Tokushima University / Educator and Researcher Directory --- Hayashi, Junji

Personal Web Page

https://researchmap.jp/7000020435 (researchmap)

https://orcid.org/0000-0001-6447-1600 (ORCID)

Field of Study

○	Enzyme Engineering, Structural Biology

Subject of Study

○

Book / Paper

Academic Paper (Judged Full Paper):

1.	Chizuru Sasaki, Satoshi Tamura, Miyuki Suzuki, Kanako Etomi, Nobuya Nii, Junji Hayashi and Kaori Kanemaru : Continuous microwave-assisted step-by-step extraction of bioactive water-soluble materials and fucoidan from brown seaweed Undaria pinnatifida waste, Biomass Conversion and Biorefinery, Vol.14, 7673-7682, 2024. (Link to Publication Site) ● Publication site (DOI): 10.1007/s13399-022-03035-6 (Link to Search Site for Scientific Articles) ● CiNii @ National Institute of Informatics (CRID): 1360298754835963008 ● Summary page in Scopus @ Elsevier: 2-s2.0-85133905658 (DOI: 10.1007/s13399-022-03035-6, CiNii: 1360298754835963008, Elsevier: Scopus)
2.	Yoshimichi Takai, Rumana Yesmin Hasi, Naoko Matsumoto, Chiho Fujita, Hanif Ali, Junji Hayashi, Ryushi Kawakami, Mutsumi Aihara, Toshiki Ishikawa, Hiroyuki Imai, Mayuko Wakida, Kazuya Ando and Tamotsu Tanaka : Degradation of glycosylinositol phosphoceramide during plant tissue homogenization, The Journal of Biochemistry, Vol.175, No.1, 115-124, 2024. (Summary) A convenient method for the determination of plant sphingolipids (glycosylinositol phosphoceramide, GIPC; glucosylceramide, GluCer; phytoceramide 1-phosphate, PC1P and phytoceramide, PCer) was developed. This method includes the extraction of lipids using 1-butanol, alkali hydrolysis with methylamine and separation by TLC. The amounts of sphingolipids in the sample were determined based on the relative intensities of standard sphingolipids visualized by primulin/UV on TLC. Using this method, we found that almost all GIPCs were degraded in response to tissue homogenization in cruciferous plants (cabbage, broccoli and Arabidopsis thaliana). The decrease in GIPCs was compensated for by increases in PC1P and PCer, indicating that GIPC was degraded by hydrolysis at the D and C positions of GIPC, respectively. In carrot roots and leaves, most of GIPC degradation was compensated for by an increase in PCer. In rice roots, the decrease in GIPCs was not fully explained by the increases in PC1P and PCer, indicating that enzymes other than phospholipase C and D activities operated. As the visualization of lipids on TLC is useful for detecting the appearance or disappearance of lipids, this method will be available for the characterization of metabolism of sphingolipids in plants. (Keyword) Glycosphingolipids / Sphingolipids / Plants / Brassica / Arabidopsis (Tokushima University Institutional Repository) ● Metadata: 118914 (Link to Publication Site) ● Publication site (DOI): 10.1093/jb/mvad080 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 37827526 ● CiNii @ National Institute of Informatics (CRID): 1520862036472955648 ● Summary page in Scopus @ Elsevier: 2-s2.0-85181760535 (Tokushima University Institutional Repository: 118914, DOI: 10.1093/jb/mvad080, PubMed: 37827526, CiNii: 1520862036472955648, Elsevier: Scopus)
3.	Ryushi Kawakami, Takami Naoki, Junji Hayashi, Kazunari Yoneda, Ohmori Taketo, Toshihisa Ohshima and Haruhiko Sakuraba : First crystal structure of an NADP+-dependent L-arginine dehydrogenase belonging to the μ-crystallin family, International Journal of Biological Macromolecules, Vol.249, 2023. (Keyword) L-Arginine dehydrogenase / μ-Crystallin/ornithine cyclodeaminase family / Crystal structure / Amino acid dehydrogenase / Site-directed mutagenesis (Tokushima University Institutional Repository) ● Metadata: 119010 (Link to Publication Site) ● Publication site (DOI): 10.1016/j.ijbiomac.2023.126070 (Link to Search Site for Scientific Articles) ● Search Scopus @ Elsevier (DOI): 10.1016/j.ijbiomac.2023.126070 (Tokushima University Institutional Repository: 119010, DOI: 10.1016/j.ijbiomac.2023.126070)
4.	Hanif Ali, Miyu Kobayashi, Katsuya Morito, Rumana Yesmin, Mutsumi Aihara, Junji Hayashi, Ryushi Kawakami, Koichiro Tsuchiya, Kazunori Sango and Tamotsu Tanaka : Peroxisomes attenuate cytotoxicity of very long-chain fatty acids, Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, Vol.1868, No.2, 159259, 2023. (Summary) One of the major functions of peroxisomes in mammals is oxidation of very long-chain fatty acids (VLCFAs). Genetic defects in peroxisomal β-oxidation result in the accumulation of VLCFAs and lead to a variety of health problems, such as demyelination of nervous tissues. However, the mechanisms by which VLCFAs cause tissue degeneration have not been fully elucidated. Recently, we found that the addition of small amounts of isopropanol can enhance the solubility of saturated VLCFAs in an aqueous medium. In this study, we characterized the biological effect of extracellular VLCFAs in peroxisome-deficient Chinese hamster ovary (CHO) cells, neural crest-derived pheochromocytoma cells (PC12), and immortalized adult Fischer rat Schwann cells (IFRS1) using this solubilizing technique. C20:0 FA was the most toxic of the C16-C26 FAs tested in all cells. The basis of the toxicity of C20:0 FA was apoptosis and was observed at 5 μM and 30 μM in peroxisome-deficient and wild-type CHO cells, respectively. The sensitivity of wild-type CHO cells to cytotoxic C20:0 FA was enhanced in the presence of a peroxisomal β-oxidation inhibitor. Further, a positive correlation was evident between cell toxicity and the extent of intracellular accumulation of toxic FA. These results suggest that peroxisomes are pivotal in the detoxification of apoptotic VLCFAs by preventing their accumulation. (Keyword) Cricetinae / Animals / Peroxisomes / Fatty Acids / CHO Cells / Cricetulus / Oxidation-Reduction (Tokushima University Institutional Repository) ● Metadata: 117825 (Link to Publication Site) ● Publication site (DOI): 10.1016/j.bbalip.2022.159259 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 36460260 ● Search Scopus @ Elsevier (PMID): 36460260 ● Search Scopus @ Elsevier (DOI): 10.1016/j.bbalip.2022.159259 (Tokushima University Institutional Repository: 117825, DOI: 10.1016/j.bbalip.2022.159259, PubMed: 36460260)
5.	Rumana Yesmin Hasi, Toshiki Ishikawa, Keigo Sunagawa, Yoshimichi Takai, Hanif Ali, Junji Hayashi, Ryushi Kawakami, Keizo Yuasa, Mutsumi Aihara, Kaori Kanemaru, Hiroyuki Imai and Tamotsu Tanaka : Nonspecific phospholipase C3 of radish has phospholipase D activity toward glycosylinositol phosphoceramide, FEBS Letters, Vol.596, No.23, 3024-3036, 2022. (Summary) Glycosylinositol phosphoceramide (GIPC) is a major sphingolipid in the plasma membranes of plants. Previously, we found an enzyme activity that produces phytoceramide 1-phosphate (PC1P) by hydrolysis of the D position of GIPC in cabbage and named this activity as GIPC-phospholipase D (PLD). Here, we purified GIPC-PLD by sequential chromatography from radish roots. Peptide mass fingerprinting analysis revealed that the potential candidate for GIPC-PLD protein was nonspecific phospholipase C3 (NPC3), which has not been characterized as a PLD. The recombinant NPC3 protein obtained by heterologous expression system in Escherichia coli produced PC1P from GIPC and showed essentially the same enzymatic properties as those we characterized as GIPC-PLD in cabbage, radish and Arabidopsis thaliana. From these results, we conclude that NPC3 is one of the enzymes that degrade GIPC. (Keyword) Phospholipase D / Raphanus / Phospholipases / Sphingolipids / Brassica / Arabidopsis (Tokushima University Institutional Repository) ● Metadata: 117824 (Link to Publication Site) ● Publication site (DOI): 10.1002/1873-3468.14520 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 36266963 ● Search Scopus @ Elsevier (PMID): 36266963 ● Search Scopus @ Elsevier (DOI): 10.1002/1873-3468.14520 (Tokushima University Institutional Repository: 117824, DOI: 10.1002/1873-3468.14520, PubMed: 36266963)
6.	Ryushi Kawakami, Tatsuya Ohshida, Junji Hayashi, Kazunari Yoneda, Toshio Furumoto, Toshihisa Ohshima and Haruhiko Sakuraba : Crystal structure of a novel type of ornithine δ-aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii, International Journal of Biological Macromolecules, Vol.208, 731-740, 2022. (Summary) Ornithine δ-aminotransferase (Orn-AT) activity was detected for the enzyme annotated as a γ-aminobutyrate aminotransferase encoded by PH1423 gene from Pyrococcus horikoshii OT-3. Crystal structures of this novel archaeal ω-aminotransferase were determined for the enzyme in complex with pyridoxal 5'-phosphate (PLP), in complex with PLP and l-ornithine (l-Orn), and in complex with N-(5'-phosphopyridoxyl)-l-glutamate (PLP-l-Glu). Although the sequence identity was relatively low (28%), the main-chain coordinates of P. horikoshii Orn-AT monomer showed notable similarity to those of human Orn-AT. However, the residues recognizing the α-amino group of l-Orn differ between the two enzymes. In human Orn-AT, Tyr55 and Tyr85 recognize the α-amino group, whereas the side chains of Thr92* and Asp93, which arise from a loop in the neighboring subunit, form hydrogen bonds with the α-amino group of the substrate in P. horikoshii enzyme. Site-directed mutagenesis suggested that Asp93 plays critical roles in maintaining high affinity for the substrate. This study provides new insight into the substrate binding of a novel type of Orn-AT. Moreover, the structure of the enzyme with the reaction-intermediate analogue PLP-l-Glu bound provides the first structural evidence for the "Glu switch" mechanism in the dual substrate specificity of Orn-AT. (Keyword) Archaea / Crystallography, X-Ray / Humans / Models, Molecular / Ornithine / Pyridoxal Phosphate / Pyrococcus horikoshii / Substrate Specificity / Transaminases (Tokushima University Institutional Repository) ● Metadata: 119006 (Link to Publication Site) ● Publication site (DOI): 10.1016/j.ijbiomac.2022.03.114 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 35337912 ● Summary page in Scopus @ Elsevier: 2-s2.0-85127367273 (Tokushima University Institutional Repository: 119006, DOI: 10.1016/j.ijbiomac.2022.03.114, PubMed: 35337912, Elsevier: Scopus)
7.	Ali Hanif, Morito Katsuya, Rumana Hasi Yesmin, Mutsumi Aihara, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru, Koichiro Tsuchiya, Sango Kazunori and Tamotsu Tanaka : Characterization of uptake and metabolism of very long-chain fatty acids in peroxisome-deficient CHO cells, Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, Vol.1867, No.2, 159088, 2022. (Summary) Fatty acids (FAs) longer than C20 are classified as very long-chain fatty acids (VLCFAs). Although biosynthesis and degradation of VLCFAs are important for the development and integrity of the myelin sheath, knowledge on the incorporation of extracellular VLCFAs into the cells is limited due to the experimental difficulty of solubilizing them. In this study, we found that a small amount of isopropanol solubilized VLCFAs in aqueous medium by facilitating the formation of the VLCFA/albumin complex. Using this solubilizing technique, we examined the role of the peroxisome in the uptake and metabolism of VLCFAs in Chinese hamster ovary (CHO) cells. When wild-type CHO cells were incubated with saturated VLCFAs (S-VLCFAs), such as C23:0 FA, C24:0 FA, and C26:0 FA, extensive uptake was observed. Most of the incorporated S-VLCFAs were oxidatively degraded without acylation into cellular lipids. In contrast, in peroxisome-deficient CHO cells uptake of S-VLCFAs was marginal and oxidative metabolism was not observed. Extensive uptake and acylation of monounsaturated (MU)-VLCFAs, such as C24:1 FA and C22:1 FA, were observed in both types of CHO cells. However, oxidative metabolism was evident only in wild-type cells. Similar manners of uptake and metabolism of S-VLCFAs and MU-VLCFAs were observed in IFRS1, a Schwan cell-derived cell line. These results indicate that peroxisome-deficient cells limit intracellular S-VLCFAs at a low level by halting uptake, and as a result, peroxisome-deficient cells almost completely lose the clearance ability of S-VLCFAs accumulated outside of the cells. (Tokushima University Institutional Repository) ● Metadata: 116579 (Link to Publication Site) ● Publication site (DOI): 10.1016/j.bbalip.2021.159088 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 34848380 ● CiNii @ National Institute of Informatics (CRID): 1360013168847983360 ● Search Scopus @ Elsevier (PMID): 34848380 ● Search Scopus @ Elsevier (DOI): 10.1016/j.bbalip.2021.159088 (Tokushima University Institutional Repository: 116579, DOI: 10.1016/j.bbalip.2021.159088, PubMed: 34848380, CiNii: 1360013168847983360)
8.	Hanif Ali, Ryouhei Yamashita, Jun-ichi Morishige, Katsuya Morito, Naoya Kakiuchi, Junji Hayashi, Mutsumi Aihara, Ryushi Kawakami, Koichiro Tsuchiya and Tamotsu Tanaka : Massspectrometric analysis of sphingomyelin with N-alfa-hydroxy fatty acyl residue in mouse tissues, Lipids, Vol.56, No.2, 181-188, 2021. (Tokushima University Institutional Repository) ● Metadata: 117826 (Link to Publication Site) ● Publication site (DOI): 10.1002/lipd.12285 (Link to Search Site for Scientific Articles) ● Search Scopus @ Elsevier (DOI): 10.1002/lipd.12285 (Tokushima University Institutional Repository: 117826, DOI: 10.1002/lipd.12285)
9.	Ryushi Kawakami, Chinatsu Kinoshita, Tomoki Kawase, Mikio Sato, Junji Hayashi, Haruhiko Sakuraba and Toshihisa Ohshima : Characterization of novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis., Bioscience, Biotechnology, and Biochemistry, Vol.85, No.7, 1650-1657, 2021. (Summary) The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as γ-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5'-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by coexpression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and nonsubstrate amino acids such as l-Arg and l-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea. (Keyword) PLP / amino acid racemase / substrate specificity / Thermococcus litoralis (Tokushima University Institutional Repository) ● Metadata: 116135 (Link to Publication Site) ● Publication site (DOI): 10.1093/bbb/zbab078 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 33942867 ● Search Scopus @ Elsevier (PMID): 33942867 ● Search Scopus @ Elsevier (DOI): 10.1093/bbb/zbab078 (Tokushima University Institutional Repository: 116135, DOI: 10.1093/bbb/zbab078, PubMed: 33942867)
10.	Junji Hayashi, Yoshiaki Ichiki, Akiko Kanda, Kazuyoshi Takagi and Mamoru Wakayama : Identification, characterization, and cloning of a novel aminoacylase, L-pipecolic acid acylase from Pseudomonas species, The Journal of General and Applied Microbiology, 2021. (Summary) L-Pipecolic acid is utilized as a vital component of specific chemical compounds, such as immunosuppressive drugs, anticancer reagents, and anesthetic reagents. We isolated and characterized a novel L-aminoacylase, N-acetyl-L-pipecolic acid-specific aminoacylase (LpipACY), from Pseudomonas sp. AK2. The subunit molecular mass of LpipACY was 45 kDa and was assumed to be a homooctamer in solution. The enzyme exhibited high substrate specificity toward N-acetyl-L-pipecolic acid and a high activity for N-acetyl-L-pipecolic acid and N-acetyl-L-proline. This enzyme was stable at a high temperature (60°C for 10 min) and under an alkaline pH (6.0-11.5). The N-terminal and internal amino acid sequences of the purified enzyme were STTANTLILRNG and IMASGGV, respectively. These sequences are highly consistent with those of uncharacterized proteins from Pseudomonas species, such as amidohydrolase and peptidase. We also cloned and overexpressed the gene coding LpipACY in Escherichia coli. Moreover, the recombinant LpipACY exhibited properties similar to native enzyme. Our results suggest that LpipACY is a potential enzyme for the enzymatic synthesis of L-pipecolic acid. This study provides the first description of the enzymatic characterization of L-pipecolic acid specific amino acid acylase. (Link to Publication Site) ● Publication site (DOI): 10.2323/jgam.2020.12.001 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 34176819 ● Search Scopus @ Elsevier (PMID): 34176819 ● Search Scopus @ Elsevier (DOI): 10.2323/jgam.2020.12.001 (DOI: 10.2323/jgam.2020.12.001, PubMed: 34176819)
11.	Tasneem Chemama, Junji Hayashi, Mamoru Wakayama and Narumol Thongwai : Characteristics of D-lactate Dehydrogenase from the High Potential D-lactic Acid Producer Leuconostoc pseudomesenteroides TC49 Isolated from Thailand, Chiang Mai Journal of Science, Vol.48, No.1, 42-55, 2021. (Link to Search Site for Scientific Articles) ● Summary page in Scopus @ Elsevier: 2-s2.0-85099648660 (Elsevier: Scopus)
12.	Chiharu Sano, Takafumi Itoh, Putthapong Phumsombat, Junji Hayashi, Mamoru Wakayama and Takao Hibi : Mutagenesis and structure-based analysis of the role of Tryptophan525 of γ-glutamyltranspeptidase from Pseudomonas nitroreducens., Biochemical and Biophysical Research Communications, 2021. (Summary) γ-Glutamyltranspeptidase (GGT) is a ubiquitous enzyme that catalyzes the hydrolysis of the γ-glutamyl linkage of γ-glutamyl compounds and the transfer of their γ-glutamyl moiety to acceptor substrates. Pseudomonas nitroreducens GGT (PnGGT) is used for the industrial synthesis of theanine, thus it is important to determine the structural basis of hydrolysis and transfer reactions and identify the acceptor site of PnGGT to improve the efficient of theanine synthesis. Our previous structural studies of PnGGT have revealed that crucial interactions between three amino acid residues, Trp385, Phe417, and Trp525, distinguish PnGGT from other GGTs. Here we report the role of Trp525 in PnGGT based on site-directed mutagenesis and structural analyses. Seven mutant variants of Trp525 were produced (W525F, W525V, W525A, W525G, W525S, W525D, and W525K), with substitution of Trp525 by nonaromatic residues resulting in dramatically reduced hydrolysis activity. All Trp525 mutants exhibited significantly increased transfer activity toward hydroxylamine with hardly any effect on acceptor substrate preference. The crystal structure of PnGGT in complex with the glutamine antagonist, 6-diazo-5-oxo-l-norleucine, revealed that Trp525 is a key residue limiting the movement of water molecules within the PnGGT active site. (Link to Publication Site) ● Publication site (DOI): 10.1016/j.bbrc.2020.11.093 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 33288198 ● Search Scopus @ Elsevier (PMID): 33288198 ● Search Scopus @ Elsevier (DOI): 10.1016/j.bbrc.2020.11.093 (DOI: 10.1016/j.bbrc.2020.11.093, PubMed: 33288198)
13.	Takafumi Itoh, Niphawan Panti, Junji Hayashi, Yosuke Toyotake, Daisuke Matsui, Shigekazu Yano, Mamoru Wakayama and Takao Hibi : Crystal structure of the catalytic unit of thermostable GH87 α-1,3-glucanase from Streptomyces thermodiastaticus strain HF3-3., Biochemical and Biophysical Research Communications, Vol.533, No.4, 1170-1176, 2020. (Summary) α-1,3-Glucan is a homopolymer composed of D-glucose (Glc) and it is an extracellular polysaccharide found in dental plaque due to Streptococcus species. α-1,3-Glucanase from Streptomyces thermodiastaticus strain HF3-3 (Agl-ST) has been identified as a thermostable α-1,3-glucanase, which is classified into glycoside hydrolase family 87 (GH87) and specifically hydrolyzes α-1,3-glucan with an endo-action. The enzyme has a potential to inhibit the production of dental plaque and to be used for biotechnological applications. Here we show the structure of the catalytic unit of Agl-ST determined at 1.16 Å resolution using X-ray crystallography. The catalytic unit is composed of two modules, a β-sandwich fold module, and a right-handed β-helix fold module, which resembles other structural characterized GH87 enzymes from Bacillus circulans str. KA-304 and Paenibacillus glycanilyticus str. FH11, with moderate sequence identities between each other (approximately 27% between the catalytic units). However, Agl-ST is smaller in size and more thermally stable than the others. A disulfide bond that anchors the C-terminal coil of the β-helix fold, which is expected to contribute to thermal stability only exists in the catalytic unit of Agl-ST. (Link to Publication Site) ● Publication site (DOI): 10.1016/j.bbrc.2020.09.133 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 33041007 ● Search Scopus @ Elsevier (PMID): 33041007 ● Search Scopus @ Elsevier (DOI): 10.1016/j.bbrc.2020.09.133 (DOI: 10.1016/j.bbrc.2020.09.133, PubMed: 33041007)
14.	Rumana Yesmin Hasi, Dai Majima, Katsuya Morito, Hanif Ali, Kentaro Kogure, Meera Nanjundan, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru and Tamotsu Tanaka : Isolation of glycosylinositol phosphoceramide and phytoceramide 1-phosphate in plants and their chemical stabilities., Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, Vol.1152, 122213, 2020. (Summary) Glycosylinositol phosphoceramide (GIPC) is a sphingophospholipid in plants. Recently, we identified that GIPC is hydrolyzed to phytoceramide 1-phosphate (PC1P) by an uncharacterized phospholipase D activity following homogenization of certain plant tissues. We now developed methods for isolation of GIPC and PC1P from plant tissues and characterized their chemical stabilities. Hydrophilic solvents, namely a lower layer of a mixed solvent system consisting of isopropanol/hexane/water (55:20:25, v/v/v) was efficient solvent for extraction and eluent in column chromatography. GIPC was isolated by Sephadex column chromatography followed by TLC. A conventional method, such as the Bligh and Dyer method, was applicable for PC1P extraction. Specifically, PC1P was isolated by TLC following mild alkali treatment of lipid extracts of plants. The yields of GIPC and PC1P in our methods were both around 50-70%. We found that PC1P is tolerant against heat (up to 125 °C), strong acid (up to 10 M HCl), and mild alkali (0.1 M KOH). In contrast, significant degradation of GIPC occurred at 100 °C and 1.0 M HCl treatment, suggesting the instability of the inositol glycan moiety in these conditions. These data will be useful for further biochemical and nutritional studies on these sphingolipids. (Tokushima University Institutional Repository) ● Metadata: 115195 (Link to Publication Site) ● Publication site (DOI): 10.1016/j.jchromb.2020.122213 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 32615533 ● Search Scopus @ Elsevier (PMID): 32615533 ● Search Scopus @ Elsevier (DOI): 10.1016/j.jchromb.2020.122213 (Tokushima University Institutional Repository: 115195, DOI: 10.1016/j.jchromb.2020.122213, PubMed: 32615533)
15.	Putthapong Phumsombat, Chiharu Sano, Hiroki Ikezoe, Junji Hayashi, Takafumi Itoh, Takao Hibi and Mamoru Wakayama : Efficient Production of L-Theanine Using Immobilized Recombinant Escherichia coli Cells Expressing a Modified γ-Glutamyltranspeptidase Gene from Pseudomonas nitroreducens, Advances in Biological Chemistry, Vol.10, No.6, 157-171, 2020. (Link to Publication Site) ● Publication site (DOI): 10.4236/abc.2020.106012 (Link to Search Site for Scientific Articles) ● Search Scopus @ Elsevier (DOI): 10.4236/abc.2020.106012 (DOI: 10.4236/abc.2020.106012)
16.	Takafumi Itoh, Rattanaporn Intuy, Wasana Suyotha, Junji Hayashi, Shigekazu Yano, Koki Makabe, Mamoru Wakayama and Takao Hibi : Structural insights into substrate recognition and catalysis by glycoside hydrolase family 87 α-1,3-glucanase from Paenibacillus glycanilyticus FH11., The FEBS Journal, 2019. (Summary) The α-1,3-glucanase from Paenibacillus glycanilyticus FH11 (Agl-FH1), a member of the glycoside hydrolase family 87 (GH87), hydrolyzes α-1,3-glucan with an endo-action. GH87 enzymes are known to degrade dental plaque produced by oral pathogenic Streptococcus species. In this study, the kinetic analyses revealed that this enzyme hydrolyzed α-1,3-tetraglucan into glucose and α-1,3-triglucan with β-configuration at the reducing end by an inverting mechanism. The crystal structures of the catalytic domain (CatAgl-FH1) complexed with or without oligosaccharides at 1.4-2.5 or 1.6 Å resolutions, respectively, are also presented. The initial crystal structure of CatAgl-FH1 was determined by native single-wavelength anomalous diffraction. The catalytic domain was composed of two modules, a β-sandwich fold module, and a right-handed β-helix fold module. The structure of the β-sandwich was similar to those of the carbohydrate-binding module family 35 members. The glycerol or nigerose enzyme complex structures demonstrated that this β-sandwich fold module is a novel carbohydrate-binding module with the capabilities to bind saccharides and to promote the degradation of polysaccharides. The structures of the inactive mutant in complexes with oligosaccharide showed that at least eight subsites for glucose binding were located in the active cleft of the β-helix fold and the architecture of the active cleft was suitable for the recognition and hydrolysis of α-1,3-glucan by the inverting mechanism. The structural similarity to GH28 and GH49 enzymes and the results of site-directed mutagenesis indicated that three Asp residues, Asp1045, Asp1068, and Asp1069, are the most likely candidates for the catalytic residues of Agl-FH1. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession numbers 6K0M (CatAgl-FH1), 6K0N (WT/nigerose), 6K0P (D1045A/nigerose), 6K0Q (D1068A/nigerose), 6K0S (D1069A/ nigerose), 6K0U (D1068A/oligo), and 6K0V (D1069A/oligo). ENZYMES: Agl-FH1, α-1,3-glucanase (EC3.2.1.59) from Paenibacillus glycanilyticus FH11. (Link to Publication Site) ● Publication site (DOI): 10.1111/febs.15161 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 31788942 ● Search Scopus @ Elsevier (PMID): 31788942 ● Search Scopus @ Elsevier (DOI): 10.1111/febs.15161 (DOI: 10.1111/febs.15161, PubMed: 31788942)
17.	Tatsuya Ohshida, Junji Hayashi, Kazunari Yoneda, Toshihisa Ohshima and Haruhiko Sakuraba : Unique active site formation in a novel galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum., Proteins, 2019. (Summary) A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction. (Link to Publication Site) ● Publication site (DOI): 10.1002/prot.25848 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 31693208 ● Search Scopus @ Elsevier (PMID): 31693208 ● Search Scopus @ Elsevier (DOI): 10.1002/prot.25848 (DOI: 10.1002/prot.25848, PubMed: 31693208)
18.	Rumana Yesmin Hasi, Makoto Miyagi, Takashi Kida, Tatsuya Fukuta, Kentaro Kogure, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru and Tamotsu Tanaka : Quantitative Analysis of Glycosylinositol Phosphoceramide and Phytoceramide 1-Phosphate in Vegetables, Journal of Nutritional Science and Vitaminology, Vol.65, No.Supplement, S175-S179, 2019. (Summary) Previously, we found an unidentified sphingolipid in cabbage, and determined it as phytoceramide 1-phosphate (PC1P). PC1P is found to be produced from glycosylinositol phosphoceramide (GIPC) by the action of phospholipase D (PLD) activity. Although GIPC is abundant sphingolipid, especially in cruciferous vegetables, amount of daily intake, digestibility and nutritional activity of GIPC are not well understood. Here, we investigated amounts of GIPC and PC1P in vegetables. GIPC was found in all vegetables examined (13 kinds) at levels 3-20 mg/100 g (wet weight). On the other hand, PC1P was present in limited vegetables which show higher GIPC-PLD activity, such as inner cabbage leaves (5.2 mg/100 g). Because PC1P is formed during homogenization by activated GIPC-PLD, level of PC1P in boiled cabbage leaves was very low. Although digestibility of GIPC is unknown at present, a portion of dietary GIPC is considered to be converted to PC1P during mastication by plant-derived GIPC-PLD activity in some vegetables. (Keyword) Brassica / Ceramides / Glycosphingolipids / Inositol / Phosphates / Phospholipase D / Plant Leaves / Sphingolipids / Vegetables (Tokushima University Institutional Repository) ● Metadata: 115197 (Link to Publication Site) ● Publication site (DOI): 10.3177/jnsv.65.S175 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 31619623 ● Summary page in Scopus @ Elsevier: 2-s2.0-85073476996 (Tokushima University Institutional Repository: 115197, DOI: 10.3177/jnsv.65.S175, PubMed: 31619623, Elsevier: Scopus)
19.	Kodchakorn Phetsri, Makoto Furukawa, Risa Yamashiro, Yuka Kawamura, Junji Hayashi, Ryuta Tobe, Yosuke Toyotake and Mamoru Wakayama : Comparative Biochemical Characterization of L-Asparaginases from Four Species of Lactic Acid Bacteria, Journal of Biotechnology and Biomedicine, Vol.2, No.3, 112-124, 2019. (Link to Publication Site) ● Publication site (DOI): 10.26502/jbb.2642-91280015 (Link to Search Site for Scientific Articles) ● Search Scopus @ Elsevier (DOI): 10.26502/jbb.2642-91280015 (DOI: 10.26502/jbb.2642-91280015)
20.	Hasi Yesmin Rumana, Makoto Miyagi, Katsuya Morito, Toshiki Ishikawa, Maki Kawai-Yamada, Hiroyuki Imai, Tatsuya Fukuta, Kentaro Kogure, Kaori Kanemaru, Junji Hayashi, Ryushi Kawakami and Tamotsu Tanaka : Glycosylinositol phosphoceramide-specific phospholipase D activity catalyzes transphosphatidylation, The Journal of Biochemistry, Vol.166, No.5, 441-448, 2019. (Summary) Glycosylinositol phosphoceramide (GIPC) is the most abundant sphingolipid in plants and fungi. Recently, we detected GIPC-specific phospholipase D (GIPC-PLD) activity in plants. Here, we found that GIPC-PLD activity in young cabbage leaves catalyzes transphosphatidylation. The available alcohol for this reaction is a primary alcohol with a chain length below C4. Neither secondary alcohol, tertiary alcohol, choline, serine nor glycerol serves as an acceptor for transphosphatidylation of GIPC-PLD. We also found that cabbage GIPC-PLD prefers GIPC containing two sugars. Neither inositol phosphoceramide, mannosylinositol phosphoceramide nor GIPC with three sugar chains served as substrate. GIPC-PLD will become a useful catalyst for modification of polar head group of sphingophospholipid. (Keyword) Biocatalysis / Brassica / Ceramides / Inositol / Molecular Structure / Phosphatidylcholines / Phospholipase D / Plant Leaves (Tokushima University Institutional Repository) ● Metadata: 113685 (Link to Publication Site) ● Publication site (DOI): 10.1093/jb/mvz056 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 31504617 ● Summary page in Scopus @ Elsevier: 2-s2.0-85073764089 (Tokushima University Institutional Repository: 113685, DOI: 10.1093/jb/mvz056, PubMed: 31504617, Elsevier: Scopus)
21.	Rattanaporn Intuy, Takafumi Itoh, Wasana Suyotha, Junji Hayashi, Shigekazu Yano, Koki Makabe, Mamoru Wakayama and Takao Hibi : X-ray crystallographic analysis of the catalytic domain of α-1,3-glucanase FH1 from Paenibacillus glycanilyticus overexpressed in Brevibacillus choshinensis., Acta Crystallographica. Section F, Structural Biology Communications, Vol.74, No.Pt 12, 770-773, 2018. (Summary) 2 (Keyword) Amino Acid Sequence / Brevibacillus / Catalytic Domain / Crystallography, X-Ray / Gene Expression Regulation, Bacterial / Gene Expression Regulation, Enzymologic / Glucans / Glycoside Hydrolases / Paenibacillus (Link to Publication Site) ● Publication site (DOI): 10.1107/S2053230X18013109 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 30511670 ● Search Scopus @ Elsevier (PMID): 30511670 ● Search Scopus @ Elsevier (DOI): 10.1107/S2053230X18013109 (DOI: 10.1107/S2053230X18013109, PubMed: 30511670)
22.	Saddam Md Hossain, Takahiro Tanaka, Junji Hayashi, Kazuyoshi Takagi, Yoichi Takeda and Mamoru Wakayama : Characterization and Thermal Denaturation Kinetic Analysis of Recombinant l-Amino Acid Ester Hydrolase from Stenotrophomonas maltophilia., Journal of Agricultural and Food Chemistry, Vol.66, No.42, 11064-11072, 2018. (Keyword) Amino Acid Sequence / Amino Acids / Carboxylic Ester Hydrolases / Cloning, Molecular / Dipeptides / Enzyme Stability / Escherichia coli / Esters / Gene Expression / Hydrogen-Ion Concentration / Kinetics / Stenotrophomonas maltophilia / Temperature / Thermodynamics / Xanthomonas (Link to Publication Site) ● Publication site (DOI): 10.1021/acs.jafc.8b04573 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 30277765 ● Search Scopus @ Elsevier (PMID): 30277765 ● Search Scopus @ Elsevier (DOI): 10.1021/acs.jafc.8b04573 (DOI: 10.1021/acs.jafc.8b04573, PubMed: 30277765)
23.	Takenori Satomura, Junji Hayashi, Tatsuya Ohshida, Haruhiko Sakuraba, Toshihisa Ohshima and Shin-Ichiro Suye : Enzymological characteristics of a novel archaeal dye-linked D-lactate dehydrogenase showing loose binding of FAD., Extremophiles, Vol.22, No.6, 975-981, 2018. (Summary) A gene-encoding a dye-linked D-lactate dehydrogenase (Dye-DLDH) homolog was identified in the genome of the hyperthermophilic archaeon Thermoproteus tenax. The gene was expressed in Escherichia coli and the product was purified to homogeneity. The recombinant protein exhibited highly thermostable Dye-DLDH activity. To date, four types of Dye-DLDH have been identified in hyperthermophilic archaea (in Aeropyrum pernix, Sulfolobus tokodaii, Archaeoglobus fulgidus, and Candidatus Caldiarchaeum subterraneum). The amino acid sequence of T. tenax Dye-DLDH showed the highest similarity (45%) to A. pernix Dye-DLDH, but neither contained a known FAD-binding motif. Nonetheless, both homologs required FAD for enzymatic activity, suggesting that FAD binds loosely to the enzyme and is easily released unlike in other Dye-DLDHs. Our findings indicate that Dye-DLDHs from T. tenax and A. pernix are a novel type of Dye-DLDH characterized by loose binding of FAD. (Keyword) Archaeal Proteins / Biosensing Techniques / Electrochemical Techniques / Flavin-Adenine Dinucleotide / Lactate Dehydrogenases / Molecular Mimicry / Sequence Homology, Amino Acid / Structural Homology, Protein / Thermoproteus (Link to Publication Site) ● Publication site (DOI): 10.1007/s00792-018-1054-3 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 30206766 ● Search Scopus @ Elsevier (PMID): 30206766 ● Search Scopus @ Elsevier (DOI): 10.1007/s00792-018-1054-3 (DOI: 10.1007/s00792-018-1054-3, PubMed: 30206766)
24.	Tatsuya Ohshida, Kohei Koba, Junji Hayashi, Kazunari Yoneda, Taketo Ohmori, Toshihisa Ohshima and Haruhiko Sakuraba : A novel bifunctional aspartate kinase-homoserine dehydrogenase from the hyperthermophilic bacterium, Thermotoga maritima., Bioscience, Biotechnology, and Biochemistry, Vol.82, No.12, 2084-2093, 2018. (Summary) The orientation of the three domains in the bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) homologue found in Thermotoga maritima totally differs from those observed in previously known AK-HseDHs; the domains line up in the order HseDH, AK, and regulatory domain. In the present study, the enzyme produced in Escherichia coli was characterized. The enzyme exhibited substantial activities of both AK and HseDH. L-Threonine inhibits AK activity in a cooperative manner, similar to that of Arabidopsis thaliana AK-HseDH. However, the concentration required to inhibit the activity was much lower (K (Keyword) Amino Acid Sequence / Aspartic Acid / Aspartokinase Homoserine Dehydrogenase / Biocatalysis / Electrophoresis, Polyacrylamide Gel / Enzyme Stability / Escherichia coli / Hot Temperature / Hydrogen-Ion Concentration / Kinetics / Protein Conformation / Recombinant Proteins / Sequence Homology, Amino Acid / Thermotoga maritima / Threonine (Link to Publication Site) ● Publication site (DOI): 10.1080/09168451.2018.1511365 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 30175674 ● Search Scopus @ Elsevier (PMID): 30175674 ● Search Scopus @ Elsevier (DOI): 10.1080/09168451.2018.1511365 (DOI: 10.1080/09168451.2018.1511365, PubMed: 30175674)
25.	Keitaro Take, Hidehisa Fujiki, Wasana Suyotha, Junji Hayashi, Kazuyoshi Takagi, Shigekazu Yano and Mamoru Wakayama : Enzymatic and molecular characterization of an acidic and thermostable chitinase 1 from Streptomyces thermodiastaticus HF 3-3., The Journal of General and Applied Microbiology, Vol.64, No.4, 190-197, 2018. (Summary) values of Chi1 for the substrate colloidal chitin were 1.23 ± 0.7 mg/mL and 6.33 ± 1.0 U/mg, respectively. Thin-layer chromatography analysis of the enzymatic reaction end products mainly detected diacetylchitobiose. We also cloned the Chi1 gene and purified the recombinant protein; the properties of the recombinant enzyme were nearly identical to those of the native enzyme. Therefore, Chi1 purified from S. thermodiastaticus HF 3-3 is unique, as it is highly stable under broad range of pH values, temperatures, and chemical exposures. Combined, these properties make this enzyme attractive for use in the industrial bioconversion of chitin. (Keyword) Amino Acid Sequence / Base Sequence / Chitin / Chitinases / Enzyme Stability / Hydrogen-Ion Concentration / Hydrolysis / Kinetics / Molecular Weight / Recombinant Proteins / Streptomyces / Substrate Specificity / Temperature (Link to Publication Site) ● Publication site (DOI): 10.2323/jgam.2017.12.002 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 29709891 ● Search Scopus @ Elsevier (PMID): 29709891 ● Search Scopus @ Elsevier (DOI): 10.2323/jgam.2017.12.002 (DOI: 10.2323/jgam.2017.12.002, PubMed: 29709891)
26.	Takenori Satomura, Junji Hayashi, Hiroaki Sakamoto, Takuro Nunoura, Yoshihiro Takaki, Ken Takai, Hideto Takami, Toshihisa Ohshima, Haruhiko Sakuraba and Shin-Ichiro Suye : d-Lactate electrochemical biosensor prepared by immobilization of thermostable dye-linked d-lactate dehydrogenase from Candidatus Caldiarchaeum subterraneum., Journal of Bioscience and Bioengineering, Vol.126, No.4, 425-430, 2018. (Summary) A stable d-lactate electrochemical sensing system was developed using a dye-linked d-lactate dehydrogenase (Dye-DLDH) from an uncultivated thermophilic archaeon, Candidatus Caldiarchaeum subterraneum. To develop the system, the putative gene encoding the Dye-DLDH from Ca. Caldiarchaeum subterraneum was overexpressed in Escherichia coli, and the expressed product was purified. The recombinant enzyme was a highly thermostable Dye-DLDH that retained full activity after incubation for 10 min at 70°C. The electrode for detection of d-lactate was prepared by immobilizing the thermostable Dye-DLDH and multi-walled carbon nanotube (MWCNT) within Nafion membrane. The electrocatalytic response of the electrode was clearly observed upon exposure to d-lactate. The electrode response to d-lactate was linear within the concentration range of 0.03-2.5 mM, and it showed little reduction in responsiveness after 50 days. This is the first report describing a d-lactate sensing system using a thermostable Dye-DLDH. (Keyword) Archaea / Archaeal Proteins / Biosensing Techniques / Electrodes / Enzyme Stability / Enzymes, Immobilized / Escherichia coli / Hot Temperature / Lactate Dehydrogenases / Lactic Acid (Link to Publication Site) ● Publication site (DOI): 10.1016/j.jbiosc.2018.04.002 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 29691195 ● Search Scopus @ Elsevier (PMID): 29691195 ● Search Scopus @ Elsevier (DOI): 10.1016/j.jbiosc.2018.04.002 (DOI: 10.1016/j.jbiosc.2018.04.002, PubMed: 29691195)
27.	Junji Hayashi, 牟田口祐太, 米田一成, 大森勇門, 大島敏久 and 櫻庭春彦 : Crystal structure of the novel amino-acid racemase isoleucine 2-epimerase from <i>Lactobacillus buchneri</i>, Vitamins, Vol.92, No.3, 121-124, 2018. (Keyword) branched-chain amino acid (BCAA) / isoleucine 2-epimerase, / <i>Lactobacillus buchneri</i> / pyridoxal 5'-phosphate (PLP) / crystal structure (Link to Publication Site) ● Publication site (DOI): 10.20632/vso.92.3_121 (Link to Search Site for Scientific Articles) ● CiNii @ National Institute of Informatics (CRID): 1390001288141268224 ● Search Scopus @ Elsevier (DOI): 10.20632/vso.92.3_121 (DOI: 10.20632/vso.92.3_121, CiNii: 1390001288141268224)
28.	Saddam Md Hossain, Takahiro Tanaka, Kazuyoshi Takagi, Junji Hayashi and Mamoru Wakayama : -derived l-amino acid ester hydrolase for synthesizing dipeptide, isoleucyl-tryptophan., 3 Biotech, Vol.8, No.3, 2018. (Summary) strongly inhibited enzyme activity. Finally, SmAEH activity was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), suggesting that the protein is a serine protease. (Link to Publication Site) ● Publication site (DOI): 10.1007/s13205-018-1195-1 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 29556427 ● Search Scopus @ Elsevier (PMID): 29556427 ● Search Scopus @ Elsevier (DOI): 10.1007/s13205-018-1195-1 (DOI: 10.1007/s13205-018-1195-1, PubMed: 29556427)
29.	Junji Hayashi, Tomonari Seto, Hironaga Akita, Masahiro Watanabe, Tamotsu Hoshino, Kazunari Yoneda, Toshihisa Ohshima and Haruhiko Sakuraba : Structure-Based Engineering of an Artificially Generated NADP⁺-Dependent d-Amino Acid Dehydrogenase, Applied and Environmental Microbiology, Vol.83, No.11, 2017. (Summary) -dependent d-amino acid dehydrogenase (DAADH) makes single-step production of d-amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d-amino acids, such as d-leucine and d-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs. (Keyword) Amino Acid Motifs / Amino Acid Oxidoreductases / Amino Acid Sequence / Bacterial Proteins / Binding Sites / Kinetics / Models, Molecular / Mutagenesis, Site-Directed / NADP / Planococcaceae / Protein Engineering / Substrate Specificity (Link to Publication Site) ● Publication site (DOI): 10.1128/AEM.00491-17 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 28363957 ● Search Scopus @ Elsevier (PMID): 28363957 ● Search Scopus @ Elsevier (DOI): 10.1128/AEM.00491-17 (DOI: 10.1128/AEM.00491-17, PubMed: 28363957)
30.	Junji Hayashi, Yuta Mutaguchi, Yume Minemura, Noriko Nakagawa, Kazunari Yoneda, Taketo Ohmori, Toshihisa Ohshima and Haruhiko Sakuraba : Crystal structure of the novel amino-acid racemase isoleucine 2-epimerase from Lactobacillus buchneri., Acta Crystallographica. Section D, Structural Biology, Vol.73, No.Pt 5, 428-437, 2017. (Summary) Crystal structures of Lactobacillus buchneri isoleucine 2-epimerase, a novel branched-chain amino-acid racemase, were determined for the enzyme in the apo form, in complex with pyridoxal 5'-phosphate (PLP), in complex with N-(5'-phosphopyridoxyl)-L-isoleucine (PLP-L-Ile) and in complex with N-(5'-phosphopyridoxyl)-D-allo-isoleucine (PLP-D-allo-Ile) at resolutions of 2.77, 1.94, 2.65 and 2.12 Å, respectively. The enzyme assembled as a tetramer, with each subunit being composed of N-terminal, C-terminal and large PLP-binding domains. The active-site cavity in the apo structure was much more solvent-accessible than that in the PLP-bound structure. This indicates that a marked structural change occurs around the active site upon binding of PLP that provides a solvent-inaccessible environment for the enzymatic reaction. The main-chain coordinates of the L. buchneri isoleucine 2-epimerase monomer showed a notable similarity to those of α-amino-ℇ-caprolactam racemase from Achromobactor obae and γ-aminobutyrate aminotransferase from Escherichia coli. However, the amino-acid residues involved in substrate binding in those two enzymes are only partially conserved in L. buchneri isoleucine 2-epimerase, which may account for the differences in substrate recognition by the three enzymes. The structures bound with reaction-intermediate analogues (PLP-L-Ile and PLP-D-allo-Ile) and site-directed mutagenesis suggest that L-isoleucine epimerization proceeds through abstraction of the α-hydrogen of the substrate by Lys280, while Asp222 serves as the catalytic residue adding an α-hydrogen to the quinonoid intermediate to form D-allo-isoleucine. (Keyword) Amino Acid Isomerases / Amino Acid Sequence / Crystallography, X-Ray / Isoleucine / Lactobacillus / Models, Molecular / Protein Conformation / Pyridoxal Phosphate / Sequence Alignment (Link to Publication Site) ● Publication site (DOI): 10.1107/S2059798317005332 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 28471367 ● Search Scopus @ Elsevier (PMID): 28471367 ● Search Scopus @ Elsevier (DOI): 10.1107/S2059798317005332 (DOI: 10.1107/S2059798317005332, PubMed: 28471367)
31.	Junji Hayashi, Kaori Yamamoto, Kazunari Yoneda, Toshihisa Ohshima and Haruhiko Sakuraba : Unique coenzyme binding mode of hyperthermophilic archaeal sn-glycerol-1-phosphate dehydrogenase from Pyrobaculum calidifontis., Proteins, Vol.84, No.12, 1786-1796, 2016. (Summary) ) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 Å. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2' of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild-type enzyme with NADPH, which suggests that the biased interactions around the C2'-phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)-dependent dehydrogenases. Proteins 2016; 84:1786-1796. © 2016 Wiley Periodicals, Inc. (Keyword) Amino Acid Motifs / Archaeal Proteins / Binding Sites / Cloning, Molecular / Coenzymes / Crystallography, X-Ray / Escherichia coli / Gene Expression / Glycerolphosphate Dehydrogenase / Hydrogen Bonding / Kinetics / Models, Molecular / NAD / NADP / Protein Binding / Protein Interaction Domains and Motifs / Protein Multimerization / Protein Structure, Secondary / Protein Subunits / Pyrobaculum / Recombinant Proteins / Thermodynamics (Link to Publication Site) ● Publication site (DOI): 10.1002/prot.25161 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 27616573 ● Search Scopus @ Elsevier (PMID): 27616573 ● Search Scopus @ Elsevier (DOI): 10.1002/prot.25161 (DOI: 10.1002/prot.25161, PubMed: 27616573)
32.	Tatsuya Ohshida, Junji Hayashi, Takenori Satomura, Ryushi Kawakami, Toshihisa Ohshima and Haruhiko Sakuraba : First characterization of extremely halophilic 2-deoxy-D-ribose-5-phosphate aldolase, Protein Expression and Purification, Vol.126, 62-68, 2016. (Summary) 2-Deoxy-d-ribose-5-phosphate aldolase (DERA) catalyzes the aldol reaction between two aldehydes and is thought to be a potential biocatalyst for the production of a variety of stereo-specific materials. A gene encoding DERA from the extreme halophilic archaeon, Haloarcula japonica, was overexpressed in Escherichia coli. The gene product was successfully purified, using procedures based on the protein's halophilicity, and characterized. The expressed enzyme was stable in a buffer containing 2 M NaCl and exhibited high thermostability, retaining more than 90% of its activity after heating at 70 °C for 10 min. The enzyme was also tolerant to high concentrations of organic solvents, such as acetonitrile and dimethylsulfoxide. Moreover, H. japonica DERA was highly resistant to a high concentration of acetaldehyde and retained about 35% of its initial activity after 5-h' exposure to 300 mM acetaldehyde at 25 °C, the conditions under which E. coli DERA is completely inactivated. The enzyme exhibited much higher activity at 25 °C than the previously characterized hyperthermophilic DERAs (Sakuraba et al., 2007). Our results suggest that the extremely halophilic DERA has high potential to serve as a biocatalyst in organic syntheses. This is the first description of the biochemical characterization of a halophilic DERA. (Tokushima University Institutional Repository) ● Metadata: 111972 (Link to Publication Site) ● Publication site (DOI): 10.1016/j.pep.2016.05.009 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 27215670 ● Summary page in Scopus @ Elsevier: 2-s2.0-84973496063 (Tokushima University Institutional Repository: 111972, DOI: 10.1016/j.pep.2016.05.009, PubMed: 27215670, Elsevier: Scopus)
33.	Junji Hayashi, Shota Inoue, Kwang Kim, Kazunari Yoneda, Yutaka Kawarabayasi, Toshihisa Ohshima and Haruhiko Sakuraba : Crystal Structures of a Hyperthermophilic Archaeal Homoserine Dehydrogenase Suggest a Novel Cofactor Binding Mode for Oxidoreductases., Scientific Reports, Vol.5, 2015. (Summary) NAD(P)-dependent dehydrogenases differ according to their coenzyme preference: some prefer NAD, others NADP, and still others exhibit dual cofactor specificity. The structure of a newly identified archaeal homoserine dehydrogenase showed this enzyme to have a strong preference for NADP. However, NADP did not act as a cofactor with this enzyme, but as a strong inhibitor of NAD-dependent homoserine oxidation. Structural analysis and site-directed mutagenesis showed that the large number of interactions between the cofactor and the enzyme are responsible for the lack of reactivity of the enzyme towards NADP. This observation suggests this enzyme exhibits a new variation on cofactor binding to a dehydrogenase: very strong NADP binding that acts as an obstacle to NAD(P)-dependent dehydrogenase catalytic activity. (Keyword) Amino Acid Sequence / Archaea / Binding Sites / Catalysis / Homoserine Dehydrogenase / Kinetics / Models, Molecular / Molecular Sequence Data / NADP / Oxidoreductases / Protein Binding / Protein Conformation / Sequence Alignment / Substrate Specificity (Link to Publication Site) ● Publication site (DOI): 10.1038/srep11674 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 26154028 ● Search Scopus @ Elsevier (PMID): 26154028 ● Search Scopus @ Elsevier (DOI): 10.1038/srep11674 (DOI: 10.1038/srep11674, PubMed: 26154028)
34.	Shin-ichi Sakasegawa, Junji Hayashi, Yoshiaki Ikura, Shigeru Ueda, Shigeyuki Imamura, Toshihiko Kumazawa, Atsuhisa Nishimura, Toshihisa Ohshima and Haruhiko Sakuraba : Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method., Analytical Biochemistry: Methods in the Biological Sciences, Vol.416, No.1, 61-66, 2011. (Summary) Pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) from the hyperthermophile Thermotoga maritima was biochemically characterized with the aim of establishing a colorimetric assay for inorganic pyrophosphate (PPi). When heterologously expressed in Escherichia coli, T. maritima PPDK (TmPPDK) was far more stable any other PPDK reported so far: it retained >90% of its activity after incubation for 1 h at 80°C, and >80% of its activity after incubation for 20 min at pHs ranging from 6.5 to 10.5 (50°C). In contrast to PPDKs from protozoa and plants, this TmPPDK showed very long-term stability at low temperature: full activity was retained even after storage for at least 2 years at 4°C. TmPPDK was successfully applied to a novel colorimetric PPi assay, which employed (i) a PPi cycling reaction using TmPPDK and nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) from Saccharomyces cerevisiae and (ii) a NAD cycling reaction to accumulate reduced nitroblue tetrazolium (diformazan). This enabled detection of 0.2 μM PPi, making this method applicable for preliminary measurement of PPi levels in PCR products in an automatic clinical analyzer. (Keyword) Colorimetry / Diphosphates / Nicotinamide-Nucleotide Adenylyltransferase / Pyruvate, Orthophosphate Dikinase / Saccharomyces cerevisiae / Thermotoga maritima (Link to Publication Site) ● Publication site (DOI): 10.1016/j.ab.2011.04.043 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 21621500 ● Search Scopus @ Elsevier (PMID): 21621500 ● Search Scopus @ Elsevier (DOI): 10.1016/j.ab.2011.04.043 (DOI: 10.1016/j.ab.2011.04.043, PubMed: 21621500)

Review, Commentary:

1.	Hironaga Akita, Junji Hayashi, Haruhiko Sakuraba and Toshihisa Ohshima : Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application., Frontiers in Microbiology, Vol.9, Aug. 2018. (Summary) -DAPDH and D-AADH, and designed mutations based on the information obtained made it possible to markedly enhance enzyme activity and to create D-AADH homologs with desired reactivity profiles. The methods described here may be an effective approach to artificial creation of biotechnologically useful enzymes. (Tokushima University Institutional Repository) ● Metadata: 115255 (Link to Publication Site) ● Publication site (DOI): 10.3389/fmicb.2018.01760 (Link to Search Site for Scientific Articles) ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 30123202 ● Search Scopus @ Elsevier (PMID): 30123202 ● Search Scopus @ Elsevier (DOI): 10.3389/fmicb.2018.01760 (Tokushima University Institutional Repository: 115255, DOI: 10.3389/fmicb.2018.01760, PubMed: 30123202)

Proceeding of International Conference:

1.	Takefumi Hattori, Katayama Megumi, Tsuzuki Hiromitsu, Okamoto Yumi, Ida Kyosuke, Yoshizumi Mariko, Abe Masanori, Ryushi Kawakami, Junji Hayashi and Masaomi Yamamura : Phenylpropanoid metabolism in basidiomycete Tricholoma matsutake -cDNA cloning of Sadenosyl-L-methionine-dependent cinnamic acid carboxyl methyltransferase-, 2nd International Lignin Symposium (Kyoto), Sep. 2024.
2.	Nakamoto Akira, Umekawa Midori, Junji Hayashi and Wakayama Mamoru : Construction of D-amino acid derivative production system using Saccharomyces cerevisiae, New Zealand Microbiological Society Conference 2017, Nov. 2017.
3.	Ogiyama Daiki, Wakayama Mamoru and Junji Hayashi : Lactovinegar production by ethanol fermentation of whey using Zymomonas mobile, New Zealand Microbiological Society Conference 2017, Nov. 2017.
4.	Ohshima Toshihisa, Junji Hayashi, Akita Hironaga and Sakuraba Haruhiko : Structuralbased engineering of an artificially created NADP-dependent D-amino acid dehydrogenase, The 3rd International Conference of D-Amino Acid Research 2017, Jul. 2017.
5.	Take Keitaro, Fujiki Hidehisa, Suyotha Wasana, Takagi Kazuyoshi, Junji Hayashi and Wakayama Mamoru : Thermostable and acidic chitinase obtained from Streptomyces thermodiastaticus, New Zealand Microbiological Society Conference 2017, 2017.
6.	Ishiyama Shinnosuke, Yamahata Naoki, Junji Hayashi and Wakayama Mamoru : Development of a new type of liquor made from whey-selections of Aspergillus species and raw materials for malt, and examination of brewing conditions, New Zealand Microbiological Society Conference 2017, 2017.
7.	Junji Hayashi, Ohshima Toshihisa and Sakuraba Haruhiko : Unique cofactor binding mode of homoserine dehydrogenase from hyperthermophilic archaeon Pyrococcus horikoshii, Extremophiles 2016, Sep. 2016.

Proceeding of Domestic Conference:

1.	Takefumi Hattori, 片山恵, Ryushi Kawakami, Junji Hayashi and Masaomi Yamamura : マツタケ由来S-アデノシル-L-メチオニン依存ケイ皮酸カルボキシルメチルトランスフェラーゼ組換え酵素の特性解明, 第74回日本木材学会大会(京都大会), Mar. 2024.
2.	伊澤命吹, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru, 大島敏久 and 櫻庭春彦 : ピロリン-5-カルボン酸レダクターゼの触媒機構の解明, 日本農芸化学会中四国支部第67回講演会, Jan. 2024.
3.	山田萌加, Junji Hayashi, Ryushi Kawakami, 金丸芳, 大島敏久 and 櫻庭春彦 : 新規二機能型融合酵素ジアミノピメリン酸脱炭酸酵素/アスパラギン酸キナーゼに関する研究, 日本農芸化学会中四国支部第67回講演会, Jan. 2024.
4.	Junji Hayashi, Ryushi Kawakami, Akira Hirata, Kaori Kanemaru, 大島敏久 and 櫻庭春彦 : 超好熱アーキア由来FAD依存性D-乳酸脱水素酵素のX線結晶構造解析, 日本農芸化学会中四国支部会2023年度中四国・西日本支部合同大会, Sep. 2023.
5.	Junji Hayashi : 酵素の特異性を立体構造から理解する, 日本農芸化学会学会創立 100 周年記念第 38 回若手研究者シンポジウム, Jul. 2023.
6.	Ryushi Kawakami, Junji Hayashi, Haruhiko Sakuraba and Toshihisa Ohshima : 超好熱アーキアPyrococcus furiosusのPLP依存性アミノ酸ラセマーゼの機能と構造の特徴, 2023年度日本ビタミン学会大会, Jun. 2023.
7.	櫻庭春彦, 高見直樹, Junji Hayashi, 米田一成, 大森勇門 and 大島敏久 : Pseudomonas veronii由来L-アルギニン脱水素酵素の構造解析, 第470回ビタミンB研究協議会, Mar. 2023.
8.	山﨑義輝, 山下晶央, Chizuru Sasaki, Junji Hayashi, Ryushi Kawakami and Kaori Kanemaru : 鳴門産スジアオノリ(Ulva prolifera)の志賀毒素吸着活性, 日本農芸化学会中四国支部第64 回講演会要旨集, Jan. 2023. (Keyword) E. coli O157 / Siga Toxin / Ulva prolifera / adsorption
9.	山下晶央, 山﨑義輝, Chizuru Sasaki, Junji Hayashi, Ryushi Kawakami and Kaori Kanemaru : アラメ(Eisenia bicyclis)によるEHEC O157 産生志賀毒素吸着, 日本農芸化学会中四国支部大会講演要旨集, 37, Sep. 2022. (Keyword) E. coli O157 / Siga Toxin / Eisenia bicyclis / adsorption
10.	Tamotsu Tanaka, Ali Hanif, KObayashi Miyu, Hasi Yesmin Rumana, Mutsumi Aihara, Junji Hayashi and Ryushi Kawakami : 極長鎖脂肪酸による毒性とその解毒装置としてのペルオキシソームの役割, 脂質栄養学, Vol.31, No.2, 143, Sep. 2022.
11.	Chizuru Sasaki, 新居美香, Junji Hayashi and Kaori Kanemaru : ワカメ非可食部の資源化を目的とした連続水熱処理による有用物質生産法の開発, 日本食品工学会2022年度大会, 岡山, Sep. 2022.
12.	中永美樹, Ryushi Kawakami, Junji Hayashi, 大島敏久 and 櫻庭春彦 : 超好熱性アーキア Thermococcus profundus 由来色素依存性 L -プロリン脱水素酵素に関する研究, 日本農芸化学会2022年度中四国支部大会 2022年9月22日, Sep. 2022.
13.	Junji Hayashi, 玉谷優奈, 小川詩緒里, Ryushi Kawakami, 里村武範, 大島敏久 and 櫻庭春彦 : 高度耐熱性FAD依存性D-乳酸脱水素酵素のX線結晶構造解析, 日本農芸化学会2022年度中四国支部大会(第63回講演会), Sep. 2022.
14.	Rumana Yesmin Hasi, Naohiro Imura, Toshiki Ishikawa, Hiroyuki Imai, Yoshimichi Takai, Hanif Ali, Mutsumi Aihara, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru and Tamotsu Tanaka : Distribution and characterization of glycosylinositol phosphoceramide specific phospholipase D in Brassica plants, 第64回日本脂質生化学会, Vol.64, 272-275, Jun. 2022.
15.	Hanif Ali, Miyu Kobayashi, Katsuya Morito, Rumana Yesmin Hasi, Mutsumi Aihara, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru, Koichiro Tsuchiya, Kazunori Sango and Tamotsu Tanaka : Peroxisomes attenuate lipotoxicity of very-long-chain fatty acids, 第64回日本脂質生化学会, Vol.64, 43-46, Jun. 2022.
16.	Junji Hayashi, 大志田達也, Ryushi Kawakami, 里村武範, 若山守, 大島敏久 and 櫻庭春彦 : 超好熱アーキア由来色素依存性D-乳酸脱水素酵素の構造解析, 日本ビタミン学会第74回大会, Jun. 2022.
17.	Hanif Ali, Miyu Kobayashi, Katsuya Morito, Rumana Yesmin Hasi, Mutsumi Aihara, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru, Koichiro Tsuchiya, Kazunori Sango and Tamotsu Tanaka : Metabolism and biological effect of very-long-chain fatty acid in peroxisome-deficient cells, 第63回日本生化学中国・四国支部例会, May 2022.
18.	Hanif Ali, Katsuya Morito, Rumana Yesmin Hasi, Mutsumi Aihara, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru, Koichiro Tsuchiya, Kazunori Sango and Tamotsu Tanaka : Characterization of uptake and metabolism of very-long-chain fatty acid in peroxisome-deficient CHO cells, 第94回日本生化学大会, Nov. 2021.
19.	Rumana Yesmin Hasi, Naohiro Imura, Toshiki Ishikawa, Hiroyuki Imai, Yoshimichi Takai, Hanif Ali, Mutsumi Aihara, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru and Tamotsu Tanaka : Production of phytoceramide 1- phosphate and inositol glycan by glycosylinositol phosphoceramide specific phospholipase D activity in plants, 第94回日本生化学大会, Nov. 2021.
20.	櫻庭春彦, 大志田達也, Ryushi Kawakami, Junji Hayashi, 米田一成 and 大島敏久 : 好熱アーキアPyrococcus horikoshii 由来オルニチンアミノトランスフェラーゼの構造解析, 第465回ビタミンB研究協議会, Nov. 2021.
21.	Hanif Ali, Katsuya Morito, Rumana Yesmin Hasi, Mutsumi Aihara, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru, Koichiro Tsuchiya, Kazunori Sango and Tamotsu Tanaka : Characterization of uptake and metabolism of very-long-chain fatty acid in peroxisome-deficient CHO cells, 第62回日本生化学中国・四国支部例会, Sep. 2021.
22.	Rumana Yesmin Hasi, Naohiro Imura, Toshiki Ishikawa, Hiroyuki Imai, Yoshimichi Takai, Hanif Ali, Mutsumi Aihara, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru and Tamotsu Tanaka : Production of phytoceramide 1- phosphate and inositol glycan by glycosylinositol phosphoceramide specific phospholipase D activity in plants, 第62回日本生化学中国・四国支部例会, Sep. 2021.
23.	Hanif Ali, Katsuya Morito, Rumana Yesmin Hasi, Mutsumi Aihara, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru, Koichiro Tsuchiya and Tamotsu Tanaka : Uptake and metabolism of very-long-chain fatty acid in animal cells, 第63回日本脂質生化学会, Jun. 2021.
24.	Junji Hayashi, 大志田達也, Ryushi Kawakami, 里村武範, 若山守, 大島敏久 and 櫻庭春彦 : 超好熱菌由来色素依存性D-乳酸脱水素酵素のX線結晶構造解析, 日本農芸化学会中四国支部第58回講演会, Jan. 2021.
25.	Ryushi Kawakami, Junji Hayashi, 木下千夏, 河瀬智紀 and 佐藤樹夫 : 超好熱アーキア Thermococcus litoralis DSM5473 のアミノ酸ラセマーゼ BAR2 の機能解析, 日本ビタミン学会第 72 回大会, Jun. 2020.
26.	Intuy Rattanaporn, Takafumi Itoh, Wasana Suyotha, Junji Hayashi, Shigekazu Yano, Koki Makabe, Yosuke Toyotake, Mamoru Wakayama and Takao Hibi : Reaction mechanism and crystallization of catalytic α-1,3-glucanase from Paenibacillus glycanilyticus FH11, Japan Society for Bioscience, Biotechnology, and Agrochemistry, Mar. 2020.
27.	大志田達也, Junji Hayashi, 米田一成, 大島敏久 and 櫻庭春彦 : 超好熱アーキアPyrobaculum aerophilum由来新規ガラクトース1-リン酸ウリジリルトランスフェラーゼに関する研究, 日本農芸化学会2020年度大会, Mar. 2020.
28.	Rattanaporn Intuy, Itoh Takafumi, Suyotha Wasana, Junji Hayashi, Yano Shigekazu, Makabe Koki, Toyotake Yosuke, Wakayama Mamoru and Hibi Takao : Reaction mechanism and crystallization of catalytic α-1,3-glucanase from Paenibacillus glycanilyticus FH11, 日本農芸化学会2020年度大会, Mar. 2020.
29.	深瀬葵, Junji Hayashi, 高木一好, 豊竹洋佑 and 若山守 : Thermococcus kodakaraensis由来組換えβ-アスパルチルトランスペプチダーゼの諸性質の検討, 日本生物工学会九州支部長崎大会, Dec. 2019.
30.	八田誠二, 野々村祐輝, Junji Hayashi, 高木一好, 豊竹洋佑 and 若山守 : Pseudomonas aeruginosa PAO1由来γ-グルタミルトランスペプチダーゼ IIのC末端領域の活性および安定性に及ぼす影響, 日本生物工学会九州支部長崎大会, Dec. 2019.
31.	能勢晶暉, 西尾啓汰, Junji Hayashi, 豊竹洋祐 and 若山守 : 脱脂菜種を原料に用いた酪醤醸造の検討, 日本生物工学会九州支部長崎大会, Dec. 2019.
32.	西田典央, Junji Hayashi, 豊竹洋祐, Junji Hayashi and 若山守 : Streptomyces thermodiastaticus HF3-3由来組換えβ-1,3-glucanaseの発現系の構築と諸性質の検討, 日本生物工学会九州支部長崎大会, Dec. 2019.
33.	田中悠磨, Junji Hayashi, 豊竹洋祐, Junji Hayashi and 若山守 : Komagataeibacter xylinus由来Superoxide dismutaseの融合型酵素の発現ならびに諸性質検討, 日本生物工学会九州支部長崎大会, Dec. 2019.
34.	加藤耀, 立花萌々子, 山畑直樹, Junji Hayashi, 豊竹洋祐 and 若山守 : Kluyveromyces lactisのRIM11欠損株の発酵特性, 日本生物工学会九州支部長崎大会, Dec. 2019.
35.	大西敦也, 田中貴大, Junji Hayashi, 豊竹洋佑 and 若山守 : Elizabethkingia sp. TT1由来L-アミノ酸エステラーゼを用いたVal-Gly合成, 日本生物工学会九州支部長崎大会, Dec. 2019.
36.	大志田達也, Junji Hayashi, 米田一成, 大島敏久 and 櫻庭春彦 : 超好熱アーキア Pyrobaculum aerophilum 由来新規ガラクトース 1-リン酸ウリジリルトランスフェラーゼの構造解析, 日本農芸化学会 2019 年度西日本・中四国支部合同沖縄大会, Nov. 2019.
37.	Tamotsu Tanaka, Rumana Yesmin Hasi, Katsuya Morito, Kentaro Kogure, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru, 今井博之 and 石川寿樹 : グリコシルイノシトールホスホセラミドの単離法の開発, 第12回セラミド研究会学術集会, Oct. 2019.
38.	Tamotsu Tanaka, Katsuya Morito, Rumana Yesmin Hasi, Junji Hayashi, Ryushi Kawakami, Kaori Kanemaru, 若山睦, 近藤千恵子, Tatsuya Fukuta and Kentaro Kogure : 食品素材に含まれるセラミドの簡便な定量方法, 日本脂質栄養学会第28回大会, Sep. 2019.
39.	Phumsombat Putthapong, Sano Chiharu, Hibi Takao, Itoh Takafumi, Junji Hayashi, Toyotake Yosuke and Wakayama Mamoru : Immobilization and site directed mutagenesis of Trp525 residue of γ-Glutamyltranspeptidase from Pseudomonas nitroreducens (PnGGT) to improve Theanine production, The Society for Biotechnology, Japan, Sep. 2019.
40.	池添浩輝, Junji Hayashi, 豊竹洋佑 and 若山守 : Pseudomonas nitroreducens由来γ-グルタミルトランスペプチダーゼの構造と機能に関する研究, 日本生物工学会, Sep. 2019.
41.	八田誠二, 野々村祐輝, Junji Hayashi, 高木一好, 豊竹洋佑 and 若山守 : Pseudomonas aeruginosa PAO1株由来γ-グルタミルトランスペプチダーゼII C末端領域の活性および安定性に及ぼす影響, 日本生物工学会, Sep. 2019.
42.	深瀬葵, Junji Hayashi, 高木一好, 豊竹洋佑 and 若山守 : Thermococcus kodakarensis由来β-アスパルチルトランスペプチダーゼ遺伝子のクローニング，発現および諸性質の解明, 日本生物工学会, Sep. 2019.
43.	山田泰蔵, Junji Hayashi, 豊竹洋佑 and 若山守 : アスパラギン合成酵素を用いたβ-アスパルチル化合物合成法の検討, 日本生物工学会, Sep. 2019.
44.	大西敦也, 田中貴大, Junji Hayashi, 豊竹洋佑 and 若山守 : Elizabethkingia sp. TT1由来組換えL-アミノ酸エステラーゼの高発現系の構築と菌体反応によるVal-Gly合成, 日本生物工学会, Sep. 2019.
45.	櫻庭春彦, 大志田達也, Junji Hayashi, 米田一成 and 大島敏久 : 超好熱菌由来ガラクトース1-リン酸ウリジリルトランスフェラーゼの構造解析, 第457回ビタミンB研究協議会, Aug. 2019.
46.	長濱伸哉, Junji Hayashi, 里村武範, 若山守, 大島敏久 and 櫻庭春彦 : D-乳酸分析用酵素の探索と機能解析, 日本農芸化学会関西支部例会第507回講演会, Feb. 2019.
47.	古川慎, Junji Hayashi and 若山守 : 食品中アクリルアミド低減に向けたL-アスパラギナーゼ高生産乳酸菌の探索及び諸性質の検討, 第25回日本生物工学会九州支部鹿児島大会, Dec. 2018.
48.	中本侃, Junji Hayashi and 若山守 : 出芽酵母によるD-アミノ酸誘導体の発酵生産法の構築, 第25回日本生物工学会九州支部鹿児島大会, Dec. 2018.
49.	石山真乃介, 山畑直樹, Junji Hayashi and 若山守 : ホエイを原料とする新規酒類の開発, 第25回日本生物工学会九州支部鹿児島大会, Dec. 2018.
50.	服部俊, 三品裕太, Junji Hayashi and 若山守 : 新規調味料「酪醬」の醸造法の検討, 第25回日本生物工学会九州支部鹿児島大会, Dec. 2018.
51.	矢幡翔太, Junji Hayashi and 若山守 : Streptomyces属由来α-1,3-グルカナーゼに関する研究, 第25回日本生物工学会九州支部鹿児島大会, Dec. 2018.
52.	櫻庭春彦, Junji Hayashi, 秋田紘長, 大森勇門 and 大島敏久 : D-アミノ酸脱水素酵素の構造情報に基づく基質特異性の改変, 第70回日本生物工学会大会, Sep. 2018.
53.	牧慶子, 田中貴大, 高木一好, Junji Hayashi and 若山守 : L -アミノ酸エステラーゼによる血管拡張作用ペプチド( T r p - H i s )の酵素合成に関する研究, 日本農芸化学会関西支部第5 0 2 回講演会, Feb. 2018.
54.	Junji Hayashi, 米田一成, 大島敏久 and 櫻庭春彦 : 超好熱アーキア由来ホモセリン脱水素酵素に見出した新奇な補酵素結合様式．, 日本農芸化学会関西支部第5 0 2 回講演会, Feb. 2018.
55.	池尻篤生, 藤原祥子, Junji Hayashi, 櫻庭春彦, 田中直孝 and 田淵光昭 : 青枯病菌エフェクター RipAY の宿主チオレドキシン依存的活性化機構の解析, 日本農芸化学会2017年度合同大阪大会, Sep. 2017.
56.	Junji Hayashi, 山本香李, 米田一成, 大島敏久 and 櫻庭春彦 : 超好熱アーキア Pyrobaculum calidifontis 由来グリセロール 1-リン酸脱水素酵素に見出した新規補酵素結合様式, 日本農芸化学会2017年度合同大阪大会, Sep. 2017.
57.	Junji Hayashi, 山本香李, 米田一成, 大島敏久 and 櫻庭春彦 : 超好熱アーキア Pyrobaculum calidifontis 由来グリセロール 1-リン酸脱水素酵素に見出した新規補酵素結合様式, 日本農芸化学会中四国支部例会, Sep. 2017.
58.	古場康平, Junji Hayashi, 大志田達也, 安部展洋, 米田一成, 大島敏久 and 櫻庭春彦 : 超好熱性細菌 Thermotoga maritima 由来アスパラギン酸キナーゼ/ホモセリンデヒドロゲナーゼ融合酵素(AK/HseDH)の分子特性, 日本農芸化学会2017年度合同大阪大会, Sep. 2017.
59.	Junji Hayashi, 峰村結芽, 牟田口祐太, 米田一成, 大森勇門, 大島敏久 and 櫻庭春彦 : 新規分岐鎖アミノ酸ラセマーゼ，イソロイシン 2-エピメラーゼの構造解析, 日本ビタミン学会第69回大会, Jun. 2017.
60.	花川大地, Junji Hayashi, 米田一成, 大島敏久 and 櫻庭春彦 : 超好熱アーキア Pyrobaculum aerophilum 由来 galactose-1-phosphate uridylyltransferase の構造解析, 日本農芸化学会2017年度大会, Mar. 2017.
61.	花川大地, Junji Hayashi, 米田一成, 大島敏久 and 櫻庭春彦 : 超好熱アーキア Pyrobaculum aerophilum 由来 galactose-1-phosphate uridylyltransferase の構造解析, 日本農芸化学会2017年度大会, Mar. 2017.
62.	大志田達也, Junji Hayashi, 里村武範, Ryushi Kawakami, 大島敏久 and 櫻庭春彦 : 高度好塩菌 Haloarcula japonica 由来 2-デオキシリボース 5-リン酸アルドラーゼに関する研究, 日本農芸化学会2017年度大会, Mar. 2017.
63.	古場康平, Junji Hayashi, 安部展洋, 米田一成, 大島敏久 and 櫻庭春彦 : 超好熱性細菌 Thermotoga maritima 由来アスパラギン酸キナーゼ / ホモセリンデヒドロゲナーゼ融合酵素(AK/HseDH)の機能解析, 日本農芸化学会2017年度大会, Mar. 2017.
64.	Junji Hayashi, 峰村結芽, 牟田口祐太, 大森勇門, 大島敏久 and 櫻庭春彦 : 新規アミノ酸ラセマーゼ，イソロイシン 2-エピメラーゼの構造解析, 第12回 D-アミノ酸学会学術講演会, Sep. 2016.
65.	大志田達也, Junji Hayashi, 里村武範, Ryushi Kawakami, 大島敏久 and 櫻庭春彦 : 高度好塩菌 Haloarcula japonica由来2-デオキシリボース-5-リン酸アルドラーゼに関する研究, 日本農芸化学会2016年度中四国支部大会, Sep. 2016.
66.	櫻庭春彦, Junji Hayashi, 牟田口祐太, 大森勇門 and 大島敏久 : Lactobacillus buchneri由来分岐鎖アミノ酸ラセマーゼの構造解析, 第445回ビタミンB研究協議会, Aug. 2016.
67.	Junji Hayashi, 牟田口祐太, 大森勇門, 大島敏久 and 櫻庭春彦 : 新規アミノ酸ラセマーゼ，イソロイシン 2-エピメラーゼの構造解析, 日本ビタミン学会第68回大会, Jun. 2016.
68.	櫻庭春彦, Junji Hayashi, 山本香李 and 大島敏久 : 好熱アーキア由来グリセロール1-リン酸デヒドロゲナーゼの構造解析, 第443回ビタミンB研究協議会, Jan. 2016.
69.	瀬戸智成, 秋田紘長, Junji Hayashi, 大島敏久 and 櫻庭春彦 : 耐熱性D-アミノ酸脱水素酵素の機能，構造解析, 日本農芸化学会中四国支部例会, Jan. 2016.
70.	Junji Hayashi, 井上翔太, 米田一成, 大島敏久 and 櫻庭春彦 : 超好熱アーキア由来ホモセリンデヒドロゲナーゼが示す新奇な補酵素結合様式, 日本農芸化学会中四国支部例会, Jan. 2016.
71.	山本香李, Junji Hayashi, 米田一成, 大島敏久 and 櫻庭春彦 : 超好熱アーキアPyrobaculum calidifontis由来グリセロール-1-リン酸デヒドロゲナーゼの機能構造解析, 日本農芸化学会中四国支部例会, Jan. 2016.
72.	Junji Hayashi, 牟田口祐太, 大森勇門, 大島敏久 and 櫻庭春彦 : 新規アミノ酸ラセマーゼ，イソロイシン 2-エピメラーゼの構造解析, 2015年度酵素・補酵素研究会, Jul. 2015.
73.	Junji Hayashi, 井上翔太, 大島敏久 and 櫻庭春彦 : 超好熱アーキア由来ホモセリンデヒドロゲナーゼが示す新奇な補酵素結合様式, 第440回ビタミンB研究協議会, Jun. 2015.
74.	津島悠斗, Junji Hayashi, 米田一成, 大島敏久 and 櫻庭春彦 : 超好熱アーキアAeropyrum pernix由来D-乳酸デヒドロゲナーゼの結晶構造解析, 日本農芸化学会中四国支部大会, Sep. 2014.
75.	Junji Hayashi, 酒瀬川信一, 櫻庭春彦 and 大島敏久 : 超好熱細菌Thermotoga maritima由来ピルビン酸オルトリン酸ジキナーゼの性質, 日本農芸化学会中四国支部大会, Sep. 2008.

Patent:

1.	櫻庭春彦, Junji Hayashi and 酒瀬川信一 : 新規なピルベートオルトホスフェートジキナーゼ，及びその製造方法, 2008-152403 (Jun. 2008), 2009-17876 (Jan. 2009), 5352788 (Sep. 2013).

Grants-in-Aid for Scientific Research (KAKEN Grants Database @ NII.ac.jp)

色素依存性D-乳酸脱水素酵素の立体構造に基づく新規有用酵素の創製 (Project/Area Number: 23K14137 )
担子菌特異的カルボキシルメチルトランスフェラーゼの反応機作の解明 (Project/Area Number: 23K05337 )
Functional and structural analysis of heat-resistant dye-dependent D-lactate dehydrogenase and its application to biosensor devices (Project/Area Number: 18K14394 )
耐熱性色素依存性D-乳酸脱水素酵素の機能・構造解析とバイオセンサー素子への応用 (Project/Area Number: 17H07257 )
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Associate Professor : Hayashi, Junji

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Grants-in-Aid for Scientific Research (KAKEN Grants Database @ NII.ac.jp)