Kyushu University Academic Staff Educational and Research Activities Database
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Noriho Kamiya Last modified date:2024.04.19

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Academic Degree
Doctor of Engineering
Country of degree conferring institution (Overseas)
Field of Specialization
Biomolecular engineering, Biochemical engineering, Bioengineering, Enzyme and Protein engineering
ORCID(Open Researcher and Contributor ID)
Total Priod of education and research career in the foreign country
Outline Activities
My research interest is focused on enzyme and protein engineering to create new protein-based biomaterials. To fully exploit the unique functions of proteins, different strategies including genetic engineering, enzyme engineering and organic synthesis should be combined, and my research is actually directed in this direction. I am now interested in post-translational, site-specific modification of functional proteins to design new nanobioarchitectures for specific functions. I am also interested in drug delivery systems for oral and transdermal administration of pharmaceutical peptides and proteins, and next-generation biopharmaceuticals such as antibody-drug conjugates. During my research, I have collaborated with many industrial companies and founded KAICO Ltd, a start-up company from Kyushu University, in collaboration with the Department of Agriculture (Prof. Kusakabe's group).
In terms of education, after completing my Ph.D., I experienced two universities, University of Tokyo & MIT, and had two undergraduate classes at the former. I spent one year at MIT as a visiting scientist and worked on the project related to protein drug delivery and protein chip technology. Now I am involved in many kinds of lectures and work at the university.
Research Interests
  • Development of easy-to-handle hydrogel compartments for single-cell analyses
    keyword : single cell culture, single cell analysis, hydrogel beads
    2020.04~2027.03We have focused on the development of a novel lipid-based drug formulation toward oral delivery of pharmaceutical peptide and protein drugs..
  • Development of next generation biopharmaceuticals including antibody-drug conjugates
    keyword : antibody-drug conjugate, site-specific labeling, antibody fragment
    2021.04~2026.03We have focused on the development of a novel lipid-based drug formulation toward oral delivery of pharmaceutical peptide and protein drugs..
  • Development of easy-to-handle compartments for biomolecular systems
    keyword : directed evolution, cell culture, proliferation
    2017.04~2026.03We have focused on the development of a novel lipid-based drug formulation toward oral delivery of pharmaceutical peptide and protein drugs..
  • Development of lipid-protein conjugates and exploration of their potential applications
    keyword : lipid-protein conjugate, artificial lipidized protein
    2018.04~2027.03We have focused on the development of a novel lipid-based drug formulation toward oral delivery of pharmaceutical peptide and protein drugs..
  • Basic research and design concept for insect biorefinery with silkworm in Kyushu University and its application
    keyword : biorefinery, insect engineering
    2013.10~2025.03Enzyme-DNA hybrids are unique and powerful bioconjugates for the ultrasensitive detection of target DNA/RNA. Here, we have developed novel enzyme-DNA/RNA hybrids that are potentially useful for a wide variety of biotechnological applications such as in situ hybridization and genomic southern blotting..
  • Design, creation and evaluation of supramolecular protein complexes.
    keyword : scaffold molecule, cellulosome, protein hybrid molecule
    2013.04~2021.03Enzyme-DNA hybrids are unique and powerful bioconjugates for the ultrasensitive detection of target DNA/RNA. Here, we have developed novel enzyme-DNA/RNA hybrids that are potentially useful for a wide variety of biotechnological applications such as in situ hybridization and genomic southern blotting..
  • Development of novel transdermal protein drug delivery systems
    keyword : protein drug delivery, pharmaceutical formulation, oral administration, vaccine
    2012.04~2018.03We have focused on the development of a novel lipid-based drug formulation toward oral delivery of pharmaceutical peptide and protein drugs..
  • Biocatalytic oxidation/reduction system in recombinant E. coli cells for the production of protein-decorated nano particles
    keyword : protein engineering, enzyme engineering, metal nanoparticle
    2011.04~2016.03In the present study, we focused on the coupling of different enzymatic activities towards the monooxygenation of one substrate to create highly efficient E. coli whole cell biocatalysts that facilitates oxygenation of hydrocarbons under mild conditions..
  • Development of novel nucleic acid-enzyme hybrid systems
    keyword : enzyme-DNA hybrid, in situ hybridization, Genomic southern blot
    2007.04~2013.03Enzyme-DNA hybrids are unique and powerful bioconjugates for the ultrasensitive detection of target DNA/RNA. Here, we have developed novel enzyme-DNA/RNA hybrids that are potentially useful for a wide variety of biotechnological applications such as in situ hybridization and genomic southern blotting. .
  • New biocatalytic systems based on the use of ionic liquids
    keyword : enzyme engineering, biocatalysis, ionic liquid, biorefinery
    2006.04~2013.03Ionic liquids are unique solvents that realize high polarity and hydrophobicity simultaneously, and its potential as novel reaction media has been recently recognized. In the present study, a new bioprocess that facilitates bicatalytic reaction in ionic liquids will be developed..
  • Protein engineering with post-translational protein modifying enzymes
    keyword : enzymatic protein engineering, site-specific modification, transglutaminase
    2002.04~2012.03Site-specific modification of proteins is often required to effectively utilize a wide variety of protein function. Recently, I have developed a novel way to conjugate recombinant proteins by combining genetic and enzyme engineering. The present enzymatic-protein-engineering approach can overcome the limit of conventional technique and is applicable to create functional protein hybrids and protein arrays..
  • Biocatalytic oxidation/reduction system in recombinant E. coli cells
    keyword : protein engineering, enzyme engineering, P450, Au nanoparticle
    2005.04~2011.03In the present study, we focused on the coupling of different enzymatic activities towards the monooxygenation of one substrate to create highly efficient E. coli whole cell biocatalysts that facilitates oxygenation of hydrocarbons under mild conditions..
  • Development of new protein drug delivery systems
    keyword : protein drug delivery, pharmaceutical formulation, oral administration, vaccine
    2003.04~2011.03We have focused on the development of a novel lipid-based drug formulation toward oral delivery of pharmaceutical peptide and protein drugs..
Academic Activities
1. Riko Nishioka, Ryo Sato, Kazuki Uchida, Rie Wakabayashi, Noriho Kamiya, Microbial transglutaminases in drug development, Academic Press, Elsevier, Transglutaminase: Fundamentals and Applications
Editors: Y. Zhang and B.K.Simpson
Chapter 9, pp.169-180 (2024)., 2024.01.
2. 神谷 典穂, 森 裕太郎, Substrate engineering of microbial transglutaminase for site-specific protein modification and bioconjugation, Springer , Transglutaminases -Multiple Functional Modifiers and Targets for New Drug Discovery-
Editors K. Hitomi, L. Fesus, S. Kojima
Springer, Chapter 17, p.373-383 (2015), 2016.01.
3. 神谷 典穂, K. MIyawaki, S. Noji, Transglutaminase-mediated in situ hybridization (TransISH) for mRNA detection in mammalian tissues, Springer Science+Business Media, NY, In Situ Hybridization Methods, vol.99 (G. Hauptmann (ed.))
Neuromethods, Chapter 29, p.549-558 (2015), 2015.04.
4. N. Kamiya, H. Abe, New fluorescent substrates of microbial transglutaminase and its application to covalent protein labeling, Humana Press, NY, Bioconjugation Protocols, Second Edition
Methods in Molecular Biology, Chapter 9, 2011.08.
1. Momoko Kitaoka, Rie Wakabayashi, Noriho Kamiya, Masahiro Goto, Solid-in-oil nanodispersions for transdermal drug delivery systems, Biotechnol. J., 11(11), 1375-1385 (2016) [Open Access], 2016.11.
2. LOPES GONCALVES GEISA APARECIDA, Noriho Kamiya, Biomolecular assembly strategies to develop potent artificial cellulosomes, Sustainable Chemical Processes, 2:19 (2014), 2014.10.
3. Cross-linking chemistry and biotechnology by transglutaminase.
4. New technoogies for oriented protein immobilization.
1. Ryutaro Ariyoshi, Takashi Matsuzaki, Ryo Sato, Kosuke Minamihata, Kounosuke Hayashi, Taisei Koga, Kensei Orita, Riko Nishioka, Rie Wakabayashi, Masahiro Goto, Noriho Kamiya, Engineering the Propeptide of Microbial Transglutaminase Zymogen: Enabling Substrate-Dependent Activation for Bioconjugation Applications., Bioconjugate chemistry, 10.1021/acs.bioconjchem.3c00544, 2024.02, Microbial transglutaminase (MTG) from Streptomyces mobaraensis is a powerful biocatalytic glue for site-specific cross-linking of a range of biomolecules and synthetic molecules that have an MTG-reactive moiety. The preparation of active recombinant MTG requires post-translational proteolytic digestion of a propeptide that functions as an intramolecular chaperone to assist the correct folding of the MTG zymogen (MTGz) in the biosynthesis. Herein, we report engineered active zymogen of MTG (EzMTG) that is expressed in soluble form in the host Escherichia coli cytosol and exhibits cross-linking activity without limited proteolysis of the propeptide. We found that the saturation mutagenesis of residues K10 or Y12 in the propeptide domain generated several active MTGz mutants. In particular, the K10D/Y12G mutant exhibited catalytic activity comparable to that of mature MTG. However, the expression level was low, possibly because of decreased chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. The K10R/Y12A mutant exhibited specific substrate-dependent reactivity toward peptidyl substrates. Quantitative analysis of the binding affinity of the mutated propeptides to the active site of MTG suggested an inverse relationship between the binding affinity and the catalytic activity of EzMTG. Our proof-of-concept study provides insights into the design of a new biocatalyst using the MTGz as a scaffold and a potential route to high-throughput screening of EzMTG mutants for bioconjugation applications..
2. Diah Anggraini Wulandari, Kyosuke Tsuru, Kosuke Minamihata, Rie Wakabayashi, Masahiro Goto, Noriho Kamiya, A Functional Hydrogel Bead-Based High-Throughput Screening System for Mammalian Cells with Enhanced Secretion of Therapeutic Antibodies., ACS Biomaterials Science & Engineering, 10.1021/acsbiomaterials.3c01386, 10, 1, 628-636, 2024.01, Droplet-based high-throughput screening systems are an emerging technology that provides a quick test to screen millions of cells with distinctive characteristics. Biopharmaceuticals, specifically therapeutic proteins, are produced by culturing cells that secrete heterologous recombinant proteins with different populations and expression levels; therefore, a technology to discriminate cells that produce more target proteins is needed. Here, we present a droplet-based microfluidic strategy for encapsulating, screening, and selecting target cells with redox-responsive hydrogel beads (HBs). As a proof-of-concept study, we demonstrate the enrichment of hybridoma cells with enhanced capability of antibody secretion using horseradish peroxidase (HRP)-catalyzed hydrogelation of tetra-thiolate poly(ethylene glycol); hybridoma cells were encapsulated in disulfide-bonded HBs. Recombinant protein G or protein M with a C-terminal cysteine residue was installed in the HBs via disulfide bonding to capture antibodies secreted from the cells. HBs were fluorescently stained by adding the protein L-HRP conjugate using a tyramide signal amplification system. HBs were then separated by fluorescence-activated droplet sorting and degraded by reducing the disulfide bonds to recover the target cells. Finally, we succeeded in the selection of hybridoma cells with enhanced antibody secretion, indicating the potential of this system in the therapeutic protein production..
3. Pugoh Santoso, Takuya Komada, Yugo Ishimine, Hiromasa Taniguchi, Kosuke Minamihata, Masahiro Goto, Toki Taira, Noriho Kamiya , Preparation of amphotericin B-loaded hybrid liposomes and the integration of chitin-binding proteins for enhanced antifungal activity, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2022.06.005, 2022.09.
4. Hiromasa Taniguchi, Yugo Ishimime, Kosuke Minamihata, Pugoh Santoso, Takuya Komada, Hendra Saputra, Kazuki Uchida, Masahiro Goto, Toki Taira, Noriho Kamiya , Liposomal Amphotericin B Formulation Displaying Lipid-Modified Chitin-Binding Domains with Enhanced Antifungal Activity, Molecular Pharmaceutics, 10.1021/acs.molpharmaceut.2c00388, 2022.09.
5. Pugoh Santoso, Kosuke Minamihata, Yugo Ishimine, Hiromasa Taniguchi, Takuya Komada, Ryo Sato, Masahiro Goto, Tomoya Takashima, Toki Taira, Noriho Kamiya , Enhancement of the Antifungal Activity of Chitinase by Palmitoylation and the Synergy of Palmitoylated Chitinase with Amphotericin B, ACS Infectious Diseases,, 8, 5, 1051-1061, 2022.04.
6. Kazuki Uchida, Hiroki Obayashi, Kosuke Minamihata, Rie Wakabayashi, Masahiro Goto, Naofumi Shimokawa, Masahiro Takagi, Noriho Kamiya, Artificial Palmitoylation of Proteins Controls the Lipid Domain-Selective Anchoring on Biomembranes and the Raft-Dependent Cellular Internalization, Langmuir, 10.1021/acs.langmuir.2c01205, 2022.08.
7. Hori, Katsutoshi; Yoshimoto, Shogo; Yoshino, Tomoko; Zako, Tamotsu; Hirao, Gen; Fujita, Satoshi; Nakamura, Chikashi; Yamagishi, Ayana; Kamiya, Noriho, Recent advances in research on biointerfaces: From cell surfaces to artificial interfaces, J. Biosci. Bioeng., 10.1016/j.jbiosc.2021.12.004, 133, 3, 195-207, 2022.03.
8. K. Minamihata, Y. Tanaka, P. Santoso, M. Goto, D. Kozome, T. Taira, N. Kamiya, Orthogonal Enzymatic Conjugation Reactions Create Chitin Binding Domain Grafted Chitinase Polymers with Enhanced Antifungal Activity, Bioconjugate Chem., 10.1021/acs.bioconjchem.1c00235, 32, 8, 1688-1698, 2021.08.
9. Ryo Sato, Kosuke Minamihata, Ryutaro Ariyoshi, Hiromasa Taniguchi, Noriho Kamiya, Recombinant production of active microbial transglutaminase in E. coli by using self-cleavable zymogen with mutated propeptide, Protein Expression and Purification, 10.1016/j.pep.2020.105730, 2020.12, Microbial transglutaminase from Streptomyces mobaraensis (MTG) has been widely used in food industry and also in research and medical applications, since it can site-specifically modify proteins by the cross-linking reaction of glutamine residue and the primary amino group. The recombinant expression system of MTG in E. coli provides better accessibility for the researchers and thus can promote further utilization of MTG. Herein, we report production of active and soluble MTG in E. coli by using a chimeric protein of tobacco etch virus (TEV) protease and MTG zymogen. A chimera of TEV protease and MTG zymogen with native propeptide resulted in active MTG contaminated with cleaved propeptide due to the strong interaction between the propeptide and catalytic domain of MTG. Introduction of mutations of K9R and Y11A to the propeptide facilitated dissociation of the cleaved propeptide from the catalytic domain of MTG and active MTG without any contamination of the propeptide was obtained. The specific activity of the active MTG was 22.7 ± 2.6 U/mg. The successful expression and purification of active MTG by using the chimera protein of TEV protease and MTG zymogen with mutations in the propeptide can advance the use of MTG and the researches using MTG mediated cross-linking reactions..
10. Wahyu Ramadhan, Yuki Ohama, Kosuke Minamihata, Kousuke Moriyama, Rie Wakabayashi, Masahiro Goto, Noriho Kamiya, Redox-responsive functionalized hydrogel marble for the generation of cellular spheroids, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2020.05.010, 130, 4, 416-423, 2020.10, Liquid marbles (LMs) have recently shown a great promise as microbioreactors to construct self-supported aqueous compartments for chemical and biological reactions. However, the evaporation of the inner aqueous liquid core has limited their application, especially in studying cellular functions. Hydrogels are promising scaffolds that provide a spatial environment suitable for three-dimensional cell culture. Here, we describe the fabrication of redox-responsive hydrogel marbles (HMs) as a three-dimensional cell culture platform. The HMs are prepared by introducing an aqueous mixture of a tetra-thiolated polyethylene glycol (PEG) derivative, thiolated gelatin (Gela-SH), horseradish peroxidase, a small phenolic compound, and human hepatocellular carcinoma cells (HepG2) to the inner aqueous phase of LMs. Eventually, HepG2 cells are encapsulated in the HMs then immersed in culture media, where they proliferate and form cellular spheroids. Experimental results show that the Gela-SH concentration strongly influences the physicochemical and microstructure properties of the HMs. After 6 days in culture, the spheroids were recovered from the HMs by degrading the scaffold, and examination showed that they had reached up to about 180 μm in diameter depending on the Gela-SH concentration, compared with 60 μm in conventional HMs without Gela-SH. After long-term culture (over 12 days), the liver-specific functions (secretion of albumin and urea) and DNA contents of the spheroids cultured in the HMs were elevated compared with those cultured in LMs. These results suggest that the developed HMs can be useful in designing a variety of microbioreactors for tissue engineering applications..
11. K. Minamihata*, Y. Hamada, G. Kagawa, W. Ramadhan, A. Higuchi, K. Moriyama, R. Wakabayashi, M. Goto, N. Kamiya*, Dual-Functionalizable Streptavidin–SpyCatcher-Fused Protein–Polymer Hydrogels as Scaffolds for Cell Culture, ACS Appl. Bio Mater.,, 3, 7734-7742, 2020.10.
12. Wahyu Ramadhan, Genki Kagawa, Kousuke Moriyama, Rie Wakabayashi, Kosuke Minamihata, Masahiro Goto, Noriho Kamiya, Construction of higher-order cellular microstructures by a self-wrapping co-culture strategy using a redox-responsive hydrogel, Scientific reports, 10.1038/s41598-020-63362-4, 10, 1, 2020.05, In this report, a strategy for constructing three-dimensional (3D) cellular architectures comprising viable cells is presented. The strategy uses a redox-responsive hydrogel that degrades under mild reductive conditions, and a confluent monolayer of cells (i.e., cell sheet) cultured on the hydrogel surface peels off and self-folds to wrap other cells. As a proof-of-concept, the self-folding of fibroblast cell sheet was triggered by immersion in aqueous cysteine, and this folding process was controlled by the cysteine concentration. Such folding enabled the wrapping of human hepatocellular carcinoma (HepG2) spheroids, human umbilical vein endothelial cells and collagen beads, and this process improved cell viability, the secretion of metabolites and the proliferation rate of the HepG2 cells when compared with a two-dimensional culture under the same conditions. A key concept of this study is the ability to interact with other neighbouring cells, providing a new, simple and fast method to generate higher-order cellular aggregates wherein different types of cellular components are added. We designated the method of using a cell sheet to wrap another cellular aggregate the ‘cellular Furoshiki’. The simple self-wrapping Furoshiki technique provides an alternative approach to co-culture cells by microplate-based systems, especially for constructing heterogeneous 3D cellular microstructures..
13. Mari Takahara, Noriho Kamiya, Synthetic Strategies for Artificial Lipidation of Functional Proteins, Chemistry - A European Journal, 10.1002/chem.201904568, 26, 21, 4645-4655, 2020.04, Biosynthesis of natural lipidated proteins is linked to important signal pathways, and therefore analyzing protein lipidation is crucial for understanding cellular functions. Artificial lipidation of proteins has attracted attention in recent decades as it allows modulation of the amphiphilic nature of the protein of interest, and is used in the design of drug-delivery systems containing antibodies anchored on a lipid bilayer carrier. However, the intrinsic hydrophobicity of lipids makes the synthesis of lipid–protein conjugates challenging with respect to the yield and selectivity of the lipidation. In this Minireview, the development of chemical and enzymatic synthetic strategies for the preparation of a range of lipid–protein conjugates that do not compromise the functions of the proteins are discussed as well as applications of the conjugates..
14. Dani Permana, Kosuke Minamihata, Ryo Sato, Rie Wakabayashi, Masahiro Goto, Noriho Kamiya, Linear Polymerization of Protein by Sterically Controlled Enzymatic Cross-Linking with a Tyrosine-Containing Peptide Loop, ACS Omega, 10.1021/acsomega.9b04163, 5, 10, 5160-5169, 2020.03, The structure of a protein complex needs to be controlled appropriately to maximize its functions. Herein, we report the linear polymerization of bacterial alkaline phosphatase (BAP) through the site-specific cross-linking reaction catalyzed by Trametes sp. laccase (TL). We introduced a peptide loop containing a tyrosine (Y-Loop) to BAP, and the Y-Looped BAP was treated with TL. The Y-Looped BAP formed linear polymers, whereas BAP fused with a C-terminal peptide containing a tyrosine (Y-tag) showed an irregular shape after TL treatment. The sterically confined structure of the Y-Loop could be responsible for the formation of linear BAP polymers. TL-catalyzed copolymerization of Y-Looped BAP and a Y-tagged chimeric antibody-binding protein, pG2pA-Y, resulted in the formation of linear bifunctional protein copolymers that could be employed as protein probes in an enzyme-linked immunosorbent assay (ELISA). Copolymers comprising Y-Looped BAP and pG2pA-Y at a molar ratio of 100:1 exhibited the highest signal in the ELISA with 26- and 20-fold higher than a genetically fused chimeric protein, BAP-pG2pA-Y, and its polymeric form, respectively. This result revealed that the morphology of the copolymers was the most critical feature to improve the functionality of the protein polymers as detection probes, not only for immunoassays but also for other diagnostic applications..
15. Rie Wakabayashi, Wahyu Ramadhan, Kousuke Moriyama, Masahiro Goto, Noriho Kamiya, Poly(ethylene glycol)-based biofunctional hydrogels mediated by peroxidase-catalyzed cross-linking reactions, Polymer Journal, 10.1038/s41428-020-0344-7, 2020.01, Biofunctional hydrogels prepared by a peroxidase, especially horseradish peroxidase (HRP), serve as an excellent class of materials or platform for the development of cellular scaffolds because their biocompatibility and mild and tunable reaction conditions provide them with desirable properties. In this focus review, we summarize our decade of research into HRP-mediated fabrication of biofunctional hydrogels and their applications, in particular cell culture scaffolds. A brief overview of potential substrates employed in HRP and improvement of the HRP hydrogelation system from the initial step until the hydrogen peroxide removal stage in an effort to meet environmental standards is discussed. We highlight our system and describe its biocompatibility and ability to functionalize molecules to support biofabrication by increasing cellular adhesiveness, retaining growth factor affinity, and finally accelerating the formation of two- and three-dimensional multicellular architectures. In the last section, we outline the adoption of hydrogelation as a self-standing, compartmentalized reaction system, i.e., the use of hydrogel marble to conduct cell-free biosynthesis. We believe that this HRP-mediated hydrogel system offers great potential not only as a cell culture scaffold but also for various biomedical applications..
16. R. Sato, K. Minamihata, R. Wakabayashi, M. Goto, N. Kamiya, PolyTag
A peptide tag that affords scaffold-less covalent protein assembly catalyzed by microbial transglutaminase, Analytical Biochemistry, 10.1016/j.ab.2020.113700, 2020.01, Assembling proteins in close vicinity to each other provides an opportunity to gain unique function because collaborative and even synergistic functionalities can be expected in an assembled form. There have been a variety of strategies to synthesize functional protein assemblies but site-specific covalent assembly of monomeric protein units without impairing their intrinsic function remains challenging. Herein we report a powerful strategy to design protein assemblies by using microbial transglutaminase (MTG). A serendipitous discovery of self-crosslinking of enhanced green fluorescent protein (EGFP) fused with StrepTag I at the C-terminus revealed that EGFP was assembled through the crosslinking of the Lys (K) residue in the C-terminus of EGFP and the Gln (Q) residue in StrepTag I (AWRHPQFGG). Site-directed mutagenesis of the residues next to the K and Q yielded EGFP assemblies with higher molecular weights. An optimized peptide tag comprised of both K and Q residues (HKRWRHYQRGG) enabled the assembly of different types of proteins of interest (POI) when it was fused to either the N- or C-terminus. The peptide tag that enabled the self-polymerization of the functional POI without a scaffold was designated as a ‘PolyTag’..
17. Wahyu Ramadhan, Genki Kagawa, Yusei Hamada, Kousuke Moriyama, Rie Wakabayashi, Kosuke Minamihata, Masahiro Goto, Noriho Kamiya, Enzymatically Prepared Dual Functionalized Hydrogels with Gelatin and Heparin to Facilitate Cellular Attachment and Proliferation, ACS Applied Bio Materials, 10.1021/acsabm.9b00275, 2, 6, 2600-2609, 2019.06, Biologically active artificial scaffolds for cell seeding are developed by mimicking extracellular matrices using synthetic materials. Here, we propose a feasible approach employing biocatalysis to integrate natural components, that is, gelatin and heparin, into a synthetic scaffold, namely a polyethylene glycol (PEG)-based hydrogel. Initiation of horseradish peroxidase-mediated redox reaction enabled both hydrogel formation of tetra-thiolated PEG via disulfide linkage and incorporation of chemically thiolated gelatin (Gela-SH) and heparin (Hepa-SH) into the polymeric network. We found that the compatibility of the type of gelatin with heparin was crucial for the hydrogelation process. Alkaline-treated gelatin exhibited superior performance over acid-treated gelatin to generate dual functionality in the resultant hydrogel originating from the two natural biopolymers. The Gela-SH/Hepa-SH dual functionalized PEG-based hydrogel supported both cellular attachment and binding of basic fibroblast growth factor (bFGF) under cell culture conditions, which increased the proliferation and phenotype transformation of NIH3T3 cells cultured on the hydrogel. Inclusion of bFGF and a commercial growth factor cocktail in hydrogel matrices effectively enhanced cell spreading and confluency of both NIH3T3 cells and HUVECs, respectively, suggesting a potential method to design artificial scaffolds containing active growth factors..
18. Mari Takahara, Rie Wakabayashi, Naoki Fujimoto, Kosuke Minamihata, Masahiro Goto, Noriho Kamiya, Enzymatic Cell-Surface Decoration with Proteins using Amphiphilic Lipid-Fused Peptide Substrates, Chemistry - A European Journal, 10.1002/chem.201900370, 25, 30, 7315-7321, 2019.05, Lipid modification of proteins plays a significant role in the activation of cellular signals such as proliferation. Thus, the demand for lipidated proteins is rising. However, getting a high yield and purity of lipidated proteins has been challenging. We developed a strategy for modifying proteins with a wide variety of synthetic lipids using microbial transglutaminase (MTG), which catalyzes the cross-linking reaction between a specific glutamine (Q) in a protein and lysine (K) in the lipid-fused peptide. The synthesized lipid substrates (lipid: fatty acids, tocopherol, lithocholic acid, cholesterol) was successfully conjugated to a protein fused with LLQG (Q-tagged protein) by an MTG reaction, yielding 90 % conversion of the Q-tagged protein in a lipidated form. The purified lipid–protein conjugates were used for labeling the cell membrane in vitro, resulting in best-anchoring ability of cholesterol modification. Furthermore, in situ cell-surface decoration with the protein was established in a simple manner: subjection of cells to a mixture of cholesterol-fused peptides, Q-tagged proteins and MTG..
19. Muhamad Alif Razi, Rie Wakabayashi, Masahiro Goto, Noriho Kamiya, Self-Assembled Reduced Albumin and Glycol Chitosan Nanoparticles for Paclitaxel Delivery, Langmuir, 10.1021/acs.langmuir.8b02809, 35, 7, 2610-2618, 2019.02, Cancer continues to pose health problems for people all over the world. Nanoparticles (NPs) have emerged as a promising platform for effective cancer chemotherapy. NPs formed by the assembly of proteins and chitosan (CH) through noncovalent interactions are attracting a great deal of interest. However, the poor water solubility of CH and low stability of this kind of NP limit its practical application. Herein, the formation of reduced bovine serum albumin (rBSA) and glycol chitosan (GC) nanoparticles (rBG-NPs) stabilized by hydrophobic interactions and disulfide bonds was demonstrated for paclitaxel (PTX) delivery. The effects of the rBSA:GC mass ratio and pH on the particle size, polydispersity index (PDI), number of particles, and surface charge were evaluated. The formation mechanism and stability of the NPs were determined by compositional analysis and dynamic light scattering. Hydrophobic and electrostatic interactions were the driving forces for the formation of the rBG-NPs, and the NPs were stable under physiological conditions. PTX was successfully encapsulated into rBG-NPs with a high encapsulation efficiency (90%). PTX-loaded rBG-NPs had a particle size of 400 nm with a low PDI (0.2) and positive charge. rBG-NPs could be internalized by HeLa cells, possibly via endocytosis. An in vitro cytotoxicity study revealed that PTX-loaded rBG-NPs had anticancer activity that was lower than that of a Taxol-like formulation at 24 h but had similar activity at 48 h, possibly because of the slow release of PTX into the cells. Our study suggests that rBG-NPs could be used as a potential nanocarrier for hydrophobic drugs..
20. Rie Wakabayashi, Ayumi Suehiro, Masahiro Goto, Noriho Kamiya, Designer aromatic peptide amphiphiles for self-assembly and enzymatic display of proteins with morphology control, Chemical Communications, 10.1039/C8CC08163H, 55, 5, 640-643, 2019.01, We herein designed bi-functional aromatic peptide amphiphiles both self-assembling to fibrous nanomaterials and working as a substrate of microbial transglutaminase, leading to peptidyl scaffolds with different morphologies that can be enzymatically post-functionalized with proteins..
21. Noriho Kamiya, Yuki Ohama, Kosuke Minamihata, Rie Wakabayashi, Masahiro Goto, Liquid Marbles as an Easy-to-Handle Compartment for Cell-Free Synthesis and In Situ Immobilization of Recombinant Proteins, Biotechnology Journal, 10.1002/biot.201800085, 13, 12, 2018.12, Liquid marble (LM), a self-standing micro-scale aqueous droplet, emerges as a micro-bioreactor in biological applications. Herein, the potential of LM as media for cell-free synthesis and simultaneous immobilization of recombinant proteins is explored. Initially, formation of hydrogel marble (HM) by using an enzymatic disulfide-based hydrogelation technique is confirmed by incorporating three components, horseradish peroxidase (HRP), a tetra-thiolated poly(ethylene glycol) derivative, and glycyl-L-tyrosine, in LM. The compatibility of the enzymatic hydrogelation with cell-free protein synthesis in LM is then validated. Although the hydrogelation reduces the level of protein synthesis in LM when compared with that in a test tube, the biosynthesis of enhanced green fluorescent protein (EGFP) is achieved. Interestingly, EGFP synthesized in LM is entrapped in the HM, and the introduction of a cysteine residue to EGFP by genetic engineering further increases the amount of protein immobilization in the hydrogel matrices. These results suggest that the cell-free synthesis and HRP-catalyzed hydrogelation can be conducted in parallel in LM, and the eventual entrapment of the key components in HM is possible. Facile recovery of macromolecular products immobilized in HM by degrading the hydrogel network under reducing conditions should lead to the design of an easy-to-handle system to screen protein functions..
22. Mari Takahara, Rie Wakabayashi, Kosuke Minamihata, Masahiro Goto, Noriho Kamiya, Design of Lipid-Protein Conjugates Using Amphiphilic Peptide Substrates of Microbial Transglutaminase, ACS App. Bio Mater., 10.1021/acsabm.8b00271, 1, 6, 1823-1829, 2018.10, Lipid modification of proteins plays a significant role in the activation of cellular signals such as proliferation. Thus, the demand for lipidated proteins is rising. However, getting a high yield and purity of lipidated proteins has been challenging. We developed a strategy for modifying proteins with a wide variety of synthetic lipids using microbial transglutaminase (MTG), which catalyzes the cross-linking reaction between a specific glutamine (Q) in a protein and lysine (K) in the lipid-fused peptide. The synthesized lipid-G3S-MRHKGS lipid (lipid: fatty acids, tocopherol, lithocholic acid, cholesterol) was successfully conjugated to a protein fused with LLQG (Q-tagged protein) by an MTG reaction, yielding >90 % conversion of the Q-tagged protein in a lipidated form. The purified lipid–protein conjugates were used for labeling the cell membrane in vitro, resulting in best-anchoring ability of cholesterol modification. Furthermore, in situ cell-surface decoration with the protein was established in a simple manner: subjection of cells to a mixture of cholesterol-fused peptides, Q-tagged proteins and MTG..
23. Safrina Dyah Hardiningtyas, Rie Wakabayashi, Momoko Kitaoka, Yoshiro Tahara, Kosuke Minamihata, Masahiro Goto, Noriho Kamiya, Mechanistic investigation of transcutaneous protein delivery using solid-in-oil nanodispersion
A case study with phycocyanin, European Journal of Pharmaceutics and Biopharmaceutics, 10.1016/j.ejpb.2018.01.020, 127, 44-50, 2018.06, Phycocyanin (PC), a water-soluble protein-chromophore complex composed of hexameric (αβ)6 subunits, has important biological functions in blue-green algae as well as pharmacological activities in biomedicine. We have previously developed a solid-in-oil (S/O) nanodispersion method to deliver biomacromolecules through the skin, although the transcutaneous mechanism has not yet been fully elucidated. To study the mechanism of transcutaneous protein delivery, we therefore enabled S/O nanodispersion by coating PC with hydrophobic surfactants and evaluated how the proteinaceous macromolecules formulated in an oil phase might permeate the skin. The extent of S/O nanodispersion of PC was dependent on the type of surfactant, suggesting that the selection of a suitable surfactant is crucial for encapsulating a large protein having a subunit structure. By measuring the intrinsic fluorescence of PC, we found that S/O nanodispersion facilitated the accumulation of PC in the stratum corneum (SC) of Yucatan micropig skin. Furthermore, after crossing the SC layer, the fluorescent recovery of PC was evident, indicating the release of the biologically active form of PC from the SC into the deeper skin layer..
24. Patma, Kosuke Minamihata, T. Tatsuke, Jae Man Lee, Takahiro Kusakabe, Noriho Kamiya, Expression and Activation of Horseradish Peroxidase–Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes, Biotechnology Journal, 10.1002/biot.201700624, 13, 1700624, 2018.06.
25. Muhamad Alif Razi, Rie Wakabayashi, Yoshiro Tahara, Masahiro Goto, Noriho Kamiya, Genipin-stabilized caseinatehitosan nanoparticles for enhanced stability and anti-cancer activity of curcumin, Colloids and Surfaces B: Biointerfaces, 10.1016/j.colsurfb.2018.01.041, 164, 308-315, 2018.04, Nanoparticles formed by the assembly of protein and polysaccharides are of great interest for the delivery of hydrophobic molecules. Herein, the formation of genipin-crosslinked nanoparticles from caseinate (CS) and chitosan (CH) is reported for the delivery of curcumin, a polyphenolic compound from turmeric, to cells. Genipin-crosslinked CS-CH nanoparticles (G-CCNPs) having a diameter of ∼250 nm and a low polydispersity index showed excellent stability over a wide pH range, as indicated by dynamic light scattering and transmission electron microscopic measurements. Cellular uptake of curcumin loaded into G-CCNPs by HeLa cells was improved, as measured by confocal laser scanning microscopy (CLSM) and fluorescence-activated cell-sorting analysis. Cell proliferation assays indicated that G-CCNPs were nontoxic and that curcumin's anticancer activity in vitro was also improved by G-CCNPs. Stability of curcumin at neutral pH was enhanced by G-CCNPs. CLSM study revealed that G-CCNPs were poorly internalized by HeLa cells, possibly because of strong cell membrane interactions and a negative zeta potential. Overall, our results suggested that the enhanced curcumin cytotoxicity might be associated with the enhanced stability of curcumin by G-CCNPs and free curcumin released from G-CCNPs into the cell. These biocompatible NPs might be suitable carriers for enhancing curcumin's therapeutic potential..
26. Lili Jia, Kosuke Minamihata, Hirofumi Ichinose, Kouhei Tsumoto, Noriho Kamiya, Polymeric SpyCatcher Scaffold Enables Bioconjugation in a Ratio-Controllable Manner, Biotechnology Journal, 10.1002/biot.201700195, 12, 12, 2017.12, Conjugating enzymes into a large protein assembly often results in an enhancement of overall catalytic activity, especially when different types of enzymes that work cooperatively are assembled together. However, exploring the proper method to achieve protein assemblies with high stability and also to avoid loss of the function of each component for efficient enzyme clustering is remained challenging. Assembling proteins onto synthetic scaffolds through varied post-translational modification methods is particularly favored since the proteins can be site-specifically conjugated together with less activity loss. Here, a SpyCatcher polymer is prepared through catalytic reaction of horseradish peroxidase (HRP) and serves as a polymeric proteinaceous scaffold for construction of protein assemblies. Taking advantage of the favorable SpyCatcher–SpyTag interaction, SpyTagged proteins can be easily assembled onto the polymeric SpyCatcher scaffold with controllable binding ratio and site specificity. Firstly, the feasibility of construction of ratio-controllable binary artificial hemicellulosomes by assembling endoxylanase and arabinofuranosidase is explored. This construct achieves higher sugar conversion than that of the free enzymes when the proportion of arabinofuranosidase is high, because the close spatial proximity of the enzymes allows them to work in a synergistic manner. Another application for biosensing is developed by conjugating SpyTagged Nanoluc and protein G onto SpyCatcher polymer. Due to the protein clustering effect, an amplified luminescent intensity is achieved by the resulting conjugates than chimera protein of Nanoluc and protein G in ovalbumin detection in ELISA..
27. Mari Takahara, Rie Wakabayashi, Kosuke Minamihata, Masahiro Goto, Noriho Kamiya, Primary Amine-Clustered DNA Aptamer for DNA-Protein Conjugation Catalyzed by Microbial Transglutaminase, Bioconjugate Chemistry, 10.1021/acs.bioconjchem.7b00594, 28, 12, 2954-2961, 2017.12, DNA-protein conjugates are promising biomolecules for use in areas ranging from therapeutics to analysis because of the dual functionalities of DNA and protein. Conjugation requires site-specific and efficient covalent bond formation without impairing the activity of both biomolecules. Herein, we have focused on the use of a microbial transglutaminase (MTG) that catalyzes the cross-linking reaction between a glutamine residue and a primary amine. In a model bioconjugation, a highly MTG-reactive Gln (Q)-donor peptide (FYPLQMRG, FQ) was fused to enhanced green fluorescent protein (FQ-EGFP) and a primary amine-clustered DNA aptamer was enzymatically synthesized as a novel acyl-acceptor substrate of MTG, whose combination leads to efficient and convenient preparation of DNA-protein conjugates with high purity. Dual functionality of the obtained DNA-EGFP conjugate was evaluated by discrimination of cancer cells via c-Met receptor recognition ability of the DNA aptamer. The DNA aptamer-EGFP conjugate only showed fluorescence toward cells with c-Met overexpression, indicating the retention of the biochemical properties of the DNA and EGFP in the conjugated form..
28. Moriyama, Kousuke, Naito, Shono, Rie Wakabayashi, Masahiro Goto, Noriho Kamiya, Enzymatically prepared redox-responsive hydrogels as potent matrices for hepatocellular carcinoma cell spheroid formation, BIOTECHNOLOGY JOURNAL, 10.1002/biot.201600087, 11, 11, 1452-1460, 2016.11.
29. Takahara, Mari, Budinova, Geisa Aparecida Lopes Goncalves, Nakazawa, Hikaru, Mori, Yutaro, Umetsu, Mitsuo, Kamiya, Noriho, Salt-Switchable Artificial Cellulase Regulated by a DNA Aptamer, BIOMACROMOLECULES, 10.1021/acs.biomac.6b01141, 17, 10, 3356-3362, 2016.10.
30. Mori, Yutaro, Budinova, Geisa Aparecida Lopes Goncalves, Nakazawa, Hikaru, Umetsu, Mitsuo, Kamiya, Noriho, One-dimensional assembly of functional proteins: toward the design of an artificial cellulosome, Mol. Syst. Des. Eng., 10.1039/C6ME90005D, 1, 1, 66-73, 2016.04, In biological systems, proteins can form well-organized, higher-order structures with unique functions that would be difficult to achieve with a single protein. These proteinaceous supramolecular structures form by self-assembly, and the spatial arrangement of the protein building blocks in them is very important. In the present study, an artificial system was developed using recombinant proteins as building blocks, which were assembled in a one-dimensional manner. The assembly of these building blocks was based on the avidin-biotin interaction. A tetrameric biotin ligand unit was designed so that the 1:4 stoichiometry of the avidin-biotin interaction was altered to a 1:2 directional interaction between the streptavidin and tetrabiotinylated protein units. In a proof-of-concept study, site-specifically tetrabiotin-labeled endoglucanase and cellulose-binding module units were prepared, then these components were self-assembled by mixing with streptavidin to mimic a natural cellulosome. The formation of one-dimensional assemblies of the protein units depended on the stoichiometry of the avidin-biotin interaction sites in the system. Interestingly, the saccharification efficiency improved when the component ratio of protein units in the assemblies was changed..
31. Yukiho HOSOMOMI, Teppei NIIDE, Rie Wakabayashi, Masahiro Goto, Noriho Kamiya, Biocatalytic Formation of Gold Nanoparticles Decorated with Functional Proteins inside Recombinant Escherichia coli Cells, ANALYTICAL SCIENCES, 32, 3, 295-300, Cover page illustration, 2016.03.
32. Hayashi, Kounosuke, JAE MAN LEE, Tomozoe, Yusuke, takahiro kusakabe, 神谷 典穂, Heme precursor injection is effective for Arthromyces ramosus peroxidase fusion protein production by a silkworm expression system, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 10.1016/j.jbiosc.2015.02.013, 120, 4, 384-386, 2015.10.
33. Lili Jia, LOPES GONCALVES GEISA APARECIDA, Yusaku Takasugi, Yutaro Mori, Shuhei Noda, Tsutomu Tanaka, Hirofumi Ichinose, 神谷 典穂, Effect of pretreatment methods on the synergism of cellulase and xylanase during the hydrolysis of bagasse, BIORESOURCE TECHNOLOGY, 10.1016/j.biortech.2015.02.041, 185, 158-164, 2015.06.
34. Kousuke Moriyama, Rie Wakabayashi, Masahiro Goto, 神谷 典穂, Enzyme-mediated preparation of hydrogels composed of poly(ethylene glycol) and gelatin as cell culture platforms, RSC ADVANCES, 10.1039/c4ra12334d, 5, 4, 3070-3073, 2015.03.
35. Kousuke Moriyama, Rie Wakabayashi, Masahiro Goto, 神谷 典穂, Characterization of Enzymatically Gellable, Phenolated Linear Poly(Ethylene Glycol) with Different Molecular Weights for Encapsulating Living Cells, BIOCHEMICAL ENGINEERING JOURNAL, 93, 25-30, 2015.01.
36. Kosuke Minamihata, Masahiro Goto, 神谷 典穂, Site-specific conjugation of an antibody-binding protein catalyzed by horseradish peroxidase creates a multivalent protein conjugate with high affinity to IgG, BIOTECHNOLOGY JOURNAL, 10.1002/biot.201400512, 10, 1, 222-226, 2015.01.
37. Kousuke Moriyama, Kosuke Minamihata, Rie Wakabayashi, Masahiro Goto, 神谷 典穂, Enzymatic preparation of a redox-responsive hydrogel for encapsulating and releasing living cells, CHEMICAL COMMUNICATIONS, 10.1039/c3cc49766f, 50, 44, 5895-5898, 2014.08, Horseradish peroxidase-mediated oxidative cross-linking of a thiolated poly(ethylene glycol) is promoted in the absence of exogenous hydrogen peroxide, by adding a small amount of phenolic compound under physiological conditions. The prepared hydrogel can encapsulate and release living mammalian cells..
38. Teppei Niide, Masahiro Goto, 神谷 典穂, Enzymatic self-sacrificial display of an active protein on gold nanoparticles, RSC ADVANCES, 10.1039/c3ra46384b, 4, 12, 5995-5998, 2014.04.
39. H. Abe, M. Goto, N. Kamiya, Protein lipidation catalyzed by microbial transglutaminase, Chem. Eur. J., 17, 14004-14008, 2011.12.
40. K. Minamihata, M. Goto, N. Kamiya, Protein heteroconjugation by the peroxidase-catalyzed tyrosine coupling reaction, Bioconjugate Chem., 22, 2332-2338, 2011.12.
41. T. Niide, M. Goto, N. Kamiya, Biocatalytic synthesis of gold nanoparticles with cofactor regeneration in recombinant Escherichia coli cells, Chem. Commun., 47, 7350-7352, 2011.05.
42. Y. Mori, M. Goto, N. Kamiya, Transglutaminase-mediated internal protein labeling with a designed peptide loop, Biochem. Biophys. Res. Commun., 410, 829-833, 2011.05.
43. Y. Mori, K. Minamihata, H. Abe, M. Goto, N. Kamiya, Protein assemblies by site-specific avidin-biotin interactions, Org. Biomol. Chem., 9, 5641-5644, 2011.05.
44. K. Minamihata, M. Goto, N. Kamiya, Site-specific protein cross-linking by peroxidase-catalyzed activation of a tyrosine-containing peptide tag, Bioconjugate Chem., 22, 74-81, 2011.04.
45. K. Moriyama, K. Sung, M. Goto, N. Kamiya, Immobilization of alkaline phosphatase on magnetic particles by site-specific and covalent cross-linking catalyzed by microbial transglutaminase, J. Biosci. Bioeng., 111, 650-653, 2011.04.
46. M. Kitaoka, Y. Tsuruda, Y. Tanaka, M. Goto, M. Mitsumori, K. Hayashi, Y. Hiraishi, K. Miyawaki, S. Noji, N. Kamiya, Transglutaminase-mediated synthesis of a novel DNA-(enzyme)n probe for highly sensitive DNA detection, Chem. Eur. J., 19, 5387-5392, 2011.03, 生体内ではほとんどのタンパク質が何らかの翻訳後修飾を受けることに着目し、翻訳後修飾過程で働く酵素の基質特異性を利用すれば、狙った部位でタンパク質を修飾できると考えた。従来の有機化学的手法では、タンパク質の狙った部位を選択的に修飾するのは極めて困難であった。そこでラベル化したい有機分子内に基質となる部位(酵素の認識部位)を設計し、これを糊代として利用することで、糊代選択的な生体分子の連結・修飾法を確立した。
47. T. Mouri, T. Shimizu, N. Kamiya,* H. Ichinose, M. Goto*, Design of a cytochrome P450BM3 reaction system linked by two-step cofactor regeneration catalyzed by a soluble transhydrogenase and glycerol dehydrogenase, Biotechnol. Prog., 25, 1372-1378, 2009.10.
48. N. Kamiya,* Y. Shiotari, M. Tokunaga, H. Matsunaga, H. Yamanouchi, K. Nakano, M. Goto, Stimuli-responsive nanoparticles composed of naturally occurring amphiphilic proteins, Chem. Commun., 5287-5289, 2009.08.
49. N. Kamiya,* H. Abe, M. Goto, Y. Tsuji, H. Jikuya, Fluorescent substrates for covalent protein labeling catalyzed by microbial transglutaminase, Org. Biomol. Chem., 7, 3407-3412, 2009.08.
50. K. Minamihata, N. Kamiya,* S. Kiyoyama, H. Sakuraba, T. Ohshima, M. Goto, Development of a novel immobilization method for enzymes from hyperthermophiles, Biotechnol. Lett., 31, 1037-1041, 2009.07.
51. N. Kamiya, S. Doi, Y. Tanaka, H. Ichinose, M. Goto, Functional immobilization of recombinant alkaline phosphatases bearing a glutamyl donor substrate peptide of microbial transglutaminase, J. Biosci. Bioeng., 104, 195-199, 2007.09.
52. Y. Tanaka, Y. Tsuruda, M. Nishi, N. Kamiya, M. Goto, Exploring enzymatic catalysis at a solid surface: a case study with transglutaminase-mediated protein immobilization, Org. Biomol. Chem., 5, 1764-1770, 2007.05.
53. J. Tominaga, N. Kamiya, M. Goto, An enzyme-labeled protein polymer bearing pendant haptens, Bioconjugate Chem., 18, 860-865, 2007.03.
54. K. Nakashima, T. Maruyama, N. Kamiya, M. Goto, Activation of lipase in ionic liquids by modification with comb-shaped poly(ethylene glycol), Sci. Technol. Adv. Mater., vol.7, 692-698 (2006), 2007.01.
55. J. Tominaga, Y. Kemori, Y. Tanaka, T. Maruyama, N. Kamiya, M. Goto, An enzymatic method for site-specific labeling of recombinant proteins with oligonucleotides, Chem. Commun., 401-403, 2007.01.
56. H. Piao, N. Kamiya, J. Watanabe, H. Yokoyama, A. Hirata, T. Fujii, I. Shimizu, S. Ito, M. Goto, Oral delivery of diclofenac sodium using a novel solid-in-oil suspension, Int. J. Pharm., vol.313, 159-162 (2006) , 2006.11.
57. K. Nakashima, T. Maruyama, N. Kamiya, M. Goto, Homogeneous Enzymatic Reaction in Ionic Liquids with Poly(ethylene glycol)-Modified Subtilisin, Org. Biomol. Chem., vol.4, 3462-3467 (2006), 2006.10.
58. T.Mouri, N.Kamiya, M.Goto, Increasing the catalytic performance of a whole cell biocatalyst harboring a cytochrome P450cam system by stabilization of an electron transfer component, Biotechnol. Lett., vol.28, 1509-1513 (2006), 2006.05.
59. T.Mouri, J.Michizoe, H.Ichinose, N.Kamiya, M.Goto, A recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration, Appl. Microbiol. Biotechnol., vol.72, 514-520 (2006), 2006.04.
60. T.Tanaka, N. Kamiya, T.Nagamune, N-terminal glycine-specific protein conjugation catalyzed by microbial transglutaminase, FEBS Letter, 10.1016/j.febslet.2005.02.064, 579, 10, 2092-2096, vol.579, 2092-2096 (2005), 2005.01.
61. N.Kamiya, S.Doi, J.Tominaga, H.Ichinose, M.Goto, Transglutaminase-mediated protein immobilization to casein nanolayers created on a plastic surface, Biomacromolecules, 10.1021/bm0494895, 6, 1, 35-38, 6, 35-38 (2005), 2005.01.
62. J. Tominaga, N. Kamiya, S. Doi, H. Ichinose, T. Maruyama, M. Goto, Design of a specific peptide tag that affords covalent and site-specific enzyme immobilization catalyzed by microbial transglutaminase, Biomacromolecules, 10.1021/bm050193o, 6, 4, 2299-2304, vol.6, 2299 -2304 (2005), 2005.01.
1. Noriho Kamiya, Biocatalysis for Biomolecular and Biomaterials engineering, The 9th International Symposium on Applied Chemistry & The 5th International Conference on Chemical & Material Engineering (ISAC-ICCME 2023), 2023.12.
2. Noriho Kamiya, Ryutaro Ariyoshi, Takashi Matsuzaki, Ryo Sato, Kosuke Minamihata, Konosuke Hayashi, Rie Wakabayashi, Masahiro Goto, Design of engineered active zymogen of microbial transglutaminase, Enzyme Engineering XXVII, 2023.10.
3. Noriho Kamiya, Riko Nishioka, Ryuya Iida, Kosuke Minamihata, Michio Kimura, Engineered Active Zymogen of Microbial Transglutaminase for Antibody-Drug Conjugation, The 16th Asian Congress on Biotechnology (ACB2023), 2023.10.
4. Noriho Kamiya, Hendra Saputra, Pugoh Santoso, Rie Wakabayashi, Toki Taira, Design of Lipid-based Bioconjugates with Antifungal Activity, Young Asian Biological Engineer’s Community (YABEC) 2023, 2023.07.
5. Noriho Kamiya, Rie Wakabayashi, Toki Taira, Enzymatic Protein Lipidation for Biomolecular Engineering at Biointerfaces, The 14th AFOB Regional Symposium (ARS 2023), 2023.04.
6. @Noriho Kamiya, Wahyu Ramadhan, Kosuke Minamihata, Rie Wakabayashi, Uju and Masahiro Goto, Functional modulation of biopolymers by biocatalysts: from bioconjugation to sustainable bioproduction, The 15th Asian Congress on Biotechnology (ACB 2022), 2022.10.
7. @Noriho Kamiya, Artificial Lipidation for Biomolecular Engineering at Biointerfaces, 2022 KSBB (Korean Society for Biotechnology and Bioengineering) Fall Meeting and International Symposium, 2022.09.
8. Pugoh Santoso, Kosuke Minamihata, Yugo Ishimine, Hiromasa Taniguchi, Ryo Sato, Masahiro Goto, Tomoya Takashima, Toki Taira, Noriho Kamiya, Synergistic antifungal activity by combining Amphotericin B with lipidated chitinase, APCChE 2022, 2022.08.
9. @Noriho Kamiya, Enzymatic Protein Lipidation for Biomolecular Engineering at Biointerfaces, The 13th AFOB Regional Symposium (ARS 2022) Taiwan series, 2022.06.
10. @Noriho Kamiya, Biomolecular engineering with microbial transglutaminase, European Society of Applied Biocatalysis (ESAB) Webinar ‘Enzyme Engineering’, 2022.02.
11. @Noriho Kamiya, Enzymatic manipulation of protein assemblies that function at biointerface, PacificChem2021, 2021.12.
12. Noriho Kamiya, Design of Bioconjugates that Function at Biological Interface, The Korean Society for Biotechnology and Bioengineering (KSBB) 2021 International Symposium, 2021.04.
13. Pugoh Santoso, Takuya Komada, Hiromasa Taniguchi, Yugo Ishimine, Ryo Sato, Kosuke Minamihata, Tomoya Takashima, Toki Taira, Noriho Kamiya, Synergistic Antifungal Action of Lipid-Modified Chitinase With Amphotericin-B, International Chemical Engineering Symposia 2021, 2021.03.
14. Noriho Kamiya, Biomolecular engineering for sustainable production of designer functional proteins, The 10th Symposium on Innovative Bioproduction Taichung (iBioT2019), 2019.11.
15. Noriho Kamiya*, Dani Permana, Wahyu Ramadhan, Kosuke Minamihata, Masahiro Goto, Biomolecular engineering by oxidative enzymatic manipulation, The 25th Young Asian Biological Engineer’s Community 2019, 2019.11.
16. Noriho Kamiya, Biomolecular engineering by biocatalysis for designer bio-based functional materials, International Symposium of Innovative Bio-production Indonesia on Biotechnology & Bioengineering (ISIBio2019), 2019.10.
17. Noriho Kamiya*, Kosuke Minamihata, Biomolecular engineering toward sustainable production of value-added functional proteins, 18th Asian Pacific Confederation of Chemical Engineering Congress (APCChE 2019), 2019.09.
18. Noriho Kamiya, Enzymatic biomolecular engineering toward designer bioconjugates and biomaterials, The 14th Asian Congress on Biotechnology (ACB 2019), 2019.07.
19. Rie Wakabayashi, Hiroki Obayashi, Noriho Kamiya, Masahiro Goto, Complemantary interaction with peptide amphiphiles guided the intracellular delivery of small molecular drugs, The 24th Symposium of Young Asian Biological Engineers' Community (YABEC2018), 2018.11.
20. Noriho Kamiya, Takashi Matsuzaki, Ryo Sato, Kounosuke Hayashi, Rie Wakabayashi, Kosuke Minamihata, Engineered active zymogen of microbial transglutaminase, The 15th Japan-China-Korea Joint Symposium on Enzyme Engineering, 2018.07.
21. Noriho Kamiya, Biocatalyst Engineering toward Biomedical Applications, ACB (Asian Congress on Biotechnology) 2017, 2017.07.
22. Noriho Kamiya, Enzyme-mediated Design of Functional Bioconjugates and Biomaterials, 2017 BEST Conference, 2017.06.
23. Noriho Kamiya, Mari Takahara, Rie Wakabayashi, Masahiro Goto, Design of novel biocatalysts by enzymatic biomolecular conjugation, The 9th AFOB Regional Symposium (ARS 2017), 2017.02.
24. 神谷 典穂, 南畑 孝介, Enzymatic Conjugation Strategy for the Design of Artificial Biomolecular Assemblies, 2016 AIChE Annual Meeting, 2016.11.
25. 神谷 典穂, Exploring biological strategies for sustainable utilization of lignocellulosic biomass, The e-ASIA Joint Research Program (e-ASIA JRP) Project Workshop, 2016.09.
26. 神谷 典穂, One-dimensional assembly of functional proteins by avidin-biotin interaction, Asian Congress on Biotechnology (ACB) 2015, 2015.11.
27. 神谷 典穂, Design of biomolecular assemblies by enzymatic protein manipulation, NANO KOREA 2015, 2015.07.
28. 神谷 典穂, Molecular design of biocatalytic assemblies for sustainable biotechnological applications, ARS 2015, 2015.05.
29. 神谷 典穂, Potential use of oxidoreductases for the fabrication of biomaterials, Active Enzyme Molecule 2014, 2014.12.
30. 神谷 典穂, Enzyme as a Catalytic Tool for Fabrication of Biomaterials, The 13th CJK Symposium on Enzyme Engineering, 2014.11.
31. 神谷 典穂, Self-sacrificial display of an active protein on gold nanoparticles, YABEC 2014, 2014.11.
32. 神谷 典穂, ENZYMATIC APPROACHES FOR ACCELERATING CELLULOSIC BIOMASS HYDROLYSIS, 16th International Biotechnology Symposium and Exhibition - IBS 2014, 2014.09.
33. 神谷 典穂, Enzyme as a catalytic tool for designing new bioconjugates, 2014 BEST Conference, 2014.06, Proteins exhibit multiple roles in living systems. In particular, enzymes facilitate metabolic pathways by catalyzing the different types of chemical reactions to sustain our life. A variety of enzyme functions have been exploited in both biochemical studies and biotechnological applications, however, there has still been a room for applying biocatalysis for the design and creation of artificial biomaterials.
In natural biological systems, proteins often form well-organized higher-order structures that associate unique functions, which cannot be accessed by a single protein unit alone. Interestingly, enzymatic post-translational modification of protein building blocks plays an important role in the formation of multi-subunit macromolecular structures.
Inspired by nature’s strategy, we are interested in configuring biocatalysis for creating new functional biomaterials. Herein, I’ll introduce our strategies which will be exemplified by three different types of enzymes (microbial trasnglutaminase, horseradish peroxidase, and glycerol dehydrogenase) to create (nano)biomaterials with distinct functions in line with their possible applications..
34. 神谷 典穂, Biomolecular Assembly by Enzymatic Conjugation and Scaffolding, 2013 KSBB Spring Meeting and International Symposium, 2014.04.
35. 神谷 典穂, Protein assembly design by enzymatic conjugation and scaffolding, 化学工学会第79年会(国際セッション), 2014.03.
36. 神谷 典穂, Assembling enzymes on a DNA scaffold for Biotechnological Applications, Asian Congress on Biotechnology (ACB-2013), 2013.12.
37. 神谷 典穂, Substrate engineering for enzymatic site-specific and covalent modification of functional proteins, Enzyme Engineering XXII: Emerging Topics in Enzyme Engineering, 2013.09.
38. 森 裕太郎, Rie Wakabayashi, Masahiro Goto, 神谷 典穂, Fabrication of higher-order protein supramolecular complexes, IGER International Symposium on Cell Surface Structures and Functions, 2013.09.
39. 神谷 典穂, Protein Supramolecular Complex Formation by Site-specific Protein Interactions and Scaffolding, IGER International Symposium on Cell Surface Structures and Functions, 2013.09, Proteins are biomacromolecules exhibiting multiple roles in living systems. A variety of protein functions have proven to be valuable in both biochemical studies and biotechnological applications. In natural biological systems, proteins often form well-organized higher-order structures that associate unique functions, which cannot be accessed by a sole protein unit. In the formation of multi-subunit protein polymers such as cell-surface pili in gram-positive bacteria, self-assembly of protein building blocks plays an important role, and interestingly, post-translational modification also facilitates the growth and stabilization of proteinaceous polymeric structures by introducing covalent bonds at specific sites of protein subunits.
Toward designer protein supramolecular complexes (PSCs), ordered protein assemblies have been designed by either site-specific ligand-receptor interaction or site-specific protein labeling onto a scaffold molecule based on a transglutaminase-catalyzed post-translational, site-specific protein modification technique with artificial substrates. For the former, strong and specific molecular interaction between a natural receptor protein, streptavidin (SA), and its small molecular ligand, biotin, was selected. By using a dimeric Escherichia coli alkaline phosphatase (AP) as a symmetric protein building block, we evaluated how the avidin-biotin interaction sites between protein units affect the formation of PSCs composed of AP and SA.[1] For the latter, we have selected nucleic acid as a polymeric scaffold, and created novel DNA- and RNA-(enzyme)n conjugate, a nucleic acid-enzyme hybrid with 1:n stoichiometry.[2] Our challenge for cellulosomal design with a nucleic acid scaffold will be also presented.[3] .
40. 神谷 典穂, Development of New Biomolecular Conjugation Techniques and Their Applications, YABEC 2013, 2013.08.
41. 神谷 典穂, Manipulating biomolecules through enzymatic post-translational protein modification, 2013 KMB's 40th Anniversary International Symposium "Recent Breakthroughs in Microbial Biotechnology: From Bench to Industry", 2013.07, Site-specific modification of proteins with a variety of organic molecules represents a valuable approach to obtain biologically active and homogeneous protein formulations. In particular, site-specific and covalent protein manipulation catalyzed by enzymes that function in post-translational modifications is practical because enzymatic transformations offer high substrate specificity under protein-friendly conditions. Recombinant proteins tagged with a short peptide, which can be post-translationally modified by a specific enzyme, have been successfully employed for this purpose.
Our group has focused on the utility of microbial transglutaminase (MTG) from Streptomyces mobaraensis in biotechnology. Transglutaminase is an enzyme that catalyzes covalent bond formation between the side chains of specific Gln and Lys residues of target peptides and proteins in post-translational modification process. By combining simple chemistry and MTG-catalyzed reaction, we have demonstrated site-specific protein conjugation with genetically introduced substrate peptide tags, site-specific protein immobilization to solid surfaces and site-specific protein labeling with new chemical entities. The basic concept has recently been extended to enzymatic conjugation of functional proteins with oligonucleotides, DNA and RNA. We are also interested in the use of oxidoreductases for enzymatic manipulation of biomolecules. Our recent efforts on biofabrication of a range of unique proteinaceous materials will be presented. .
42. 神谷 典穂, Hiroki Abe, Masahiro Goto, Controlling protein localization by enzymatic protein lipidation, YABEC 2012, 2012.10.
43. 神谷 典穂, 中元亜耶, Uju, Masahiro Goto, Chiaki Ogino, Nobuhiro Ishida, Potential of pyridinium ionic liquids in a cellulosic biomass pretreatment process, 15th International Biotechnology Symposium and Exhibition, 2012.09.
44. 神谷 典穂, Designing biocatalysis for protein engineering through enzymatic post-translational modification
, The 2nd International Conference on Molecular and Functional Catalysis, 2012.07.
45. 神谷 典穂, Momoko Kitaoka, Kounosuke Hayashi, A novel methodology for multiple enzyme labeling on nucleic acid scaffolds, 12th Japan-China-Korea Joint Symposium on Enzyme Engineering, 2012.05.
Membership in Academic Society
  • Japanese Society for Biomaterials
  • Research on biomolecular engineering using enzyme-catalyzed crosslinking reactions
  • Tailing DNA aptamers with a functional protein by two-step enzymatic reaction, J. Biosci. Bioeng., 116 (6), 660 (2013)
  • 機能性タンパク質の部位特異的且つ共有結合的修飾のための酵素基質エンジニアリング
  • New fluorescent substrates designed for covalent protein labeling catalyzed by microbial transglutaminase
  • Functionalization of the cytochrome P450cam monooxygenase system in the cell-like aqueous compartments of water-in-oil emulsions, J. Biosci. Bioeng., 99, 12-17 (2005)
Other Educational Activities
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Professional and Outreach Activities
As a faculty member of the Centre for Future Chemistry (Director for 2019-2023), I've collaborated with many companies, provide consultancy services, organised public lectures for citizens, and given lectures through research activities, mainly at FiaS (Industry-Academia Collaboration and Exchange Centre). I am also actively accepting international students from overseas for the "International Course for Chemistry of Molecular Systems", a special support framework for government-sponsored international students funded by the Ministry of Education, Culture, Sports, Science and Technology (MEXT). As part of international exchange activities, I've served as a board member and Vice President (Japan) of AFOB (Asian Federation of Biotechnology). From October 2013 to September 2016, I served as the Japanese representative of YABEC (Young Asian Biological Engineer's Community), and from June 2019 to May 2023, I served as Editor-in-Chief of the Journal of Bioscience and Bioengineering, the fragship journal of the Japanese Society for Biological Engineering. In April 2018, I've been involved in founding KAICO Co. Ltd. (founding member), and from 2022, I am also contributing to 'Itoshima Meneki-mura Project' as a new form of collaboration between the University and Itoshima City..