Language：English   Publishing type：Research paper (scientific journal)   Publisher：BMFH Press  

DOI： 10.12938/bmfh.2020-003

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jbiosc.2020.06.004

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jbiosc.2019.11.009

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jbiosc.2019.07.004

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jbiosc.2019.06.017

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Language：Japanese   Publisher：(公社)日本生物工学会  

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Language：Japanese   Publisher：(公社)日本生物工学会  

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Publishing type：Research paper (scientific journal)   Publisher：BMFH Press  

DOI： 10.12938/bmfh.18-015

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Authorship：Last author,　Corresponding author   Language：English   Publisher：BMFH Press  

<p>Co-culture of lactic acid bacteria (LAB) and yeast induces specific responses that are not observed in pure culture. Gene expression profiles of <i>Lactobacillus paracasei</i> ATCC 334 co-cultured with <i>Saccharomyces cerevisiae</i> IFO 0216 were analyzed by DNA microarray, and the responses induced by direct contact with the yeast cells were investigated. Coating the LAB cells with recombinant DnaK, which acts as an adhesive protein between LAB and yeast cells, enhanced the ratio of adhesion of the LAB cells to the yeast cells. The signals induced by direct contact were clarified by removal of the LAB cells unbound to the yeast cells. The genes induced by direct contact with heat-inactivated yeast cells were very similar to both those induced by the intact yeast cells and those induced by a soluble mannan. The top 20 genes upregulated by direct contact with the heat-inactivated yeast cells mainly encoded proteins related to exopolysaccharide synthesis, modification of surface proteins, and transport systems. In the case of the most upregulated gene, LSEI_0669, encoding a protein that has a region homologous to polyprenyl glycosylphosphotransferase, the expression level was upregulated 7.6-, 11.0-, and 8.8-fold by the heat-inactivated yeast cells, the intact yeast cells, and the soluble mannan, respectively, whereas it was only upregulated 1.8-fold when the non-adherent LAB cells were not removed before RNA extraction. Our results indicated that the LAB responded to direct contact with the yeast cells through recognition of mannan on the surface of the yeast.</p>

DOI： 10.12938/bmfh.BMFH-2016-015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing Ltd  

Pigmentation in mammals is important for protection of skin and eyes from ultraviolet radiation. Dysregulation of pigmentation is often associated with other conditions that are not directly linked to pigmentation. Here, we isolated spontaneously occurring hypopigmented mice that occasionally experienced severe diarrhea during lactation. Treatment of these mice with dextran sulfate sodium salt, a conventional method to induce acute colitis, caused chronic diarrhea with granulomatous colitis. Gene mapping and sequencing revealed that the mice had a nonsense mutation in the Hermansky–Pudlak syndrome (Hps)5 gene. As some HPS patients can develop granulomatous colitis, the simple induction of chronic colitis in spontaneously mutated Hps5-deficient mice may become an invaluable model for exploring treatment options in patients with HPS as well as other patients with inflammatory bowel disease.

DOI： 10.1111/pcmr.12504

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Language：Japanese   Publisher：Japanese Society for Engineering Education  

DOI： 10.20549/jseeja.2016.0_334

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Language：Japanese  

CiNii Books

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INT INST ANTICANCER RESEARCH  

Background/Aim: The aim of the present study was to establish the strategy for producing a single-chain variable fragment (scFv) antibody fused with interleulin-2 (IL2) by Pichia pastoris and to optimize production during fed-batch cultivation in a 5-l fermenter. Materials and Methods: We constructed a fusion sequence consisting of an scFv gene derived from a mouse monoclonal antibody against a tumor-associated antigen (designated MK-1 antigen) and human interleulin-2 (IL-2) gene, ligated the sequences to expression vector pPICZ alpha-A and separately transformed the constructs into Pichia pastoris strains GS115 and KM71H. Results: The highest concentration of secreted fusion protein, 738 +/- 44 mg/l, was obtained after a 60-h induction. To investigate the specific binding activity of the partially purified fusion protein, we used an enzyme-linked immunosorbent assay and antigen from a whole-cell lysate. Student's t-test showed that the specific binding activity of the partially-purified fusion protein to the lysate of Chinese hamster ovary cell lines expressing the MK-1 antigen was significantly higher than that of the lysate of CHO cell lines that do not express MK-1. Conclusions: The method described here permits the production of substantial amounts of the fusion protein for conducting functional studies on the biological role of these fusion proteins.

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Scientific Research Publishing, Inc.  

DOI： 10.4236/jsbs.2013.32020

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Other Link： http://www.scirp.org/journal/doi.aspx?DOI=10.4236/jsbs.2013.32020

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-β-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the α-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture. Expression of the A. oryzae endoglucanase CelB and two heterologous endoglucanases (TrEglI and TrEglIII from Trichoderma reesei) under the control of the hlyA promoter were also found to be stronger than under the control of the amyA promoter in A. oryzae grown in wheat bran solid-state culture, suggesting that the hlyA promoter may be useful for the overproduction of other proteins as well. In wheat bran solid-state culture, the productivity of the hlyA promoter in terms of protein produced was high when the cultivation temperature was 30°C or 37°C, when the water content was 0.6 or 0.8 ml/g wheat bran, and from 48 to 72 h after inoculation. Because A. oryzae sporulated actively under these conditions and because hemolysin has been reported to play a role in fungal fruiting body formation, high-level expression of hlyA may be related to sporulation. © 2011 Springer-Verlag.

DOI： 10.1007/s00253-011-3446-5

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Other Link： http://link.springer.com/article/10.1007/s00253-011-3446-5/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

A strategy for preventing reduction of ethanol yield in fermentation caused by bacterial contamination was developed. In solid-state ethanol fermentation in which saccharification, fermentation and ethanol recovery are performed simultaneously, the addition of exogenous ethanol to the fermentation mixture at the start of fermentation at 50gkg-1 prevented contamination, and the ethanol yield reached 0.50gg-1. © 2010 The Society for Biotechnology, Japan.

DOI： 10.1016/j.jbiosc.2010.11.012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

To save cost and input energy for bioethanol production, a consolidated continuous solid-state fermentation system composed of a rotating drum reactor, a humidifier, and a condenser was developed. Biomass, saccharifying enzymes, yeast, and a minimum amount of water are introduced into the system. Ethanol produced by simultaneous saccharification and fermentation is continuously recovered as vapor from the headspace of the reactor, while the humidifier compensates for the water loss. From raw corn starch as a biomass model, 95 +/- 3, 226 +/- 9, 458 +/- 26, and 509 +/- 64 gl(-1) of ethanol solutions were recovered continuously when the ethanol content in reactor was controlled at 10-20, 30-50, 50-70 and 75-85 g kg-mixture(-1), respectively. The residue showed a lesser volume and higher solid content than that obtained by conventional liquid fermentation. The cost and energy for intensive waste water treatment are decreased, and the continuous fermentation enabled the sustainability of enzyme activity and yeast in the system.

DOI： 10.1007/s00253-010-2716-y

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：SOC FIBER SCIENCE TECHNOLOGY  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Fluorescent-labeled invertase, a hyperglycosylated mannoprotein from Saccharomyces cerevisiae, was found to bind to Lactococcus lactis IL1403 at acidic pH. Proteins on the cell wall of the bacterium affinity-purified using invertase as a ligand were identified to be heat shock proteins such as DnaK and GroEL and glycolytic enzymes such as pyruvate kinase and glyceraldehyde-3- phosphate dehydrogenase. DnaK bound to both the bacterium and yeast at pH 4 and aggregated them at above 0.1 mg/ml, whereas no significant difference between the circular dichroism spectra of DnaK at pH 4 and 7 was observed. These results indicate that the cytosolic proteins, including DnaK displayed on the cell wall, cause the lactic acid bacterium to adhere to the yeast. © 2009 Springer-Verlag.

DOI： 10.1007/s00253-009-2295-y

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Other Link： http://link.springer.com/article/10.1007/s00253-009-2295-y/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Generally, mammalian cells utilize glucose and glutamine as primary energy sources. To investigate the effect of energy sources on metabolic fluxes and antibody production, glucose- or glutamine-limited serum-free continuous culture of hybridoma 3A21 cells, which produce anti-ribonuclease A antibody, was carried out. The cell volume and dry cell weight were evaluated under various steady-state conditions. The specific consumption and production rates were evaluated on the basis of dry cell weight. On the basis of these results, the fluxes of the metabolic pathway were calculated. It was found that increasing the specific growth rate causes the specific ATP and antibody production rates to decrease. The fluxes between malate and pyruvate also decreased with the increase in specific growth rate. To increase the ATP production rate under steady-state conditions by the enhancement of fluxes between malate and pyruvate, the reduced metabolic fluxes were increased by an intermediate (pyruvate, malate, and citrate) addition. As a result, higher specific ATP and antibody production rates were achieved following the intermediate addition at a constant dilution rate.

DOI： 10.1007/s00449-009-0351-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

Single-chain Fv antibodies (scFv) genetically fused with polystyrene-binding peptides (PS-tags, (PS19-1; RAFIASRRIRRP, PS19-6; RIIIRRIRR)) were generated by recombinant Escherichia coli for direct and site-specific immobilization of scFv on polystyrene supports with high antigen-binding activity. PS-tag-fused scFvs (scFv-PS-tags) specific for human C-reactive protein (CRP) were successfully over-expressed as an inclusion body and were refolded using the batch-dilution method. When scFv-PS-tags were immobilized on a hydrophilic PS (phi-PS) plate in the presence of Tween 20, they showed high antigen-binding activity comparable to, or greater than, that of a whole monoclonal antibody (mAb) on a hydrophobic PS (pho-PS) plate, which has been the exclusive method for enzyme-linked immunosorbent assay (ELISA). Furthermore, when a scFv-PS-tag was used as a ligand antibody in one-and two-step ELISA, the assay time was reduced without loss of sensitivity. These results indicate that strong and specific attachment of PS-tags onto the phi-PS surface prevented scFv conformational changes and consequently, the high antigen-binding activities of scFvs were preserved. Nearly identical results were obtained by use of PS-tag-fused scFvs with different VH/VL pairs. Therefore, a variety of scFvs could be functionalized onto phi-PS plates by genetic fusion of PS-tags. ScFv-PS-tags, which possess high antigen-binding activity on the phi-PS plate, are more useful ligand antibodies than whole mAbs. Thus, scFv-PS-tags are applicable in both clinical diagnosis and proteomic research.

DOI： 10.1007/s00216-009-2999-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

The difference in responses to osmotic stress between the laboratory and sake-brewing strains of Saccharomyces cerevisiae at the translational level was compared by two-dimensional polyacrylamide gel electrophoresis. Proteins, whose production was significantly changed by the osmotic stress, were identified by peptide mass fingerprinting. In the laboratory strain, translation of Hor2p, the protein responsible for glycerol biosynthesis, and Ald6p, related to acetate biosynthesis, was induced under high osmotic pressure conditions. In addition, production of proteins related to translation and stress response was also changed under this condition. On the other hand, in the sake-brewing strain, translation of Hor2p, Hsp26p, and some stress-related proteins was upregulated. The change in the production of enzymes related to glycolysis and ethanol formation was small; however, the production of enzymes related to glycerol formation increased in both strains. These results suggest that enhancement of glycerol formation due to enhancement of the translation of proteins, such as Hor2p, is required for growth of S. cerevisiae under high osmotic pressure condition. This is the first report on the analysis of responses of a sake-brewing strain to high osmotic pressure stress based on proteomics. (C) 2009 Published by Elsevier Ltd.

DOI： 10.1016/j.procbio.2009.02.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

For substantial cell growth and exopolysaccharide (EPS) production of Bifidobacterium longum JBL05, anaerobic conditions (dissolved oxygen concentrations below 0.05 ppm) and the supplement Of CO2 (&gt;= 20%) were required. Under these conditions, B. longum JBL05 produced EPS amounts of up to 2.9 g/l. (c) 2009, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2008.12.015

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2009.0.658.0

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2009f.0.1078.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and gamma-hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was 200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value, whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained. Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally as the ratio of gamma-hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies, a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating variable, and was successfully proved by the experiments.

DOI： 10.1007/s10295-008-0427-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Hydrolysates of lignocelluloses hydrolyzed by diluted sulfuric acid contain toxic compounds that inhibit ethanol production by Saccharomyces cerevisiae and the ethanologenic recombinant Escherichia coli KO11. We investigated the biological detoxification of a hydrolysate of waste house wood (WHW) by a thermophilic bacterium, Ureibacillus thermosphaericus. When the hydrolysate was treated with this bacterium at 50 degrees C for 24 h, the ethanol production rate by S. cerevisiae increased markedly and was comparable to that for the hydrolysate treated with an excess amount of calcium hydroxide (overliming). Chromatographic analysis of synthetic hydrolysates containing furfural or 5-hydroxymethyl furfural that are considered to be major toxic compounds in hydrolysates revealed that U. thermosphaericus degrades these compounds. In the WHW hydrolysates, however, the concentrations of these compounds were not decreased markedly by the bacterium. These results suggest that the bacterium degrades minor but more toxic compounds or phenolic compounds in the WHW hydrolysates. The combination of bacterial and overliming treatments of hydrolysates minimized significantly the decrease in ethanol production rate by E. coli KO11 as fermentation proceeded. Because the bacterium grows rapidly and does not consume sugars, our biological detoxification should be useful for bioethanol production from acid hydrolysates of lignocelluloses.

DOI： 10.1263/jbb.106.128

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 fused to the linker region and the starch-binding domain (SBD) of the a-amylase of Streptococcus bovis 148 was produced intracellularly in Escherichia coli. The fusion protein (CPH-SBD) was able to bind to the cell surface of Lactobacillus casei NRRL B-441 and to corn starch. Therefore, adhesion of cells to corn starch was mediated by the fusion protein. At a cell density of 10(9) cfu/ml and a starch concentration of 5 mg/ml, CPH-SBD-displaying L. casei cells aggregated with corn starch, whereas the free cells of L. casei did not form any aggregates with corn starch. After incubation in simulated gastric juice (pH 3.0, 1 h), the survival percentages of free cells, amylose-coated free cells, and free cells mixed with corn starch were 0.074%, 7.2%, and 3.1% respectively. When CPH-SBD-displaying bacteria aggregated with corn starch, their survival percentage was 8% higher than that of free cells mixed with corn starch. The survival of the amylose-coated CPH-SBD-displaying L. casei cells was comparable to that of amylose-coated free cells, whereas the survival percentage of amylose-coated aggregates of CPH-SBD-displaying bacteria with corn starch was 28% higher than that of amylose-coated mixture of free cells with corn starch. These results demonstrate the potential usefulness of the cell-surface display technique for enhancement of the delivery of viable microorganisms to the intestinal tract.

DOI： 10.1263/jbb.105.503

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

A rational strategy for the rapid establishment of a sensitive sandwich enzyme linked immunosorbant assay was developed. The kinetic properties required for the solid-phase and enzyme-conjugated antibodies of sandwich ELISA were determined rationally on the basis of a kinetic model describing antibody-antigen interaction. Some antibodies possessing the required kinetic properties against a model antigen, C-reactive protein (CRP), were successfully isolated from a phage antibody library under the screening conditions that were designed on the basis of simulation results. The best combination of solid-phase and enzyme-conjugated antibodies that gives the most sensitive sandwich ELISA was determined by simulation on the basis of the apparent association and dissociation rate constants of the isolated antibodies. It was confirmed by experiment that the sandwich ELISA using the best combination of antibodies was actually the most sensitive one. Our strategy would be useful for the rapid establishment of sensitive sandwich ELISAs compared with the traditional hybridoma method in which the best condition is determined by trial and error.

DOI： 10.1263/jbb.105.261

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

In this paper, we report a simultaneous realization of both efficient ethanol production and saving medium nutrient (corn steep liquor ICSL]) during bioethanol fermentation of overliming-treated hydrolysate of waste house wood (WHW) using ethanologenic Escherichia coli K011. In cultivation using WHW hydrolysate supplemented with 4% (v/v) CSL and 0.2 g-dry cell weight (DCW)/l E. coli K011 cells, the overall ethanol yield reached 84% of the theoretical value at 61 h. When we conducted the cultivation with 1% CSL to reduce the supplemental medium cost, the overall ethanol yield remained in the range of 66-72% even at 90 h. We proposed two alternative methods for increasing the overall yield even with 1% CSL. The first method involved increasing the inoculum size of E. coli K011 up to 0.8 g-DCW/l, where 83% of the overall yield was attained at 60 h of cultivation. The second method involved the coculture of 0.2 g-DCW/l E. coli K011 together with 0.02 g-DCW/l of Saccharomyces cerevisiae TJ1, and the overall yield reached 81% at 47 h of cultivation.

DOI： 10.1263/jbb.105.90

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The C-terminal region of the peptidoglycan hydrolase (CPH) of Laetococcus lactis IL1403 produced intracellularly in Escherichia coli was able to attach to the surface of cells of Lactobacillus casei NRRL B-441, Bacillus subtilis 168, E. coli XL1-blue and Saccharomyces cerevisiae IFO0216. Therefore, this domain is a suitable fusion partner for the adhesion of proteins to cell surfaces. The production of cell-surface adhesive proteins using this domain in Pichia pastoris is particularly attractive, because this organism has better capability to allow the correct folding of the recombinant proteins than prokaryotic hosts. However, when this domain is produced in this yeast, its cell-surface binding activity may be limited by glycosylation. In this study, therefore, we constructed a CPH mutant (CPHM) devoid of the potential N-glycosylation sites by site-directed mutagenesis. CPHM was successfully expressed extracellularly in P pastoris (GS115) using the methanol inducible AOX1 promoter with an a-mating factor signal sequence, whereas the native CPH was not produced in this host. Western blot analysis revealed that the apparent molecular size of CPHM was 18 kDa greater than that of CPH produced in E. coli (32 kDa), which is attributed to O-glycosylation. However, CPHM produced in P pastoris was capable of binding to the cell surfaces despite its modification by the yeast, and its dissociation rate constant from the surface of L. casei NRRL B-441 cells was 3.5-fold lower than that of CPH produced in E. coli. These results demonstrate the applicability of the constructed domain (CPHM) for the production of cell-surface adhesive proteins in P pastoris.

DOI： 10.1263/jbb.105.134

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

With the aim of constructing an efficient protein display system for lactic acid bacteria (LABs), the effect of fusion direction on the cell-surface binding activity of the C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 was studied. CPH fused to the a-amylase (AMY) of Streptococcus bovis 148 either at its C-terminus (CPH-AMY) or at its N-terminus (AMY-CPH) was expressed intracellularly in Escherichia coli. This domain was able to direct binding of AMY to the surface of L. lactis ATCC 19435 in both constructs. However, the number of bound molecules per cell and the specific activity for starch digestion in the case of CPH-AMY were 3 and 14 times greater than those in the case of AMY-CPH, respectively. Of the LABs tested, L. lactis ATCC 19435 showed the highest binding capability for CPH-AMY, up to 6 x 10(4) molecules per cell, with a dissociation rate constant of 5.00 x 10(-5) s(-1). The binding of CPH-AMY to the surface of Lactobacillus delbrueckii ATCC 9649 cells was very stable with a dissociation rate constant of 6.96 x 10(-6) s(-1). The production of CPH-AMY in the soluble form increased 3-fold as a result of coexpression with a molecular chaperone, trigger factor. The results of this study suggest the usefulness of CPH as a bidirectional anchor protein for the production of cell-surface adhesive enzymes in E. coli. Furthermore, the importance of the fusion direction of CPH in determining cell-surface binding and enzymatic activities was shown.

DOI： 10.1263/jbb.105.116

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2008f.0.727.0

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2008f.0.986.0

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2008.0.416.0

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2008.0.415.0

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2008.0.389.0

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Publisher：The Society of Chemical Engineers, Japan  

DOI： 10.11491/scej.2008f.0.993.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

During industrial production process using yeast, cells are exposed to the stress due to the accumulation of ethanol, which affects the cell growth activity and productivity of target products, thus, the ethanol stress-tolerant yeast strains are highly desired. To identify the target gene(s) for constructing ethanol stress tolerant yeast strains, we obtained the gene expression profiles of two strains of Saccharomyces cerevisiae, namely, a laboratory strain and a strain used for brewing Japanese rice wine (sake), in the presence of 5% (v/v) ethanol, using DNA microarray. For the selection of target genes for breeding ethanol stress tolerant strains, clustering of DNA microarray data was performed. For further selection, the ethanol sensitivity of the knockout mutants in each of which the gene selected by DNA microarray analysis is deleted, was also investigated. The integration of the DNA microarray data and the ethanol sensitivity data of knockout strains suggests that the enhancement of expression of genes related to tryptophan biosynthesis might confer the ethanol stress tolerance to yeast cells. Indeed, the strains overexpressing tryptophan biosynthesis genes showed a stress tolerance to 55 ethanol. Moreover, the addition of tryptophan to the culture medium and overexpression of tryptophan permease gene conferred ethanol stress tolerance to yeast cells. These results indicate that overexpression of the genes for trypophan biosynthesis increases the ethanol stress tolerance. Tryptophan supplementation to culture and overexpression of the tryptophan permease gene are also effective for the increase in ethanol stress tolerance. Our methodology for the selection of target genes for constructing ethanol stress tolerant strains, based on the data of DNA microarray analysis and phenotypes of knockout mutants, was validated. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jbiotec.2007.05.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

This paper aims to establish the methodology for building object-oriented metabolic pathway models which describe metabolic flux change due to the cultivation conditions change such as from normal one to high osmotic pressure (sugar concentration) based on the genomic data analysis, and also to apply the method for metabolic pathway recruiting in an improved strain producing useful compounds which can play important roles for green chemistry. The model will be developed using the data of transcriptome, proteome and metabolites, including metabolic flux analysis. Practically, profiling data of transcriptome and proteome have been taken at high osmolarity condition. And a better-improved strain based on the analysis was obtained. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bej.2006.06.012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

In a batch coculture of kefiran-producing lactic acid bacteria Lactobacillus kefiranofaciens and lactate-assimilating yeast Saccharomyces cerevisiae, lactate accumulation in the medium was observed, which inhibited kefiran production. To enhance kefiran productivity by preventing lactate accumulation, we conducted lactose-feeding batch operation with feedforward/feedback control during the coculture, so that the lactate production rate of L. kefiranofaciens was balanced with the lactate consumption rate of S. cerevisiae. The lactate concentration was maintained at less than 6 g V throughout the fed-batch coculture using a 5 l jar fermentor, although the concentration reached 33 g l(-1) in the batch coculture. Kefiran production was increased to 6.3 g in 102 h in the fed-batch coculture, whereas 4.5 g kefiran was produced in 97 It in the batch coculture. The kefiran yield on lactose basis was increased up to 0.033 g g(-1) in the fed-batch coculture, whereas that in the batch coculture was 0.027 g g(-1).

DOI： 10.1263/jbb.103.557

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

During saline stress, Saccharomyces cerevisiae changes its metabolic fluxes through the direct accumulation of metabolites such as glycerol and trehalose, which in turn provide tolerance to the cell against stress. Previous research shows that the various controls at both transcriptional and translational levels decide the phenomenon of stress, but details about such transition is still not very clear. This paper attempts to extract some hidden features through the information extraction approach from DNA microarray data during transition to osmotic tolerance, which are expected to be important in directing to the tolerance stage upon encountering osmotic stress in yeast. Time course of DNA microarray data during osmotic tolerance was analyzed by computational approach 'self-organizing map (SOM) extended with hierarchical clustering'. Since eukaryotic gene expression is governed by short regulatory sequences found upstream in promoter regions, therefore clusters containing the similar profiles obtained by SOM were further analyzed for overrepresentation of known regulatory binding sites in promoter region. It was found that apart from known and expected 'STRE' during osmotic stress, the 'GCN4' binding site is also found to be significant. Hence, it was suggested that the process of osmotic tolerance proceeds through a stage of amino acid starvation. The intracellular amino acids pool also found to be depleted during transition and restoration is faster in brewing strain than laboratory strain. Experiments involving supplementation of amino acids helps in reducing the lag time for recovery, which was found to be similar to that of brewing strain.

DOI： 10.1007/s00253-007-0837-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The limit of detection (LOD) and range of quantitation (ROQ) of competitive enzyme-linked immunosorbent assay (ELISA) were determined from a model describing the calibration curve and precision profile of the assay. The calibration curve is given by solving the differential equations describing the change in the concentrations of an antigen-antibody complex and an enzyme-conjugated antigen-antibody complex by a Runge-Kutta method. The precision profile is described in terms of possible error sources such as the pipetting volumes of the analyte, enzyme-conjugated antigen, antibody and substrate solutions, calibration curve and inherent absorbances between the wells in an ELISA plate. An appropriate concentration of the enzyme-conjugated antigen that balances a low detection limit and sufficient color development was found to be in a narrow range, which is consistent with the empirical rule. The optimum conditions for competitive ELISA using an antibody with a kinetic property can be designed from our model.

DOI： 10.1263/jbb.103.427

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The production of 1,4-dihydroxy-2-naphthoic acid (DHNA) was investigated using a fed-batch culture of Propionibacterium freudenreichii ET-3. DHNA is a precursor of menaquinone (MK) and is transformed to MK by combination with an isoprenoid unit. We found that ET-3 stopped MK production and increased DHNA production in an anaerobic fed-batch culture by maintaining the lactose concentration at approximately zero. The maximum DHNA concentration observed in the anaerobic fed-batch culture was markedly higher than the maximum DHNA concentration observed in an anaerobic batch culture. Moreover, MK or DHNA production was affected by the lactose feeding rate; this suggests that lactose metabolism participates in the syntheses of these products. On the other hand, accumulation of propionate was found to inhibit DHNA production in the fed-batch culture. Based on the fact that ET-3 increases DHNA production in an aerobic culture by consuming propionate, we carried out a cultivation experiment in which an anaerobic fed-batch culture was switched to an anaerobic batch culture and found that the DHNA production was increased to a greater extent than the DHNA production in an anaerobic fed-batch culture. These results suggest that DHNA production by ET-3 is markedly influenced by carbon source limitation and the oxygen supply.

DOI： 10.1128/AEM.01307-06

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

This is the first study showing the successful application of waste house wood (WHW) to the pilot-scale production of bioethanol by hydrolysis using diluted acid and fermentation using the ethanologenic recombinant Escherichia coli KO11. The major sugars in the WHW hydrolysate were glucose, mannose and xylose; the percentages were approximately 35%, 35% and 20% (w/w), respectively. In anaerobic fermentation using a 5-l reactor in which the oxygen transfer rate (OTR) was 0 mmol/(l. h), KO11 consumed only 25% of the xylose in the WHW hydrolysate over the examined fermentation time of 100 h; however, hexoses such as glucose and mannose were consumed completely. Microaeration at an OTR of 4 mmol/(l-h) enhanced the xylose utilization ratio of KO11 to 100%, at which the ethanol concentration was 35.4 g/l and the ethanol yield was 0.42, although the maximum ethanol concentrations were 28.8 and 26.6 g/l at OTRs of 0 mmol/(l. h) and 15 mmol/(l. h), respectively. Moreover, this microaerobic fermentation at OTR of 4 mmol/(l.h) was applied to 1000-l scale bioethanol production using the WHW hydrolysate. The xylose utilization ratio reached 100% and the ethanol yield was determined to be 0.45 for a 63-h fermentation, which were comparable to those obtained from the laboratory-scale fermentation.

DOI： 10.1263/jbb.103.350

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC ANALYTICAL CHEMISTRY  

The mathematical model for predicting the precision, limit of detection (LOD) and range of quantitation (ROQ) in a competitive enzyme-linked immunosorbent assay (ELISA) proposed by Hayashi et al. (Anal. Chem., 2004, 76, 1295) was validated. The model describes the relative standard deviation (RSD) of concentration estimates by the RSDs of pipetting volumes of analyte, enzyme-conjugated antigen, antibody and substrate solutions, and the standard deviation (SD) of inherent absorbances between the wells in an ELISA plate. For 6 kinds of direct competitive ELISA kits, the LOD and ROQ predicted by the model agreed well with those obtained by experiments with real samples. It was also confirmed that the model is applicable to the prediction of uncertainty that depends on the pipetting error of the viscous antiserum solution. The model was demonstrated to be useful for estimating the LOD and ROQ of competitive ELISA.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The specific ethanol production rate of raw starch by arming yeast cells displaying a-amylase and glucoamylase increased significantly when the cells and starch granules settled together. The specific ethanol production rate also increased when the size distribution of starch granules was almost same as that of the yeast cells. These results indicate that the surface contact between starch granules and yeast cells is important for increasing the apparent specific activity of alpha-amylase, which was the rate-limiting factor of the direct fermentation.

DOI： 10.1263/jbb.103.95

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Transcriptional responses of laboratory and brewing strains of Saccharomyces cerevisiae to osmotic stresses induced by adding either NaCl or sorbitol to their cultures were compared by clustering DNA microarray data. Our results suggest that the difference in the transcriptional responses of the two strains between NaCl and sorbitol additions is small when the dynamics of the total change in gene expression are similar.

DOI： 10.1263/jbb.102.568

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

In the direct ethanol fermentation of raw starch by arming yeast with alpha-amylase and glucoamylase, it is preferable to use a flocculent yeast because it can be recovered without centrifugation. Three types of arming yeast system, I (nonflocculent), II (mildly flocculent), and III (heavily flocculent), were constructed and their fermentation performances were compared. With an increase in the degree of flocculation, specific ethanol production rate for soluble starch decreased (0.19, 0.17, and 0.12 g g-dry-cell(-1) h(-1) for systems I, II, and III, respectively), but that for raw starch did not decrease as much as expected (0.06, 0.06, and 0.04 g g-dry-cell(-1) h(-1) for systems I, II and III, respectively). Microscopic observation revealed that many starch granules were captured in the yeast flocs in system III during the direct ethanol fermentation of raw starch. It was suggested that the capture of starch granules increases apparent substrate concentration for amylolytic enzymes in arming yeast cell flocs; thus, the specific ethanol production rate of system III was kept at a level comparable to those of the other systems.

DOI： 10.1007/s00253-006-0454-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

To investigate the effects of oxygen supply on Propionibacterium freudenreichii ET-3 metabolism and 1,4-dihydroxy-2-naphthoic acid (DHNA) production in detail, the strain was cultured by switching from anaerobic condition to aerobic condition at 72 h (termed anaerobic-aerobic switching culture hereafter) employing different oxygen transfer rates (OTRs) in the range of 0.08-0.90 mg/(l(.)h). It was found that a 0.08 mg/(l(.)h) OTR could not change the metabolism or improve the DHNA production of P.freudenreichii ET-3. When the OTR was in the range of 0.23-0.66 mg/(l. h), propionate, which inhibits DHNA production significantly, was consumed during the aerobic phase. Final DHNA concentration increased to 0.22 mM, irrespective of OTR. When the OTR was 0.90 mg/(l(.)h), a sudden increase in dissolved oxygen (DO) concentration during the aerobic phase resulted in a sudden decrease in DHNA concentration. To attenuate the stresses caused by propionate and oxygen exposure, we designed an optimal cultivation in which the anaerobic and aerobic phases were repeated three times alternately. As a result, propionate concentration was maintained below the level that inhibits DHNA production, and no DO concentration was detected throughout the culture. The final DHNA concentration in this culture was 0.33 mM, which is 2.7-fold that in the anaerobic culture and 1.5-fold that in the anaerobic-aerobic switching culture.

DOI： 10.1263/jbb.102.198

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

This is the first report on the production of both 1,4-dihydroxy-2-naphthoic acid (DHNA) and menaquinone by Propionibacterium freudenreichii ET-3. DHNA can be a stimulator of bifidogenic growth, and menaquinone has important roles in blood coagulation and bone metabolism. During anaerobic culture, DHNA and menaquinone concentrations reached 0.18 mM and 0.12 mM, respectively. The molar ratio between these products was approximately 3:2, which was not affected by culture pH and temperature over the ranges of 6.0-7.0 and 31-35 degrees C, respectively. As for organic acid, propionate and acetate accumulated at concentrations of 0.3 M and 0.15 M, respectively, and the propionate accumulation particularly inhibited further production of DHNA. To improve DHNA production, we switched from anaerobic condition to aerobic condition during the culture when lactose was depleted. DHNA concentration continued to increase even after lactose exhaustion, reaching 0.24 mM. In contrast to DHNA production, menaquinone production stopped after the switch to aerobic condition. The total molar production of DHNA and menaquinone was 0.3 mM irrespective of aerobic culture and anaerobic-aerobic switching culture. Therefore, the anaerobic-aerobic switching culture could increase the production ratio of DHNA to menaquinone. The DHNA concentration obtained from the anaerobic-aerobic switching culture was 1.3-fold higher than that in the anaerobic culture, because P. freudenreichii ET-3 utilized propionate accumulated in the medium via the reversed methylmalonyl CoA pathway under aerobic condition. The culture method proposed in this study could be applicable to industrial-scale fermentation using 1000 1 of media, by which 0.23 mM DHNA was produced.

DOI： 10.1263/jbb.101.464

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s00253-005-0101-z

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Other Link： http://link.springer.com/article/10.1007/s00253-005-0101-z/fulltext.html

Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s00253-005-0192-6

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Other Link： http://link.springer.com/article/10.1007/s00253-005-0192-6/fulltext.html

Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2006f.0.931.0

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2006f.0.1005.0

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KOREAN SOC BIOTECHNOLOGY & BIOENGINEERING  

The effect of carbon dioxide on yeast growth was investigated during the cultivation of pH 5.0 and pH 6.8, by replacing the nitrogen part with carbon dioxide under aerobic conditions. The values of the specific growth rate under pH 5.0 and pH 6.8 conditions became 64.0% and 46.9%, respectively, compared to those before the change in gas composition. This suggests that the effect of carbon dioxide was greater pronounced in pH 6.8 than in pH 5.0. The genome-wide transcriptional response to elevated carbon dioxide was examined using a DNA microarray. As for upregulated genes, it was noteworthy that 3 genes were induced upon entry into a stationary phase and 6 genes were involved in stress response. Of 53 downregulated genes, 22 genes were involved in the ribosomal biogenesis and assembly and 5 genes were involved in the lipid metabolism. These facts suggest that carbon dioxide could bring the cell conditions partially to a stationary phase. The ALD6 gene encoding for cytosolic acetalclehyde dehydrogenase was downregulated, which would lead to a lack of cell components for the growth. The downregulation of ALD6 was greater in pH 6.8 than in pH 5.0, consistent with physiological response. This suggests that it might be the most effective factor for growth inhibition.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The effects of sulfur sources on the desulfurization activity of Rhodococeus erythropolis KA2-5-1 were investigated by using an exponential fed-batch culture technique. The feed rate of a sulfur source was controlled independently of the feed rate of ethanol, which was used as a carbon and energy source. Among the sulfur sources examined were dibenzothiophene (DBT), ammonium sulfate, (L)-cysteine, (L)-methionine, and 2-amino-ethanesulfonic acid. When the fed-medium contained DBT as the sole sulfur source, KA2-5-1 cells showed a maximum desulfurization activity of approximately 130 mmol 2-HBP kg-cell(-1)h(-1). Similar levels of enzyme activity were also achieved with inexpensive ammonium sulfate by using the exponential fed-batch culture technique. In addition, higher levels of desulfurization activity were achieved by increasing the dosage of the DBT desulfurization (dsz) operon and dszD gene in R. erythropolis KA2-5-1. The recombinant strain showed a maximum desulfurization activity of approximately 250 mmol 2-HBP kg-cell(-1)h(-1) in the exponential fed-batch cultures.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Focusing on drug metabolism in liver, we constructed and evaluated a drug-metabolizing bioartificial liver (BAL) support system. In a previous study, we constructed ammonia-metabolizing CHO and hepatoma-derived HepG2 cell lines by recombination of the glutamine synthetase (GS) gene. For further mimicking of liver metabolism, the human hepatoma-derived cell line HepG2 was transformed by the pBudCE-GS-CYP3A4 vector, which contains GS and drug-metabolizing CYP 3A4 genes. The constructed GS-3A4-HepG2 cell line showed 3A4 activity higher than that of human primary hepatocytes. The drug-metabolizing activity of BAL (BAL clearance) was evaluated using this cell line. The estimated clearance was higher than that of the human hepatocyte system.

DOI： 10.1021/bp049757a

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2005.0.313.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

To establish the ammonia-metabolizing cell lines for a bioartificial liver support system, CHO-K1 and HepG2 were transformed with pBK-CMV-GS vector that contains glutamine synthetase (gs) gene. The recombinant cell lines were selected under the various concentrations of glutamine synthetase inhibitor, methionine sulfoximine (MSX). The host CHO-K1 and HepG2 cell lines produces ammonia, but the both MSX tolerable CHO (GS-CHO) and HepG2 (GS-HepG2) cell lines endowed with the high GS activity could metabolize the ammonium from medium. The ammonia-metabolizing activity of CHO and HepG2 cell was about one-fourth of that of primary hepatocyte. (C) 2004 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.enzmictec.2004.08.027

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

To isolate phages displaying a practical and useful antibody with a high k(on) value and/or a low k(off) value from phage display antibody libraries, we developed a rational strategy based on a kinetic model. In the model, the recovery of a phage displaying an antibody after a round of biopanning is expressed as a function of five parameters, the apparent association rate constant of the phage antibody to the immobilized antigen (k(on)'), the apparent dissociation rate constant of the phage antibody from the immobilized antigen (k(off)'), the effective antigen concentration (C), the time for the binding process (t(b)) and the time for the washing process (t(w)). An optimum set of operating parameters (C, t(b) and t(w)) for isolating phages displaying an antibody with a high km value was designed based on the model. Three rounds of biopanning were carried out under the designed conditions, against a phage library in which the hypervariable regions of an original antibody were randomized. All isolated phages displayed an antibody with a higher k(on) value and one displayed an antibody with a 30-fold greater km value than that of the original antibody. Experimental conditions which improve the efficiency of conventional off-rate selections are also described. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.molcatb.2004.02.016

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Language：English   Publisher：The Society of Chemical Engineers, Japan  

Gene amplification has been developed as an indispensable technique for the bio-pharmaceutical production by recombinant mammalian cells. The most commonly used technique is likely dihydroforate reductase (<I>dhfr</I>) gene amplification system in Chinese hamster ovary (CHO) cell line. In this gene amplification system, high productive CHO cells can be selected by "time-consuming" stepwise increases of methotrexiate (MTX) concentration in culture medium. However, it is of great significance to shorten the time for constructing the stable and productive recombinant CHO cell line in the industrial process. In this study, we focused on a rapid construction method for gene-amplified cell line. We employed the human granulocyte-macrophage colony stimulating factor (hGM-CSF) as a model of glycoprotein and investigated the relationship among the productivity and stability of amplified gene, the location of the amplified gene and the MTX concentration. The distribution of amplified gene on the chromosome was analyzed using fluorescence <I>in situ</I> hybridization (FISH). Based on the FISH image analysis, we speculated the specific chromosome location suitable for gene amplification.

DOI： 10.11491/apcche.2004.0.488.0

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Language：English   Publisher：The Society of Chemical Engineers, Japan  

To improve metabolic pathways for enhancement of a targeted product systematically, quantitative systems analysis in bionetworks of gene, protein, and metabolic pathways is highly desired. Quantification of effect of perturbation of genes and proteins on metabolic fluxes enables to elucidate the parameters responsible for the control of flux in metabolic pathways. In this study, mechanism of flux redistribution at a key branch point in glutamate production by coryneform bacteria, <I>Corynebacterium glutamicum</I> and <I>Corynebacterium efficiens</I> is studied. The quantitative analysis at a key branch point, namely, 2-oxoglutarate(αKG), was performed by comparison of flux distribution before and after addition of the triggering operations such as depletion of biotin and addition of Tween 40. The flux redistribution from the central pathway to glutamate synthetic pathway obviously occurred after the decrease in activity of 2-oxoglutarate dehydrogenase complex, ODHC, while activities of other enzymes at the branch point of isocitrate dehydrogenase, ICDH, and glutamate dehydrogenase, GDH, were not significantly changed. The comparative studies on flux distribution of a wild type strain of <I>C. glutamicum</I> and two genetically engineered strains which harbor a plasmid with genes encoding ICDH and GDH, respectively, also reached the same conclusion that the ODHC is the most crucial factor for the control of flux distribution at the 2-oxoglutarate branch point. The difference in glutamate production between <I>C. glutamicum</I> and <I>C. efficiens</I> is due to a result of the difference in the degree of decrease in ODHC activity. A model with kinetic parameters well explained the phenomena of flux redistribution at the 2-oxoglutarate branch point and predicted flux redistribution.

DOI： 10.11491/apcche.2004.0.423.0

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Language：English   Publisher：The Society of Chemical Engineers, Japan  

Growth properties and gene expression profiles by using slide glass-based DNA microarray under high osmotic pressure condition (addition of NaCl) were compared between the two yeast strains, a laboratory strain and a brewing one used for Japanese rice wine (sake) brewing. Growth properties in both strains after addition of 1 M of NaCl revealed that the brewing strain was more tolerant to osmotic stress than the laboratory one. DNA microarray analysis data indicated that the genes encoding the enzymes involved in acetate, glycerol and trehalose synthetic pathways were up-regulated in both strains under the stress conditions. Moreover, genes encoding sodium ion pump and cupper ion-binding proteins were more up-regulated in brewing strain than in the laboratory strain. Up-regulation of these genes might characterize the osmotic stress-tolerance of the brewing strain. We constructed a glycerol-overproducing strain by expressing <I>GPD1</I> gene encoding glycerol-3-phosphate dehydrogenase constitutively in the laboratory strain and evaluated the osmotic stress tolerance of this strain. This improved strain showed more tolerant to osmotic stress than the parent strain, but glycerol was immediately exported outside of the cells. We are constructing further improved strain by disrupting the gene encoding glycerol exporter protein and we will evaluate again the stress tolerance of this strain.

DOI： 10.11491/apcche.2004.0.422.0

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Language：English   Publisher：The Society of Chemical Engineers, Japan  

In this report, present status of a project for a process development based on genomics going on in our groups is explained. Goals of the project supported by the New Energy and Industrial Technology Development Organization (NEDO) is to build object-oriented metabolic pathway models which describe metabolic flux change due to the cultivation conditions change such as from normal one to high osmotic pressure (sugar concentration), and also to apply the model for effectively designing an improved strain producing useful compounds which can play important roles for green chemistry. The model will be developed using the data of transcriptome, proteome and metabolites. In the project, the analysis system which was composed of bioreactor system for taking the on-line/off-line data of metabolic flux analysis, NMR analysis system and software system for data mining from transcriptome as well as proteome, has been set up and took data for high sodium chloride concentration as a model of high osmolarity condition. At the same time, three sub-projects have been proceeded; (1) Basic techniques for metabolic engineering in order to control the targeted pathway flux in <I>Saccharomyces cerevisiae</I> have been developed. (2) Profiling data of transcriptome and proteome have been taken at high sodium chloride concentrations. (3) As one of clustering techniques of the profiling data, SOM (self organizing map) has been extensively studied, applied to the transcriptome data obtained in this project. The project has been now on the stage of strain improvement based on the analysis of the genome information.

DOI： 10.11491/apcche.2004.0.416.0

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Language：English   Publisher：The Society of Chemical Engineers, Japan  

It is one serious problem that high sugar concentration at an initial phase and/or accumulation of product during cultivation causes osmotic stress to cells in fermentation process using yeast, <I>Saccharomyces cerevisiae</I>. In this study, we analyzed the protein expression of yeast using two-dimensional electrophoresis under a high osmotic pressure condition and investigated the physiological change in yeast cell. A laboratory strain FY834 and brewing one IFO2347 were grown in YPD medium. High osmotic pressure condition was prepared by adding NaCl at final concentration of 1 M in mid-log phase. Although the growth of FY834 was stopped and that of IFO2347 was decreased, the growth of both strains was recovered later. The period between addition of NaCl and recovery of growth was 4 hours for FY834 and 2 hours for IFO2347, respectively, indicating that IFO2347 was more tolerant to osmotic stress than FY834. Protein samples were extracted from cells before and after adding NaCl and applied to two-dimensional electrophoresis. Protein expression of FY834 compared to the sample before addition of NaCl was changed after 30 min from adding NaCl, while that of IFO2347 was changed after 15 min. These results suggest that IFO2347 responds more quickly to osmotic pressure than FY834. From the view point of the metabolic pathway, the expression of proteins related to glycerol synthesis were increased after addition of NaCl in both strains, suggesting that yeast strains adapt to high osmotic pressure by synthesis of glycerol. It was consistent to the results of metabolic flux analysis.

DOI： 10.11491/apcche.2004.0.431.0

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Language：English   Publisher：The Society of Chemical Engineers, Japan  

Genome-wide expression profiles in the yeast <I>Saccharomyces cerevisiae</I> responding to environmental stresses were analyzed using microarray. Two yeast strains, a laboratory strain (FY834) and a brewing one (IFO2347) which is more osmotic tolerant than FY834 strain were analyzed and the differences between the two strains in response to several environmental stresses were elucidated. Based on the analyses of DNA microarray data, the information for breeding osmosis tolerance strain was extracted. To analyze microarray data, a Self-Organizing map (SOM) was introduced to cluster gene expression data. The range of experimental errors was determined by multiple measurements of a same sample, and the effect of expected error on the clustering results of the SOM was simulated. The clusters obtained by the SOM were further clustered until the influence of experimental errors on the clustering results falls to a certain criterion. A hierarchical clustering was adopted to cluster the SOM results, and we could obtain the clustering results which cannot be further subdivided in consideration with the experimental errors. By introducing some other information such as sequences of transcription factors, gene ontology data and metabolic pathways, we found that in the obtained clusters the frequency distributions of some factors, such as genes having specific transcription factors and genes categorized to specific functions, are significantly deviate from those computed by randomized data. Moreover we identified some clusters which represent significant up-regulation or down-regulation only in the specific conditions. For example, a gene cluster in which gene expressions were up-regulated only in IFO2347 strain was identified, and which represents the difference in osmotic tolerance between these strains.

DOI： 10.11491/apcche.2004.0.778.0

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Language：English   Publisher：The Society of Chemical Engineers, Japan  

Single step bioconversion of raw starch to ethanol has raised considerable interest in recent years. We have developed yeast strains displaying α-amylase and glucoamylase on yeast cell surface for this purpose. The advantage of this system is that the displayed enzymes can be recovered easily with the yeast after fermentation compared to the case that these amylolytic enzymes are secreted into medium. However, display of α-amylase on cell surface may reduce the apparent association rate of the enzyme to the substrate because both of them are huge molecules, resulting a lower apparent conversion rate. The recovery of the yeast cells together with the amylolytic enzymes becomes easier when a flocculating yeast strain is used as the host strain. However, flocculation of the arming yeast may again reduce the apparent conversion rate. To establish an industrial ethanol production process with a good cost performance ratio, the advantages and disadvantages of each strategy should be evaluated quantitatively. Therefore, from a viewpoint of kinetics, we compared the performance of the fermentation systems in the case that α-amylase is displayed on the cell surface or secreted in the medium, and in the cases that a flocculating or non-flocculating strain is used as the host strain. A kinetic model has been developed to simulate the direct ethanol fermentation from raw corn starch by arming yeast. The simulation results were found agree well with experimental data obtained from direct ethanol fermentation.

DOI： 10.11491/apcche.2004.0.789.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC CHEMICAL ENG JAPAN  

We investigated the composting process of an aquatic plant for developing an effective method of their recycling. The changes in microflora were monitored by temperature gradient gel electrophoresis (TGGE) of the DNA fragments amplified by reverse transcription PCR from the 16S ribosomal RNA extracted from the compost. The decrease in the cellulose content of aquatic plant compost was found to be faster than that of other composts. During the phase when the specific activity of cellulase was high, the decrease in the cellulose content was rapid. In the TGGE analysis, a few bands were found that changed in intensity in relation to that of the specific activity of cellulase. The bacteria responsible for the bands were isolated and, based on the DNA sequences of the 16S rRNA, identified as Bacillus spp. For an effective composting process, we examined the use of the composted material as a seed. When the resultant compost was used as a seed for the next cycle of composting, the decrease in cellulose content during the second composting cycle occurred faster than that during the first cycle.

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2003f.0.141.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A fuzzy control system was applied to a bioreactor in which the flocculent yeast Saccharomyces diastaticus ATCC 60715 was immobilized by entrapment in a reticulated polyurethane foam cube (6 nun x 6 mm x 6 mm, biomass support particle (BSP)). The fuzzy inference algorithm was used to calculate the suitable level of glucose feed rate from the monitored data of the CO2 evolution rate (CER) and the integrated amount of CO2 evolution. One of the main concerns is the mass transfer of glucose in the BSP in which S. diastaticus was immobilized. With the BSPs, glucose diffusivity becomes lower and starvation of the cells can occur if the control system is not quick enough to respond to the starvation. The fuzzy control system was successful in controlling the glucose feed pump to maintain a suitable glucose concentration for the smooth continuation of ethanol production. (C) 2002 Elsevier Science B.V. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

A kinetic model describing the affinity selection process of phage display libraries was established and verified experimentally using ribonuclease A (RNase A) and anti-RNase A single chain Fv phage antibody. Desorption of target molecules from a solid phase and orientation of the epitopes of adsorbed target molecules are taken into account in this model. The ratio of the effective antigen to the total antigen was estimated to be 0.0127(+/-)0.0018 when RNase A was immobilized on polystyrene beads by passive adsorption. The model can faithfully describe the recovery of the phage antibody in a round of biopanning based on the following parameters; the effective concentration of RNase A on the beads, the desorption rate constant of RNase A from the beads, the dissociation constant and dissociation rate constant of the phage antibody from RNase A, and the time for blocking, binding and washing in the biopanning process. The causes of the typical problems in biopanning are explained quantitatively by the model and the practical solutions for the problems are discussed.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：INT INST INFORMATICS & SYSTEMICS  

The affinity of a ligand to its target has often been evaluated by its dissociation constant (K-D). However, K-D is a static parameter that assumes the equilibrium of a ligand reaction with a target. Since, in practical ligand applications, the reaction time is usually limited, the properties of ligands must be evaluated dynamically. A phage display system is one of the most powerful tools for isolating specific ligands such as antibodies. We established kinetic models that describe antibody-antigen reactions that incorporate the behaviors of phage antibodies into a process that isolates them from phage display libraries. Based on this model, we rationally designed experimental conditions for isolating antibodies with a high association rate constant (k(on)), thus enabling the rapid assay of their antigens. From a customized phage antibody library in which two hypervariable regions of an original antibody are randomized, some antibodies with higher k(on) values than the original antibody were successively isolated under the rationally designed conditions.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

A simple and rapid method based on enzyme-linked immunosorbent assay (ELISA) was developed for measuring the association rate constant of antibody-antigen interactions. An antibody and its antigen are mixed in a solution to initiate the equilibrium reaction. At different time intervals, the amount of the free antibody in the reaction mixture is estimated by an indirect ELISA. The association rate constant was estimated by nonlinear regression against an equation introduced from the derivation of the mass balance of antigen-antibody interaction. This method can determine the association rate constant of antibodies with a dissociation rate constant up to 5 x 10(-3) s(-1). The association rate constant of a single-chain Fv (scFv) to its antigen, bovine pancreatic ribonuclease A (RNase A), determined by the present method agreed well with those determined by the fluorescence polarization method and surface plasmon resonance method. No significant difference in the association rate constant was found between the soluble anti-RNase A scFv and the same scFv displayed on a phage (5.65+/-0.54x10(4) M-1 s(-1) and 5.96+/-0.56x10(4) M-1 s(-1), respectively).

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS INC  

In previous work, we clarified the relationship between the productivity and stability of gene-amplified cells and the location of the amplified gene. The location of the amplified gene enabled us to classify resistant cells into two types. One type of resistant cell group, in which the amplified genes were observed near the telomeric region, was named the "telomere type." The other type of cell group, in which the amplified genes were observed in other chromosomal regions, was named the "other type." The phenotypes of these two types of cells are very different. In this experiment, using a fluorescein isothiocyanate-labeled methotrexate (F-MTX) reagent with flow cytometry, we were easily able to distinguish between highly productive cells and the other types of cells. The level of fluorescence differed according to the difference in resistance to MTX. Based on this new finding, highly productive gene-amplified cells could be isolated from heterogeneous gene-amplified cell pools more easily than by the method of limiting-dilution assay. The limiting-dilution method requires several months to obtain highly productive gene-amplified cells, while our flow-cytometry-based method of selection requires only a few weeks. (C) 2001 John Wiley & Sons, Inc.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

A phage display library is a powerful tool for screening ligands such as antibodies and peptides that specifically recognize a target. In this study, we established a kinetic model describing the affinity selection process of phage display libraries and verified the model experimentally. Desorption of target molecules from a solid phase and orientation of the epitopes of adsorbed target molecules are taken into account in this model. The ratio of the effective antigen density to the total antigen density was estimated to be 0.0127(+/-)0.0018 when bovine pancreatic ribonuclease A (RNase A) adsorbed on polystyrene beads was recognized by an anti-RNase A single-chain Fv phage antibody. The model can faithfully describe the recovery of the phage antibody in a round of biopanning based on the effective concentration of RNase A on the beads, the desorption rate constant of RNase A from the beads, the dissociation constant and dissociation rate constant of the phage antibody from RNase A, and the time for blocking, equilibrium and washing in the biopanning process. A recommended biopanning protocol based on the model is also described.

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Constrained optimization for microbial fermentation was studied. For optimization, we used not the maximum principle but a nonlinear programming method because of the need to consider many metabolic reactions. In the case of L-lysine fermentation, the optimization problem in L-lysine production was formulated as a nonlinear programming problem. In general, the state equations based on material balances are represented as differential equations, but such equations which are dependent on time can not be applied to a nonlinear programming problem. Therefore, the state equations were made discrete in a time base, and a new single vector which is not dependent on time was substituted. From these formulae, the objective function and the constraints using nonlinear programming problem were defined as the amount of L-lysine produced, and as a metabolic reaction model and empirical equations, respectively. Computer program was developed to solve this constrained nonlinear programming problem. The applied algorithm of the computer programming was a sequential quadratic programming method (SQP method). When the constrained nonlinear programming problem is solved using the SQP method, the maximum amount of L-lysine produced and the optimal feeding rate of L-threonine could be calculated. From the calculated results, it was clear that introduction of the equality and inequality constraints was easy. L-Lysine at a concentration up to 75.3 g/l could be produced when the fermentation was carried out under optimal conditions.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

DNA aptamers that bind to hematoporphyrin IX (HPIX) were isolated using an in vitro selection technique. Most aptamers obtained after the 7th and 10th rounds contained guanine-rich sequences. Binding assay using fluorescence polarization technique and structural analysis by CD spectra revealed that the parallel guanine-quartet structure of the aptamer participates in the recognition of HPIX. (C) 2000 Elsevier Science Ltd. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The effect of L-threonine feeding in the production phase on L-lysine production by Brevibacterium flavum, which requires L-homoserine or L-threonine for cell growth, was investigated considering the concerted inhibition by L-threonine plus L-lysine, and the metabolism related to lysine production. Exponential feeding of L-threonine increased L-lysine production to 70 g/l about three times that without feeding. From the analysis of the metabolic flux, carbon flux of L-lysine synthesis pathway in the production phase after L-threonine feeding was higher than that in the growth phase. The results show that feeding of an inhibitory substance may increase the production, especially when the substance is necessary for the continuation of cell growth and/or production.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

The binding of a monoclonal antibody to an epitope peptide was controlled by the conformational change of the epitope peptide induced by anions, We synthesized peptides in which the epitope sequence DTYRYI for the monoclonal antibody AU1 is located between amphiphilic peptides (KKLL)(n) and (LLKK)(n). In the absence of an appropriate anion, the peptide was in a random coil state and the epitope was linear. In contrast, in the presence of an appropriate anion, the peptide exhibited an anti-parallel a-helical structure and the epitope was subsequently 'bent', In the presence of 41 muM triphosphate, the association constant between the antibody and the peptide was decreased by one order of magnitude in the case of n = 3 and at least three orders of magnitude in the case of n = 4 or 5, Oligo-DNAs, as anions, dissociated the antibody-peptide complex, whereas triphosphate did not. The DNA concentrations required for 50% dissociation of the antibody-peptide complex at pH 7.5 were 4x10(-8), 1x10(-7) and 6x10(-6) M for decamer, octamer and hexamer DNA, respectively.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Previously, we established an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese hamster ovary (CHO) cell line. With a gradual increase in methotrexate (MTX) concentration, gene-amplified cell pools had high and stable specific growth and production rates. Moreover, the phenotype of gene-amplified cells seemed to be affected by the location of the amplified gene in chromosomal DNA. We suspected that various kinds of gene-amplified cells might appear during the long-term selection to construct gene-amplified cell pools. To clarify the behavior of gene-amplified cell pools during a stepwise increase of MTX concentration, me isolated gene-amplified clones derived from gene-amplified cell pools. We compared the characteristics of isolated clones, such as the productivity of recombinant protein, stability of amplified genes, and the location of amplified genes. As a result, telomere-type clones, in which the amplified gene was located near the telomeric region, were found to be more stable and productive than other types of clones. Telomere-type clones had over 100 copies of amplified genes in the chromosomal DNA. In contrast, a large number of other types of clones had less than 10 copies of amplified genes. During long-term cultivation in the absence of MTX, in other types of clones, amplified genes rapidly decreased in the chromosomal DNA.

DOI： 10.1021/bp000114e

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KLUWER ACADEMIC PUBL  

In order to establish an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese Hamster Ovary (CHO) cell line, various kinds of stepwise methotrexate (MTX) selection were carried out. The specific growth and production rates of the cell were compared with each other, and the distribution of the amplified gene location was determined using fluorescence in situ hybridization (FISH). The specific growth and production rates of the cell pool reached the highest levels under the selection condition in which the stepwise increase in the MTX concentration was most gradual; about 82% of amplified genes were observed near the telomeric region. During long-term cultivation without MTX, the percentage of amplified genes near the telomeric region hardly changed, but that of amplified genes at other regions decreased. Based on these results, stable and highly productive cell pools could be easily and quickly constructed and amplified and gradual stepwise increase of the MTX concentration. In addition, the FISH technique was powerful tool to evaluate highly productive and stable gene-amplified cells based on the chromosomal location of the amplified gene.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE SA  

An expert system was used to achieve the high production of desulfurizing cells of Rhodococcus erythropolis KA 2-5-1. By adding a proper amount of sulfur containing component with the aid of the expert system, we could avoid excess feeding which resulted in the lowering of desulfurizing activity and starvation which caused serious damage to cell growth. In order to determine the addition amount by the expert system, the data of the amount of chemical elements contained in the cells were used as a reference for comparison with the medium components present. Culture experiments were carried out using a 51 jar fermenter with several kinds of media whose components were determined based on the inferred results with the aid of the expert system. We could restrict the amount of the sulfur component addition so that sulfur was a growth-limiting factor; in contrast, the amounts of other elements were sufficient for growth.
As a result, the maximum specific production rate of 2-hydroxy biphenyl (2HBP) and the maximum cell concentration were 20 mmol kg-drycells(-1) h(-1) and of 45 g-drycells l(-1), respectively. At 100 h of cultivation, 1 g/l of dibenzothiophene (DBT) was converted to 2HBP within 20 h, i.e., 10 mmol kg-drycells(-1) h(-1) of specific desulfurization activity, and the specific activity remained stable for a long period in the culture experiment. (C) 2000 Elsevier Science S.A. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

On line control coupled with an expert system was constructed for the control of a fed-batch culture with the aim of achieving a high fell density. During the cultivation, the expert system could monitor the extents of sufficiencies in the amounts of chemical elements in the medium every 10 min, and suggest a modification to the feeding control policy if the amount of a particular component was inadequate for cell growth. However, we often encounter such a kind of cultivations in which some particular carbon source such as glucose and ethanol should be controlled at low concentration in many culture processes, because the excess feeding might cause the growth inhibition by its own accumulation in the culture broth, or the biproduct accumulation like as lactate or acetate, unsuitable substances for smoothed growth. In this study, we developed an online control system based on production rules which managed the glucose feed rate from DO signal. The online control was carried our by the control computer connected with the other computer for expert system, because a relatively long time (several minutes) was needed for the inference of the expert system and the influence of the starvation of carbon source on the cell growth is not negligible even in several minutes. The on-line control system with expert system was applied to the production of cell mass of E, coli W3110 and the final cell concentration reached 137 g-dry cell weight/l.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Rhodococcus erythropolis KA2-5-1 is one of the best strains for the desulfurization of dibenzothiophene (DBT) via a sulfur-specific pathway in which DBT is converted to the end product, 2-hydroxybiphenyl, by releasing sulfite via DBT-sulfone and 2-(2'-hydroxyphenyl) benzene sulphinate. The objective of this research is to develop a culture method in order to attain a high cell density with a high level of specific desulfurization activity. Compared with glucose or glycerol, ethanol was found to be a preferable carbon source for obtaining a high specific activity (SA) of desulfurization. When the amount of DBT fed was restricted by feeding 2.9 mg-DBT/g-ethanol solution, the maximum SA and final cell concentration were 135.5 (mmol-2HBP/kg-dry cell weight.h) and 37 (g-dry cell weight/l), respectively. On the other hand, when glucose or glycerol was used as a carbon source, the SA was lower than 50 (mM-2HBP/kg-dry cell weight.h) and the final cell concentration was also lower than 27 (g-dry cell weight/l). The activities of the desulfurization enzymes in R. erythropolis KA2-5-1 grown on ethanol were remarkably higher than when the strain was grown on glucose or glycerol. It was also suggested that NADH, which is produced by the biochemical reaction of NAD with ethanol catalyzed by alcohol dehydrogenase, might contribute to the conversion of FMN to FMNH2, which is a coenzyme for the activities of desulfurization enzymes.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

A human lysozyme expression system by Pichia pastoris was constructed with the expression vector of pPIC9, which contains the a-factor signal peptide known for high secretion efficiency. P.pastoris expressed the human lysozyme at about 300 mg/I broth, but four extra residues (Glu(-4)-Ala(-3)-Glu(-2)-Ala(-1)-) were added at the N-terminus of the expressed protein (EAEA-lysozyme), To determine the effect of the four extra residues on the stability, structures and folding of the protein, calorimetry, X-ray crystal analysis and GuHCl denaturation experiments were performed. The calorimetric studies showed that the EAEA-lysozyme was destabilized by 9.6 kJ/mol at pH 2.7 compared with the wild-type protein, mainly caused by the substantial decrease in the enthalpy change (Delta H). On the basis of structural information on the EAEA-lysozyme, thermodynamic analyses show that (1) the addition of the four residues slightly affected the conformation in other parts far from the N-terminus, (2) the large decrease in the enthalpy change due to the conformational changes would be almost compensated by the decrease in the entropy change and (3) the decrease in the Gibbs energy change between the EAEA and wild-type human lysozymes could be explained by the summation of each Gibbs energy change contributing to the stabilizing factors concerning the extra residues.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

The specific antibody production rate is closely related to the specific ATP production rate. In order to increase the specific antibody production rate by increasing the specific ATP production rate, the effects of oxygen concentration reinforcement (4.7, 6.9 and 10.3 mgl(-1)) or metabolic intermediate addition (pyruvate, malate and citrate) on ATP production were investigated under glutamine-limited continuous cultivation with a constant specific growth rate. As a result, the specific ATP production rate decreased under high oxygen concentration cultivation. However, the metabolic fluxes related to ATP production were increased and higher specific ATP and antibody production rates were achieved by addition of metabolic intermediates.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

In order to investigate the effect of the sugar composition of the medium on IgG glycosylation in mouse hybridoma cultivation, the batch culture of mouse hybridoma 3A21 (RCB 1285) was carried out under various sugar compositions. The macroheterogeneity (relative ratio of glycosylated antibody) was analyzed by an enzyme-linked lectin binding assay during cultivation. The relative ratio of glycosylated antibody decreased as cultivation progressed. However, in mannose-enriched cultivation, the relative ratio was maintained at a high level. The microheterogeneity of each oligosaccharide chain was analyzed by two-dimensional mapping techniques. The high-mannose type oligosaccharide chain content was high in mannose-enriched cultivation. The content of oligosaccharide chains terminating in galactose residues, which plays an important role in the immune system, was shown to be high in galactose-enriched cultivation. Using galactose- and mannose-enriched cultivations, the number of oligosaccharide chains terminating in galactose residues could be increased.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

Tn order to obtain a stable and highly productive gene-amplified recombinant Chinese Hamster Ovary (CHO) cell line, five binds of stepwise methotrexate (MTX) selection were carried out. The specific growth and production rates of the cells were compared with each other, and the distribution of the location of amplified genes was determined using fluorescence in situ hybridization (FISH). The specific growth and production rates of the cell line obtained under the selection condition in which the stepwise increase in the MTX concentration was most gradual reached the highest levels and under this condition about 80% of the amplified genes were observed near the telomeric site. During long-term cultivation without MTX, the percentage of amplified genes near the telomeric site hardly changed, but that of amplified genes at other sites decreased. The specific production rate gradually decreased during cultivation without MTX To clarify the relationship between the specific production rate and the location of amplified genes, a cloned cell line, DR1000L4N, was obtained. This cell line showed higher productivity, and the amplified genes were all situated near the telomeric site.

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：SOC CHEMICAL ENG JAPAN  

In order to establish a cell line suitable for a hybrid artificial liver support system, an ammonia metabolizing activity is given to a Chinese Hamster Ovary (CHO) cell line using genetic engineering techniques. CHO-K1 cells were transformed with pSV 2-GS vector which contain glutamine synthetase gene connected with SV 40 promoter. The recombinant cell line was selected under various concentrations of glutamine synthetase inhibitor, methionine sulfoximine (MSX). The 800 mu M MSX tolerable cell line has high glutamine synthetase activity and can remove ammonium from the medium. The kinetic parameters of this cell line were investigated under 0, 1, or 2 mM ammonia concentration. Ammonia consumption occurred during the middle phase of cultivation (growth phase). The specific ammonium consumption rate of the established cell line during growth phase is about one fifth of that of primary hepatocyte.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The methylotrophic yeast Pichia pastoris is one of the best hosts for the production of foreign proteins because of the presence of the strong AOX1 promoter induced by methanol. Methanol feeding during the production phase of the foreign proteins is important because methanol not only induces protein production but also provides energy source for the host cells. Excess methanol inhibits the growth of host cells, while an insufficient amount of energy source and/or methanol starvation lead to poor growth and production. We constructed a simple methanol control system consisting of a semiconductor gas sensor and a relay. Using this system, we studied the effect of methanol concentration on the production of a model foreign protein, human beta(2)-glycoprotein I domain V. The methanol concentrations were kept constant at 1.5, 10, 17, or 31 g.l(-1) (+/-5%) during the production phase. Although the specific rates of growth and methanol consumption decreased with increase in the methanol concentration, the specific production rates increased, indicating that the energy for the production competed with that for cell growth. Accordingly, we provided glycerol as an extra energy source during the production phase, with the result that the specific production rate increased two times. Our simple and inexpensive system will help bioengineering studies on the production of recombinant proteins in P. pastoris, the growth and production of objective proteins in which are dependent on the methanol concentration.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Peptide ligands which bound to a model monomeric protein, bovine pancreatic ribonuclease A, could be isolated from a constrained random hexapeptide phage library. Selection was successful in a low ionic strength buffer (10 mM sodium phosphate, pH 6.0), whereas it failed in TBS (50 mM Tris-Cl, 150 mM NaCl, pH 7.5). Two of the displayed amino acid sequences from among the clones isolated were AEGACEQLDYNC and AEGACLWHDQLC. Electrostatic interaction appeared to play an important role in the binding because these phages could not bind to RNase A at a high ionic strength. The results suggest that selection in low ionic strength buffers could make possible the isolation of peptide ligands against proteins of interest which do not originally interact with another peptide or protein.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：PERGAMON PRESS LTD  

An investigation was made into the water quality and hydrology in paddy fields adjacent to Lake Yogo, Japan, where a circular irrigation system with an oxidation ditch for removal of nitrogen, phosphorus, and COD was established in 1997 with the aim of saving irrigation water and reducing pollutants in drainage water. A simulation was made, and evaluated by comparing the simulated values for nitrogen and phosphorus in the drainage water from the agricultural land with observed data. It was concluded from the evaluation that the simulation could predict a reduction in the nutrient load entering the lake. The circular irrigation system could contribute to a reduction of the pollutant load by 44.2% for total phosphorus and 26.9% of for total nitrogen compared with the conventional irrigation system by operating the circulation pumps for 7 hours a day during the irrigation period. Copyright (C) 1998 IFAC.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

We investigated the relationship between the specific production rate and the concentration of the toxic drug methotrexate (MTX), inhibitory to nucleic acid synthesis in the medium, during the construction of recombinant CHO cells. The results of selection of MTX tolerant cells when the MTX concentration was increased under 10 different stepwise conditions showed that the number of days required to obtain cells resistant to 1000 nM MTX depended on the selection conditions. Tolerant cells were obtained most rapidly at an MTX concentration of 10nM. With increasing MTX concentration, the specific production rate increased, while the specific growth rate gradually decreased. In the case of a gradual increase in the MTX concentration, the specific production rate reached a higher level. We also investigated the stability of the specific production rate during cultivation over a long period with or without MTX. In long-term cultivation, the specific production rate decreased, while the specific growth rate remained at the same level. The specific production rate in a culture without MTX decreased more rapidly than that in a culture with MTX.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

To investigate the effect of culture conditions on glycosylation patterns, we carried out the batch cultivation (using serum-containing or serum-free media) and glucose-limited continuous cultivation (serum-free medium). Con A lectin blotting showed that N-linked glycosylation occurred under all the culture conditions used. The sugar composition of the oligosaccharide chain in each culture was studied using gas-chromatography and was found to be different under each culture condition. The detailed structure of each oligosaccharide chain was then analyzed by two-dimensional mapping techniques using pyridylamination method. The oligosaccharide chain content in human IgG was higher in batch cultivation using a serum medium than that in serum-free cultures. However, oligosaccharide chain content with a galactose residue at the chain end, which plays an important role in the immune system, was shown to be high in glucose-limited continuous cultivation under a low specific growth rate.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：PERGAMON PRESS LTD  

Glucose or glutamine-limited continuous cultivation of hybridoma cells was carried out. Under steady-state conditions, the specific consumption and production rates were calculated at various specific growth rates. On the basis of these results, the fluxes of the metabolic pathway were calculated. It was found that increasing the specific growth rate caused the specific ATP and antibody production rates to decrease. An estimation of the ATP consumption rates for cell growth and antibody production showed that the proportion of ATP consumed in antibody production decreased at a high specific growth rate. In order to increase the ATP production while keeping the specific growth rate high, the reduced metabolic fluxes were increased by the addition of pyruvate. As a result, higher specific ATP and antibody production rates were achieved at a high specific growth rate. Copyright (C) 1998 IFAC.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The interaction between a single chain Fv (sFv) of the monoclonal antibody 3A21 and its antigen, bovine pancreatic ribonuclease A (RNase A), was studied by site-directed mutagenesis of the hypervariable regions and fluorescence polarization analysis. The affinity constants of wild-type sFv and a mutant sFv D31A (Asp(31) of heavy chain was replaced by Ala) for RNase A were found to be 2.7 x 10(7) and 4.7 x 10(6)M(-1) in PBS at pH 7.2 and 37 degrees C, respectively. Whereas the affinity constant of D31A is not affected by NaCl concentration, that of wild-type sFv is almost the same as that of D31A in the presence of more than 1 M NaCl. These results demonstrate that Asp(31) of the heavy chain interacts electrostatically with a positively charged amino acid residue of RNase A. (C) 1998 Elsevier Science Ltd. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The interaction between a single-chain Fv (sFv) of the monoclonal antibody 3A21 and its antigen, bovine pancreatic ribonuclease A (RNase A), was studied by site-directed mutagenesis of the hypervariable regions and fluorescence polarization analysis. The affinity constants of wild-type sFv and a mutant sFv D31A (Asp(31) of heavy chain was replaced by Ala) for RNase A were found to be 2.7 x 10(7) and 4.7 x 10(6) M-1 in PBS at pH 7.2 and 37 degrees C, respectively. While the affinity constant of D31A is not affected by NaCl concentration, that of wild-type sFv is almost the same as that of D31A in the presence of more than 1 M NaCl. These results demonstrate that Asp(31) of the heavy chain interacts electrostatically with a positively charged amino acid residue of RNase A. (C) 1997 Published by Elsevier Science Ltd. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

In order to elucidate the mechanism of binding of beta(2)-glycoprotein I (beta(2)-GPI) to cardiolipin (CL), we constructed a high-level expression system for the C-terminal domain (Domain V) of beta(2)-GPI using Pichia pastoris and studied its conformation and liposome-binding activity. Purified Domain V was found to have the native disulfide bonds. It had a compactly folded conformation, judging from the circular dichroism spectrum, and exhibited a cooperative unfolding transition induced by pH or urea. Also, it bound liposomes containing CL. Commercially available human beta(2)-GPI is known to be selectively cleaved between Lys 317 and Thr 318. We found that bovine factor Xa weakly but specifically cleaves the corresponding site of recombinant Domain V, i.e., the peptide bond between Lys 77 and Thr 78. The conformation of the ''nicked'' Domain V, which was cleaved at this site, was examined by circular dichroism and fluorescence measurements, and concluded to be similar to that of the intact protein. The stability of the nicked Domain V to urea was slightly lower than that of the intact protein. Although both Domains V bound to liposomes containing CL, the affinity of the nicked Domain V was greatly reduced in comparison with the intact protein, indicating that the cleavage of the peptide bond between Lys 77 and Thr 78 controls the binding to CL. In addition, analysis of the fluorescence spectra in the presence and absence of CL liposomes indicated that Trp 76 is involved in the binding site. These results suggest that the region including Trp 76, Lys 77, and Thr 78 has a critical role in binding to CL.

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：KLUWER ACADEMIC PUBL  

Glucose or glutamine - limited continuous cultivation was carried out. At various steady state conditions, the specific antibody and ATP production rates were calculated under various specific growth rates. On the basis of these results, the ATP consumption rate for cell growth and antibody production was estimated. With the decrease of specific growth rate, the specific antibody production rate and the ATP proportion consumed in antibody production increased. In order to verify these analyses, the fluxes of the metabolic pathway in ATP production were calculated.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC FERMENTATION BIOENGINEERING, JAPAN  

Complementary DNA of the anti-bovine ribonuclease A monoclonal antibody 3A21 variable regions was cloned, sequenced and expressed as a single chain Fv phage antibody to investigate antibody-antigen interaction. The results of amino acid replacement experiments and changes in the affinity constant of the antibody 3A21 with change in pH, indicated that at least one electrostatic interaction participates in this antigen-antibody interaction.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN BIOCHEMICAL SOC  

A genomic DNA clone for protein disulfide isomerase (PDI) of Saccharomyces cerevisiae was isolated by hybridization with synthesized oligonucleotide probes based on a partial amino acid sequence of yeast PDI. The introduction of a multiple copy plasmid carrying this fragment into yeast caused a tenfold increase in PDI specific activity and in the amount of PDI antigen in the extract. The gene on this fragment was named PDI1. The nucleotide sequence of the gene predicts a polypeptide of 522 amino acids with about 30% identity to mammalian PDIs. The predicted amino acid sequence contains an N-terminal signal peptide-like sequence, the C-terminal putative endoplasmic reticulum retention signal of yeast (HDEL), and two putative active site sequences of PDI (WCGHCK). The predicted polypeptide is acidic and contains five putative glycosylation sites, consistent with the molecular properties of the purified yeast PDI [T. Mizunaga et al. (1990) J. Biochem. 108, 846-851]. The PDI1 gene was mapped on chromosome III. A gene disruption experiment revealed that the PDI1 gene is essential for cell growth.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN BIOCHEMICAL SOC  

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Language：English   Publisher：JAPAN SOC BIOSCI BIOTECHN AGROCHEM  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

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Language：Japanese   Publisher：Japanese Society for Engineering Education  

CiNii Books

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Language：Japanese  

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Other Link： http://search.jamas.or.jp/link/ui/2016232244

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CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2016232058

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Language：Japanese  

CiNii Books

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CiNii Books

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CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2014247597

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Language：Japanese  

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Language：Japanese   Publisher：日本生物工学会  

CiNii Books

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Other Link： http://dl.ndl.go.jp/info:ndljp/pid/10517661

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Language：Japanese   Publisher：Kansai University  

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Other Link： http://hdl.handle.net/10112/3225

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：Japanese Society for Engineering Education  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2009066368

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Language：Japanese  

DOI： 10.11491/scej.2007f.0.996.0

J-GLOBAL

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：English  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2008290915

Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

CiNii Books

J-GLOBAL

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

DOI： 10.11491/scej.2005.0.249.0

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：日本生物工学会  

CiNii Books

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Language：Japanese  

CiNii Books

J-GLOBAL

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese  

CiNii Books

J-GLOBAL

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：English  

CiNii Books

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Language：Japanese  

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese  

CiNii Books

J-GLOBAL

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese  

CiNii Books

J-GLOBAL

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Language：Japanese  

DOI： 10.11491/scej.2003f.0.127.0

J-GLOBAL

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Language：Japanese  

DOI： 10.11491/scej.2003f.0.128.0

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：Japanese   Publisher：日本生物工学会  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：The Society for Biotechnology, Japan  

CiNii Books

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Language：Japanese   Publisher：一般社団法人日本機械学会  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu1962.40.336

CiNii Books

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Other Link： https://jlc.jst.go.jp/DN/JALC/00153683075?from=CiNii

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Language：Japanese  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2003139643

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CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2003139657

Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese  

CiNii Books

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Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：化学同人  

CiNii Books

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Language：Japanese   Publisher：日本生物工学会  

CiNii Books

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=300692

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：シ-エムシ-  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：化学同人  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：化学同人  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：化学同人  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：化学同人  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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CiNii Books

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CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：化学同人  

CiNii Books

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Language：Japanese   Publisher：日本生物工学会  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：化学同人  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：化学同人  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：化学同人  

CiNii Books

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Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：化学同人  

CiNii Books

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Language：Japanese   Publisher：日本生物工学会  

CiNii Books

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Language：Japanese  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2001214568

Language：Japanese  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2001223902

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CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2001213096

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CiNii Books

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Language：Japanese  

CiNii Books

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CiNii Books

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER CHEMICAL SOC  

Web of Science

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER CHEMICAL SOC  

Web of Science

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Language：Japanese  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2001235521

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Language：Japanese  

CiNii Books

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CiNii Books

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CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2001248104

Language：Japanese  

CiNii Books

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Language：Japanese   Publisher：The Biophysical Society of Japan General Incorporated Association  

CiNii Books

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Language：Japanese  

CiNii Books

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Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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CiNii Books

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CiNii Books

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CiNii Books

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CiNii Books

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CiNii Books

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：JAPAN BIOCHEMICAL SOC  

Web of Science

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Venue：オンライン（日本）  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：岡山大学  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：岡山大学  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：岡山大学  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：岡山大学  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：岡山大学  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：岡山大学  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：新潟大学五十嵐キャンパス  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：新潟大学旭町キャンパス  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：大手町ファ−ストスクエアカンファレンス  

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Language：Japanese   Presentation type：Poster presentation  

Venue：佐久平  

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