担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press ({OUP})  

DOI： 10.1093/bbb/zbab199

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掲載種別：研究論文（学術雑誌）  

DOI： 10.1039/d1ra07623j

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jbiosc.2021.08.013

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/bbb/zbab079

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.2323/jgam.2019.11.006

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/mra.00273-20

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担当区分：最終著者,　責任著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/MRA.01204-19

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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/femsle/fny042

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1107/S1399004715017939

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/prot.24722

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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  

Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for 'disarming' the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, thiohemiacetal intermediate, a thioacyl intermediate and an NAD(+)-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp(3)-to-sp(2) transition during enzyme-mediated oxidation.

DOI： 10.1038/ncomms6935

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担当区分：筆頭著者,　責任著者   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  

Two novel aerobic p-n-nonylphenol-degrading bacterial strains were isolated from seawater obtained from the coastal region of Ogasawara Islands, Japan. The 16S rRNA gene sequence analysis indicated that the strains are affiliated with the order Alteromonadales within the class Gammaproteobacteria. One isolate, strain KU41G2, is most closely related to Maricurvus nonylphenolicus (99.2 % similarity), and is tentatively identified as M. nonylphenolicus. The other isolate, strain KU41G(T), is also most closely related to M. nonylphenolicus; however, the 16S rRNA gene sequence similarity was only 94.7 %. Cells of strain KU41G(T) are Gram-negative rods with a single polar flagellum. The predominant respiratory lipoquinone was ubiquinone-8, and the major cellular fatty acids were C-17:1 omega 8c (24.2 %); C-15:0 iso 2-OH; and/or C-16:1 omega 7c (16.3 %), C-15:0 (10.3 %), C-11:0 3-OH (9.5 %), C-9:0 3-OH (6.7 %), C-10:0 3-OH (6.4 %), and C-18:1 omega 7c (5.5 %). The DNA G+C content was 53.3 mol%. On the basis of physiological, chemotaxonomic, and phylogenetic data, strain KU41G(T) is suggested to represent a novel species of a new genus, for which we propose the name Pseudomaricurvus alkylphenolicus gen. nov., sp. nov. The type strain of P. alkylphenolicus is KU41G(T) (=JCM 19135(T) = KCTC 32386(T)).

DOI： 10.1007/s00284-013-0455-x

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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  

There are few entries of carbon-carbon bond hydrolases (EC 3.7.1.-) in the ExPASy database. In microbes, these enzymes play an essential role in the metabolism of alicyclic or aromatic compounds as part of the global carbon cycle. CpdC is a omega-pentadecalactone hydrolase derived from the degradation pathway of cyclopentadecanol or cyclopentadecanone by Pseudomonas sp. strain HI-70. CpdC was purified to homogeneity and characterized. It is active as a dimer of 56,000 Da with a subunit molecular mass of 33,349. Although CpdC has the highest activity and reaction rate (k(cat)) toward omega-pentadecalactone, its catalytic efficiency favors lauryl lactone as a substrate. The melting temperature (T-m) of CpdC was estimated to be 50.9 +/- 0.1 degrees C. The half-life of CpdC at 35 degrees C is several days. By virtue of its high level of expression in Escherichia coli, the intact CpdC-encoding gene and progressive 3'-end deletions were employed in the construction of a series of fusion plasmid system. Although we found them in inclusion bodies, proof-of-concept of overproduction of three microbial cutinases of which the genes were otherwise expressed poorly or not at all in E. coli was demonstrated. On the other hand, two antigenic proteins, azurin and MPT63, were readily produced in soluble form.

DOI： 10.1128/AEM.02435-13

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担当区分：筆頭著者,　責任著者   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  

A novel aerobic, Gram-negative, non-motile, pleomorphic, and rod-shaped bacterium designated KU5D5(T) was isolated from seawater that was obtained from the coastal region of the Goto Islands, Japan, on the basis of its ability to utilize cyclohexylacetate as the sole source of carbon and energy. Strain KU5D5(T) grew at pH 6.0-8.0 and 10-35 A degrees C in the presence of 1.0-5.0 % (w/v) NaCl. Analysis of the 16S rRNA gene sequence revealed that this strain was affiliated to the family Rhodobacteraceae in the class Alphaproteobacteria and was related most closely to Lutimaribacter saemankumensis (96.6 % similarity) and Oceanicola pacificus (96.6 %). The predominant respiratory lipoquinone was ubiquinone-10 and the major cellular fatty acids were C-18:1 omega 7c (66.7 %), C-16:0 (7.7 %), C-12:1 3-OH (6.1 %), and C-17:0 (6.1 %). The DNA G+C content was 58.9 mol %. On the basis of physiological, chemotaxonomic, and phylogenetic data, strain KU5D5(T) is suggested to represent a novel species of the genus Lutimaribacter, for which the name Lutimaribacter litoralis sp. nov. is proposed. It is also proposed that O. pacificus should be transferred to the genus Lutimaribacter as Lutimaribacter pacificus comb. nov. The type strain of L. litoralis is KU5D5(T) (=JCM 17792(T) = KCTC 23660(T)) and the type strain of L. pacificus is W11-2B(T) (=CCTCC AB 208224(T) = LMG 24619(T) = MCCC 1A01034(T)).

DOI： 10.1007/s00284-013-0321-x

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担当区分：筆頭著者   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  

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (-) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (K-m = 32 mu M), and it catalyzes the reduction of flavin mononucleotide (FMN) (K-m = 3.6 mu M; k(cat) = 283 s(-1)) in preference to flavin adenine dinucleotide (FAD) (K-m = 19 mu M; k(cat) = 128 s(-1)). Sequence determination of similar to 40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE(25-1) for 2,5-DKCMO-1 and camE(25-2) for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE(36)). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and similar to 533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono-and bicyclic ketone substrates.

DOI： 10.1128/AEM.03958-12

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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：PUBLIC LIBRARY SCIENCE  

Cyclohexylamine oxidase (CHAO) is a flavoprotein first described in Brevibacterium oxydans strain IH-35A that carries out the initial step of the degradation of the industrial chemical cyclohexylamine to cyclohexanone. We have cloned and expressed in Escherichia coli the CHAO-encoding gene (chaA) from B. oxydans, purified CHAO and determined the structures of both the holoenzyme form of the enzyme and a product complex with cyclohexanone. CHAO is a 50 kDa monomer with a PHBH fold topology. It belongs to the flavin monooxygenase family of enzymes and exhibits high substrate specificity for alicyclic amines and sec-alkylamines. The overall structure is similar to that of other members of the flavin monooxygenase family, but lacks either of the C- or N-terminal extensions observed in these enzymes. Active site features of the flavin monooxygenase family are conserved in CHAO, including the characteristic aromatic cage. Differences in the orientations of residues of the CHAO aromatic cage result in a substrate-binding site that is more open than those of its structural relatives. Since CHAO has a buried hydrophobic active site with no obvious route for substrates and products, a random acceleration molecular dynamics simulation has been used to identify a potential egress route. The path identified includes an intermediate cavity and requires transient conformation changes in a shielding loop and a residue at the border of the substrate-binding cavity. These results provide a foundation for further studies with CHAO aimed at identifying features determining substrate specificity and for developing the biocatalytic potential of this enzyme.

DOI： 10.1371/journal.pone.0060072

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/bi300635b

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/AEM.07694-11

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担当区分：筆頭著者,　責任著者   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  

An aerobic, Gram-negative bacterial strain, designated KU27E1(T), which degrades phthalate and dimethylphthalate, was isolated from seawater obtained from the coastal region of Ishigaki Island, Japan. Cells are motile rods with polar flagella. Strain KU27E1(T) grew at 15-30A degrees C, pH 6.0-8.0, in the presence of 1.0-2.0% (w/v) NaCl. The 16S rRNA gene sequence analysis revealed that this strain was affiliated with the family Rhodobacteraceae in the class Alphaproteobacteria, and was most closely related to Tropicibacter naphthalenivorans (96.8%). The predominant respiratory lipoquinone was ubiquinone-10, and the major cellular fatty acid was C-18:1 omega 7c (88.5%). The G+C content of genomic DNA was 58.7 mol%. Based on the physiological, chemotaxonomic, and phylogenetic data, strain KU27E1(T) is suggested to represent a novel species of the genus Tropicibacter, for which the name Tropicibacter phthalicus sp. nov. is proposed. The type strain of Tropicibacter phthalicus is designated as KU27E1(T) (=JCM 17793(T) = KCTC 23703(T)).

DOI： 10.1007/s00284-012-0085-8

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担当区分：筆頭著者,　責任著者   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  

Eleven phthalate-degrading bacterial strains were isolated from seawater collected off the coast of Japan. The isolates were found to be most closely related to the marine bacterial genera Alteromonas, Citreicella, Marinomonas, Marinovum, Pelagibaca, Rhodovulum, Sulfitobacter, Thalassobius, Thalassococcus, Thalassospira, and Tropicibacter. For the first time, members of these genera were shown to be capable of growth on phthalate. The plate assay for visual detection of phthalate dioxygenase activity and PCR detection of a possible gene encoding 4,5-dihydroxyphthalate decarboxylase indicated that phthalate is degraded via 4,5-dihydroxyphthalate to protocatechuate in all the isolates.

DOI： 10.1007/s11274-011-0925-x

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担当区分：筆頭著者,　責任著者   掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press (OUP)  

DOI： 10.1111/j.1574-6968.2011.02471.x

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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：CANADIAN SCIENCE PUBLISHING, NRC RESEARCH PRESS  

The biocatalytic performance of a cloned cyclohexylamine oxidase derived from Brevibacterium oxydans fl-1-35A towards structurally different amines was investigated. Cycloalkyl primary amines, alkyl aryl amines, and a-carbon-substituted aliphatic amines were identified as suitable substrates for the biocatalyst based on an activity assay. Kinetic resolutions of several amines by either recombinant whole cells or crude enzyme extracts prepared therefrom gave enantiomerically pure (R)-amines besides the corresponding ketones. When cyclohexylamine oxidase in combination with a boraneammonia complex as reducing agent was applied to the deracemization of several substrates, excellent enantiomeric ratios (>99:1) and good isolated yields (62%-75%) of the corresponding (R)-amines were obtained.

DOI： 10.1139/V11-086

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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 CHEMICAL SOC  

Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O(2) as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP(+) in two distinct states, to resolutions of 2.3 and 2.2 angstrom. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.

DOI： 10.1021/ja9010578

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担当区分：筆頭著者   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  

Six cyclohexylacetic acid-degrading strains were isolated from soil samples in Japan and identified as members of the genera Cupriavidus (strain KUA-1), Rhodococcus, and Dietzia by 16S rRNA gene sequence analysis. For the first time members of these genera were shown to be capable of degrading cyclohexylacetic acid. A selected strain, KUA-1, which is the first reported Gram-negative organism capable of growth on cyclohexylacetic acid, was identified as a Cupriavidus metallidurans, based on morphologic and physiologic characteristics and its 16S rRNA gene sequence. Metabolite analysis by HPLC-MS indicated that 1-cyclohexenylacetic acid is an intermediate of cyclohexaneacetic acid metabolism in strain KUA-1.

DOI： 10.1007/s00284-008-9158-0

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担当区分：筆頭著者  

学術研究助成基金

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL PUBLISHING  

A gram-negative bacterium, strain KU-46, was isolated from agricultural soil contaminated with pesticides and was found to utilize 2,4-dinitrophenol as the sole source of carbon and nitrogen. Based on 16S rRNA gene sequence analysis and its morphological, biochemical, and physiological characteristics, strain KU-46 was identified as a Burkholderia sp. Metabolite analyses by HPLC and liquid chromatography-MS indicated that 4-nitrophenol, 1,4-benzoquinone, and nitrite are the intermediates of 2,4-dinitrophenol metabolism, and 2,4-dinitrophenol is metabolized via 4-nitrophenol to 1,4-benzoquinone by strain KU-46. The 2,4-dinitrophenol degradation pathway enzymes are induced by both 2,4-dinitrophenol and 4-nitrophenol.

DOI： 10.1111/j.1574-6968.2007.00816.x

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担当区分：筆頭著者   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  

Pseudomonas fluorescens strain KU-7 is a prototype microorganism that metabolizes 2-nitrobenzoate (2-NBA) via the formation of 3-hydroxyanthranilate (3-HAA), a known antioxidant and reductant. The initial two steps leading to the sequential formation of 2-hydroxy/aminobenzoate and 3-HAA are catalyzed by a NADPH-dependent 2-NBA nitroreductase (NbaA) and 2-hydroxylaminobenzoate mutase (NbaB), respectively. The 216-amino-acid protein NbaA is 78% identical to a plasmid-encoded hypothetical conserved protein of Polaromonas strain JS666; structurally, it belongs to the homodimeric NADH:flavin mononucleotide (FMN) oxidoreductase-like fold family. Structural modeling of complexes with the flavin, coenzyme, and substrate suggested specific residues contributing to the NbaA catalytic activity, assuming a ping-pong reaction mechanism. Mutational analysis supports the roles of Asn40, Asp76, and Glu113, which are predicted to form the binding site for a divalent metal ion implicated in FMN binding, and a role in NADPH binding for the 10-residue insertion in the beta 5-alpha 2 loop. The 181-amino-acid sequence of NbaB is 35% identical to the 4-hydroxylaminobenzoate lyases (PnbBs) of various 4-nitrobenzoate-assimilating bacteria, e.g., Pseudomonas putida strain TW3. Coexpression of nbaB with nbaA in Escherichia coli produced a small amount of 3-HAA from 2-NBA, supporting the functionality of the nbaB gene. We also showed by gene knockout and chemotaxis assays that nbaY, a chemoreceptor NahY homolog located downstream of the nbaA gene, is responsible for strain KU-7 being attracted to 2-NBA. NbaY is the first chemoreceptor in nitroaromatic metabolism to be identified, and this study completes the gene elucidation of 2-NBA metabolism that is localized within a 24-kb chromosomal locus of strain KU-7.

DOI： 10.1128/JB.01098-06

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担当区分：筆頭著者   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  

Bacterial strain KU-15, identified as a Burkholderia terrae by 16S rRNA gene sequence analysis, was one of 11 new isolates that grew on 2-nitrobenzoate as sole source of carbon and nitrogen. Strain KU-15 was also found to grow on anthranilate, 4-nitrobenzoate, and 4-aminobenzoate. Whole cells of strain KU-15 were found to accumulate ammonia in the medium, indicating that the degradation of 2-nitrobenzoate proceeds through a reductive route. Metabolite analyses by high-performance liquid chromatography indicated that 3-hydroxyanthranilate, anthranilate, and catechol are intermediates of 2-nitrobenzoate metabolism in strain KU-15. Enzyme studies suggested that 2-nitrobenzoate degradation occurs via the formation of 2-hydroxylaminobenzoate and that the pathway branches at this point to form two different aromatic intermediates: anthranilate and 3-hydroxyanthranilate. PCR amplifications and DNA sequencing revealed DNA fragments encoding a polypeptide homologous to 2-amino-3-carboxymuconate 6-semialdehyde decarboxylase and anthranilate 1,2-dioxygenase.

DOI： 10.1271/bbb.60419

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/bi060108c

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担当区分：筆頭著者   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  

Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that offer the prospect of high chemo-, regio-, and enantioselectivity in the organic synthesis of lactones or esters from a variety of ketones. In this study, we have cloned, sequenced, and overexpressed in Escherichia coli a new BVMO, cyclopentadecanone monooxygenase (CpdB or CPDMO), originally derived from Pseudomonas sp. strain HI-70. The 601-residue primary structure of CpdB revealed only 29% to 50% sequence identity to those of known BVMOs. A new sequence motif, characterized by a cluster of charged residues, was identified in a subset of BVMO sequences that contain an N-terminal extension of similar to 60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 mu mol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains similar to 1 mol flavin adenine dinucleotide per mole of protein. A deletion mutant suggested the importance of the N-terminal 54 amino acids to CPDMO activity. In addition, a Ser261Ala substitution in a Rossmann fold motif resulted in an improved stability and increased affinity of the enzyme towards NADPH compared to the wild-type enzyme (K-m = 8 mu M versus K-m = 24 mu M). Substrate profiling indicated that CPDMO is unusual among known BVMOs in being able to accommodate and oxidize both large and small ring substrates that include C-11 to C-15 ketones, methyl-substituted C-5 and C-6 ketones, and bicyclic ketones, such as decalone and beta-tetralone. CPDMO has the highest affinity (K-m = 5.8 mu M) and the highest catalytic efficiency (k(cat)/K-m ratio of 7.2 x 10(5) M-1 s(-1)) toward cyclopentadecanone, hence the Cpd designation. A number of whole-cell biotransformations were carried out, and as a result, CPDMO was found to have an excellent enantioselectivity (E > 200) as well as 99% S-selectivity toward 2-methylcyclohexanone for the production of 7-methyl-2-oxepanone, a potentially valuable chiral building block. Although showing a modest selectivity (E = 5.8), macrolactone formation of 15-hexadecanolide from the kinetic resolution of 2-methylcyclopentadecanone using CPDMO was also demonstrated.

DOI： 10.1128/AEM.72.4.2707-2720.2006

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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 CHEMICAL SOC  

The enzyme alpha-amino-beta-carboxy-muconic-epsilon-semialdehyde decarboxylase (ACMSD) plays an important role in the biodegradation of 2-nitrobenzoic acid in microorganisms and in tryptophan catabolism in humans. We report that the overexpressed ACMSD enzyme from Pseudomonas fluorescens requires a divalent metal, such as Co(II), Fe(II), Cd(II), or Mn(II), for catalytic activity and that neither a redox reagent nor an organic cofactor is required for the catalytic function. The metal ions can be taken up in either cell or cell-free preparations for generating the active form of ACMSD. The kinetic parameters and enzyme specific activity are shown to depend on the metal ion present in the enzyme, suggesting a catalytic role of the metal center. EPR spectrum of Co(II)-ACMSD provides a high-spin (S = 3/2) mononuclear metal ion in a non-heme, noncorrinoid environment with a mixed nitrogen/oxygen ligand field. We observe hyperfine interactions due to Co-59 nucleus at temperatures below 5 K but not at higher temperatures. Ten hyperfine lines are present in the g-L region, and three equivalent nitrogen hyperfine couplings are required to simulate the resonances in the EPR spectrum. The results for the metal binding site are also assessed using the copper-substituted enzyme, and the EPR spectral assignments for both cobalt and copper proteins give strong support for a distorted trigonal bipyramidal geometry of the metal center. Ultimately, these results suggest for the first time that ACMSD is a metal-dependent enzyme that catalyzes a novel nonoxidative decarboxylation.

DOI： 10.1021/ja0532234

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1264/jsme2.20.144

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Trichosporon cutaneum strain KUY-6A, a cyclohexanecarboxylic acid-utilizing yeast, was able to grow on cyclopentanol (CPOL) as a sole source of carbon and energy. Growth experiments revealed the strain, KUY-6A, could utilize up to 42 mM of CPOL with an optimum at 24 mM. Optimal growth was found between pH 4.0 to 9.0. The generation time under optimal growth conditions on CPOL was 3.0 h. Analysis indicated that cyclopentanone (CPON) and glutaric acid were intermediates of CPOL metabolism in strain KUY-6A. The results of growth and enzyme experiments are consistent with the degradation of CPOL via CPON, 5-valerolactone, 5-hydroxyvaleric acid, and glutaric acid. © 2004, Japanese Society of Microbial Ecology &amp
The Japanese Society of Soil Microbiology. All rights reserved.

DOI： 10.1264/jsme2.19.241

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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：ELSEVIER SCIENCE BV  

Cyclopentanone monooxygenase (CPMO) from Comamonas sp. NCIMB 9872 expressed in E. coli was evaluated as a potential new bioreagent for Baeyer-Villiger oxidations of 4-alkoxy- and halo-substituted cyclohexanones (10 examples). The results were compared with those obtained in oxidations catalyzed by an engineered E coli strain expressing cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. CPMO was found to have modest to good stereoselectivity and broader substrate acceptability than CHMO. The stereoselectivities of the two enzymes were generally opposite. It appears, therefore, that the two engineered strains can be useful and complementary reagents for enantioselective Baeyer-Villiger oxidations of certain prochiral ketones. (C) 2003 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S1381-1177(03)00036-5

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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  

The 2-nitrobenzoic acid degradation pathway of Pseudomonas fluorescens strain KU-7 proceeds via a novel 3-hydroxyanthranilate intermediate. In this study, we cloned and sequenced a 19-kb DNA locus of strain KU-7 that encompasses the 3-hydroxyanthranilate meta-cleavage pathway genes. The gene cluster, designated nbaEX HJIGFCDR, is organized tightly and in the same direction. The nbaC and nbaD gene products were found to be novel homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate6-semialdehyde decarboxylase, respectively. The NbaC enzyme carries out the oxidation of 3-hydroxyanthranilate to 2-amino-3-carboxymuconate-6-semialdehyde, while the NbaD enzyme catalyzes the decarboxylation of the latter compound to 2-aminomuconate-6-semialdehyde. The NbaC and NbaD proteins were overexpressed in Escherichia coli and characterized. The substrate specificity of the 23.8-kDa NbaC protein was found to be restricted to 3-hydroxyanthranilate. In E. coli, this enzyme oxidizes 3-hydroxyanthranilate with a specific activity of 8 U/mg of protein. Site-directed mutagenesis experiments revealed the essential role of two conserved histidine residues (His52 and His96) in the NbaC sequence. The NbaC activity is also dependent on the presence of Fe2+ but is inhibited by other metal ions, such as Zn2+, Cu2+, and Cd2+. The NbaD protein was overproduced as a 38.7-kDa protein, and its specific activity towards 2-amino-3-carboxymuconate-6-semialdehyde was 195 U/mg of protein. Further processing of 2-aminomuconate-6-semialdehyde to pyruvic acid and acetyl coenzyme A was predicted to proceed via the activities of NbaE, NbaF, NbaG, NbaH, NbaI, and NbaJ. The predicted amino acid sequences of these proteins are highly homologous to those of the corresponding proteins involved in the metabolism of 2-aminophenol (e.g., AmnCDEFGH in Pseudomonas sp. strain AP-3). The NbaR-encoding gene is predicted to have a regulatory function of the LysR family type. The function of the product of the small open reading frame, NbaX, like the homologous sequences in the nitrobenzene or 2-aminophenol metabolic pathway, remains elusive.

DOI： 10.1128/AEM.69.3.1564-1572.2003

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER CHEMICAL SOC  

We have now determined the complete gene sequence of the cyclohexanol (chn) degradation pathway in Acinetobacter sp. NCIMb 9871 as well as the putative genes for the beta-oxidation of adipic acid to acetyl-CoA and succinyl-CoA. In addition, a new insertion sequence, potentially useful in strain characterization, was identified. Knowledge of the nucleotide sequence of the chn genes was used to construct clones of Escherichia coli that would overproduce the requisite biocatalysts: a flavin monooxygenase (ChnB; cyclohexanone 1,2-monooxygenase [CHMO]), a ring-opening hydrolase (ChnC; epsilon-caprolactone hydrolase) and three oxido-reductases (ChnA, cyclohexanol dehydrogenase; ChnD, 6-hydroxyhexanoate dehydrogenase; and ChnE, 6-oxohexanoate dehydrogenase). Besides the well known application of CHMO as a Baeyer-Villiger biocatalyst that carries out stereoselective oxidations of a wide variety of ketones to the corresponding lactones, potential applications of the Chn biocatalysts in the development of "green" bioprocesses such as an "envirocompatible" synthesis of adipic acid are discussed.

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担当区分：筆頭著者   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  

Cyclopentanone 1,2-monooxygenase, a flavoprotein produced by Pseudomonas sp. strain NCIMB 9872 upon induction by cyclopentanol or cyclopentanone (M. Griffin and P. W. Trudgill, Biochem. J. 129:595-603, 1972), has been utilized as a biocatalyst in Baeyer-Villiger oxidations. To further explore this biocatalytic potential and to discover new genes, we have cloned and sequenced a 16-kb chromosomal locus of strain 9872 that is herein reclassified as belonging to the genus Comamonas. Sequence analysis revealed a cluster of genes and six potential open reading frames designated and grouped in at least four possible transcriptional units as (orf11-orf10-orf9)-(cpnE-cpnD-orf6-cpnC)-(cpnR-cpnB-cpnA)-(orj3-orf4 [partial 3' end]). The cpnABCDE genes encode enzymes for the five-step conversion of cyclopentanol to glutaric acid catalyzed by cyclopentanol dehydrogenase, cyclopentanone 1,2-monooxygenase, a ring-opening 5-valerolactone hydrolase, 5-hydroxyvalerate dehydrogenase, and 5-oxovalerate dehydrogenase, respectively. Inactivation of cpnB by using a lacZ-Km(r) cassette resulted in a strain that was not capable of growth on cyclopentanol or cyclopentanone as a sole carbon and energy source. The presence of sigma(54)-dependent regulatory elements in front of the divergently transcribed cpnB and cpnC genes supports the notion that cpnR is a regulatory gene of the NtrC type. Knowledge of the nucleotide sequence of the cpn genes was used to construct isopropyl-beta-thio-D-galactoside-inducible clones of Escherichia coli cells that overproduce the five enzymes of the cpn pathway. The substrate specificities of CpnA and CpnB were studied in particular to evaluate the potential of these enzymes and establish the latter recombinant strain as a bioreagent for Baeyer-Villiger oxidations. Although frequently nonenantioselective, cyclopentanone 1,2-monooxygenase was found to exhibit a broader substrate range than the related cyclohexanone 1,2-monooxygenase from Acinetobacter sp. strain NCIMB 9871. However, in a few cases opposite enantioselectivity was observed between the two biocatalysts.

DOI： 10.1128/AEM.68.11.5671-5684.2002

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1139/V02-035

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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  

Baeyer-Villiger cyclohexanone 1,2-monooxygenase (CHMO) was purified 17.1-fold from cell extracts of the fungus Exophiala jeanselmei grown on cyclohexanol to electrophoretically homogeneity by serial chromatographies. The molecular mass of the native enzyme was approximately 74 kDa by gel filtration and SDS-PAGE. Some enzymic characterizations were studied. The NH2-terminal amino acid residues were Ala-Lys-Ser-Leu-Asp-Val-Leu-Ile-Val-Gly-Ala-Gly-Phe-Gly-Gly-Ile-TyrGln-Leu-, with similarity to the bacterial CHMOs of FAD-binding and NADPH-dependent type Baeyer-Villiger monooxygenases.

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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：ELSEVIER SCIENCE BV  

A bacterial strain KU-7, identified as a Pseudomonas fluorescens by 16S rDNA sequencing, was one of the 12 new isolates that are able to grow on 2-nitrobenzoate as a sole source of carbon, nitrogen, and energy. Resting cells of KU-7 were found to accumulate ammonia in the medium indicating that degradation of 2-NBA proceeds through a reductive route. Metabolite analyses by thin layer chromatography and high pressure liquid chromatography indicated that 3-hydroxyanthranilate is an intermediate of 2-nitrobenzoate metabolism in KU-7 cells. This offers an alternative route to 2-nitrobenzoate metabolism since anthranilate (2-aminobenzoate) or catechol were detected as intermediates in other bacteria. Crude extracts of KU-7 cells converted 2-nitrobenzoate to 3-hydroxyanthranilate with oxidation of 2 mol of NADPH. Ring cleavage of 3-hydroxyanthranilate produced a transient yellow product, identified as 2-amino-3-carboxymuconic 6-semialdehyde, that has a maximum absorbance at 360 nm. The initial enzymes of the 2-nitrobenzoate degradation pathway were found to be inducible since succinate-grown cells produced very low enzyme activities. A pathway for 2-nitrobenzoate degradation in KU-7 was proposed. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

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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  

N,N-Dimethylformamidase (DMFase) from Alcaligenes sp. strain KUFA-1, a bacterium that can grow on N,N-dimethylformamide (DMF) as the sole carbon and nitrogen source, catalyzes the first step of the DMF degradation. The DMFase gene dmfA1A2 was cloned in Escherichia coli, and its nucleotides were sequenced. The deduced amino acid sequence of the enzyme consisted of two alpha- and two beta-subunits with 132 and 762 amino acids, respectively, and had little similarity to sequences in protein databases, including various amidases. The protein may be a new kind of amidase. DMFase activity was detected in E. coli cells transformed with an expression plasmid of the cloned DMFase gene. The properties of recombinant DMFase purified from E. coli were identical to those of Alcaligenes DMFase.

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担当区分：筆頭著者  

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Cyclohexylamine oxidase (CHAO) from a cell extract of Brevibacterium grown on cyclohexylamine was purified 50.2-fold, to electrophoretic homogeneity, by serial chromatographies. The molecular mass of the native enzyme was estimated to be approximately 50 kDa by gel filtration and SDS-PAGE. The optimum pH was 7.4 and the stable pH range was 6.0 to 7.0. The enzyme was thermostable up to 30 degrees C. The enzyme was found to be highly specific for the deamination of alicyclic monoamines such as cyclopentylamine, cycloheptylamine, and N-methylcyclohexylamine and aliphatic monoamines, such as sec-butylamine. The apparent K-m value for cyclohexylamine was 1.23 mM. The enzyme was inhibited by flavin enzyme inhibitors such as quinine and quinacrine. The N-terminal 27 amino acid residues were determined as Gly-Ser-Val-Thr-Pro-Asp-Pro-Asp-Val-Asp-Val-Ile-His-Gly-Ala-Gly-Ser-Gly-Ser-Ala-Leu, revealing homology to conventional flavin-containing amine oxidases (EC 1.4.3.4).

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担当区分：筆頭著者   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 bacterial strain capable of growing on cyclohexylamine (CHAM) was isolated by using enrichment and isolation techniques. The strain isolated, strain IH-35A, was classified as a member of the genus Brevibacterium. The results of growth and enzyme studies are consistent with degradation of CHAM via cyclohexanone (CHnone), 6-hexanolactone, 6-hydroxyhexanoate, and adipate. Cell extracts obtained from this strain grown on CRAM contained CRAM oxidase, and the model for CRAM oxidation by this enzyme was similar to the model for deamino oxidation of amine by amine oxidase.

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開催年月日： 2018年1月

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開催年月日： 2017年9月

開催地：大阪（大阪府立大学）  

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開催年月日： 2017年9月

開催地：東京（早稲田大学）  

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開催年月日： 2017年3月

開催地：京都  

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開催年月日： 2017年3月

開催地：京都  

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開催年月日： 2017年3月

開催地：京都  

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開催地：富山  

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開催地：札幌  

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開催地：札幌  

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開催地：千里ライフサイエンスセンター  

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開催地：千里ライフサイエンスセンター  

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開催年月日： 2015年5月

開催地：東京海洋大学品川キャンパス  

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開催年月日： 2015年4月

記述言語：英語  

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開催年月日： 2015年3月

開催地：Boston, MA  

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開催年月日： 2015年3月

開催地：岡山  

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開催地：岡山  

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開催地：岡山  

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開催年月日： 2013年9月

開催地：県立広島大学（広島）  

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開催年月日： 2013年9月

開催地：県立広島大学（広島）  

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開催年月日： 2013年3月

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開催地：仙台  

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開催地：仙台  

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開催年月日： 2013年1月

開催地：関西大学, 吹田  

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開催年月日： 2013年1月

開催地：関西大学, 吹田  

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開催年月日： 2012年10月

開催地：神戸ポートピアホテル（兵庫県）  

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開催年月日： 2012年9月

開催地：京都学園大学  

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開催年月日： 2012年3月

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開催年月日： 2012年3月

開催地：京都女子大学  

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開催年月日： 2012年3月

開催地：京都女子大学  

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開催年月日： 2012年3月

開催地：京都女子大学  

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開催年月日： 2012年3月

開催地：京都女子大学  

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開催年月日： 2012年1月

開催地：関西大学, 吹田  

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開催年月日： 2012年

記述言語：日本語  

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開催年月日： 2011年8月

開催地：千里ライフサイエンスセンター，豊中  

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開催年月日： 2011年8月

開催地：千里ライフサイエンスセンター，豊中  

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開催年月日： 2011年8月

開催地：千里ライフサイエンスセンター，豊中  

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開催年月日： 2011年3月

開催地：京都女子大学  

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開催年月日： 2011年3月

開催地：京都女子大学  

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開催年月日： 2011年3月

開催地：京都女子大学  

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開催年月日： 2011年3月

開催地：京都女子大学  

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開催年月日： 2011年3月

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開催年月日： 2010年10月

開催地：近畿大学農学部  

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開催年月日： 2010年9月

開催地：University of Massachusetts Boston, Boston, Massachusetts, USA  

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開催年月日： 2010年3月

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開催年月日： 2010年

記述言語：英語  

From the first crystal structures of a prototypical cyclohexanone monooxygenase (CHMO) complexed with both FAD and NADP(+) cofactors to whole genome mining of new microbial Baeyer-Villiger monooxygenases (BVMOs), this review highlights the recent progress in Baeyer-Villiger biooxidation technology. Protein engineering of BVMOs as enantioselective enzymes in asymmetric catalysis to accommodate new substrates is an active pursuit. Cofactor recycling appears to no longer be an issue, and we have gained a greater understanding of whole-cell biotransformation dynamics and limitations, leading us towards industrial scale realization. In terms of substrate profiling, new naturally-occurring substrates have been found in addition to a seeming rebirth of interest in BVMO-mediated oxidation of steroids.

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開催年月日： 2009年11月

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開催年月日： 2009年9月

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開催年月日： 2009年3月

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開催年月日： 2009年1月

開催地：関西大学  

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開催年月日： 2009年

記述言語：日本語  

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開催年月日： 2009年

記述言語：日本語  

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開催年月日： 2008年9月

開催地：京都学園大学  

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開催年月日： 2007年6月

開催地：大阪大学  

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開催年月日： 2007年6月

開催地：大阪大学  

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開催年月日： 2007年5月

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開催年月日： 2006年10月

開催地：京都工芸繊維大学  

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開催年月日： 2006年10月

開催地：京都工芸繊維大学  

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開催年月日： 2006年10月

開催地：京都工芸繊維大学  

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開催年月日： 2006年9月

開催地：大阪大学豊中キャンパス  

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開催年月日： 2006年1月

開催地：関西大学  

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開催年月日： 2006年

記述言語：日本語  

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開催年月日： 2005年9月

開催地：大阪大学  

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開催年月日： 2005年9月

開催地：大阪大学  

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開催年月日： 2004年9月

開催地：名城大学  

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開催年月日： 2004年3月

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開催年月日： 2004年3月

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開催年月日： 2004年1月

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開催年月日： 2004年

記述言語：日本語  

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開催年月日： 2003年9月

ReactIRTM 4000 is a state-of-the-art tool that allows realtime, in-situ analysis of a biotransformation and it can provide a characteristic fingerprint of specific molecularinformation that reflects changes in a reaction as a function of time. We have applied this technique to monitor the progress of a Baeyer-Villiger monooxygenase (BVMO) reaction. BVMOs areflavoproteins and they are nature's alternative to the abiotic Baeyer-Villiger organic reaction transformations of (cyclic) ketones into esters or lactones catalyzed by peracids. The latter chemicals are strong oxidants and can produce undesirable side reactions. BVMO applicationsinclude the synthesis of natural products andproduction of chiral building blocks for possible drug synthesis and value-added products. As part of a biocatalyst ”toolkit” development program, we have cloned a cyclododecanone monooxygenase (CDMO)-encoding gene (cdnB) from a soil microorganism, Pseudomonas putida strain HI-70. In a phylogenetic analysis, the predicted amino acid sequence of CDMO is found in a different cluster than that of the majority of the cloned BVMOs (e.g. cyclohexanone monooxygenase [CHMO] of Acinetobacter spp; cyclopentanone monooxygenase [CPMO] of Comamonas sp. NCIMB 9872). CDMO is active towards cyclic ketones, ranging from C10 to C16. This is different from the specificity shown by CHMO or CPMO that favours short-chain cyclic compounds. Using an overexpression clone of cdnB gene (pCD100B) in Escherichia coli strain BL21, the production of lauryl lactone from cyclododecanone was evaluated. ReactIR was found to be an useful monitoring tool in the development of this biocatalytic process. This study includes also a preliminary development of a two-phase bioreactor system that addresses low reactant solubility and product recovery.

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開催年月日： 2003年9月

2-nitrobenzoic acid (2-NBA) via the novel formation of 3-hydroxyanthranilic acid (3-HAA) instead of anthranilate or 2-aminobenzoate as anintermediate. We previously cloned and analyzed the 3-HAA catabolic gene cluster (nbaEXHJIGFCD), responsible for the conversion of 3-HAA into Krebs cycle intermediate (Muraki et al., Appl. Environ. Microbiol., 69:1564-1572, 2003). In order to identify the initial genes of the nbapathway, we further screened 24,000 transconjugants and isolated 37 mutants strains that were unable to grow on solid medium containing 2-NBA as a sole source of carbon and energy. DNA regions flanking the transposon in these mutant strains were isolated by Inverse PCR and individual DNA sequences were determined. As a result, we identified two mutant strains, named KUM-19 and KUM-33, in which the potential nbaA and nbaB genes were mutagenized respectively. The predicted primary structure of the nbaAencoded protein was found to have 31% sequence identity to a FMN-binding protein (MTH152, PDB ID: 1EJE) of Methanobacterium thermoautrophicum dH. The predicted amino acid sequence of the nbaB-encoded protein was found to have 34% sequence identity to the 4-hydroxylaminobenzoate lyase (PnbB) of 4-nitrobenzoate assimilating Pseudomonas putida TW3. Pairwise alignment of NbaA and MTH152 sequences indicated the presence of 7 conserved amino acid residues that may be involved in binding of FMN molecule. The nbaA and nbaB ORFs were separately cloned into the IPTG-inducible expression vector pSD80, and crude extracts of transformed E. coli cells werefound to show expression of the expected molecular size as well as the expected activity of 2-nitrobenzoate nitroreductase (NbaA) and 2-hydroxylaminobenzoate mutase (NbaB). In summary, at least ten catabolic genes and one regulatory gene are necessary for the metabolism of 2-NBA in strain KU-7. Within the 23-kb sequenced locus there are at least 13 other ORFs, many of which remain to be characterized.

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開催年月日： 2003年5月

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開催年月日： 2001年10月

3-hydroxyanthranilate 3,4-dioxygenase, a novel eukaryotic homolog in the degradation pathway of 2-nitrobenzoate by Pseudomonas fluorescens strain KU-7

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開催年月日： 2001年10月

Cyclopentanone 1,2-monooxygenase:Old wine, new bottle

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開催年月日： 2001年9月

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開催年月日： 2000年2月

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開催年月日： 2000年

記述言語：日本語  

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開催年月日： 1999年

記述言語：日本語  

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開催年月日： 1999年

記述言語：日本語  

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開催年月日： 1999年

記述言語：日本語  

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開催年月日： 1998年

記述言語：日本語  

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開催年月日： 1998年

記述言語：日本語  

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開催年月日： 1998年

記述言語：日本語  

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開催年月日： 1997年

記述言語：日本語  

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開催年月日： 1997年

記述言語：日本語  

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開催年月日： 1996年

記述言語：日本語  

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開催地：Web開催  

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開催地：Web開催  

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