IWAKI, Hiroaki |
|---|
Faculty, Department/Institute
- Faculty of Chemistry, Materials and Bioengineering Department of Life Science and Biotechnology
Academic status (qualification)
- Professor Apr. 1,2016
Undergraduate Degrees・University
- Kansai University Faculty of EngineeringDepartment of Biotechnology 1995 Graduated
Graduate Degrees・University
- Kansai University Doctor's Degree Program Biotechnology major 2000 Completed
Academic Degrees
- Doctor of engineering Mar. 2000 Kansai University
Homepage Address, E-mail Address
- Homepage Address：https://wps.itc.kansai-u.ac.jp/eme/
Research fields
| Research fields | keyword |
|---|---|
| Environmental microbiotechnology | |
| Applied microbiology |
Research topics
| research topic | |
|---|---|
| Study theme state | International Joint Research |
| research duration | |
| Research Programs | |
| keyword | |
| Research field | |
| Research Topics Overview |
| research topic | |
|---|---|
| Study theme state | International Joint Research |
| research duration | |
| Research Programs | |
| keyword | |
| Research field | |
| Research Topics Overview |
Research Career
- National Research Council of Canada/Biotechnology Research Institute 2000/9～2003年/3
- 2003/4/1～2006年/3/31
- 2006/4/1～2009年/3/31
- Associate Professor
Academic Associations
| 所属学会・団体名 | 役職名 (役職在任期間) |
|---|---|
| American Society for Microbiology |
Research Publications
Academic presentationOtherCo-author;;;;2022/10/20～
Academic presentationOther;;;;;2022/10/18～
FieldworkIn refereedIn-house publicationSingle-AuthorIWAKI,Hiroaki2022/8～
PapersIn refereedAcademic JournalCo-authored;;2022/7～
FieldworkIn refereedIn-house publicationSingle-AuthorIWAKI,Hiroaki2022/7～
Academic presentationUnrefereedOtherCo-authored;;;;2022/3/23～
PapersIn refereedAcademic JournalCo-authored;;2022/2～10.1093/bbb/zbab199
PapersIn refereedAcademic JournalCo-authored;;;;;2022/1～
PapersIn refereedAcademic JournalInternational coauthorship;;;;2021/12～10.1016/j.jbiosc.2021.08.013
FieldworkIn refereedAcademic JournalSingle-AuthorIWAKI,Hiroaki2021/11～
Academic presentationUnrefereedOtherCo-authored;;;IWAKI,Hiroaki2021/10/29～
PapersIn refereedAcademic JournalCo-authored;;;;2021/7～10.1093/bbb/zbab079
FieldworkIn refereedAcademic JournalSingle-AuthorIWAKI,Hiroaki2020/12～
PapersIn refereedAcademic JournalCo-authored;;;;2020/11～10.2323/jgam.2019.11.006
FieldworkIn refereedAcademic JournalSingle-AuthorIWAKI,Hiroaki2020/10～
PapersIn refereedAcademic JournalCo-authored;;;2020/5/7～
PapersIn refereedAcademic JournalCo-authored;;;2019/11/7～10.1128/MRA.01204-19
LectureUnrefereedOtherSingle-AuthorIWAKI,Hiroaki2019/9/26～
Academic presentationUnrefereedOtherCo-authored;;2019/9/22～
OrganizerIWAKI,Hiroaki2019/8/30～
Research reportUnrefereedOtherCo-author;;IWAKI,Hiroaki;2019/1/24～
FieldworkIn refereedAcademic JournalSingle-AuthorIWAKI,Hiroaki2018/12～
Academic presentationOtherCo-author;IWAKI,Hiroaki;;2018/9/5～
PapersIn refereedAcademic JournalCo-authoredIWAKI,Hiroaki;;2018/4～10.1093/femsle/fny042
Academic presentationUnrefereedOtherCo-author;;2018/3/17～
OrganizerIWAKI,Hiroaki2018/1/29～
Research reportUnrefereedOtherCo-author;IWAKI,Hiroaki;2018/1/18～
PapersIn refereedIn-house publicationSingle-AuthorIWAKI,Hiroaki2017/12～
Magazine articleUnrefereedAcademic JournalSingle-AuthorIWAKI,Hiroaki2017/10～
Academic presentationUnrefereedOtherCo-author;IWAKI,Hiroaki;2017/9/22～
Academic presentationUnrefereedCo-author;IWAKI,Hiroaki;2017/9/14～
OrganizerIWAKI,Hiroaki2017/8/21～
Academic presentationIsolation and genetic characterization of cyclododecanone degrading bacteriumUnrefereedOtherCo-author;;2017/3/20～
Academic presentationUnrefereedOtherCo-author;;2017/3/20～
Academic presentationUnrefereedOtherCo-author;IWAKI,Hiroaki;2017/3/18～
Research reportUnrefereedOtherCo-author;;2017/1/19～
Academic presentationUnrefereedOtherCo-author;IWAKI,Hiroaki;2016/9/30～
OrganizerUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2016/9/29～
Academic presentationUnrefereedOtherCo-author;;2016/3/28～
Academic presentationUnrefereedOtherCo-author;;2016/3/28～
Academic presentationUnrefereedOtherCo-author;;2016/3/28～
PapersIn refereedAcademic JournalInternational coauthorship;;;;;;;;;;;;;;;;;2015/11～10.1107/S1399004715017939
Academic presentationUnrefereedOtherCo-author;;;;2015/9/1～
Academic presentationUnrefereedOtherCo-author;;;2015/9/1～
Academic presentationUnrefereedOtherCo-author;;;2015/5/31～
International academic conferenceUnrefereedAcademic JournalCo-author;;;;;;;;2015/3/30～
Academic presentationUnrefereedOtherCo-author;;;2015/3/27～
Academic presentationUnrefereedOtherCo-author;;;2015/3/27～
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PapersIn refereedAcademic JournalInternational coauthorship;;;;;;;;;2015/1/7～doi:10.1038/ncomms6935
PapersIn refereedAcademic JournalInternational coauthorship;;;;;;;;;2015/1～
PapersIn refereedAcademic JournalInternational coauthorship;;;;;;;;;2014/4～
PapersIn refereedAcademic JournalCo-authorIWAKI,Hiroaki;;;2014/2～doi: 10.1007/s00284-013-0455-x
PapersIn refereedAcademic JournalInternational coauthorship;;;;;;;;2013/11～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2013/9/6～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;;2013/9/6～
PapersIn refereedAcademic JournalCo-authorIWAKI,Hiroaki;;;;;2013/6～
PapersIn refereedAcademic JournalInternational coauthorshipIWAKI,Hiroaki;;;;;;;2013/5～
Research reportUnrefereedIn-house publicationCo-authorIWAKI,Hiroaki;;;;;;;;;;;;2013/3/31～
PapersIn refereedAcademic JournalInternational coauthorship;;;;;;;;2013/3/26～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2013/3/26～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;;2013/3/25～
Research reportUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2013/1/29～
Research reportUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2013/1/29～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;;2012/10/24～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;;2012/9/29～
PapersIn refereedAcademic JournalInternational coauthorship;;;;;;;;;;2012/7～
PapersIn refereedAcademic JournalCo-authorIWAKI,Hiroaki;;;2012/4～
PapersIn refereedAcademic JournalInternational coauthorship;;;;;;;;;;2012/4～
Research reportUnrefereedIn-house publicationCo-authorIWAKI,Hiroaki;;;;;;;;;;;2012/3/31～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2012/3/25～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;;2012/3/24～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2012/3/24～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;;;2012/3/24～
PapersIsolation and characterization of marine bacteria capable of utilizing phthalateIn refereedAcademic JournalCo-authorIWAKI,Hiroaki;;;2012/3～
Research reportUnrefereedOtherSingle-AuthorIWAKI,Hiroaki;2012/1/24～
PapersIn refereedAcademic JournalInternational coauthorship;;;;;;2012/1～
PapersIn refereedAcademic JournalCo-authorIWAKI,Hiroaki;;;2012/1～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;;2011/8/31～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2011/8/31～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2011/8/30～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2011/3/27～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;;2011/3/27～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2011/3/27～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2011/3/27～
Research reportUnrefereedIn-house publicationCo-author;;;;;;;;;;;2011/3～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2010/10/3～
International academic conferenceThe enzymatic oxidation of ketones and amines: a tale of two newly cloned classes of biocatalystUnrefereedOtherCo-authorIWAKI,Hiroaki;;;;;;2010/9/30～
In refereedOtherCo-authorIWAKI,Hiroaki;;;;;;;;2010/8/11～
Research reportUnrefereedIn-house publicationCo-author;;;;;;;;;;;2010/3～
Academic presentationCo-authorIWAKI,Hiroaki;;;;2009/11/22～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;;;2009/10/31～
Academic presentationOtherCo-authorIWAKI,Hiroaki;;;;;;;2009/10/31～
Academic presentationOtherCo-authorIWAKI,Hiroaki;;;;;2009/10/31～
Academic presentationCo-authorIWAKI,Hiroaki;;;2009/9/24～
Academic presentationCo-authorIWAKI,Hiroaki;;;;2009/9/14～
PapersCrystal Structures of Cyclohexanone Monooxygenase Reveal Complex Domain Movements and a Sliding CofactorIn refereedAcademic JournalCo-authorI. Ahmad Mirza;Brahm J. Yachnin;Shaozhao Wang;Stephan Grosse;Helene Bergeron;Akihiro Imura;Hiroaki Iwaki;Yoshie Hasegawa;Peter C. K. Lau;Albert M. Berghuis;2009/7～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 A. 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.
Research reportUnrefereedIn-house publicationCo-author;;;;;;;;;;;2009/3～
Research reportUnrefereedOtherSingle-AuthorIWAKI,Hiroaki;;2009/1/15～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2008/9/13～
PapersIsolation and characterization of new cyclohexylacetic acid-degrading bacteriaIn refereedAcademic JournalCo-authorHiroaki Iwaki;Emiko Nakai;Shota Nakamura;Yoshie Hasegawa;2008/8～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. Members of these genera were for the first time demonstrated 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 morphological and physiological 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.Kansai University Research Grants 20060401-200703
PapersDegradation of Monohydroxylated Benzoates by Strain KUFI-6N of the Yeast-like Fungus Exophiala jeanselmeiIn refereedAcademic JournalCo-authorHiroaki Iwaki;Tao Zhang;Yoshie Hasegawa;World J Microbiol Biotechnol24(2)2008/1/19～Exophiala jeanselmei strain KUFI-6N, a cyclohexanol-utilizing yeast-like fungus, was found to grow on three isomers of hydroxybenzoate as a sole source of carbon. p- and m-Hydroxybenzoate was converted to protocatechuate by separate and highly specific hydroxylases. o-Hydroxybenzoate, on the other hand, was converted to catechol.Kansai University Research Grants
PapersIsolation and characterization of a new 2,4-dinitrophenol-degrading bacterium Burkholderia sp. strain KU-46 and its degradation pathwayIn refereedAcademic JournalCo-authorHiroaki Iwaki;Kazuya Abe;Yoshie Hasegawa;FEMS Microbiol. Lett.274(1):112-1172007/9～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. Strain KU-46 was identified as a member of Burkholderia sp. based on its morphological and physiological characteristics and 16S rRNA gene sequence analysis. Metabolite analyses by high-performance liquid chromatography and liquid chromatography-mass spectrometry indicated that 4-nitrophenol, 1,4-benzoquinone, and nitrite are intermediates of 2,4-dinitrophenol metabolism, and 2,4-dinitrophenol is metabolized via 4-nitrophenol to 1,4-benzoquinone in strain KU-46. The 2,4-dinitrophenol degradation pathway enzymes are induced by both 2,4-dinitrophenol and 4-nitrophenol.Kansai University Research Grants 20060401-200703
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2007/6～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2007/6～
International academic conferenceBroad Substrate Spectrum and Crystal Structures of a New Baeyer-Villiger Cyclohexanone Monooxygenase of Rhodococcus OriginUnrefereedOtherInternational coauthorshipS. Wang;A. Mirza;B. Yachnin;A. Berghuis;H. Iwaki;Y. Hasegawa;A. Imura;S. Grosse;H. Bergeron;P.C.K. Lau;90th Canadian Chemistry Conference and Exhibition in Winnipeg, Manitoba2007/5/27～90th Canadian Chemistry Conference and Exhibition in Winnipeg, Manitoba
PapersCharacterization of a Pseudomonad 2-Nitrobenzoate Nitroreductase and its Catabolic Pathway Associated 2-Hydroxylaminobenzoate Mutase and a Chemoreceptor Involved in 2-Nitrobenzoate ChemotaxisIn refereedAcademic JournalInternational coauthorshipHIROAKI IWAKI;TAKAMICHI MURAKI;SHUN ISHIHARA;YOSHIE HASEGAWA;KATHRYN N. RANKIN;TRAIAN SULEA;JASON BOYD;PETER C.K. LAU;J. Bacteriol.189(9):3502-3514 (with cover photograph)2007/5～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-hydroxylaminobenzoate (2-HABA) and 3-HAA are catalyzed by a NADPH-dependent 2-nitrobenzoate nitroreductase (NbaA) and 2-HABA 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: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 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 5-2 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., P. putida strain TW3.
Co-expression 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 assay that nbaY, a chemoreceptor NahY homolog located downstream of the nbaA gene, is responsible for strain KU-7 to be attracted toward 2-NBA. NbaY is the first identified chemoreceptor in nitroaromatic metabolism, and this study completes the gene elucidation of 2-NBA metabolism that is localized within a 24-kb chromosomal locus of strain KU-7.Kansai University Research Grants 20060401-200703
PapersDegradation of 2-Nitrobenzoate by Burkholderia terrae Strain KU-15In refereedAcademic JournalCo-authorHiroaki IWAKI;Yoshie HASEGAWA;Biosci. Biotechnol. Biochem.71(1), 145-1512007/1～Bacterial strain KU-15, identified as a Burkholderia terrae by 16S rRNA gene sequence analysis, was one of eleven new isolates that grew on 2-nitrobenzoate as a 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 chromatograph 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 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.Kansai University Research Grants 200504-200603
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2006/10/1～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2006/10/1～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2006/10/1～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2006/9/11～
Papersα -Amino- β -Carboxymuconic- ε -Semialdehyde Decarboxylase (ACMSD) Is a New Member of the Amidohydrolase SuperfamilyIn refereedAcademic JournalInternational coauthorshipTingfeng Li;Hiroaki Iwaki;Rong Fu;Yoshie Hasegawa;Hong Zhang;Aimin Liu;Biochemistry45(21):6628-66342006/5/30～The enzymatic activity of Pseudomonas fluorescens α -Amino- β -Carboxymuconic- ε -Semialdehyde decarboxylase (ACMSD) is critically dependent on a transition metal ion [Li, T., Walker, A. L., Iwaki, H., Hasegawa, Y., and Liu, A. (2005) J. Am. Chem. Soc. 127, 12282-12290]. Sequence analysis in this study further suggests that ACMSD belongs to the amidohydrolase superfamily, whose structurally characterized members comprise a catalytically essential metal cofactor. To identify ACMSD's metal ligands and assess their functions in catalysis, a site-directed mutagenesis analysis was conducted. Alteration of His-9, His-177, and Asp-294 resulted in a dramatic loss of enzyme activity, substantial reduction of the metal-binding ability, and an altered metallocenter electronic structure. Thus, these residues are confirmed to be the endogenous metal ligands. His-11 is implicated in metal binding because of the strictly conserved HxH motif with His-9. Mutations at the 228 site yielded nearly inactive enzyme variants H228A and H228E. The two His-228 mutant proteins, however, exhibited full metal-binding ability and a metal center similar to that of the wild-type enzyme as shown by EPR spectroscopy. Kinetic analysis on the mutants indicates that His-228 is a critical catalytic residue along with the metal cofactor. Since the identified metal ligands and His-228 are present in all known ACMSD sequences, it is likely that ACMSD proteins from other organisms contain the same cofactor and share similar catalytic mechanisms. ACMSD is therefore the first characterized member in the amidohydrolase superfamily that represents a C-C breaking activity.
PapersPseudomonad Cyclopentadecanone Monooxygenase Displaying an Uncommon Spectrum of Baeyer-Villiger Oxidations of Cyclic KetonesIn refereedAcademic JournalInternational coauthorshipHiroaki Iwaki;Shaozhao Wang;Stephan Grosse;Helene Bergeron;Ayako Nagahashi;Jittiwud Lertvorachon;Jianzhong Yang;Yasuo Konishi;Yoshie Hasegawa;;Peter C. K. Lau;Appl. Enviorn. Microbiol.72(4):2707-27202006/4～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 60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 ?mol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains 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 (Km = 8 ?M versus Km = 24 ?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 C11 to C15 ketones, methyl-substituted C5 and C6 ketones, and bicyclic ketones, such as decalone and ?-tetralone. CPDMO has the highest affinity (Km = 5.8 ?M) and the highest catalytic efficiency (kcat/Km ratio of 7.2 x 105 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.
Research reportUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2006/1～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2005/9/30～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;2005/9/30～
PapersKinetic and Spectroscopic Characterization of ACMSD from Pseudomonas fluorescens Reveals a Pentacoordinate Mononuclear MetallocofactorIn refereedAcademic JournalInternational coauthorshipTingfeng Li;Antoinette L. Walker;Hiroaki Iwaki;Hong Zhang;Yoshie Hasegawa;;Aimin Liu;Journal of the American Chemical Society127/35 12282-122902005/9/7～The enzyme 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (ACMSD) plays an important role in two metabolic events: the kynurenine pathway present primarily in mammals and the 2-nitrobenzoic acid pathway in microorganisms. The step catalyzed by ACMSD determines the
flow of metabolic intermediates to either acetyl CoA or quinolinic acid in these pathways. Previous investigations of this enzyme failed to reveal the conserved protein sequence motifs
and the cofactor critical for the enzyme activity. In this study, multiple sequence alignment and protein database search suggest that ACMSD is most closely related to dihydroorotase of the amidohydrolase superfamily with a TIM-barrel fold. ACMSD isolated from Pseudomonas fluorescens requires divalent metal such as Co(II), Fe(II), Cd(II), or Mn(II) for catalytic activity. On the other hand, Cu(II), Zn(II), or Ca(II)/Mg(II) ions are not able to activate the metal-free enzyme, either in the absence or presence of 5 mM reduced glutathione. When different metal ion reconstituted ACMSD is used, the kinetic constant Km varies. Ultimately, these results suggest that ACMSD is a metaldependent enzyme and the metallocenter plays a catalytic role in the non-oxidative C-C bond cleavage. We propose that ACMSD is a new member in the amidohydrolase superfamily, representing a new metal-dependent activity.
PapersCloning and sequence analysis of the 4-hydroxybenzoate 3-hydroxylase gene from a cyclohexanecarboxylate-degrading gram-positive bacterium, ”Corynebacterium cyclohexanicum” strain ATCC 51369In refereedAcademic JournalCo-authorHiroaki Iwaki;Hiroshi Saji;Kazuya Abe;Yoshie Hasegawa;Microbes and Environments20/3 144-1502005/9～A DNA fragment that carries the gene encoding 4-hydroxybenzoate 3-hydroxylase (pobA) together with genes encoding a potential regulator (pobR) and a potential transporter (pobK) was cloned from ”Corynebacterium cyclohexanicum” strain ATCC 51369, which is herein reclassified as belonging to the genus Arthrobacter. Nucleotide sequencing revealed that the deduced amino acid sequence encoded by the Arthrobacter pobA gene exhibits 42.0-46.7% identity with that of gram-negative bacteria. The gene organization of the pob cluster differs from that of gram-negative bacteria. The pobA gene product (PobA), expressed in Escherichia coli, preferred NADH over NADPH similar to 4-hydroxybenzoate 3-hydroxylase of ATCC 51369. The Arthrobacter pobA gene was inactivated by insertion of pK19mob. The resultant mutant strain, POBA1, grew on neither cyclohexanecarboxylate nor 4-hydroxybenzoate. These results strongly suggest that the cloned pobA gene plays an essential role in the catabolism not only of 4-hydroxybenzoate but also of cyclohexanecarboxylate in strain ATCC 51369
PapersDegradation of cyclopentanol by Trichosporon cutaneum strain KUY-6AIn refereedAcademic JournalCo-authorHiroaki Iwaki;Hiroshi Saji;Emiko Nakai;Yoshie Hasegawa;Microbes and Environments19(3) pp.241-2432004/9～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.
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;;2004/9～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;;2004/3～
Academic presentationUnrefereedOtherCo-authorIWAKI,Hiroaki;;;;2004/3～
Biocatalysts and microorganisms as environmental and sustainability toolsOtherCo-authorP.C.K.Lau;H.Bergeron;J.Boyd;S.Grosse;S.Z.Wang;J.Z.Yang;K.Rankin;T.Sulea;A.Imura;A.Miyadera;H.Iwaki;T.Muraki;Y.Hasegawa;2004/1～
UnrefereedIn-house publicationCo-authorIWAKI Hiroaki;;2003/11～
International academic conferenceA REACT-IRTM ANALYSIS OF LAURYL LACTONE PRODUCTION BY CYCLODODECANONE MONOOXYGENASE DERIVED FROM Pseudomonas putida STRAIN HI-70OtherCo-authorAyako Nagahashi;Hiroaki Iwaki;Yoshie Hasegawa;Jianzhong Yang;Stephan Grosse;Helene Bergeron;Jason Boyd;Peter C. K. Lau;Pseudomonas 20032003/9～Pseudomonas 2003ReactIRTM 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 molecular
information 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 are
flavoproteins 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 applications
include the synthesis of natural products and
production 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.
International academic conferenceCLONING AND CHARACTERIZATION OF THE INITIAL 2-NITROBENZOATE NITROREDUCTASE AND 2-HYDROXYLAMINOBENZOATE MUTASE ENCODING GENES FOR 2-NITROBENZOATE CATABOLISM IN PSEUDOMONAS FLUORESCENS STRAIN KU-7UnrefereedOtherInternational coauthorshipTakamichi Muraki;Ayako Nagahashi;Masami Taki;Hiroaki Iwaki;Yoshie Hasegawa;Peter C.K. Lau;Pseudomonas 20032003/9～Pseudomonas 20032-nitrobenzoic acid (2-NBA) via the novel formation of 3-hydroxyanthranilic acid (3-HAA) instead of anthranilate or 2-aminobenzoate as an
intermediate. 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 nba
pathway, 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 were
found 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.
PapersMonooxygenase-catalysed Baeyer-Villger oxidations: CHMO versus CPMOIn refereedAcademic JournalInternational coauthorshipS.Wang;M.M.Kayser;H.Iwaki;P.C.K.Lau;Cyclohexanone monooxygenase;Cyclopentanone monooxygenase;Baeyer-villiger oxidations;J. Mol. Cat. B: Enzymatic22/34,2112003/6/2～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.colistrain 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.
International academic conferenceA ReactIR study of cyclododecanone monooxygenase-catalyzed reaction for lauryl lactone productionOtherCo-authorJ.Yang;H.Iwaki;H.Bergeron;PCK.Lau;Annual general meeting, Canadian Society of Microbiologists2003/5～
PapersProkaryotic homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase in the degradation pathway of 2-nitrobenzoate by Pseudomonas fluorescens strain KU-7In refereedAcademic JournalInternational coauthorshipT.Muraki;M.Taki;Y.Hasegawa;H.Iwaki;P.C.K.Lau;adipic acid;green chemistry;Acinetobacter;gene cloning;Biodegradation;Cyclohexanol;Appl. Enviorn. Microbiol.69/3, 1564-15722003/3～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 enocompasses the 3-hydroxyanthranilate meta-cleavage pathway genes.The gene cluster, designated nbaEX HJIGFCDR, is organized tightly and in the same derection.The nbaC and nbaDgene products were found to be novel homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3carboxymuconate-6semialdehyde 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 coliand 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-derected 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 Fe┏sup┓2+┏/sup┓but is inhibited by other metal ions, such as Zn┏sup┓2+┏/sup┓, Cu┏sup┓2+┏/sup┓, and Cd┏sup┓2+┏/sup┓.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.
PapersCyclohexanol biodegradation genes: A pathway of opportunitiesIn refereedAcademic JournalInternational coauthorshipH.Iwaki;Y.hasegawa;M.Teraoka;T.Tokuyama;L.Bernard;P.C.K.Lau;Biodegradation;gene cloning;Pseudomonas fluorescens;3-hydroxyanthranilate;2-nitrobenzoic acid;Biocatalysis in Polymer Science, ACS Symposium Series840, 802003～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 β -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; ε -caprolactone hydrolase)and three oxido-reductases(ChnA, cyclohexanol dehydrogenase; ChnD, 6-hydroxyhexanoate dehydrogenase;and ChnE,6-oxohexanoate dehydrogenase).Besides the will 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.
PatentsA gene encoding cyclododecanone monooxygenaseCo-authorH.Iwaki;Y.Hasegawa;PCK.Lau;WO03025164, EP1430118, CA24604992003～The invention relates to a new strain of Pseudomonas putida (designated as HI-70) and to the isolation, cloning, and sequencing of a cyclododecanone monooxygenase-encoding gene (named cdnB) from said strain. The invention also relates to a new cyclododecanone monooxygenase and to a method of use of the cyclododecanone monooxygenase-encoding gene.
PapersCloning and characterization of a gene cluster involved in cyclopentanol metabolism in Comamonas sp. strain NCIMB 9872 and biotransformations effected by Escherichia coli-expressed cyclopentanone 1,2-monooxygenaseIn refereedAcademic JournalInternational coauthorshipH. Iwaki;Y.Hasegawa.;S.Wang;M.M.Kayser;P.C.K.Lau;Baeyer-Villiger oxidations;Cyclopentanone 1,2-monooxygenase;biotransformations;Cyclopentanol;gene cloning;Comamonas;Appl. Environ. Microbiol.68/11, 5671-56842002/11～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)-(orf3-orf4[partial 3' end]).The cpnABCDEgenes 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 cpnBby using a lacZ-Kmrcassette resulted in a strain that was not capable of growth on cyclopentanol or cyclopentanoe as a sole carbon and energy source.The presence of σ 54-dependent regulatory elements in front of the divergently transcribed cpnBandcpnCgenes supports the notion that cprR is a regulatory gene of the NtrC type.Knowledge of the nucleotide sequence of the cpn genes was used to construct isopropyl- β -thio-D-galactoside-inducible clones of Escherichia coli cells that overproduce the five enzymes of the cpnpathway.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.
PapersBaeyer-Villiger oxidations catalyzed by engineered microorganisms:enantioselective synthesis of δ -valerolactones with functionalized chainsIn refereedAcademic JournalInternational coauthorshipS.Wang;G.Chen;M.M.Kayser;H.Iwaki;P.C.K.Lau;Y.Hasegawa;optically pure 2-substituted Cyclopentanones;optically pure lactons;Cyclohexanone monooxygenase;Cyclohexanone monooxygenase;biotransformation;Baeyer-Villger oxidations;Can. J. Chem.80/6, 6132002/6～Abstract: Cyclohexanone monooxygenase(CHMO)from Acinetobacter sp NCIMB 9871 expressed in baker's yeast and in E. coli and cyclopentanone monooxygenase(CPMO)from Comamonas(previously Pseudomonas)sp.NCIMB 9872 expressed in E. coli are new bioreagents for Baeyer-Villiger oxidations.These engineered microorganisms, requiring neither biochemical expertise nor equipment beyond that found in chemical laboratories, were evaluated as reagents for Baeyer-Villiger oxidations of cyclopentanones substituted at the 2-position with polar and nonpolar chains suitable for further modifications.Two such functionalized substrates that can be transformed into highly enantiopure lactones were identifide.The perfomance and the potential of these bioreagents are discussde.
PatentsCloning, sequencing, and expression of a Comamonas cyclopentanone 1,2-monooxygenase encoding gene in Escherichia coliCo-authorH.Iwaki;Y.Hasegawa;PCK.Lau;WO0206452, CA24181552002～Cyclopentanone 1,2-monooxygenase (CPMO) from Comamonas (previously Pseudomonas) sp. strain NCIMB 9872 carries out the second step of a degradation pathway that allows the bacterium to use cyclopentanol as a sole carbon source for growth. In the present invention there is reported the localization of the CPMO-encoding gene (cpnB) on a 4.3-kb SphI fragment, the determination of its sequence. The 550-amino acid CPMO polypeptide (Mr, 62,111) encoded by the gene was found to have 36.5% identity with the sequence of cyclohexanone 1,2-monooxygenase (CHMO) of Acinetobacter sp. strain NCIMB 9871. The 62-kDa CPMO was expressed in E. coli as an IPTG-inducible protein.
International academic conference3-hydroxyanthranilate 3,4-dioxygenase, a novel eukaryotic homolog in the degradation pathway of 2-nitrobenzoate by Pseudomonas fluorescens strain KU-7OtherCo-authorT.Muraki;M.Taki;Y.Hasegawa;H.Iwaki;P.C.K.Lau;ASM conference on Biodegradation, Biotransformation and Biocatalysis2001/10～American Society for Microbiology3-hydroxyanthranilate 3,4-dioxygenase, a novel eukaryotic homolog in the degradation pathway of 2-nitrobenzoate by Pseudomonas fluorescens strain KU-7
International academic conferenceCyclopentanone 1,2-monooxygenase:Old wine, new bottleOtherCo-authorH. Iwaki;Y. Hasegawa;S.Wang;M.Kayser;P.C. Lau;ASM conference on Biodegradation, Biotransformation and Biocatalysis2001/10～ASM conference on Biodegradation, Biotransformation, and BiocatalysisCyclopentanone 1,2-monooxygenase:Old wine, new bottle
International academic conferenceBiodegradation of cycloaliphatic compounds: New genes and opportunitiesOtherCo-authorH.Iwaki;Y.Hasegawa;PCK. Lau;Pseudomonas 20012001/9～Pseudomonas 2001
PapersPurification and characterization of cyclohexanone 1,2-monooxygenase from Exophiala jeanselmei KUFI-6NIn refereedAcademic JournalCo-authorY.Hasegawa;Y.Nakai;T.Tokuyama;H.Iwaki;Exophiala jeanselmei;Cyclohexanone monooxygenase;Baeyer-Villger oxidation;Biosci. Biotechnol. Biochem.64/12, 26962000/12～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-Tyr-Gln-Leu-, with similarity to the bacterial CHMOs of FAD-binding and NADPH-dependent type Baeyer-Villiger monooxygenases.
PapersA novel degradation pathway of 2-nitrobenzoate via 3-hydroxyanthranilate in Pseudomonas fluoresens strain KU-7In refereedAcademic JournalInternational coauthorshipY.Hasegawa;T.Muraki;T.Tokuyama;H.Iwaki;M.Tatsuno;P.C.K.Lau;Pseudomonas;nitoroaromatic componud;3-hydroxyanthranilate;biodegradation;FEMS Microbiol. Lett.190/2, 1852000/9/15～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 indecating that degradation of 2-NBA proceeds through a reductive route.Metabolite analyses by thin layer chromatography and high pressure liquid chromatography nidicated 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 abosorbance 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.
International academic conferenceA fistful of BVMOs (Baeyer-Villiger monooxygenase)OtherCo-authorH.Iwaki;Y.Hasegawa;T.Tokuyama;A.Imura;H.Bergeron;PCK.Lau;IBC's 5th Annual World Congress on Enzyme Technologies 20002000/2～IBC's 5th Annual World Congress on Enzyme Technologies 2000
PapersCloning and expression of N,N-demethylformamidase gene from Alcaligenes sp.KUFA-1In refereedAcademic JournalCo-authorY.Hasegawa;T.Tokuyama;H.Iwaki;N,N-dimethylformamide;DMF;gene cloning;Alcaligenes;N,N-dimethylformamidase;Biosci. Biotechnol. Biochem.63/12, 20911999/12～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 α -and two β -subunits with 132 and 762 amino acids, respectively, and had little similarity to sequences in protein datebases, including various amidases.The protein may be a new kind of amidase.DMFase activity was detected in E. coli cells tramsformed 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.
PapersIdentification of a transcriptional activetor (ChnR) and 6-oxohexanoate dehydrogenase (ChnE) in the cyclohexanol catabolic pathway in Acinetobacter sp. NCIMB 9871, and Localization of their encoding genesIn refereedAcademic JournalInternational coauthorshipH.Iwaki;Y.Hasegawa;M.Teraoka;T.Tokuyama;H.Bergeron;P.C.K.Lau;Acinetobacter;AraC/XylS type;transcriptional activator;Cyclohexanol degradation;Appl. Environ. Microbiol.65/11, 51581999/11～We identified chnR, a gene encoding an AraC-XylS type of transcriptional activator that regulates the expression of chnB, the structural gene for cyclohexanone monooxygenase(CHMO) in Acinetobactersp.strain NCIMB 9871.The gene sequence of chnE, which encodes an NADP+-linked 6-oxohexanoate dehydrogenase, the enzyume catalyzing the fifth step of cyclohexanol degradation, was also determinec The gene arrangement is chnB-chnE-chnR.The predicted molecular masses of the three polypeptides were verified by radiolabeling by using the T7 expression system.Inducible expression of cloned chnB in Escherchia coli dependent upon the presence of chnR.A tramscriptional chnB::lacZ fusion experiment revealed that cyclohexanone induces chnBexpression in E. coli, in which a 22-fold increase in activity was observed.
PapersPurification and characterization of a novel cyclohexylamine oxidase from the cyclohexylamine-degrading Brevibacterium IH-35AIn refereedAcademic JournalCo-authoredH.Iwaki;M.Shimizu;T.Tokuyama;Y.Hasegawa;FAD-containing amine oxidase;Brevibacterium;Cyclohexylamine oxidase;Cyclohexylamine degration;J. Biosci. Bioeng.88/3, 2641999/9～Cyclohexylamine oxidase(CHAO) from a cell extract of Brevibacterium grown on cyclohexylamine was purified 50.2-fold, to clectrophoretic 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 ℃ .The enzyme was found to be highly specific for the deamination of alicyclic monoamines, such as cyclopentylamine, cycloheptylamine, and N-methylcycoihexylamine and plophatic monoamines, sudh as sec-butylamine.The apparent Km value for cyclohexylamine was 1.23 mM.The enzyume was inhibited by flavin enzyume 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-Ile-His-Gly-Ala-Gly-Ile-Ser-Gly-Ser-Ala-Ala-Ala-Lys-Ala-Leu-,revealing homology to conventional flavin-containig amine oxidases(EC 1.4.3.4).
PapersBiodegradation of cyclohexylamine by Brevibacteium oxydans IH-35AIn refereedAcademic JournalCo-authorH.Iwaki;M.Shimizu;T.Tokuyama;Y.Hasegawa;Cyclohexanone monooxygenase;Cyclohexanone;Brevibacterium;Cyclohexylamine degradation;Appl. Enviorn. Microbiol.65/5, 23221999/5～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 CHAM contained CHAM oxidase, and the model for CHAM oxidation by this enzyme was similar to the model for deamino oxidation of amine by amine oxidase.
Research Activities Overseas
- Study abroadSep. 2000-Mar. 2003Canada Biotechnology Research Institute/National Research Council of Canada
Participation in International Conferences
- Pseudomonas 2003 Sep.2003-Sep. 2003
- ASM conference on Biodegradation, Biotransformation, Biocatalysis Oct.2001-Oct. 2001
Courses Taught
- Experiments of Chemistry
- Biology I
- Freshmen Seminar
- Fundamental Experiments of Life Science
- Genetic Engineering
- Experiments of Biology
- Experiments of Biotechnology
- Exercises in Thesis Projects I and II
- Thesis Projects I
- Thesis Projects II
- Science and Technology English I
- SeminarI（Biotechnology)
- SeminarII（Biotechnology)
- SeminarIII（Biotechnology)
- SeminarIV（Biotechnology)
- Advanced Environmental Microbiology
- Advanced Environmental Microbiology
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