penicillamine, a copper-specific metal chelator, remarkably suppressed the growth of Bacillus subtilis 168 when added to a synthetic medium under Cu2+ limitation. DNA microarray and screening of 2,602 knockout mutants showed that the zosA gene was de-repressed in the presence of 0.1% dl-penicillamine, and that the zosA mutant was sensitive to dl-penicillamine medium. The zosA mutant delayed the growth under Cu-limitation even without the chelator, and the sensitivity to dl-penicillamine was reversed by induction using 0.3mM IPTG and the Pspac promoter inserted directly upstream of the zosA gene. Furthermore, the zosA mutant showed elevated tolerance of excessive Cu2+ but not of excessive Zn2+ added to LB and synthetic media. Homology modeling of the ZosA protein suggested that the protein can fold itself into essential domains for constituting a metal transporting ATPase. Our study suggests that zosA is a candidate gene involved in copper uptake.

DOI： 10.1080/09168451.2015.1107462

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：英語   出版者・発行元：TAYLOR & FRANCIS LTD  

Biosynthesis of selenocysteine-containing proteins requires monoselenophosphate, a selenium-donor intermediate generated by selenophosphate synthetase (Sephs). A non-radioactive assay was developed as an alternative to the standard [8-C-14] AMP-quantifying assay. The product, AMP, was measured using a recombinant pyruvate pyrophosphate dikinase from Thermus thermophilus HB8. The K-M and k(cat) for Sephs2-Sec60Cys were determined to be 26M and 0.352min(-1), respectively.

DOI： 10.1080/09168451.2016.1200458

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記述言語：日本語  

DOI： 10.20632/vso.90.1_39

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

DOI： 10.20632/vso.89.10_495

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：岡山大学農学部  

Pyranose oxidase (EC 1.1.3.10 ; PROD) catalyzes the oxidation of aldopyranoses at the position C‒2to yield the corresponding 2‒keto-aldoses and H2O2 , using oxygen as an electron acceptor. The enzyme shows broad substrate specificity as well as reactivity for 1,5‒anhydro‒d‒glucitol (1,5‒AG), which is known as a clinical glycemic marker. It is considered that the reactivity of PROD for 1,5‒AG is useful in the development of an amperometric-type biosensor, which is a convenient diagnostic device for selfmonitoringblood glucose (SMBG). However, the levels of dissolved oxygen in blood affect biosensorsystems that are equipped with an artificial electron mediator. In the present study, we attempted to develop an O2‒insensitive oxidase that would improve the dye-mediated dehydrogenase activity. We performed site-directed mutagenesis on PROD isolated from basidiomycetous fungus No. 52, which generated 11 mutants. The amino acid substitution Q421A exhibited a significant decrease (8.8% of wild type) in its oxidase activity, whereas it maintained its dehydrogenase activity (67% of wild type). In this study, we characterized PROD mutants from basidiomycetous fungus No. 52, which showed improved dye-mediated dehydrogenase activity.

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.88.9_481

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.89.3_148

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記述言語：日本語   出版者・発行元：日本生物工学会  

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その他リンク： http://search.jamas.or.jp/link/ui/2016232175

記述言語：日本語   出版者・発行元：日本生物工学会  

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その他リンク： http://search.jamas.or.jp/link/ui/2016232036

記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：(公社)日本ビタミン学会  

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記述言語：日本語   出版者・発行元：岡山大学農学部  

L‒Glutamate oxidase (LGOX) from Streptomyces sp. X‒119‒6 has strict substrate specificity towardL‒glutamate. Recently, we solved the X‒ray crystal structure of LGOX and this revealed that Arg305 inthe active site is the key residue involved in substrate recognition. Therefore, we created 19 mutantenzymes of R305X‒LGOX by saturation mutagenesis. One of them R305D‒LGOX, Arg305 substitutedwith Asp exhibited oxidase activity for L‒Arg. Optimum pH of R305D‒LGOX mutant enzyme was pH8.5. Interestingly, the activity of R305D‒LGOX toward L‒Arg was inhibited by phosphate. And furthermore,the substrate specificity of R305D‒LGOX was affected by using buffer. The results of inhibitionanalysis suggest, that phosphate is a competitive inhibitor of R305D‒LGOX when L‒Arg is used assubstrate. Kinetic analysis of R305D‒LGOX showed that Km value and kcat value of R305D‒LGOX towardl-Arg were 0.68 mM and 6.7 s-1 respectively. In this study, we showed that R305D‒LGOX mutantenzyme is a novel l-arginine oxidase and useful for l-arginine biosensor.

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記述言語：日本語   出版者・発行元：日本生物工学会  

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その他リンク： http://search.jamas.or.jp/link/ui/2016045849

記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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J-GLOBAL

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.87.1_51

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.87.8_463

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：岡山大学農学部  

The gene encoding a thermostable amidase (EC 3.5.1.4) from thermophilic bacterium Thermus sp.O-3-1, was cloned and expressed in Escherichia coli JM109. The cloned amidase gene (ami) is 930 bp and encodes a protein composed of 310 amino acids. The protein is predicted to have a molecular mass of 33,089 Da. The amidase from Thermus sp.O-3-1 was purified by heat treatment and DEAE Toyopearl 650M column chromatography. The molecular mass of the native enzyme was estimated to be about 70 kDa by gel filtration chromatography, indicating that the enzyme has a homodimeric structure. The purified enzyme was stable up to 80°C and within a pH range from 7.0 to 10.0. The optimum temperature and pH for enzyme activity were 90°C, and 9.0, respectively. The enzyme was strongly inhibited by the metal-chelating compound EDTA. The activity of the EDTA-treated enzyme was reactivated by the addition of Co(2+), Ni(2+) and Mn(2+) ions. Therefore the enzyme was predicted to be metalloenzyme. Finally,as a result of investigation into substrate specificity, the purified enzyme was suggested to be D-amino acid specific amidase, as it showed higher activity toward D-Leu-pNA than L-Leu-pNA.好熱性細菌Thermus sp.O-3-1 由来の耐熱性アミダーゼ遺伝子を大腸菌中にクローニングし，その塩基配列を決定した．ami 遺伝子は930 bp からなり，310アミノ酸をコードしていた．本酵素の分子量は33，089 Daであると予想された．Thermus sp.O-3-1 由来アミダーゼを大腸菌で生産させ，熱処理とDEAE-トヨパール650M陰イオン交換カラム等により精製した．ゲル濾過クロマトグラフィーとSDS-PAGE の結果から本酵素は分子質量33 kDa のサブユニット2分子からなるダイマー構造を有していることが明らかとなった．精製酵素の熱安定性は80℃まで，pH 安定性は7.0～10.0であり，安定性の高い酵素であった．最適温度は90℃，最適 pH は9.0であった．EDTA により活性が著しく阻害され，Co(2+)やNi(2+)，Mn(2+)によって活性の回復，向上が見られたため，本酵素は金属酵素であることが示唆された．基質特異性の検討の結果，L-Leu-pNA よりもD-Leu-pNA に対して高い活性を示したため，本酵素がＤ-アミノ酸基質に特異性を持つアミダーゼであることが判明した．本酵素は耐熱性を有するユニークなＤ-アミノ酸アミダーゼであり，今後産業利用が期待される．

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.85.8_446

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：岡山大学農学部  

L-Methionine decarboxylase [EC 4.1.1.57] catalyzes the decarboxylation of L-methionine and is a pyridoxal 5'-phosohate(PLP)-dependent enzyme. L-Methionine decarboxylase has been purified 630-fold by DEAE-Toyopearl 650M, Phenyl-Toyopearl 650M and Sephacryl S-300 column chromatographies from Streptomyces sp.590. The enzyme has a dimeric structure with identical subunits of Mr 60,000. This enzyme shows optimum activity at pH7.0 and 45°C, and is stable between pH5.7 and pH9.0. L-Methionine decarboxylase has antitumor activity against RERF-LC-AI and HeLa cells. Ten N-terminal amino acid sequence of L-methionine decarboxylase was determined, and the sequence showed no homology with other reported proteins.

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

DOI： 10.20632/vso.85.1_23

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.85.3_155_1

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：日本生化学会  

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記述言語：英語  

Sinefungin, a nucleoside antibiotic with potent antifungal, antiviral, and anti-trypanosome activities, has been a target for production enhancement in the past decades through medium optimization and strain improvement. For the purpose of introducing a more rational approach, we induced rpoB mutation in the producer strain, Streptomyces incarnatus NRRL 8089, by optimized UV-irradiation, and a resulting rifampicin-resistant strain rif-400 increased the sinefungin production by 7-fold. The growth and melanin production were obviously accelerated in the rifampicin-resistant high-producer mutant, while the morphological differentiation such as aerial mycelia and spiked-spore formation was retained. Molecular cloning and DNA sequencing identified a single mutation A1340G in the rpoB gene, which encodes the beta-subunit of RNA polymerase, and the resulting amino acid substitution Asp447Gly corresponded to one of mutations that reportedly allowed the transcriptional up-regulation of actinorhodin production in S. coelicolor A3(2). Sinefungin production was further enhanced by resting cell system using the rpoB mutant strain in the presence of 10 mM L-Arg. D-Arg or L-ornithine did not enhance the sinefungin production, and >50 mM urea strongly suppressed the nucleoside antibiotic production, supporting the proposed biosynthetic mechanism by which urea is liberated from the guanidino-group-bearing intermediate that is produced by enzymatic condensation of L-Arg and ATP.

DOI： 10.1016/j.jbiosc.2009.10.017

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：岡山大学農学部  

Sinefungin is a nucleoside antibiotic, in which a molecule of L-ornithine is linked to the 5' end of adenosine through a C-C bond. The antibiotic was isolated from the culture broth of Streptomyces incarnatus. For the purpose of detecting intermediate of sinefungin biosynthesis, resting cell suspensions were incubated with supplemental L-arginine, and L-ornithine. 50mM Arginine was converted to a compound X that has low polarity. 50mM ornithine was not converted and remained in reaction solution. Compound X was purified using HPLC, and analyzed using (1)H-NMR and FAB-MS. These analyses showed that a compound X is "ornithine-lactam" (Mw＝114), which has a structure of circularized ornithine. These results indicated that S. incarnatus has an enzyme that converts arginine to ornithine-lactam. Such an enzyme has never been reported, and suggested that it may be relevant to sinefungin biosynthesis.シネフンギンは抗真菌，抗マラリア活性を有する核酸系抗生物質であり，放線菌 S. incarnatus により生合成される．シネフンギンはアデノシンとオルニチンがＣﾝＣ結合した構造であり，無細胞抽出液での取り込み実験からＬﾝアルギニンと ATP から生合成されると推測される．Ｌﾝアルギニン，Ｌﾝオルニチンを S. incarnatus の休止菌体反応系への投与を行いシネフンギン中間体の探索を行った．その結果50ﾋアルギニンは24時間以内に低極性化合物へと変換された．一方50ﾋオルニチンは変換されず反応液中に残存した．HPLC で化合物を精製し，1HﾝNMR，FABﾝMS での分析の結果オルニチン環状モノペプチド，「オルニチンラクタム」（分子量114）であることを明らかにした．この結果は S. incarnatus がアルギニンからオルニチンラクタムへの変換酵素を有する事を示唆する．このような酵素の報告例はこれまでになく，ニ次代謝酵素であることが示唆され，シネフンギン生合成との関連性に興味が持たれる．

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記述言語：日本語   出版者・発行元：岡山大学農学部  

The gene encoding a cystathionine γ-synthase from Sulfolobus tokodaii was cloned and expressed in Escherihia coli Rosetta-gami (DE3). Cystathionine γ-synthase ［EC 2. 5. 1. 48］ from Sulfolobus tokodaii (stCGS) was purified by heat treatment, DEAE- Toyopearl 650M and Sephacryl S-300 column chromatographies from E. coli transformants. stCGS shows optimum activity at pH 7.0, and is stable between pH5.0 and pH9.0. The optimum temperature of stCGS is above 100℃, and the enzyme showed the remaining activity of almost 100% up to 70℃. The K(m) and V(max) with O-phospho-L- homoserine as a substrate are 0.82 mM and 2.42 U/mg. To analyze the role of Phe 97 in the active site of stCGS, we constructed F97Y, R99C, and F97Y-R99C mutant enzymes. Although native stCGS has no activity toward l-methionine, F97Y mutant enzyme gained the elimination activity toward L-methionine.好熱好酸性アーキア Sulfolobus tokodaii 由来シスタチオニンγﾝシンターゼ（stCGS）遺伝子を pET-11a に組み込み pET-stCGS を構築した．このベクターでE. coli Rosettaﾝgami（DE3）を形質転換し，本遺伝子を発現させ，精製及び性質検討を行った．大腸菌で発現したシスタチオニンγﾝシンターゼの活性が無細胞抽出液で確認できた．S. tokodaii シスタチオニンγﾝシンターゼを70℃熱処理 DEAEﾝトヨパールイオン交換カラム等により単一精製した．精製酵素の最適温度は100℃以上であり，熱安定性は60分間処理で70℃までほぼ100ｵの残存活性を示した．また，最適pHについてはリン酸緩衝液やブリトンﾝロビンソン広域緩衝液の場合はpH7.0の時が最も活性が高く，トリス塩酸緩衝液の場合はpH9.0が最適であった．pH安定性についてはpH5.0～9.0において安定であった．O-ホスホ-l-ホモセリンに対するKm，Vmaxは，それぞれ0.82mM，2.42U/㎎であった．アポ酵素のホロ化実験により，本酵素活性がPLP に依存していることが明らかとなった．更に本酵素の脱離反応での基質特異性の検討を行った．変異酵素を用いた実験により，stCGSの基質特異性には，活性中心に存在するPhe97を含む領域が深く関わっていることが示唆された．

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

DOI： 10.20632/vso.83.11_632_2

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記述言語：日本語   出版者・発行元：日本生物工学会  

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その他リンク： http://search.jamas.or.jp/link/ui/2011082793

記述言語：英語   出版者・発行元：WILEY-BLACKWELL  

L-Glutamate oxidase (LGOX) from Streptomyces sp. X- 119- 6, which catalyzes the oxidative deamination of L-glutamate, has attracted increasing attention as a component of amperometric L-glutamate sensors used in the food industry and clinical biochemistry. The precursor of LGOX, which has a homodimeric structure, is less active than the mature enzyme with an alpha(2)beta(2)gamma(2) structure; enzymatic proteolysis of the precursor forms the stable mature enzyme. We solved the crystal structure of mature LGOX using molecular replacement with a structurally homologous model of l- amino acid oxidase ( LAAO) from snake venom: LGOX has a deeply buried active site and two entrances from the surface of the protein into the active site. Comparison of the LGOX structure with that of LAAO revealed that LGOX has three regions that are absent from the LAAO structure, one of which is involved in the formation of the entrance. Furthermore, the arrangement of the residues composing the active site differs between LGOX and LAAO, and the active site of LGOX is narrower than that of LAAO. Results of the comparative analyses described herein raise the possibility that such a unique structure of LGOX is associated with its substrate specificity.

DOI： 10.1111/j.1742-4658.2009.07103.x

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：岡山大学農学部  

An alanine racemase (EC 5.1.1.1) from an extreme thermophilic bacterium Thermus thermophilus HB8, was purified and characterized, and its gene was cloned. The cloned alanine racemase gene (alr) was expressed in Escherichia coli JM 109. The alr gene is composed of a 1080 bp and encoded a 360 aminoacid, and was predicted to have a molecular weight of 38,596. The enzyme was purified by heat shock at 70°C for 10min and DEAE Toyopearl 650M column chromatography. The purified enzyme had an optimum pH9.0∼10.0 and an optimum temperature of 55°C∼60°C. Enzyme activity was retained 100% after incubation of the enzyme at 70°C for 10min. Alanine racemase from Thermus thermophilus HB8 is a monomeric enzyme with a molecular mass of 39 kDa.高度好熱性細菌 Thermus thermophilus HB8 由来アラニンラセマーゼ遺伝子を大腸菌中にクローニングし、発現させた後に、精製及び性質検討を行った。alr遺伝子は1080bpからなり360アミノ酸残基 HB8をコードしていたので、本酵素は38,596Daの分子量であると予想された。alr遺伝子の [ G + C ] 含量は、72%であり、Tm値は98.8℃であった。T.thermophilus HB8由来アラニンセマーゼを中等度好熱性細菌 Geobacillus stearothermophilus 及び赤痢菌 Shigella sonnei 由来アラニンラセマーゼと一次配列の比較をしたところ、G.stearothermophilus 由来のものと33%、赤痢菌由来の酵素を28%の相同性を示した。T.thermophilus HB8 由来アラニンラセマーゼを、70℃で10分間の熱処理後、DEAE-トーヨーパール陰イオン交換カラム等により精製した。精製酵素の最適温度は、d-アラニンからl-アラニンへの反応で55℃、l-アラニンからd-アラニンへの反応では60℃であり、最適pHは、9.0〜10.0であった。また、70℃で30分インキュベーションを行った後にも、活性の低下は見受けられず耐熱性を示した。更に、本酵素は分子量38,000モノマー酵素であると推定され、その反応機構に興味が持たれる。

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記述言語：英語   出版者・発行元：CELL PRESS  

Insects and fungi share a long history of association in various habitats, including the wood-decomposition niche [7]. Fungal mimicry of termite eggs is one of the most striking evolutionary consequences of insect-fungus association [2, 3]. Termites of the genus Reticulitermes often harbor fungal sclerotia, called "termite balls," along with eggs in nursery chambers, whereby the fungus gains a competitor-free habitat in termite nests. Sophisticated morphological and chemical camouflage are needed for the fungus to mimic termite eggs [3]. However, the mechanism of chemical egg mimicry by the fungus is unknown. Here, we show that the fungus mimics termite eggs chemically by producing the cellulose-digesting enzyme beta-glucosidase. We found that the termite egg-recognition pheromone consists of beta-glucosidase and lysozyme. Both enzymes are major salivary compounds in termites and are also produced in termite eggs. Termite balls were tended by termites only when the fungus produced beta-glucosidase. Our results demonstrated that the overlap of the cellulose digestion niche between termites and the fungus sharing the same chemicals provided the opportunity for the origin of termite egg mimicry by the fungus. This suggests that pheromone compounds might have originally evolved within other life history contexts, only later gaining function in chemical communication.

DOI： 10.1016/j.cub.2008.11.030

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DOI： 10.3209/saj.SAJ230205

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DOI： 10.11299/brte.20.226

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記述言語：日本語  

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：(公社)日本生化学会  

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記述言語：英語   出版者・発行元：TAYLOR & FRANCIS LTD  

The cysteinyl residue at the active site Of L-methionine gamma-lyase from Pseudomonas putida (MGL_Pp) is highly conserved among the heterologous MGLs. To determine the role of Cys116, we constructed 19 variants of C116X MGL_Pp by saturation mutagenesis. The Cys116 mutants possessed little catalytic activity, while their affinity for each substrate was almost the same as that of the wild type. Especially, the C116S, C116A, and C116H variants composed active site catalytic function as measured by the kinetic parameter k(cat) toward L-methionine. Furthermore, the mutagenesis of Cys116 also affected the substrate specificity of MGL_Pp at the active center. Substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a decrease in that toward L-methionine. Propargylglycine inactivated the WT MGL, C116S, and C116A mutants. Based on these results, we postulate that Cys116 plays an important role in the gamma-elimination reaction of L-methionine and in substrate recognition in the MGLs.

DOI： 10.1271/bbb.80015

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記述言語：日本語  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：英語   出版者・発行元：OXFORD UNIV PRESS  

Escherichia coli growing under anaerobic conditions produce H(2) and CO(2) by the enzymatic cleavage of formate that is produced from pyruvate at the end of glycolysis. Selenium is an integral part of formate dehydrogenase H (FDH(H)), which catalyses the first step in the formate hydrogen lyase (FHL) system. The genes of FHL system are transcribed only under anaerobic conditions, in the presence of a sigma(54)-dependent transcriptional activator Fh1A that binds formate as an effector molecule. Although the formate addition to the nutrient media has been an established procedure for inducing high FDHH activity, we have identified a low-salt nutrient medium containing <0.1% NaCl enabled constitutive, high expression of FDH(H) even without formate and D-glucose added to the medium. The novel conditions allowed us to study the effects of disrupting genes like trxB (thioredoxin reductase) or gor (glutathione reductase) on the production of FDH(H) activity and also reductive assimilation of selenite (SeO(3)(2-)) into the selenoprotein. Despite the widely accepted hypothesis that selenite is reduced by glutathione reductase-dependent system, it was demonstrated that trxB gene was essential for FDH(H) production and for labelling the FDH(H) polypeptide with (75)Se-selenite. Our present study reports for the first time the physiological involvement of thioredoxin reductase in the reductive assimilation of selenite in E. coli.

DOI： 10.1093/jb/mvm247

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記述言語：日本語   出版者・発行元：岡山大学農学部  

The isoamylase gene from Rhodothermus marinus was cloned into and expressed in Escherichia coliTop 10. As a result of characterization of purified R. marinus isoamylase. the enzyme had an optimumpH of 4.0 and optimum temperature of 70℃. Thermal inactivation studies of the purified R. marinusisoamylase revealed the enzymatic activity to be uninfluenced after one hour incubation at 60℃. Theseresults suggest that R. marinus isoamylase has high thermostability. The crystallization and crystalstructure analysis of R. marinus isoamylase was performed. The three-dimensional structure at 1.9Åresolution was determined in complex with the panose. R. marinus isoamylase is composed of threedomains N, A and C, and, has a (β/α)8-barrel in domain A. The secondary structural alignments of theR. marinus isoamylase and P. amyloderamosa isoamylase was carried out. They have the four active-siteconsensus regions characteristic of the α-amylase family. And the essential residue of the α-amylasefamily (D359, E395, and D467) was conserved in these enzymes. R. marinus isoamylase has shorter loopsthan P. amyloderamosa isoamylase. And R. marinus isoamylase had no Ca2+ binding site. These resultsare thought to be factors of thermostability of R. marinus isoamylase.Rhodothermus marinus 由来イソアミラーゼ遺伝子を組み込んだプラスミド pBX２を使用し，大腸菌 Top10株を形質転換し，16時間の前培養，24時間の本培養後，菌体破砕し，得られた無細胞抽出液を熱処理（80℃，10 min），50ｵ硫安分画，陰イオン交換カラムクロマトグラフィー（DEAEﾝトヨパール），ハイドロキシアパタイトカラムクロマトグラフィーに供して本酵素の精製を行った．本精製酵素の性質検討を行った結果，本酵素の最適反応温度は70℃，pH４であり，また本酵素は60℃で１時間処理しても活性が低下することが無く，Pseudomonas amyloderamosa 由来イソアミラーゼよりも高い耐熱性を有することが判明した．本酵素の結晶化・X線結晶構造解析を行った結果，本酵素は P. amyloderamosa 由来イソアミラーゼと同様Ｎドメイン・ＡドメインＣドメインの３つのドメインから構成されており，活性残基（Ｄ359，Ｅ395，Ｄ467）など活性中心付近のアミノ酸残基も P. amyloderamosa 由来イソアミラーゼと同様，高度に保存されていた．本酵素の熱安定性が P. amyloderamosa 由来イソアミラーゼよりも高い要因として，P. amyloderamosa 由来イソアミラーゼよりもループの長さが全体的に短いことと，カルシウムイオン結合サイトの欠如が挙げられた．今後さらに構造解析を進めることにより，本酵素の熱安定性機構，反応機構など更なる知見が得られることが期待される．

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記述言語：英語   出版者・発行元：WILEY-LISS  

The crystal structure of Acidithiobacillus thiooxidans isocitrate dehydrogenase complexed with NAD(+) and citrate has been solved to a resolution of 1.9 angstrom. The protein fold of this NAD(+)-dependent enzyme shares a high similarity with those of NADP(+-)dependent bacterial 1CDHs. The NAD(+) and the citrate are clearly identified in the active site cleft with a well-defined electron density. Asp-357 is the direct cofactor-specificity determinant that interacts with 2'-OH and 3'-OH of the adenosine ribose. The adenosine ribose takes a C2'-endo puckering conformation as previously reported for an NAD(+-)specific isopropylmalate dehydrogenase. The nicotinamide moiety of NAD(+) has the amide NH2 group oriented in cis conformation with respect to the C4 carbon of the nicotinamide ring, slanted toward the bound citrate molecule with a dihedral angle of -21 degrees. The semi-empirical molecular orbital calculation suggests that the pro-R hydrogen atom at C4 of NADH would bear the largest negative charge when the amide NH2 group is in such conformation, suggesting that the amide group has a catalytically significant role in stabilizing the transition state as NADH is being formed during the hydride transter catalysis.

DOI： 10.1002/prot.21486

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：英語   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Social insects rely heavily on pheromone communication to maintain their sociality. Egg protection is one of the most fundamental social behaviours in social insects. The recent discovery of the termite-egg mimicking fungus 'termite-ball' and subsequent studies on termite egg protection behaviour have shown that termites can be manipulated by using the termite egg recognition pheromone (TERP), which strongly evokes the egg-carrying and -grooming behaviours of workers. Despite the great scientific and economic importance, TERP has not been identified because of practical difficulties. Herein we identified the antibacterial protein lysozyme as the TERP. We isolated the target protein using ion-exchange and hydrophobic interaction chromatography, and the MALDI-TOF MS analysis showed a molecular size of 14.5 kDa. We found that the TERP provided antibacterial activity against a gram-positive bacterium. Among the currently known antimicrobial proteins, the molecular size of 14.5 kDa limits the target to lysozyme. Termite lysozymes obtained from eggs and salivary glands, and even hen egg lysozyme, showed a strong termite egg recognition activity. Besides eggs themselves, workers also supply lysozyme to eggs through frequent egg-grooming, by which egg surfaces are coated with saliva containing lysozyme. Reverse transcript PCR analysis showed that mRNA of termite lysozyme was expressed in both salivary glands and eggs. Western blot analysis confirmed that lysozyme production begins in immature eggs in queen ovaries. This is the first identification of proteinaceous pheromone in social insects. Researchers have focused almost exclusively on hydrocarbons when searching for recognition pheromones in social insects. The present finding of a proteinaceous pheromone represents a major step forward in, and result in the broadening of, the search for recognition pheromones. This novel function of lysozyme as a termite pheromone illuminates the profound influence of pathogenic microbes on the evolution of social behaviour in termites.

DOI： 10.1371/journal.pone.0000813

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：(公社)日本ビタミン学会  

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記述言語：英語  

DOI： 10.1093/jb/mvm055

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記述言語：日本語   出版者・発行元：岡山大學農學部  

ほ乳類チオレドキシン還元酵素はC末端配列-Gly-Cys-SeCys-Gly（end）の後ろから2番目にセレノシステイン（SeCys）残基を持つ。SeCysをシステインに変換すると酵素の活性は大きく低下するので、SeCys残基が触媒活性に必須であることが分かる。これに対してキイロショウジョウバエのチオレドキシン還元酵素（Dm-rxR）のC末端配列にはセレンが含まれず、システイン残基の対が2つのセリンに挟まれた配列-Ser-Cys-Cys-Ser（end）を持つ。それでもDm-rxRはほ乳類のセレン含有酵素と同程度の触媒能を示す。われわれはヒト肺チオレドキシン還元酵素にDm-rxRのC末端テトラペプチド配列を導入してその効果を調べた。しかし、酵素活性はまったく上昇せず、Dm-rxRのC末端のテトラペプチド配列-Ser-Cys-Cys-SerだけではCys残基のチオール基を活性化する効果はなかった。そこで、分子軌道計算MOPACを用いて酸化還元機能を担うためのC末端配列モチーフを探索した。その結果、テトラペプチドにさらに2つ先のプロリンまでを含めたPro-X-Ser-Cys-Cys-Ser（end）により初めて酸化還元モチーフとして機能する可能性が示唆された。Proを含むこの配列モチーフはミツバチや蚊などほかの昆虫のrxRでも保存されていた。

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：日本生物工学会  

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その他リンク： http://search.jamas.or.jp/link/ui/2008290885

記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：日本生物工学会  

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その他リンク： http://search.jamas.or.jp/link/ui/2008290902

記述言語：英語   出版者・発行元：ELSEVIER SCIENCE SA  

Large-scale production Of L-glutamate oxidase (L-GlOx) from Streptomyces sp. X-119-6 was conducted with the use of an Escherichia coli expression system. The active alpha(2)beta(2)gamma(2)-subunit structure of the enzyme was obtained by proteolysis of a precursor form using metalloendopeptidase. The performance of this recombinant enzyme was tested in an amperometric sensing system to determine the L-glutamate concentration. To this end, a thin-film, three-electrode system was formed. Hydrogen peroxide produced by an enzymatic reaction was detected by using a platinum working electrode. The sensitivity of the L-glutamate sensor was 220 nA/mM, and the lower detection limit was 3 mu M (S/N = 3). Up to 800 mu M, a linear relationship was observed between the output current and the L-glutamate concentration. Furthermore, we used a microanalysis system to determine the activities of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) and, ultimately, to test whether the recombinant enzyme can additionally be used to analyze enzyme activities. A polydimethylsiloxane (PDMS) flow channel with a mixing compartment was also used. The substrate solution and the sample solution for either GOT or GPT were mixed as they were being removed from the flow channel. As time elapsed, the L-glutamate concentration in the mixed solution increased because of the enzymatic reaction of GOT or GPT; consequently, a constant increase in current was observed. The relation between the slope of the response curve and the enzyme activity was linear up to 127 U/l for GOT and to 88 U/l for GPT. The results of this series of experiments demonstrate that recombinant L-GlOx can be used to construct micro sensors and microanalysis systems. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.snb.2006.01.008

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記述言語：英語   出版者・発行元：WILEY-LISS  

DOI： 10.1002/prot.21011

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：英語   出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Catalase plays a key role in protecting cells against toxic reactive oxygen species. Here we report on the cloning, purification and characterization of a catalase (KatA, DR1998) from the extremely radioresistant bacterium Deinococcus radiodurans. The size of purified D. radiodurans KatA monomer was 65 kDa while gel filtration revealed that the size of the enzyme was 240 kDa, suggesting that KatA formed a homotetramer in solution. Purified KatA displayed a final specific activity of 68,800 U/mg of protein. The catalase activity of KatA was inhibited by sodium azide, sodium cyanide and 3-amino-1,2,4-triazole. The absorption spectrum of KatA exhibited a Soret band at 408 nm. The position of the spectral peak remained unchanged following reduction of KatA with dithionite. No peroxidase activity was found for KatA. These results demonstrate that D. radiodurans KatA is a typical monofunctional heme-containing catalase. The stability of KatA with respect to H2O2 stress was superior to that of commercially available Aspergillus niger and bovine liver catalases. The relative abundance of KatA in cells in addition to the H2O2 resistance property may play a role in the survival strategy of D. radiodurans against oxidative damage.

DOI： 10.1263/jbb.101.315

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記述言語：日本語   出版者・発行元：岡山大学農学部  

The mehcanism of azo dyes decolorization by Candida sp. MK-1, Aeromonas sp. B-5 and Actinobacillus sp. B-11 were analyzed. The maximal decolorization activity was observed at pH 7.5 and 30℃ on Candida sp. MK-1, at alkaline and at 35℃ on Aeromonas sp. B-5 and Actinobacillus sp. B-11. The HPLC analysis of the supernatant of the Acid Red 27 detected in the blank. The retention time of this peak matched that of a reference standard compound of 4-amino-1-naphthalenesulfonate, produced by reductive cleavage of Acid Red 27. The decolorization of azo dyes with cell free extract of Candida sp. MK-a was promoted by the addition of several coenzymes or lawsone. The remarkable promotion of decolorization was observed by the addition of glutamate dehydrogenase with α-ketoglutarate and NH4+. Therefore, it was suggested that Candida sp. MK-1 azoreductase catalyzed decolorization of azo dye by NADPH dependent reductive cleavage.

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：岡山大學農學部  

Acidihiobacillus hiooxidans由来アコニターゼ遺伝子を大腸菌中にクローニングし、発現させ、精製及び性質検討を行った。A. hiooxidansアコニターゼはEscherichia coli JM109株で発現させ、活性が無細胞抽出液で確認された。646アミノ酸から成っており、イソクエン酸脱水素酵素遺伝子（icd）の上流に位置していた。またブタ心筋由来アコニターゼと大腸菌アコニターゼA型と高いアミノ酸配列相同性があり、ブタ心筋アコニターゼで触媒反応、基質の結合に関与するとされている27アミノ酸のうち25アミノ酸の保存が確認された。A. hiooxidansアコニターゼはDEAE-oyopearl 650M等により精製された。精製酵素は至適pH7.5、至適温度60℃であった。また40℃で1時間インキュベートを行った後にも、活性の低下は見受けられなかった。pH6.0-8.0で1時間インキュベートした後も、100％の活性を維持していた。更に本酵素は、分子量66000のモノマー酵素であることが明らかになった。なお、本論文中に記載したA. hiooxidansのアコニターゼ遺伝子を含む塩基配列はDDBJデータベースに登録されており、アクセション番号AB196983で検策できる。

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その他リンク： http://agriknowledge.affrc.go.jp/RN/2010710742

記述言語：日本語   出版者・発行元：微量栄養素研究会事務局  

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記述言語：日本語   出版者・発行元：公益社団法人 日本ビタミン学会  

Selenium is an essential trace element for mammals, birds, and some bacteria. Its remarkable biological effects in eukaryotes may be related to unique functions of various selenoproteins. A^<75>Se-labeled protein was purified from a human lung adenocarcinoma cell line, NCI-H441 . A homodimer of 57-kDa subunits contained selenium in the form of selenocysteine. The selenoprotein contained FAD as a prosthetic group and catalyzes NADPH-dependent reduction of insulin in the presence of thioredoxin. The subunit composition and catalytic properties were identical to those of mammalian thioredoxin reductase (TrxR). This was the first report on the biochemical relevance between selenium and Trx-mediated metabolic regulation. Interestingly, human lung adenocarcinoma cells produced the selenoenzyme TrxR over hundred times that of normal mammalian cells. A Iabile selenium donor compound monoselenophosphate, required for selenoprotein biosynthesis, is synthesized from selenide and ATP by selenophosphate synthetase (SPS).

DOI： 10.20632/vso.79.3_143

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その他リンク： http://search.jamas.or.jp/link/ui/2005136810

記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：英語   出版者・発行元：NATL ACAD SCIENCES  

A labile selenium donor compound monoselenophosphate is synthesized from selenide and ATP by selenophosphate synthetase (SPS). In the present study, Sps1 and Sps2 were cloned from a cDNA library prepared from human lung adenocarcinoma cells (NCI-H441). The human lung Sps1 has been cloned as an ORF of 1,179 bp, identical in sequence to that of the recently revised human liver Sps1. The in-frame TGA codon of the lung Sps2 was genetically altered to TGT (Cys) to obtain the Sps2Cys gene. Expression of the recombinant plasmids containing Spsi or Sps2Cys was highly toxic to Escherichia coli host cells grown aerobically. Accordingly, the human lung Sps homologs were characterized by an in vivo complementation assay using a selD mutant strain. An added selenium source and a low salt concentration (0.1-0.25% NaCl) in the medium were required for reproducible and sensitive in vivo complementation. Sps2Cys effectively complemented the selD mutant, and the resulting formate dehydrogenase H activity was as high as that of WT E. coli MC4100. In contrast, only a weak complementation of the selD mutant by the Sps1 gene was observed when cells were grown in selenite media. Better complementation with added L-selenocysteine suggested involvement of a selenocysteine lyase for mobilization of selenium. Based on this apparent substrate specificity of the Sps1 and Sps2 gene products we suggest that the Sps1-encoded enzyme depends on a selenium salvage system that recycles L-selenocysteine, whereas the Sps2 enzyme can function with a selenite assimilation system.

DOI： 10.1073/pnas.0406313101

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：岡山大学農学部  

Some properties of D-amino scid acetyltransferase purified from Saccharomyces cerevisiae were investigated. The enzyme was purified to homogeneity by ammonium sulfate fractionation, column chromatographies on DEAE-Toyopearl 650M, Sephacryl S-200, QAE-Toyopearl 550C and affinity chromatography with D-glutamate as a ligand. The molecular weight was estimated to be about 53,000 by gel filtration. Relative molecular mass studies indicated that the enzyme was a monomer structure. The purified enzyme had an optimum pH of 8.4 and an optimum temperature of 40C. The Km values of the purified enzyme determined with tryptophan and acetyl-CoA were 4.5 * 10 -3M, respectively. The 20 residues of N-terminal amino acid sequence were analyzed.

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：英語   出版者・発行元：JAPANESE BIOCHEMICAL SOC  

L-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 is a protein of 150 kDa that has hexamer structure alpha(2)beta(2)gamma(2). The gene encoding LGOX was cloned and heterologously expressed in Escherichia coli. LGOX isolated from the E. coli transformant had the structure of a one chain polypeptide. Although the recombinant LGOX exhibited catalytic activity, it was inferior to the LGOX isolated from Streptomyces sp. X-119-6 in catalytic efficiency. The recombinant LGOX exhibited low thermostability compared to the LGOX isolated from Streptomyces sp. X-119-6 and was an aggregated form. Proteolysis of the recombinant LGOX with the metalloendopeptidase from Streptomyces griseus (Sgmp) improved its catalytic efficiency at various pH. Furthermore, the Sgmp-treated recombinant LGOX had a subunit structure of alpha(2)beta(2)gamma(2) and nearly the same enzymological character as the LGOX isolated from Streptomyces sp. X-119-6. A higher molecular species observed for the recombinant LGOX was not detected for the Sgmp-treated recombinant LGOX. These results prove that proteolysis by Sgmp is involved in the stabilization of the recombinant LGOX.

DOI： 10.1093/jb/mvg206

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記述言語：英語   出版者・発行元：ELSEVIER SCIENCE BV  

Streptomyces cattleya NRRL 8057 produces monofluoroacetate and 4-fluorothreonine from inorganic fluoride. Mutants blocked in fluorometabolite production were prepared by chemical mutagenesis, and cosynthesis experiments with these blocked mutants were carried out by suspending cells of one blocked mutant in the supernatant broth of another blocked mutant. The harvest age of the cells, pH of the buffer, potassium fluoride concentration and glycerol supplementation were optimized for the monofluoroacetate production by a resting-cell suspension of S. cattleya. Successful cosynthesis with pairs of the mutants characterized four distinctive blocked sites in the order N-82, N-44, N-43 and N-47. Additional preparation of blocked mutants by UV irradiation and their cosynthesis assay confirmed that U-303, U-304, U-400 and U-500 were blocked in later steps than N-47. O'Hagan et al. recently proposed that fluoroacetaldehyde, the hypothetical precursor of monofluoroacetate and 4-fluorothreonine, derives from 5'-fluoro-5-deoxyadenosine, the first fluorinated metabolite synthesized from S-adenoSyl-L-methionine and inorganic fluoride by the novel enzyme 'fluorinase'. We were able to detect fluorinase activity in crude extracts of wild type and N-47 mutant strains, but not in the other mutant strains whose blocked steps flanked that of N-47. (C) 2003 Elsevier B.V. All rights reserved.

DOI： 10.1016/S1381-1177(03)00088-2

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記述言語：英語  

DOI： 10.1016/S1381-1177(03)00089-4

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記述言語：日本語   出版者・発行元：岡山大學農學部  

ヒトのチオレドキシン還元酵素（TrxR）のカルボキシル末端配列の酸化還元に伴うエンタルピー変化を計算した。半経験的分子軌道計算WinMOPAC 3.5Proを用いてモデルペプチド、N-Ac-Ser-Ile-Leu-Gln-Ala-Gly-X 1-X2-Glyの生成熟を算出した。X1、X2のアミノ酸配列は-SeCys-Cys-、-Cys-SeCys-、-SeCys-SeCys-、-Cys-Cys-のいずれかで、それぞれ酸化状態と還元状態を計算し、そのエンタルピー差を求めた。ハミルトニアンAM1とPM3は同じ傾向の計算結果を示し、セレノスルフィドまたはジセレニドを形成するペプチドは酸化状態でより安定化するのに対して-Cys-Cys-の配列を持つペプチドは還元形の方が安定であることを示した。ほ乳類のTrxRのSeCys498をCysに置換すると酵素活性が1％程度にまで低下することが報告されている。これは、変異酵素が-Cys497-Cys498-間で酸化的架橋を形成にくいからと考察されていたが、今回の分子軌道計算の結果は、この仮説を支持している。

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記述言語：日本語   出版者・発行元：岡山大學農學部  

CGATCGを認識するAfa22MI制限修飾系遺伝子を好酸性細菌Acidocella facilis 22Mより、クローニングし、塩基配列を決定した。その結果、Ｃ５－シトシンメチラーゼに特徴的なモチーフが保存されたM.Afa22MI遺伝子、その上流に、M.Afa22MI遺伝子とは逆方向に、very short patch repair endonuclease様タンパク質遺伝子（Afa22MI vsr）、M.Afa22MI遺伝子の下流にM.Afa22MI遺伝子と同方向に、制限酵素（R.Afa22MI）遺伝子と推定されるオープンリーディングフレームが見出された。M.Afa22MIの推定アミノ酸配列は、Xanthomonas oryzae pv. oryzae由来M.XorIIの配列と全体で約63％、variable region内で約53％の高い配列類似性を示した。また、Afa22MI vsrの推定アミノ酸配列も、M.XorIIに付随するXorII vsrの配列と約66％の高い類似性を示した。

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その他リンク： http://eprints.lib.okayama-u.ac.jp/10584

記述言語：英語   出版者・発行元：ELSEVIER SCIENCE BV  

An isocitrate dehydrogenase (ICDH) with an unique coenzyme specificity from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The native enzyme was homodimeric with a subunit of M-r 45 000 and showed a 78-fold preference for NAD(+) over NADP(+). The cloned ICDH gene (icd) was expressed in an icd-deficient strain of Escherichia coli EB106; the activity was found in the cell extract. The gene encodes a 429-amino acid polypeptide and is located between open reading frames encoding a putative aconitase gene (upstream of icd) and a putative succinyl-CoA synthase beta-subunit gene (downstream of icd). A. thiooxidans ICDH showed high sequence similarity to bacterial NADP(+)-dependent ICDH rather than eukaryotic NAD(+)-dependent ICDH, but the NAD(+)-preference of the enzyme was suggested due to residues conserved in the coenzyme binding site of the NAD(+)-dependent decarboxylating dehydrogenase. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-1097(02)00857-1

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：英語   出版者・発行元：TAYLOR & FRANCIS LTD  

A specific inhibitor of DNA cytosine C-5-methylases would be useful for studying genomic imprinting, X-chromosome inactivation, carcinogenesis, and regulation of tissue-specific gene expression, for these physiological phenomena appears to be regulated through DNA methylation in promoter sequences. This paper reports a novel convenient in vitro assay method for screening DNA cytosine C-5-Methylase inhibitor. Our method uses a commercially available Hae III methylase (cytosine C-5 methylase), its corresponding Hae III endonuclease, and lambda DNA as their substrate.

DOI： 10.1080/1057563029001/4809

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記述言語：英語   掲載種別：書評論文，書評，文献紹介等   出版者・発行元：ACADEMIC PRESS INC  

DOI： 10.1016/S0076-6879(02)47029-2

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記述言語：日本語   出版者・発行元：岡山大学  

好酸性従属栄養細菌Acidocella facilis AIU409株は、基質としてソルビタンモノエステルであるTweenを含む培地中で培養された時に、菌体外に熱安定性の高い酸性エステラーゼを生産する。エステラーゼをコードする遺伝子(estA)をA. facilis AIU409株のゲノムライブラリーから単離し、大腸菌MV1184にクローニングし、その遺伝子の全塩基配列を決定した。その結果、estAの構造遺伝子が、1881塩基対であることが明らかになった。酸性エステラーゼ遺伝子のオープンリーディングフレーム(ORF)は、627アミノ酸残基（計算された分子量は64、140ダルトン）をコードしていた。ロー囚子非依存性の転写終結シグナルが終止コドンであるTGAのすぐ下流に存在していた。そのN末端予想アミノ酸配列より、酸性エステラーゼの前駆体は、N末端に23個のアミノ酸残基から成るシグナルペプチドを有していた。また、リパーゼのコンセンサス配列であるG-X-S-X-Sの配列が存在することが明らかとなっ。酸性エステラーゼの予想アミノ酸配列から計算された分子量は61,486であり、これはSDS-PAGEによって予想されていた分子量より、やや低い値であった。また、Acidocella facilis酸性エステラーゼの予想アミノ酸配列は、Aeromonas hydrophila由来のacyltrans-ferase12、13)やXenorhabdus luminescens由来のリパーゼ14)と高い相同性を示した。

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その他リンク： http://eprints.lib.okayama-u.ac.jp/10603

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記述言語：英語   出版者・発行元：FFIジャ-ナル編集委員会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：英語   出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN  

We purified an acid trehalase (EC 3.2.1.28, a,alpha,alpha'-trehalose glucohydrolase) from an acidophilic bacterium, Acidobacterium capsulatum. The enzyme was homogeneous based on polyacrylamide gel electrophoresis, and was composed of a single polypeptide chain with a molecular mass of 57 kDa. Maximum trehalase activity was observed at pH 2.5. The acid trehalase exhibited an apparent K-m of 1.0 mM for trehalose at 30 degreesC and pH 3.0, The trehalase was located in the periplasmic space. The activity of the enzyme was activated by 1.0 mM MnCl2 or CoCl2, and inhibited by 1.0 mM PbCl2, HgCl2, NiCl2, p-chloromercuribenzoate, N-ethylmaleimide, monoiodoacetate, or EDTA. The enzyme showed high specificity for trehalose. It was found that an equimolar mixture of alpha -D-glucose and beta -D-glucose was formed on hydrolysis of trehalose by the trehalase.

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記述言語：英語   出版者・発行元：微量栄養素研究会事務局  

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：英語   出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN  

We purified to homogeneity an alanine racemase (EC 5.1.1.1) from Thermus thermophilus HB8, an extreme thermophile. Interestingly, the enzyme possessed a monomeric structure with a molecular weight of about 38,000. The enzyme was most active at pH 8 and 75 degreesC, and remained active after incubation at 80 degreesC for 30 min.

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記述言語：英語  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：英語  

A strain of Bacillus sphaericus isolated from a local soil sample has been found to use β,β-dimethyl-DL-cysteine (DL-penicillamine) as the sole nitrogen source. Crude cell extract of the bacterium showed potent penicillamine-consuming activity only in the presence of NAD, which, however, was not used as an electron acceptor. Characterization of reaction products revealed that penicillamine was derivatized to a phosphoramide adduct with the ADP moiety of NAD, whereas the nicotinamide-ribose group was released and hydrolyzed spontaneously to ribose and nicotinamide. The phosphoramide product, ADP-penicillamine, caused potent product inhibition on the purified enzyme, and adenylate deaminase was found to be effective in converting the inhibitory product into inosine-diphosphate-penicillamine and thereby maintained the catalysis for several hours. The novel enzyme, termed as NAD:penicillamine ADP transferase, showed a single band on SDS-polyacrylamide gel electrophoresis with a mass of approximately 42 kDa. The native enzyme was monomeric. The enzyme showed high substrate specificity to NAD (K(m) = 13.0 mM) and L-penicillamine (K(m) = 6.5 mM)
other nucleotides such as NADP, NAD(P)H, AMP, ADP, and ADP-ribose did not substitute for NAD, and L-valine, L-cysteine, L-homocysteine, L-cystine, L-leucine, and L-isoleucine did not serve as the substrate. Kinetic studies suggested an Ordered Bi Bi mechanism, with NAD as the first substrate to bind and ADP-L-penicillamine as the last product released. The novel NAD-dependent enzyme may catalyze the first step in penicillamine degradation in the strain of B. sphaericus.

DOI： 10.1074/jbc.274.2.795

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：英語  

The gene xynA encoding an acid endo-β-1,4-xylanase from an acidophilic bacterium, Acidobacterium capsulatum 161, was cloned and expressed in Escherichia coli. The nucleotide sequence of the 1.6-kb DNA fragment containing xynA was analyzed, revealing an open reading frame of 1,215 bp encoding a peptide of 405 amino acid residues. The deduced amino acid sequence of XynA was very similar to other xylanases that are from the glycosyl hydrolase family 10. XynA was purified to homogeneity by SDS-polyacrylamide gel electrophoresis from E. coli transformants. The molecular mass and isoelectric point of XynA were 41 kDa and 7.3, respectively. The xylanase activity of the cloned XynA is an endo-acting enzyme that shows optimal activity at pH 5.0 and 65°C, and is stable pH between 3.0 and 8.0. The Km and Vmax with oat spelt xylan as a substrate at pH 5.0 and 30°C are 3.5 mg/ml and 403 μmol/min/mg. © 1998 Taylor &amp
Francis Group, LLC.

DOI： 10.1271/bbb.62.1061

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記述言語：英語  

An alanine racemase (EC 5.1.1.1) from an acidophilic heterotrophic bacterium, Acidiphilium organovorum 13H, was purified and characterized. The enzyme had a dimeric structure with identical subunits of M, 33,000 each. Although A. organovorum 13H is an acidophile, the enzyme had its maximum velocity at pH 9, corresponding to its location in the cytoplasm. Activity was maximum between 50 and 60°C. For an enzyme from a mesophile, it was stable to heat, showing no loss of activity after a 30-min incubation at 65°C. The enzyme needed pyridoxal 5′. phosphate (PLP) as a cofactor for its activity, as seen from the loss of activity upon dialysis against PLP-free buffer containing hydroxylamine and its absorption maximum at 420 nm. Activity was ihhibited by common inhibitors of PLP-dependent enzymes. PLP content studies found that 1 mole of enzyme contained 2 moles of PLP. The enzyme catalyzed the symmetric reversible racemization of alanine exclusively. © 1998, Taylor &amp
Francis Group, LLC. All rights reserved.

DOI： 10.1271/bbb.62.242

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記述言語：英語   掲載種別：書評論文，書評，文献紹介等  

We describe here a sensitive and straightforward method for characterizing the methylation specificity of type II DNA methyltransferase (MTase) using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. DNA substrate, prepared by ligation of a commercially available oligonucleotide, was modified by the subject MTase, and was derivatized to a mixture of single-stranded oligonucleotides through endonuclease treatment, heat-denaturation and limited digestion by 3'-terminus-specific phosphodiesterase I. MALDI-TOF mass spectrometry was used to determine the mass differences between the digestion products, and the methylated nucleotide was explicitly identified by the mass increase of 14 Da due to the base modification. The method was applicable to the three representative MTases M.EcoRI, M.BamHI and M.HaeIII.

DOI： 10.1093/nar/25.20.4162

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記述言語：日本語   出版者・発行元：化学工業社  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：日本語   出版者・発行元：日本ビタミン学会  

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記述言語：英語   出版者・発行元：日本生物工学会  

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J-GLOBAL

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記述言語：日本語   出版者・発行元：社団法人日本農芸化学会  

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記述言語：日本語   出版者・発行元：社団法人日本農芸化学会  

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記述言語：日本語   出版者・発行元：社団法人日本農芸化学会  

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記述言語：英語  

We report the isolation and characterization of a new selenoprotein from a human lung adenocarcinoma cell line, NCI-H441. Cells were grown in RPMI 1640 medium containing 10% (vol/vol) fetal bovine serum and 0.1 μM [75Se]selenite. A 75Se-labeled protein was isolated from sonic extracts of the cells by chromatography on DE-23, phenyl-Sepharose, heparin-agarose, and butyl-Sepharose. The protein, a homodimer of 57-kDa subunits, was shown to contain selenium in the form of selenocysteine
hydrolysis of the protein alkylated with either iodoacetate or 3-bromopropionate yielded Se- carboxymethyl-selenocysteine or Se-carboxyethyl-selenocysteine, respectively. The selenoprotein showed two isoelectric points at pH 5.2 and pH 5.3. It was distinguished from selenoprotein P by N-glycosidase assay and by the periodate-dansylhydrazine test, which indicated no detectable amounts of glycosyl groups on the protein. The selenoprotein contains FAD as a prosthetic group and catalyzes NADPH-dependent reduction of 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB), and reduction of insulin in the presence of thioredoxin (Trx). The specific activity was determined to be 31 units/mg by DTNB assay. Apparent K(m) values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 μM, respectively. DTNB reduction was inhibited by 0.2 mM arsenite. Although the subunit composition and catalytic properties are similar to those of mammalian thioredoxin reductase (TR), the human lung selenoprotein failed to react with anti-rat liver TR polyclonal antibody in immunoblot assays. The selenocysteine-containing TR from the adenocarcinoma cells may be a variant form distinct from rat liver TR.

DOI： 10.1073/pnas.93.3.1006

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記述言語：英語  

We report the isolation and characterization of a new selenoprotein from a human lung adenocarcinoma cell line, NCI-H441. Cells were grown in RPMI 1640 medium containing 10% (vol/vol) fetal bovine serum and 0.1 μM [75Se]selenite. A 75Se-labeled protein was isolated from sonic extracts of the cells by chromatography on DE-23, phenyl-Sepharose, heparin-agarose, and butyl-Sepharose. The protein, a homodimer of 57-kDa subunits, was shown to contain selenium in the form of selenocysteine
hydrolysis of the protein alkylated with either iodoacetate or 3-bromopropionate yielded Se- carboxymethyl-selenocysteine or Se-carboxyethyl-selenocysteine, respectively. The selenoprotein showed two isoelectric points at pH 5.2 and pH 5.3. It was distinguished from selenoprotein P by N-glycosidase assay and by the periodate-dansylhydrazine test, which indicated no detectable amounts of glycosyl groups on the protein. The selenoprotein contains FAD as a prosthetic group and catalyzes NADPH-dependent reduction of 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB), and reduction of insulin in the presence of thioredoxin (Trx). The specific activity was determined to be 31 units/mg by DTNB assay. Apparent K(m) values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 μM, respectively. DTNB reduction was inhibited by 0.2 mM arsenite. Although the subunit composition and catalytic properties are similar to those of mammalian thioredoxin reductase (TR), the human lung selenoprotein failed to react with anti-rat liver TR polyclonal antibody in immunoblot assays. The selenocysteine-containing TR from the adenocarcinoma cells may be a variant form distinct from rat liver TR.

DOI： 10.1073/pnas.93.3.1006

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記述言語：日本語   出版者・発行元：日本酪農科学会  

Bioassay-directed screening for antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) led us to the culture broth of Enterococcus faecalis TH 10, which was isolated from a Malaysian fermentation food, Temph. The anti-MRSA component was readily extracted with ethyl acetate from the culture broth at pH 3, and the extract retained the activity when incubated overnight with various proteolytic enzymes. In addition, the extract showed poor antimicrobial activity against various lactic acid bacteria, including Enterococcus faecalis. These properties strongly suggest that the active compound is distinct from bacteriocin, an extracellularly released peptide or protein which shows potent activity against species closely related to the bacteriocin-producing strain. E. faecalis has been reported to produce bacteriocin which has strong hemolytic activity on mammalian erythrocytes, but the active component of the TH 10 strain did not mediate the lysis of rabbit and human erythrocytes.

DOI： 10.11465/milkscience.45.A-93

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その他リンク： https://jlc.jst.go.jp/DN/JLC/20016775976?from=CiNii

記述言語：日本語   出版者・発行元：日本生物工学会  

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記述言語：日本語   出版者・発行元：日本酪農科学会  

Bioassay-directed screening for antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) led us to the culture broth of Enterococcus faecalis TH 10, which was isolated from a Malaysian fermentation food, Temph. The anti-MRSA component was readily extracted with ethyl acetate from the culture broth at pH 3, and the extract retained the activity when incubated overnight with various proteolytic enzymes. In addition, the extract showed poor antimicrobial activity against various lactic acid bacteria, including Enterococcus faecalis. These properties strongly suggest that the active compound is distinct from bacteriocin, an extracellularly released peptide or protein which shows potent activity against species closely related to the bacteriocin-producing strain. E. faecalis has been reported to produce bacteriocin which has strong hemolytic activity on mammalian erythrocytes, but the active component of the TH 10 strain did not mediate the lysis of rabbit and human erythrocytes.

DOI： 10.11465/milkscience.45.A-93

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その他リンク： https://jlc.jst.go.jp/DN/JLC/20016775976?from=CiNii

記述言語：日本語  

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記述言語：日本語   出版者・発行元：岡山大学  

Selenium belongs to the Ⅵb group of the periodic table,and possesses both metallic and nonmetallic characteristics. Physicochemical properties of selenium resemble more or less those of sulfur,and selenium may be indiscriminately incorporated in place of sulfur in cellular constituents and disturb metabolism. Alkali disease and blind stagger disease of livestock are caused by selenium-polluted grass. Carcinogenic effect is also one of the marked biological properties of selenium. In spite of the toxic and carcinogenic effects,selenium is actually an essential trace element for bacteria,fish,and mammals. Life has exploied the high reactivity and unique characteristics of organoselenium compounds,especially in the form of selenoenzymes. Mammalian glutathione peroxidase(EC 1.11.1.9) has selenocysteine residue at the active site, and the enzyme plays a central role in the biological defense system against oxidative challenge by activated oxygens and radicals. The author has studied the low redox potential and high reactivity of selenium-containing compounds, and developed glutathione peroxidase mimics. Glutaselenone, a selenium analog of glutaheione, catalyzes a glutathione peroxidase like reaction in vitro. Studies on the mechanisum of glutaselenone-catalyzed reaction revealed that glutaselenone is converted to a selenosulfide conjugate with glutathione in its catalysis. Thioredoxins contain a conserved sequence,Cys-Gly-Pro-Cys, which is known to form intramolecular disulfide bond with consecutive β-turn conformations. THe peptide is expected to serve as an template for intramolecular selenosulfide bond formation when either the cysteine is replaced by selenocystein. A tetrapeptide,Secys-Gly-Pro-Cys, was synthesized chemically. It showed glutathione peroxidase-like activity three times as high as glutaselenone. The high catalytic activity is ascribed to an intramolecular selenosulfide bond formation in the catalytic reaction.セレンは2-、0、2+、4+、6+と多彩な酸化還元状態をとるという点で金属的な性質を持つ。また有機セレン化合物として求核的・求電子的な反応性に関与できるという点で酸素や硫黄などの典型元素のような振る舞いをする。生命は進化の過程で、セレンのユニークな化学的特性をうまく利用し、グルタチオンペルオキシダーゼなどのセレン酵素を獲得してきたと考えられる。セレン酵素の研究に触発されて、セレン酵素化合物を医薬品として利用する研究が進められており注目を集めている。このような背景のもと、グルタセレノンとテトラペプチドを合成した。とくにテトラペプチドはグルタセレノンの反応機構研究から生まれたペプチドである。今後さらに高い活性を持つ人工酵素がデザインされ、合成されるだろう。その時、鋳型として蛋白質や核酸、オリゴ糖などある特定のコンフォメーションをとる生体物質が利用されると思われる。

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その他リンク： http://ousar.lib.okayama-u.ac.jp/11140

記述言語：英語  

Streptomyces cattleya produces fluoroacetate and 4-fluorothreonine from inorganic fluoride added to the culture broth. We have shown by 19F nuclear magnetic resonance (NMR) spectrometry that fluoroacetate is accumulated first in the culture broth and that accumulation of 4-fluorothreonine is next. To show precursors of the carbon skeleton of fluoroacetate, we carried out tracer experiments with various 14C- and 13C-labeled compounds. Radioactivity of [U-14C]glucose, [U-14C]glycerol, [U-14C]serine, and [U-14C]β-hydroxypyruvate was incorporated into fluoroacetate to an extent of 0.2 to 0.4%, whereas [3-14C]pyruvate, [2,3-14C]succinate, and [U- 14C]aspartate were less efficiently incorporated (0.04 to 0.08%). The addition of [2-13C]glycerol to the mycelium suspension of Streptomyces cattleya caused exclusive enrichment of the carboxyl carbon of fluoroacetate with 13C
about 40% of carboxyl carbon of fluoroacetate was labeled with 13C. We studied the radioactivity incorporation of [3-14C]-, [U-14C]-, and [1-14C]β-hydroxypyruvates to show that C-2 and C-3 of β-hydroxy pyruvate are exclusively converted to the carbon skeleton of fluoroacetate. These results suggest that the carbon skeleton of fluoroacetate derives from C-1 and C-2 of glycerol through β-hydroxypyruvate, whose hydroxyl group is eventually replaced by fluoride.

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記述言語：英語  

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記述言語：英語   掲載種別：書評論文，書評，文献紹介等   出版者・発行元：IOS PRESS  

The recent discovery that mammalian thioredoxin reductase is a selenoprotein furnishes an additional explanation of the mutual sparing roles of selenium and vitamin E in cellular antioxidant systems. Thioredoxin reductases isolated from human lung adenocarcinoma cells, human Jurkat T-cells and HeLa cells contain selenocysteine which is located in a C- terminal tripeptide, -Cys-SeCys-Gly.

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記述言語：英語  

We synthesized the selenium analog of glutathione disulfide by a liquid phase method and named it glutaselenone (i.e., γ-L-glutamyl-L-selenocysteinylglycine) diselenide. The selenol of selenocysteine was protected by the p-methoxybenzyl group, which was removed by acidolysis with trifluoroacetic acid in the presence of thioanisol. The overall yield of the final product, glutaselenone diselenide, was about 9% based on the starting compound, Se-(p-methoxybenzyl)-L-selenocysteine. Glutaselenone diselenide showed a broad absorption band between 270 and 400 nm and circular dichroism bands around 270 nm (positive) and 330 nm (negative), which were attributable to diselenide bond. © 1993 Academic Press, Inc.

DOI： 10.1006/abio.1993.1021

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記述言語：日本語  

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