研究者詳細 - 石橋　松二郎

YAMASAKI Shunsuke, ISHIBASHI Matsujiro, TANAKA Ryoichi, TOKUNAGA Hiroko, ARAKAWA Tsutomu, TOKUNAGA Masao . General Salting-out and Distinctive Mg-ion Effects on Processing and Maturation of Halophilic Thermolysin-like Proteinase, Salilysin . Salt and Seawater Science & Technology6 ( 0 ) 12 - 20 2025年

記述言語：英語出版者・発行元：The Society of Sea Water Science, Japan

Pro-form of halophilic thermolysin-like proteinase, salilysin, was processed three times to form intermediate 1 (I1), intermediate 2 (I2) and final mature form (M). Halophilic enzymes are generally characterized by enhanced stability and catalytic activity in the presence of high concentration of salts. Autolytic processing of I1-form salilysin to I2-form and M-form in vitro was enhanced with the addition of 1.0 M and 0.5 M salts in parallel to valency and general salting-out effects of salts: NaCl ⁄ KCl < NH4Cl < (NH4)2SO4 ⁄ Na2SO4 ⁄ MgSO4. Mg salts at lower concentration, 0.1 M, regardless of salting-out (MgSO4: exerting stabilizing effects on protein structures) or salting-in (MgCl2: exerting destabilizing effects) salts, showed the highest efficiency in enhancing autolysis of I1 to mature form, suggesting importance of Mg ion in addition to the general salting-out effects of high concentration of salts. Even 5 ～ 10 mM Mg salts clearly enhanced autolysis of I1 and processing of inactive pro-E167A salilysin by wild-type mature salilysin, thus again suggesting distinct effects of Mg ion. Processing and maturation of halophilic salilysin was both enhanced by general salting-out effects of various salts and by specific effects, most likely possible binding to salilysin, of Mg ion.

DOI： 10.11457/ssst.6.0_12

3-1-3 Isolation and characterization of peanut nodule-associated bacteria in Myanmar（3-1 土壌生物の生態と機能 2024年度福岡大会） . 日本土壌肥料学会講演要旨集70 ( 0 ) 25 - 25 2024年8月

TOKUNAGA Masao, AKUTA Teruo, TOKUNAGA Yuhei, TOMIOKA Yui, ARAKAWA Tsutomu, ISHIBASHI Matsujiro . Agarose Gel Electrophoresis Analysis of Chemically Cross-linked ClpB Chaperone Protein from Moderate Halophile . Salt and Seawater Science & Technology5 ( 0 ) 22 - 26 2024年

記述言語：英語出版者・発行元：The Society of Sea Water Science, Japan

Chemical cross-linking combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a simple way to analyze oligomerization and complex formation of proteins. However, cross-linked proteins with a large molecular mass, for example with molecular weight greater than ∼400 kDa, show slow mobility and tend to stack in loading area with poor resolution. Agarose gel electrophoresis (AGE) may be more suitable, due to a large pore size of the agarose, to analyze proteins as well as DNA fragments, especially for large macromolecules and their complexes. Here, we expressed and purified recombinant ClpB chaperone protein (homologue of eukaryotic HSP104) derived from moderate halophile and analyzed the purified protein by SDS-PAGE and SDS-AGE. Chemical cross-linking and SDS-AGE analysis clearly demonstrated hexameric assembly of ClpB subunit protein. Heat-denaturation profile of the ClpB with native-AGE analysis done without SDS suggested that ClpB protein was denatured at around 50 °C.

DOI： 10.11457/ssst.5.0_22

Htwe A.Z., Yamakawa T., Ishibashi M., Tsurumaru H. . Isolation and characterization of mung bean (Vigna radiata L.) rhizobia in Myanmar . Symbiosis94 ( 1 ) 51 - 63 2024年

記述言語：日本語出版者・発行元：Symbiosis

We collected soil samples from six major mung bean cropping regions in Myanmar: Sagaing, Mandalay, Nay Pyi Taw, and Magway in the tropical savanna climate zone and Bago and Yangon in the tropical monsoon climate zone. All fields grew mung bean for at least 5 years and had no history of rhizobial inoculation. Mung bean ‘Yezin-11’, a popular cultivar in Myanmar, was inoculated with soil suspensions. From the nodules formed on the roots, we isolated 55 rhizobial strains. Identification of the isolates revealed the dominant species of indigenous rhizobia in each region. We identified 53 Bradyrhizobium strains and 2 Ensifer strains. Bradyrhizobium yuanmingense was dominant in the tropical savanna zone and Bradyrhizobium sp. (B. liaoningense or B. diversitatis) and B. centrosematis were dominant in the tropical monsoon zone. Principal component analysis indicates that the dominance of B. yuanmingense in the tropical savanna zone might be due to high concentration of NO3-N and P2O5 in the soil. It also indicates that the dominance of B. centrosematis in the tropical monsoon zone might be caused by drastically low pH and high concentration of NH4 in the soil. Bradyrhizobium centrosematis YGN-M9, B. yuanmingense SGG-M3, and Bradyrhizobium sp. BGO-M5 significantly increased nodulation (nodule number and nodule dry weight), acetylene reduction activity, and shoot dry weight, respectively, relative to Ensifer terangae MDY-M6. Co-inoculation with these three strains increased nodulation significantly compared with single inoculation of BGO-M5. The characterization of mung bean rhizobia and selection of microbial inoculant candidates will be useful for the development of microbial inoculants in Myanmar.

DOI： 10.1007/s13199-024-01013-2

3-1-4 Isolation and Characterization of Mung bean Rhizobia in Myanmar（3-1 土壌生物の生態と機能 2023年度愛媛大会） . 日本土壌肥料学会講演要旨集69 ( 0 ) 28 - 28 2023年9月

Kirishima M., Yokoyama S., Matsuo K., Hamada T., Shimokawa M., Akahane T., Sugimoto T., Tsurumaru H., Ishibashi M., Mataki Y., Ootsuka T., Nomoto M., Hayashi C., Horiguchi A., Higashi M., Tanimoto A. . Gallbladder microbiota composition is associated with pancreaticobiliary and gallbladder cancer prognosis . BMC Microbiology22 ( 1 ) 147 2022年12月

記述言語：日本語出版者・発行元：BMC Microbiology

Background: The microbial population of the intestinal tract and its relationship to specific diseases has been extensively studied during the past decade. However, reports characterizing the bile microbiota are rare. This study aims to investigate the microbiota composition in patients with pancreaticobiliary cancers and benign diseases by 16S rRNA gene amplicon sequencing and to evaluate its potential value as a biomarker for the cancer of the bile duct, pancreas, and gallbladder. Results: We enrolled patients who were diagnosed with cancer, cystic lesions, and inflammation of the pancreaticobiliary tract. The study cohort comprised 244 patients. We extracted microbiome-derived DNA from the bile juice in surgically resected gallbladders. The microbiome composition was not significantly different according to lesion position and cancer type in terms of alpha and beta diversity. We found a significant difference in the relative abundance of Campylobacter, Citrobacter, Leptotrichia, Enterobacter, Hungatella, Mycolicibacterium, Phyllobacterium and Sphingomonas between patients without and with lymph node metastasis. Conclusions: There was a significant association between the relative abundance of certain microbes and overall survival prognosis. These microbes showed association with good prognosis in cholangiocarcinoma, but with poor prognosis in pancreatic adenocarcinoma, and vice versa. Our findings suggest that pancreaticobiliary tract cancer patients have an altered microbiome composition, which might be a biomarker for distinguishing malignancy.

DOI： 10.1186/s12866-022-02557-3

Ura T., Kameda T., Laksmi F.A., Ishibashi M., Arakawa T., Shiraki K., Hirano A. . Affinity of phenolic compounds for transition metal ions immobilized on cation-exchange columns . Journal of Chromatography A1676 463277 2022年8月

記述言語：日本語出版者・発行元：Journal of Chromatography A

Immobilized metal ion affinity chromatography (IMAC) is useful in purification of histidine-tagged or histidine-rich proteins and peptides from a variety of hosts. However, phenolic compounds including polyphenols interfere with IMAC due to their high affinities for the transition metals immobilized on the column resins, which hampers the purification of proteins from plant-based host systems. In contrast to extensive knowledge of the mechanism of the interactions between phenolic compounds and transition metal ions in solution, an understanding of the interactions on the columns, where transition metal ions are immobilized on the resins, remains elusive. This study systematically investigated the affinity of phenolic compounds for transition metal ions by varying the number and position of phenolic hydroxyl groups (OH groups) and using different transition metals—Fe(II), Cu(II) and Ni(II)—on various IMACs, in which the columns were fabricated by equilibrating the cation-exchange column with transition metal solutions. It was found that the more OH groups the aromatic compounds have, the higher the affinity for transition metal ions; in particular, methyl gallate and pyrogallol were permanently bound to the IMAC column, which reflected coordinate bond formation with the transition metal ions. Importantly, the phenolic compounds showed no obvious affinity for the Ni(II)-IMAC column, in contrast to the Fe(II)- and Cu(II)-IMAC columns, whereas imidazole and histidine-tagged proteins showed evident binding to the Ni(II)-IMAC column. Ni(II)-IMAC should thus be especially effective in isolating histidine-tagged and histidine-rich species from phenolic compound-containing systems. These results indicate that the affinity between phenolic compounds and transition metal ions on the column is consistent with the results in solution. They also provide a comprehensive view for devising strategies to improve IMAC purification of target proteins and peptides from samples containing phenolic compounds.

DOI： 10.1016/j.chroma.2022.463277

Tomioka Y., Arakawa T., Akuta T., Nakagawa M., Ishibashi M. . Analysis of proteins by agarose native gel electrophoresis in the presence of solvent additives . International Journal of Biological Macromolecules198 26 - 36 2022年2月

記述言語：日本語出版者・発行元：International Journal of Biological Macromolecules

Solvent additives, including NaCl, arginine hydrochloride (ArgHCl), glycine and sucrose, are used to enhance protein stability or reduce protein aggregation. Here, we studied the effects of these additives on proteins using agarose native gel electrophoresis. Since these additives are used at relatively high concentration, we first confirmed that they do not interfere with the performance of the native gel electrophoresis. Agarose native gel electrophoresis showed that aggregation of bovine serum albumin (BSA) induced by heating was slightly reduced by NaCl and ArgHCl. On the contrary, glycine and sucrose had marginal effects. ArgHCl and NaCl promoted heat aggregation of monoclonal antibody (mAb), while glycine and sucrose stabilized the native mAb. Arginine methyl ester inhibited heat aggregation of lysozyme and, to a much lesser extent, BSA. These results show that agarose native gel electrophoresis can be used to analyze the effects of solvent additives on proteins subjected to heat stresses. SYPRO Orange that stains only unfolded proteins confirmed unfolded structures of soluble aggregates.

DOI： 10.1016/j.ijbiomac.2021.12.084

Ishibashi M. . Salt Mediated Modulation of Autolysis of Thermolysin-Like Proteinase, Salilysin, Isolated from a Moderate Halophile, Chromohalobacter salexigens DSM3043 . Protein Journal40 ( 2 ) 223 - 233 2021年4月

記述言語：日本語出版者・発行元：Protein Journal

Halophilic salilysin is first synthesized as a pro-form, which has been shown autolysis activity to process pro-region (55 amino acids long) three times to form intermediate 1 (I1), intermediate 2 (I2) and final mature (M) salilysin. The autolysis of I1- to M-form salilysin in vitro was significantly accelerated with increasing NaCl concentration up to 4 M. Strong salting-out salts, (NH4)2SO4, Na2SO4 and MgSO4, were more effective, suggesting that autolysis is enhanced by inter-molecular association or structure compaction or both. However, MgCl2, a salting-in salt, was also effective, suggesting that other mechanisms, such as charge shielding and ionic binding to this halophilic protein, operated. Autolytic cleavage at site 3 resulted in mixed formation of correctly and incorrectly processed mature forms in the absence of salt, indicating that salt affected the accuracy of autolytic cleavage reaction. Far UV circular dichroism (CD) measurements indicated that E167A pro-salilysin showed an identical CD spectrum to the wild-type mature salilysin, suggesting pro-form has a proper fold for proteolytic activity. Thermal scanning indicated that E167A pro-salilysin was more heat-stable by ~ 10 °C than mature form. The CD spectra, thermal stability and modeling structure of salilysin clearly suggested that pro-salilysin is folded to the same structure as native form and is functional for autolysis.

DOI： 10.1007/s10930-021-09964-x

Matsujiro Ishibashi· Ryoichi Tanaka· Shunsuke Yamasaki· Hiroko Tokunaga1 · Tsutomu Arakawa·Masao Tokunaga . Salt Mediated Modulation of Autolysis of Thermolysin-Like Proteinase,　Salilysin, Isolated from a Moderate Halophile, Chromohalobacter salexigens DSM3043 . The Protein Journal 2021年1月査読

Tokunaga Masao, Arakawa Tsutomu, Tokunaga Yuhei, Ishibashi Matsujiro . Efficient Expression and Refolding of Insolubilized Halophilic Starch-Binding Domain with Aggregation-Prone Peptide . Salt and Seawater Science & Technology2 ( 0 ) 1 - 2 2021年

記述言語：英語出版者・発行元：日本海水学会

Soluble expression of halophilic starch-binding domain （SBD1） from Kocuria α-amylase was low due to its instability in recombinant host Escherichia coli cells. Here, the stable SBD1 protein was expressed efficiently with an aggregation-prone peptide tag in insoluble E. coli inclusion bodies （IBs）. Insoluble SBD1 protein with the tag, thus obtained, was easily solubilized with 8 M urea and refolded into an active form by simple urea dilution. The yield of active SBD1 with the tag after the solubilization / dilution method was 15 ～ 30 times higher than that of direct expression of SBD1 in soluble fraction.

DOI： 10.11457/ssst.2.0_1

Tanaka R. . Salt-enhanced processing, proteolytic activity and stability of halophilic thermolysin-like proteinase, salilysin, isolated from a moderate halophile, Chromohalobacter salexigens DSM3043 . International Journal of Biological Macromolecules164 77 - 86 2020年12月

Fina Amreta Laksmi, Shigeki Arai, Tsutomu Arakawa, Hirohito Tsurumaru,Yoshitaka Nakamura, Budi Saksono, Masao Tokunaga, Matsujiro Ishibashi . Expression and characterization of L-arabinose isomerase from Geobacillus stearothermophilus for improved activity under acidic condition. . Protein Expression and Purification175 2020年11月査読

Laksmi F.A. . Expression and characterization of L-arabinose isomerase from Geobacillus stearothermophilus for improved activity under acidic condition . Protein Expression and Purification175 105692 2020年11月

Nakamura Y. . Microbial Community Analysis of Digested Liquids Exhibiting Different Methane Production Potential in Methane Fermentation of Swine Feces . Applied Biochemistry and Biotechnology191 ( 3 ) 1140 - 1154 2020年7月

中村　嘉孝 . 豚糞を基質とした高効率メタン発酵に関する研究 . 2020年3月査読

Fina Amreta Laksmi . Study on industrial application of enzymes from extremophiles . 2020年3月査読

Laksmi F.A. . Expression, Folding, and Activation of Halophilic Alkaline Phosphatase in Non-Halophilic Brevibacillus choshinensis . Protein Journal39 ( 1 ) 46 - 53 2020年2月

Arai S. . Catalytic mechanism and evolutionary characteristics of thioredoxin from Halobacterium salinarum NRC-1 . Acta Crystallographica Section D: Structural Biology76 ( Pt 1 ) 73 - 84 2020年1月

Fina Amreta Laksmi, Hikari Imamura, Hirohito Tsurumaru, Yoshitaka Nakamura, Hiroshi Hanagata, Shigeki Arai, Masao Tokunaga, Matsujiro Ishibashi . Expression, folding, and activation of halophilic alkaline phosphatase in non-halophilic Brevibacillus choshinensis. . The Protein Journal39 46 - 53 2020年査読

SHOBUDANI MIKU, AUNGZAWHTWE, YAMAKAWA TAKEO, ISHIBASHI MATSUJIRO, TSURUMARU HIROHITO . Mutants Disrupted in the Type Ⅲ Secretion System of Bradyrhizobium elkanii BLY3-8 Overcame Nodulation Restriction by Rj₃-genotype Soybean . Ｍｉｃｒｏｂｅｓ　ａｎｄ　ｅｎｖｉｒｏｎｍｅｎｔｓ35 ( 2 ) 2020年

出版者・発行元：日本微生物生態学会 / 日本土壌微生物学会 / Taiwan Society of Microbial Ecology / 植物微生物研究会 / 極限環境微生物学会

Bradyrhizobium elkanii BLY3-8 does not form nodules on the roots of Rj3-genotype soybean (cultivar D-51). This is a cultivar-specific nodulation restriction. The genes A6X20_40975 and A6X20_41030 in strain BLY3-8 were predicted to encode the transcriptional activator and apparatus of the type III secretion system (T3SS) (the proteins TtsI and RhcJ), respectively. Mutants disrupted in these genes overcame the nodulation restriction. These results suggest that an effector injected via T3SS into Rj3-genotype soybean is involved in nodulation restriction by Rj3-genotype soybean.

DOI： 10.1264/jsme2.ME19151

Tokunaga M. . Insoluble expression of highly soluble halophilic metal binding protein for metal ion biosorption: Application of aggregation-prone peptide from hen egg white lysozyme . Protein Expression and Purification156 50 - 57 2019年4月

ARAKAWA Tsutomu, TOKUNAGA Hiroko, ISHIBASHI Matsujiro, TOKUNAGA Masao . Metal Binding Properties of His-Asp Repeat Peptide from Halophilic His-rich Metal Binding Protein Monitored by Circular Dichroism Measurement . 日本海水学会誌73 ( 3 ) 159 - 164 2019年

出版者・発行元：日本海水学会

A His-Asp repeat peptide sequence was identified in a His-rich metal binding protein （HP） produced by a halophilic marine bacterium, Chromohalobacter salexigens DSM 3043T. A synthetic peptide, PepB20, containing 8 His-Asp repeats showed binding to an Ni-loaded metal-chelating column and elution around 150-200 mM imidazole in the presence of both low （0.15 M） and high （1.5 M） concentrations of NaCl. Addition of Ni or Zn ions at 0.1 to 1 mM resulted in changes in secondary structure of PepB20, leading to metal-induced insoluble aggregation at 1 mM. PepB20 showed different circular dichroism （CD） profiles induced by the binding of Ni or Zn ions, suggesting a formation of different secondary structures by these metal ions. We described metal binding properties and consequent structure changes of PepB20 examined by CD measurements and discuss the possible physiological importance of the His-rich metal binding insertion sequence found in HP.

DOI： 10.11457/swsj.73.3_159

Fina Amreta Laksmi, Shigeki Arai, Hirohito Tsurumaru, Yoshitaka Nakamura,Budi Saksono, Masao Tokunaga, Matsujiro Ishibashi . Improved substrate specificity for D-galactose of L-arabinose isomerase for industrial application . Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics1866 ( 11 ) 1084 - 1091 2018年11月査読

Laksmi F.A. . Improved substrate specificity for D-galactose of L-arabinose isomerase for industrial application . Biochimica et Biophysica Acta - Proteins and Proteomics1866 ( 11 ) 1084 - 1091 2018年11月

Mizukami M. . Efficient production of Trastuzumab Fab antibody fragments in Brevibacillus choshinensis expression system . Protein Expression and Purification150 109 - 118 2018年10月

Ishibashi M. . Expression and characterization of the Renilla luciferase with the cumulative mutation . Protein Expression and Purification145 39 - 44 2018年5月

Matsujiro Ishibashi, Ryo Kawanabe, Norie Amaba, Shigeki Arai, Fina Amreta Laksmi, Kenta Komori, Masao Tokunaga . Expression and characterization of the Renilla luciferase with the cumulative mutation . Protein Expression and Purification145 39 - 44 2018年査読

Megumi Shigehisa, Norie Amaba, Shigeki Arai, Chisato Higashi, Ryo Kawanabe, Ayano Matsunaga, Fina Amreta Laksmi, Masao Tokunaga, Matsujiro Ishibashi . Stabilization of luciferase from Renilla reniformis using random mutations . Protein Engineering, Design and Selection30 ( 1 ) 7 - 13 2017年1月査読

Shigehisa M, Amaba N, Arai S, Higashi C, Kawanabe R, Matsunaga A, Laksmi FA, Tokunaga M, Ishibashi M . Stabilization of luciferase from Renilla reniformis using random mutations. . Protein engineering, design & selection : PEDS30 ( 1 ) 7 - 13 2017年1月

尾辻大樹, 黒木未知瑠, 徳永廣子, 山口類, 徳永雄平, 石橋松二郎, 荒川力, 徳永正雄 . 好塩性ヒスチジン高含有金属結合タンパク質の結合容量と塩環境下での重金属回収の可能性 . 日本海水学会誌71 ( 5 ) 308 - 314 2017年

出版者・発行元：日本海水学会

好塩性海洋細菌Chromohalobacter salexigens由来ヒスチジン高含有金属結合タンパク質（HP）は，Treponema pallidum由来のTroAなどの良く研究されている他の金属結合タンパク質のアミノ酸配列と比較した場合，46アミノ酸残基からなるユニークな挿入配列（多くのヒスチジン残基を含むIS46配列）を持つという特徴がある．この研究では，Zn，Cu，NiもしくはMnを含んだ緩衝液に対してHPの平衡透析を行い，HPの金属結合容量を定量した．その結果，飽和条件下で1分子のHPに最大約10分子のZn，Cu，もしくはNiイオンが結合することが分った．HPに対する金属イオンの親和性は，Zn＞Cu＞Ni＞Mnの順であった．さらに，HPを利用した塩環境下での重金属回収の可能性を試した．His-tagを付与したHP（His-tagged HP）を固定化したSepharoseカラムを作成し，金属結合量を定量したところ1分子のHis-tagged HPあたり約9.5個のNiイオンが結合した．これは塩環境下における重金属の吸着・回収にHPが利用できる可能性を示したものと考えられる．

DOI： 10.11457/swsj.71.5_308

Shigehisa M. . Stabilization of luciferase from Renilla reniformis using random mutations . Protein Engineering, Design and Selection30 ( 1 ) 7 - 13 2017年

NISHIMOTO Airi, OTSUJI Daiki, TOKUNAGA Hiroko, ISHIBASHI Matsujiro, ARAKAWA Tsutomu, TOKUNAGA Masao . Characterization of Metal Binding Protein ZP and its His-rich Peptides from a Halophilic Marine Bacterium Chromohalobacter salexigens . 日本海水学会誌71 ( 4 ) 252 - 253 2017年

出版者・発行元：日本海水学会

We cloned a gene, Csal-0188, from genomic DNA of Chromohalobacter salexigens DSM 3043T. This gene was annotated to encode Zn-binding protein（ZP）, which showed high homology with the His-rich metal binding protein（HP）we reported. His-tag-ZP was successfully expressed in Escherichia coli and purified. ZP was constructed with a thrombin digestion site so that His-tag could be removed. ZP and chemically synthesized oligo-peptides from the His-rich insertion sequence of ZP showed several metal binding activities. This His-rich peptide was found to bind to Ni column in the presence of 8 M urea, showing clear binding under denaturation conditions, like conventional His-tag.

DOI： 10.11457/swsj.71.4_252

TOKUNAGA Masao, TOKUNAGA Hiroko, ISHIBASHI Matsujiro, ARAKAWA Tsutomu . Detergent-tolerant Halophilic α-amylase Isolated from a Moderate Halophile Nesterenkonia halobia . 日本海水学会誌71 ( 3 ) 189 - 190 2017年

出版者・発行元：日本海水学会

We examined the effects of several detergents on the stability of halophilic Nesterenkonia halobia α-amylase（NhAmy）. NhAmy was purified to homogeneity from the culture supernatant by simple one-step dextrin affinity column chromatography. The purified enzyme was found to be highly resistant to several detergents, including sodium dodecyl sulfate, which is one of the strongest protein denaturants.

DOI： 10.11457/swsj.71.3_189