Publications by authors named "Katsuhiro Kojima"

35 Publications

Strategic design and improvement of the internal electron transfer of heme b domain-fused glucose dehydrogenase for use in direct electron transfer-type glucose sensors.

Biosens Bioelectron 2021 Mar 17;176:112911. Epub 2020 Dec 17.

Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC27599, USA. Electronic address:

A fusion enzyme composed of an Aspergillus flavus-derived flavin adenine dinucleotide glucose dehydrogenase (AfGDH) and an electron transfer domain of Phanerochaete chrysosporium-derived cellobiose dehydrogenase (Pcyb) was previously reported to show the direct electron transfer (DET) ability to an electrode. However, its slow intramolecular electron transfer (IET) rate from the FAD to the heme, limited the sensor signals. In this study, fusion FADGDH (Pcyb-AfGDH) enzymes were strategically redesigned by performing docking simulation, following surface-electrostatic potential estimation in the predicted area. Based on these predictions, we selected the amino acid substitution on Glu324, or on Asn408 to Lys to increase the positive charge at the rim of the interdomain region. Pcyb-AfGDH mutants were recombinantly produced using Pichia pastoris as the host microorganism, and their IET was evaluated. Spectroscopic observations showed that the Glu324Lys (E324K) and Asn408Lys (N408K) Pcyb-AfGDH mutants showed approximately 1.70- and 9.0-fold faster IET than that of wildtype Pcyb-AfGDH, respectively. Electrochemical evaluation revealed that the mutant Pcyb-AfGDH-immobilized electrodes showed higher DET current values than that of the wildtype Pcyb-AfGDH-immobilized electrodes at pH 6.5, which was approximately 9-fold higher in the E324K mutant and 15-fold higher in the N408K mutant, than in the wildtype. Glucose enzyme sensors employing N408K mutant was able to measure glucose concentration under physiological condition using artificial interstitial fluid at pH 7.4, whereas the one with wildtype Pcyb-AfGDH was not. These results indicated that the sensor employed the redesigned mutant Pcyb-AfGDH can be used for future continuous glucose monitoring system based on direct electron transfer principle. (247 words).
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http://dx.doi.org/10.1016/j.bios.2020.112911DOI Listing
March 2021

Creation of a novel DET type FAD glucose dehydrogenase harboring Escherichia coli derived cytochrome b as an electron transfer domain.

Biochem Biophys Res Commun 2020 09 29;530(1):82-86. Epub 2020 Jul 29.

Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC27599, USA. Electronic address:

Fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) are the most popular and advanced enzymes for SMBG sensors because of their high substrate specificity toward glucose and oxygen insensitivity. However, this type of FADGDH hardly shows direct electron transfer (DET) ability. In this study, we developed a new DET-type FADGDH by harboring Cytochrome b (cyt b) derived from Escherichia coli as the electron transfer domain. The structural genes encoding fusion enzymes composed of cyt b at either the N- or C-terminus of fungal FADGDH, (cyt b-GDH or GDH-cyt b), were constructed, recombinantly expressed, and characteristics of the fusion proteins were investigated. Both constructed fusion enzymes were successfully expressed in E. coli, as the soluble and GDH active proteins, showing cyt b specific redox properties. Thusconstructed fusion proteins showed internal electron transfer between FAD in FADGDH and fused cyt b. Consequently, both cyt b-GDH and GDH-cyt b showed DET abilities toward electrode. Interestingly, cyt b-GDH showed much rapid internal electron transfer and higher DET ability than GDH-cyt b. Thus, we demonstrated the construction and production of a new DET-type FADGDH using E.coli as the host cells, which is advantageous for future industrial application and further engineering.
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http://dx.doi.org/10.1016/j.bbrc.2020.06.132DOI Listing
September 2020

Engineered Glucose Oxidase Capable of Quasi-Direct Electron Transfer after a Quick-and-Easy Modification with a Mediator.

Int J Mol Sci 2020 Feb 8;21(3). Epub 2020 Feb 8.

Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA.

Glucose oxidase (GOx) has been widely utilized for monitoring glycemic levels due to its availability, high activity, and specificity toward glucose. Among the three generations of electrochemical glucose sensor principles, direct electron transfer (DET)-based third-generation sensors are considered the ideal principle since the measurements can be carried out in the absence of a free redox mediator in the solution without the impact of oxygen and at a low enough potential for amperometric measurement to avoid the effect of electrochemically active interferences. However, natural GOx is not capable of DET. Therefore, a simple and rapid strategy to create DET-capable GOx is desired. In this study, we designed engineered GOx, which was made readily available for single-step modification with a redox mediator (phenazine ethosulfate, PES) on its surface via a lysine residue rationally introduced into the enzyme. Thus, PES-modified engineered GOx showed a quasi-DET response upon the addition of glucose. This strategy and the obtained results will contribute to the further development of quasi-DET GOx-based glucose monitoring dedicated to precise and accurate glycemic control for diabetic patient care.
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http://dx.doi.org/10.3390/ijms21031137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036908PMC
February 2020

Rational engineering of Aerococcus viridansl-lactate oxidase for the mediator modification to achieve quasi-direct electron transfer type lactate sensor.

Biosens Bioelectron 2020 Mar 18;151:111974. Epub 2019 Dec 18.

Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC, 27599, USA. Electronic address:

The l-lactate oxidase (LOx) based lactate sensors are widely used for clinical diagnostics, sports medicine, and food quality control. However, dissolved oxygen interference and electroactive interferent effects are inherent issues of current lactate sensors. In this paper, a quasi-direct electron transfer (quasi-DET) type lactate sensor was developed using rationally engineered Aerococcus viridans LOx (AvLOx) modified with amine-reactive phenazine ethosulfate (PES). Since the modification of wild type AvLOx by PES did not result quasi-DET, engineered AvLOx with additional Lys residue was designed. The additional Lys residue was introduced by substituting residue locating on the surface of AvLOx, and within 20 Å of the isoalloxazine ring of FMN. Among several constructed mutants, Ala96Leu/Asn212Lys double mutant showed the highest dye-mediated dehydrogenase activity with negligible oxidase activity, showing quasi-DET properties after PES modification, when the enzyme was immobilized on screen printed carbon electrode. The constructed electrode did not show oxygen interference in cyclic voltammetric analysis and distinct catalytic current with 20 mM l-lactate. The sensor performance of a chronoamperometric l-lactate sensor employing PES modified Ala96Leu/Asn212Lys AvLOx, marked with linear range between 0 and 1 mM, with sensitivity of 13 μA/mM∙cm, and a limit of detection of 25 μM for l-lactate. By applying -200 mV vs. Ag/AgCl, l-lactate could be monitored with negligible interference from 170 μM ascorbic acid, 1.3 mM acetaminophen, 1.4 mM uric acid or 20 mM glucose. These results indicated that a quasi-DET type lactate sensor was developed that did not suffer from the interference of oxygen and representative electroactive ingredient compounds.
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http://dx.doi.org/10.1016/j.bios.2019.111974DOI Listing
March 2020

Isolation and Screening of Indigenous Plant Growth-promoting Rhizobacteria from Different Rice Cultivars in Afghanistan Soils.

Microbes Environ 2019 Dec 14;34(4):347-355. Epub 2019 Sep 14.

Institute of Agriculture, Tokyo University of Agriculture and Technology.

To develop biofertilizers for rice in Afghanistan, 98 plant growth-promoting rhizobacteria were isolated from rice plants and their morphological and physiological characteristics, such as indole-3-acetic acid production, acetylene reduction, phosphate and potassium solubilization, and siderophore production, were evaluated. The genetic diversity of these bacteria was also analyzed based on 16S rRNA gene sequences. Of 98 bacteria, 89.7% produced IAA, 54.0% exhibited nitrogenase activity, and 40% showed phosphate solubilization and siderophore production. Some isolates assigned to Pseudomonas (brassicacearum, chengduensis, plecoglossicida, resinovorans, and straminea) formed a relationship with rice, and P. resinovorans and P. straminea showed nitrogen fixation. Rhizobium borbori and R. rosettiformans showed a relationship with rice plants and nitrogen fixation. Among the isolates examined, AF134 and AF137 belonging to Enterobacter ludwigii and P. putida produced large amounts of IAA (92.3 μg mL) and exhibited high nitrogenase activity (647.4 nmol CH h), respectively. In the plant growth test, more than 70% of the inoculated isolates showed significantly increased root and shoot dry weights. Highly diverse bacterial isolates showing promising rice growth-promoting traits were obtained from Afghanistan alkaline soils.
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http://dx.doi.org/10.1264/jsme2.ME18168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934389PMC
December 2019

X-ray structure of the direct electron transfer-type FAD glucose dehydrogenase catalytic subunit complexed with a hitchhiker protein.

Acta Crystallogr D Struct Biol 2019 Sep 28;75(Pt 9):841-851. Epub 2019 Aug 28.

Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.

The bacterial flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase complex derived from Burkholderia cepacia (BcGDH) is a representative molecule of direct electron transfer-type FAD-dependent dehydrogenase complexes. In this study, the X-ray structure of BcGDHγα, the catalytic subunit (α-subunit) of BcGDH complexed with a hitchhiker protein (γ-subunit), was determined. The most prominent feature of this enzyme is the presence of the 3Fe-4S cluster, which is located at the surface of the catalytic subunit and functions in intramolecular and intermolecular electron transfer from FAD to the electron-transfer subunit. The structure of the complex revealed that these two molecules are connected through disulfide bonds and hydrophobic interactions, and that the formation of disulfide bonds is required to stabilize the catalytic subunit. The structure of the complex revealed the putative position of the electron-transfer subunit. A comparison of the structures of BcGDHγα and membrane-bound fumarate reductases suggested that the whole BcGDH complex, which also includes the membrane-bound β-subunit containing three heme c moieties, may form a similar overall structure to fumarate reductases, thus accomplishing effective electron transfer.
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http://dx.doi.org/10.1107/S2059798319010878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719666PMC
September 2019

Elevated glutathione synthesis in leaves contributes to zinc transport from roots to shoots in Arabidopsis.

Plant Sci 2019 Jun 13;283:416-423. Epub 2018 Nov 13.

Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan; Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan. Electronic address:

Glutathione (GSH) is a vital compound involved in several plant metabolic pathways. Our previous study indicated that foliar GSH application can increase zinc (Zn) levels in leafy vegetables. The objective of this study was to determine the mode of action of GSH as it relates to Zn transport from roots to shoots. Two types of transgenic Arabidopsis plants with genes for GSH synthesis, including StGCS-GS or AtGSH1 driven by the leaf-specific promoter of chlorophyll a/b-binding protein (pCab3) gene were generated. Both types of transgenic Arabidopsis plants showed significant increases in shoot GSH concentrations compared to the wild type (WT). Monitoring Zn movement by positron-emitting tracer imaging system (PETIS) analysis indicated that the Zn amount in the shoots of both types of transgenic Arabidopsis plants were higher than that in the WT. GSH concentration in phloem sap was increased significantly in WT with foliar applications of 10 mM GSH (WT-GSH), but not in transgenic Arabidopsis with elevated foliar GSH synthesis. Both types of transgenic Arabidopsis with elevated foliar GSH synthesis and WT-GSH exhibited increased shoot Zn concentrations and Zn translocation ratios. These results suggest that enhancement of endogenous foliar GSH synthesis and exogenous foliar GSH application affect root-to-shoot transport of Zn.
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http://dx.doi.org/10.1016/j.plantsci.2018.11.003DOI Listing
June 2019

Complete Genome Sequence of Plant Growth-Promoting Bacillus pumilus TUAT1.

Microbiol Resour Announc 2019 May 23;8(21). Epub 2019 May 23.

Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan

TUAT1 was isolated from soil in a university research field. Strain TUAT1 has the ability to promote the growth of plants, including that of rice, and has been commercialized as a biofertilizer. Here, we sequenced and annotated the genome of TUAT1 to understand the molecular mechanisms underlying its plant growth promotion.
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http://dx.doi.org/10.1128/MRA.00076-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533381PMC
May 2019

Burkholderia and Paraburkholderia are Predominant Soybean Rhizobial Genera in Venezuelan Soils in Different Climatic and Topographical Regions.

Microbes Environ 2019 Mar 15;34(1):43-58. Epub 2019 Feb 15.

Institute of Agriculture, Tokyo University of Agriculture and Technology (TUAT).

The climate, topography, fauna, and flora of Venezuela are highly diverse. However, limited information is currently available on the characterization of soybean rhizobia in Venezuela. To clarify the physiological and genetic diversities of soybean rhizobia in Venezuela, soybean root nodules were collected from 11 soil types located in different topographical regions. A total of 395 root nodules were collected and 120 isolates were obtained. All isolates were classified in terms of stress tolerance under different concentrations of NaCl and Al. The tolerance levels of isolates to NaCl and Al varied. Based on sampling origins and stress tolerance levels, 44 isolates were selected for further characterization. An inoculation test indicated that all isolates showed the capacity for root nodulation on soybean. Based on multilocus sequence typing (MLST), 20 isolates were classified into the genera Rhizobium and Bradyrhizobium. The remaining 24 isolates were classified into the genus Burkholderia or Paraburkholderia. There is currently no evidence to demonstrate that the genera Burkholderia and Paraburkholderia are the predominant soybean rhizobia in agricultural fields. Of the 24 isolates classified in (Para) Burkholderia, the nodD-nodB intergenic spacer regions of 10 isolates and the nifH gene sequences of 17 isolates were closely related to the genera Rhizobium and Bradyrhizobium, respectively. The root nodulation numbers of five (Para) Burkholderia isolates were higher than those of the 20 α-rhizobia. Furthermore, among the 44 isolates tested, one Paraburkholderia isolate exhibited the highest nitrogen-fixation activity in root nodules.
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http://dx.doi.org/10.1264/jsme2.ME18076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440732PMC
March 2019

Evaluation of Immune Responses Induced by Simultaneous Inoculations of Soybean (Glycine max [L.] Merr.) with Soil Bacteria and Rhizobia.

Microbes Environ 2019 Mar 5;34(1):64-75. Epub 2019 Feb 5.

Institute of Agriculture, TUAT.

Legumes form root nodules and fix atmospheric nitrogen by establishing symbiosis with rhizobia. However, excessive root nodules are harmful to plants because of the resulting overconsumption of energy from photosynthates. The delay of an inoculation of the soybean super-nodulation mutant NOD1-3 with Bradyrhizobium diazoefficiens USDA110 by 5 d after an inoculation with several soil bacteria confirmed that one bacterial group significantly decreased root nodules throughout the study period. Moreover, no significant changes were observed in nitrogen fixation by root nodules between an inoculation with USDA 110 only and co-inoculation treatments. To clarify the potential involvement of PR proteins in the restriction of nodule formation in the plants tested, the relative expression levels of PR-1, PR-2, PR-5, and PDF1.2 in NOD1-3 roots were measured using real-time PCR. One group of soil bacteria (Gr.3), which markedly reduced nodule numbers, significantly induced the expression of PR-1, PR-5 and PDF1.2 genes by day 5 after the inoculation. By days 7, 10, and 20 after the inoculation, the expression levels of PR-2 and PR-5 were lower than those with the uninoculated treatment. Inoculations with this group of soil bacteria resulted in lower root nodule numbers than with other tested soil bacteria exerting weak inhibitory effects on nodulation, and were accompanied by the induction of plant defense-related genes. Thus, PR genes appear to play important roles in the mechanisms that suppresses nodule formation on soybean roots.
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http://dx.doi.org/10.1264/jsme2.ME18110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440728PMC
March 2019

Designer fungus FAD glucose dehydrogenase capable of direct electron transfer.

Biosens Bioelectron 2019 Jan 26;123:114-123. Epub 2018 Jul 26.

Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan; Ultizyme International Ltd., 1-13-16, Minami, Meguro, Tokyo 152-0013, Japan; Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA. Electronic address:

Fungi-derived flavin adenine dinucleotide glucose dehydrogenases (FADGDHs) are currently the most popular and advanced enzymes for self-monitoring of blood glucose sensors; however, the achievement of direct electron transfer (DET) with FADGDHs is difficult. In this study, a designer FADGDH was constructed by fusing Aspergillus flavus derived FADGDH (AfGDH) and a Phanerochaete chrisosporium CDH (PcCDH)-derived heme b-binding cytochrome domain to develop a novel FADGDH that is capable of direct electron transfer with an electrode. A structural prediction suggested that the heme in the CDH may exist in proximity to the FAD of AfGDH if the heme b-binding cytochrome domain is fused to the AfGDH N-terminal region. Spectroscopic observations of recombinantly produced designer FADGDH confirmed the intramolecular electron transfer between FAD and the heme. A decrease in pH and the presence of a divalent cation improved the intramolecular electron transfer. An enzyme electrode with the immobilized designer FADGDH showed an increase in current immediately after the addition of glucose in a glucose concentration-dependent manner, whereas those with wild-type AfGDH did not show an increase in current. Therefore, the designer FADGDH was confirmed to be a novel GDH that possesses electrode DET ability. The difference in the surface electrostatic potentials of AfGDH and the catalytic domain of PcCDH might be why their intramolecular electron transfer ability is inferior to that of CDH. These relevant and consistent findings provide us with a novel strategic approach for the improvement of the DET properties of designer FADGDH. (241 words).
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http://dx.doi.org/10.1016/j.bios.2018.07.027DOI Listing
January 2019

Engineered fungus derived FAD-dependent glucose dehydrogenase with acquired ability to utilize hexaammineruthenium(III) as an electron acceptor.

Bioelectrochemistry 2018 Oct 10;123:62-69. Epub 2018 Apr 10.

Department of Industrial Technology and Innovation, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan; Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan; Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA; Ultizyme International Ltd., 1-13-16, Minami, Meguro, Tokyo 152-0013, Japan. Electronic address:

Fungal FAD-dependent glucose dehydrogenases (FADGDHs) are considered to be superior enzymes for glucose sensor strips because of their insensitivity to oxygen and maltose. One highly desirable mediator for enzyme sensor strips is hexaammineruthenium(III) chloride because of its low redox potential and high storage stability. However, in contrast to glucose oxidase (GOx), fungal FADGDH cannot utilize hexaammineruthenium(III) as electron acceptor. Based on strategic structure comparison between FADGDH and GOx, we constructed a mutant of Aspergillus flavus-derived FADGDH, capable of utilizing hexaammineruthenium(III) as electron acceptor: AfGDH-H403D. In AfGDH-H403D, a negative charge introduced at the pathway-entrance leading to the FAD attracts the positively charged hexaammineruthenium(III) and guides it into the pathway. The corresponding amino acid in wild-type GOx is negatively charged, which explains the ability of GOx to utilize hexaammineruthenium(III) as electron acceptor. Electrochemical measurements showed a response current of 46.0 μA for 10 mM glucose with AfGDH-H403D and hexaammineruthenium(III), similar to that with wild-type AfGDH and ferricyanide (47.8 μA). Therefore, AfGDH-H403D is suitable for constructing enzyme electrode strips with hexaammineruthenium(III) chloride as sole mediator. Utilization of this new, improved fungal FADGDH should lead to the development of sensor strips for blood glucose monitoring with increased accuracy and less stringent packing requirements.
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http://dx.doi.org/10.1016/j.bioelechem.2018.04.007DOI Listing
October 2018

AtOPT6 Protein Functions in Long-Distance Transport of Glutathione in Arabidopsis thaliana.

Plant Cell Physiol 2018 Jul;59(7):1443-1451

Institute of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo, Japan.

The involvement of the Arabidopsis oligopeptide transporter AtOPT6, which was previously shown to take up glutathione (GSH) when expressed in yeast cells or in Xenopus laevis oocytes, in GSH transport was analyzed using opt6 knockout mutant lines. The concentration of GSH in flowers or siliques was lower in opt6 mutants relative to wild-type plants, suggesting involvement of AtOPT6 in long-distance transport of GSH. The GSH concentration in phloem sap was similar between opt6 mutants and wild-type plants. These results, combined with earlier reports showing expression of AtOPT6 in the vascular bundle, especially in the cambial zone, suggest that AtOPT6 functions to transport GSH into cells surrounding the phloem in sink organs. The opt6 mutant plants showed delayed bolting, implying the importance of AtOPT6 for regulation of the transition from vegetative to reproductive growth. After cadmium (Cd) treatment, the concentration of the major phytochelatin PC2 was lower in flowers in the opt6 mutants and Cd was accumulated in roots of opt6 mutant plants compared with wild-type plants. These results suggest that AtOPT6 is likely to be involved in transporting GSH, PCs and Cd complexed with these thiols into sink organs.
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http://dx.doi.org/10.1093/pcp/pcy074DOI Listing
July 2018

Elucidation of the intra- and inter-molecular electron transfer pathways of glucoside 3-dehydrogenase.

Bioelectrochemistry 2018 Aug 7;122:115-122. Epub 2018 Mar 7.

Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture & Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Electronic address:

Glucoside 3‑dehydrogenase (G3DH) is a flavin adenine dinucleotide (FAD)-containing oxidoreductase that catalyzes the oxidation of the hydroxy group on the C-3 position of pyranose and shows broad substrate specificity by oxidizing many saccharides. Due to unique site specificity and wide substrate specificity, G3DHs can be used for synthesis of sugar derivatives, anodic catalysis in biofuel cells, multi-sugar analysis using enzyme electrode, and for enzymatic detection of 1,5‑anhydro‑d‑glucitol, a clinical marker for diabetes. However, few studies have focused on the fundamental biochemical properties of G3DH, including its electron transfer pathway. In this study, we isolated the G3DH gene from Rhizobium radiobacter, a homologue of marine bacterial G3DH, and reported that the isolated gene fragment contains the genes encoding the G3DH catalytic subunit (subunit I), G3DH hitch-hiker subunit (subunit II), and cytochrome c-like molecule (CYTc). Furthermore, we report the recombinant expression of G3DH from R. radiobacter in Escherichia coli, the characterization of recombinant G3DH and the investigation of the molecular electron pathway of G3DH. We first prepared the G3DH subunit I-II complex using a co-expression vector for both subunits. The G3DH subunit I-II complex showed dye-mediated G3DH activity toward methyl‑α‑d‑glucoside (MαG). Electron paramagnetic resonance (EPR) and inductively coupled plasma optical emission spectroscopy (ICP-OES) analyses revealed that subunit I contains an iron-sulfur cluster. We, then, prepared recombinant CYTc and revealed that it is capable of accepting electrons from the catalytic subunit of G3DH by absorption spectrum analysis. These results suggested that R. radiobacter G3DH possesses an iron‑sulfur cluster that may play an important role in the electron transfer from FAD to cytochrome c like molecule, which is an external electron acceptor of G3DH. Furthermore, we demonstrated that CYTc mediate the electron transfer from G3DH to electrode without the artificial electron mediator.
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http://dx.doi.org/10.1016/j.bioelechem.2018.03.001DOI Listing
August 2018

Mutagenesis Study of the Cytochrome c Subunit Responsible for the Direct Electron Transfer-Type Catalytic Activity of FAD-Dependent Glucose Dehydrogenase.

Int J Mol Sci 2018 Mar 21;19(4). Epub 2018 Mar 21.

Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture & Technology, Koganei, Tokyo 184-8588, Japan.

The FAD-dependent glucose dehydrogenase from (FADGDH) is a hetero-oligomeric enzyme that is capable of direct electron transfer (DET) with an electrode. The cytochrome (cyt ) subunit, which possesses three hemes (heme 1, heme 2, and heme 3, from the N-terminal sequence), is known to enable DET; however, details of the electron transfer pathway remain unknown. A mutagenesis investigation of the heme axial ligands was carried out to elucidate the electron transfer pathway to the electron mediators and/or the electrode. The sixth axial ligand for each of the three heme irons, Met109, Met263, and Met386 were substituted with His. The catalytic activities of the wild-type (WT) and mutant enzymes were compared by investigating their dye-mediated dehydrogenase activities and their DET abilities toward the electrode. The results suggested that (1) heme 1 with Met109 as an axial ligand is mainly responsible for the electron transfer with electron acceptors in the solution, but not for the DET with the electrode; (2) heme 2 with Met263 is responsible for the DET-type reaction with the electrode; and (3) heme 3 with Met386 seemed to be the electron acceptor from the catalytic subunit. From these results, two electron transfer pathways were proposed depending on the electron acceptors. Electrons are transferred from the catalytic subunit to heme 3, then to heme 2, to heme 1 and, finally, to electron acceptors in solution. However, if the enzyme complex is immobilized on the electrode and is used as electron acceptors, electrons are passed to the electrode from heme 2.
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http://dx.doi.org/10.3390/ijms19040931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979317PMC
March 2018

Minimizing the effects of oxygen interference on l-lactate sensors by a single amino acid mutation in Aerococcus viridansl-lactate oxidase.

Biosens Bioelectron 2018 Apr 14;103:163-170. Epub 2017 Dec 14.

Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan; Ultizyme International Ltd, 1-13-16, Minami, Meguro, Tokyo 152-0013, Japan; Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 3-8-1 Harumi-cho, Fuchu, Tokyo 183-8538, Japan. Electronic address:

l-lactate biosensors employing l-lactate oxidase (LOx) have been developed mainly to measure l-lactate concentration for clinical diagnostics, sports medicine, and the food industry. Some l-lactate biosensors employ artificial electron mediators, but these can negatively impact the detection of l-lactate by competing with the primary electron acceptor: molecular oxygen. In this paper, a strategic approach to engineering an AvLOx that minimizes the effects of oxygen interference on sensor strips was reported. First, we predicted an oxygen access pathway in Aerococcus viridans LOx (AvLOx) based on its crystal structure. This was subsequently blocked by a bulky amino acid substitution. The resulting Ala96Leu mutant showed a drastic reduction in oxidase activity using molecular oxygen as the electron acceptor and a small increase in dehydrogenase activity employing an artificial electron acceptor. Secondly, the Ala96Leu mutant was immobilized on a screen-printed carbon electrode using glutaraldehyde cross-linking method. Amperometric analysis was performed with potassium ferricyanide as an electron mediator under argon or atmospheric conditions. Under argon condition, the response current increased linearly from 0.05 to 0.5mM l-lactate for both wild-type and Ala96Leu. However, under atmospheric conditions, the response of wild-type AvLOx electrode was suppressed by 9-12% due to oxygen interference. The Ala96Leu mutant maintained 56-69% of the response current at the same l-lactate level and minimized the relative bias error to -19% from -49% of wild-type. This study provided significant insight into the enzymatic reaction mechanism of AvLOx and presented a novel approach to minimize oxygen interference in sensor applications, which will enable accurate detection of l-lactate concentrations.
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http://dx.doi.org/10.1016/j.bios.2017.12.018DOI Listing
April 2018

Mediator Preference of Two Different FAD-Dependent Glucose Dehydrogenases Employed in Disposable Enzyme Glucose Sensors.

Sensors (Basel) 2017 Nov 16;17(11). Epub 2017 Nov 16.

Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.

Most commercially available electrochemical enzyme sensor strips for the measurement of blood glucose use an artificial electron mediator to transfer electrons from the active side of the enzyme to the electrode. One mediator recently gaining attention for commercial sensor strips is hexaammineruthenium(III) chloride. In this study, we investigate and compare the preference of enzyme electrodes with two different FAD-dependent glucose dehydrogenases (FADGDHs) for the mediators hexaammineruthenium(III) chloride, potassium ferricyanide (the most common mediator in commercial sensor strips), and methoxy phenazine methosulfate (mPMS). One FADGDH is a monomeric fungal enzyme, and the other a hetero-trimeric bacterial enzyme. With the latter, which contains a heme-subunit facilitating the electron transfer, similar response currents are obtained with hexaammineruthenium(III), ferricyanide, and mPMS (6.8 µA, 7.5 µA, and 6.4 µA, respectively, for 10 mM glucose). With the fungal FADGDH, similar response currents are obtained with the negatively charged ferricyanide and the uncharged mPMS (5.9 µA and 6.7 µA, respectively, for 10 mM glucose), however, no response current is obtained with hexaammineruthenium(III), which has a strong positive charge. These results show that access of even very small mediators with strong charges to a buried active center can be almost completely blocked by the protein.
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http://dx.doi.org/10.3390/s17112636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712826PMC
November 2017

Novel fungal FAD glucose dehydrogenase derived from Aspergillus niger for glucose enzyme sensor strips.

Biosens Bioelectron 2017 Jan 18;87:305-311. Epub 2016 Aug 18.

Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 3-8-1 Harumi-cho, Fuchu, Tokyo 183-8538, Japan; Diabetes Research Institute, Mills-Peninsula Health Services, 100 South San Mateo Drive, San Mateo, CA 94401, USA.

In this study, a novel fungus FAD dependent glucose dehydrogenase, derived from Aspergillus niger (AnGDH), was characterized. This enzyme's potential for the use as the enzyme for blood glucose monitor enzyme sensor strips was evaluated, especially by investigating the effect of the presence of xylose during glucose measurements. The substrate specificity of AnGDH towards glucose was investigated, and only xylose was found as a competing substrate. The specific catalytic efficiency for xylose compared to glucose was 1.8%. The specific activity of AnGDH for xylose at 5mM concentration compared to glucose was 3.5%. No other sugars were used as substrate by this enzyme. The superior substrate specificity of AnGDH was also demonstrated in the performance of enzyme sensor strips. The impact of spiking xylose in a sample with physiological glucose concentrations on the sensor signals was investigated, and it was found that enzyme sensor strips using AnGDH were not affected at all by 5mM (75mg/dL) xylose. This is the first report of an enzyme sensor strip using a fungus derived FADGDH, which did not show any positive bias at a therapeutic level xylose concentration on the signal for a glucose sample. This clearly indicates the superiority of AnGDH over other conventionally used fungi derived FADGDHs in the application for SMBG sensor strips. The negligible activity of AnGDH towards xylose was also explained on the basis of a 3D structural model, which was compared to the 3D structures of A. flavus derived FADGDH and of two glucose oxidases.
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http://dx.doi.org/10.1016/j.bios.2016.08.053DOI Listing
January 2017

Genotypic difference in (137)Cs accumulation and transfer from the contaminated field in Fukushima to azuki bean (Vigna angularis).

J Environ Radioact 2016 Jul 19;158-159:138-47. Epub 2016 Apr 19.

Instutute of Agriculture, Tokyo University of Agriculture and Technology, Saiwaicho 3-5-8, Fuchu, Tokyo, 183-8509, Japan. Electronic address:

The screening of mini-core collection of azuki bean accessions (Vigna angularis (Willd.) Ohwi & Ohashi) for comparative uptake of (137)Cs in their edible portions was done in field trials on land contaminated by the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident. Ninety seven azuki bean accessions including their wild relatives from a Japanese gene bank, were grown in a field in the Fukushima prefecture, which is located approximately 51 km north of FDNPP. The contamination level of the soil was 3665 ± 480 Bq kg(-1) dry weight ((137)Cs, average ± SD). The soil type comprised clay loam, where the sand: silt: clay proportion was 42:21:37. There was a significant varietal difference in the biomass production, radiocaesium accumulation and transfer factor (TF) of radiocaesium from the soil to edible portion. Under identical agricultural practice, the extent of (137)Cs accumulation by seeds differed between the accessions by as much as 10-fold. Inter-varietal variation was expressed at the ratio of the maximum to minimum observed (137)Cs transfer factor for seeds ranged from 0.092 to 0.009. The total biomass, time to flowering and maturity, and seed yield had negative relationship to (137)Cs activity concentration in seeds. The results suggest that certain variety/varieties of azuki bean which accumulated less (137)Cs in edible portion with preferable agronomic traits are suitable to reduce the (137)Cs accumulation in food chain on contaminated land.
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http://dx.doi.org/10.1016/j.jenvrad.2016.04.011DOI Listing
July 2016

Growth and (137)Cs uptake and accumulation among 56 Japanese cultivars of Brassica rapa, Brassica juncea and Brassica napus grown in a contaminated field in Fukushima: Effect of inoculation with a Bacillus pumilus strain.

J Environ Radioact 2016 Jun 14;157:27-37. Epub 2016 Mar 14.

Institute of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan. Electronic address:

Fifty six local Japanese cultivars of Brassica rapa (40 cultivars), Brassica juncea (10 cultivars) and Brassica napus (6 cultivars) were assessed for variability in growth and (137)Cs uptake and accumulation in association with a Bacillus pumilus strain. Field trial was conducted at a contaminated farmland in Nihonmatsu city, in Fukushima prefecture. Inoculation resulted in different responses of the cultivars in terms of growth and radiocesium uptake and accumulation. B. pumilus induced a significant increase in shoot dry weight in 12 cultivars that reached up to 40% in one B. rapa and three B. juncea cultivars. Differences in radiocesium uptake were observed between the cultivars of each Brassica species. Generally, inoculation resulted in a significant increase in (137)Cs uptake in 22 cultivars, while in seven cultivars it was significantly decreased. Regardless of plant cultivar and bacterial inoculation, the transfer of (137)Cs to the plant shoots (TF) varied by a factor of up to 5 and it ranged from to 0.011 to 0.054. Five inoculated cultivars, showed enhanced shoot dry weights and decreased (137)Cs accumulations, among which two B. rapa cultivars named Bitamina and Nozawana had a significantly decreased (137)Cs accumulation in their shoots. Such cultivars could be utilized to minimize the entry of radiocesium into the food chain; however, verifying the consistency of their radiocesium accumulation in other soils is strongly required. Moreover, the variations in growth and radiocesium accumulation, as influenced by Bacillus inoculation, could help selecting well grown inoculated Brassica cultivars with low radiocesium accumulation in their shoots.
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http://dx.doi.org/10.1016/j.jenvrad.2016.02.024DOI Listing
June 2016

An Fe-S cluster in the conserved Cys-rich region in the catalytic subunit of FAD-dependent dehydrogenase complexes.

Bioelectrochemistry 2016 Dec 2;112:178-83. Epub 2016 Feb 2.

Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture & Technology, Koganei, Tokyo, Japan; Ultizyme International Ltd., Meguro, Tokyo, Japan.

Several bacterial flavin adenine dinucleotide (FAD)-harboring dehydrogenase complexes comprise three distinct subunits: a catalytic subunit with FAD, a cytochrome c subunit containing three hemes, and a small subunit. Owing to the cytochrome c subunit, these dehydrogenase complexes have the potential to transfer electrons directly to an electrode. Despite various electrochemical applications and engineering studies of FAD-dependent dehydrogenase complexes, the intra/inter-molecular electron transfer pathway has not yet been revealed. In this study, we focused on the conserved Cys-rich region in the catalytic subunits using the catalytic subunit of FAD dependent glucose dehydrogenase complex (FADGDH) as a model, and site-directed mutagenesis and electron paramagnetic resonance (EPR) were performed. By co-expressing a hitch-hiker protein (γ-subunit) and a catalytic subunit (α-subunit), FADGDH γα complexes were prepared, and the properties of the catalytic subunit of both wild type and mutant FADGDHs were investigated. Substitution of the conserved Cys residues with Ser resulted in the loss of dye-mediated glucose dehydrogenase activity. ICP-AEM and EPR analyses of the wild-type FADGDH catalytic subunit revealed the presence of a 3Fe-4S-type iron-sulfur cluster, whereas none of the Ser-substituted mutants showed the EPR spectrum characteristic for this cluster. The results suggested that three Cys residues in the Cys-rich region constitute an iron-sulfur cluster that may play an important role in the electron transfer from FAD (intra-molecular) to the multi-heme cytochrome c subunit (inter-molecular) electron transfer pathway. These features appear to be conserved in the other three-subunit dehydrogenases having an FAD cofactor.
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http://dx.doi.org/10.1016/j.bioelechem.2016.01.010DOI Listing
December 2016

Structural analysis of fungus-derived FAD glucose dehydrogenase.

Sci Rep 2015 Aug 27;5:13498. Epub 2015 Aug 27.

Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan.

We report the first three-dimensional structure of fungus-derived glucose dehydrogenase using flavin adenine dinucleotide (FAD) as the cofactor. This is currently the most advanced and popular enzyme used in glucose sensor strips manufactured for glycemic control by diabetic patients. We prepared recombinant nonglycosylated FAD-dependent glucose dehydrogenase (FADGDH) derived from Aspergillus flavus (AfGDH) and obtained the X-ray structures of the binary complex of enzyme and reduced FAD at a resolution of 1.78 Å and the ternary complex with reduced FAD and D-glucono-1,5-lactone (LGC) at a resolution of 1.57 Å. The overall structure is similar to that of fungal glucose oxidases (GOxs) reported till date. The ternary complex with reduced FAD and LGC revealed the residues recognizing the substrate. His505 and His548 were subjected for site-directed mutagenesis studies, and these two residues were revealed to form the catalytic pair, as those conserved in GOxs. The absence of residues that recognize the sixth hydroxyl group of the glucose of AfGDH, and the presence of significant cavity around the active site may account for this enzyme activity toward xylose. The structural information will contribute to the further engineering of FADGDH for use in more reliable and economical biosensing technology for diabetes management.
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http://dx.doi.org/10.1038/srep13498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642536PMC
August 2015

Stabilization of fungi-derived recombinant FAD-dependent glucose dehydrogenase by introducing a disulfide bond.

Biotechnol Lett 2015 May 4;37(5):1091-9. Epub 2015 Feb 4.

Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Nakamachi, Koganei, Tokyo, Japan.

Objective: To improve the stability of E. coli-produced non-glycosylated fungal FAD-glucose dehydrogenase induced a disulfide bond by site-directed mutagenesis based on structural comparisons with glucose oxidases.

Results: The FAD-glucose dehydrogenase (GDH) mutant Val149Cys/Gly190Cys, which was constructed based on a comparison with the three dimensional structure of glucose oxidase, showed a 110 min half-life of thermal inactivation at 45 °C, which is 13-fold greater than that of the wild-type enzyme. The considerable increase in thermal stability was further supported by Eyring plot analysis. The kinetic parameters of Val149Cys/Gly190Cys (k cat = 760 s(-1), Km = 35 mM, and catalytic efficiency (k cat/Km) = 22 s(-1 )mM(-1)) were almost identical to those of the wild-type enzyme (k cat = 780 s(-1), Km = 35 mM, k cat/Km = 22 s(-1 )mM(-1)). The substrate specificity of Val149Cys/Gly190Cys is indistinguishable from that of the wild type.

Conclusion: The constructed mutant, Val149Cys/Gly190Cys, had significantly increased structural stability without changing the catalytic activity and kinetic parameters of FAD-GDH, including its characteristic substrate specificity.
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http://dx.doi.org/10.1007/s10529-015-1774-8DOI Listing
May 2015

The development and characterization of an exogenous green-light-regulated gene expression system in marine cyanobacteria.

Mar Biotechnol (NY) 2015 Jun 1;17(3):245-51. Epub 2015 Feb 1.

Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan.

A green-light-regulated gene expression system derived from Synechocystis sp. PCC 6803 was constructed and introduced into the marine cyanobacterial strain Synechococcus sp. NKBG 15041c. The regulation system was evaluated using gfp uv as a reporter gene under red-light illumination and under simultaneous red- and green-light illumination. Expression of the reporter gene was effectively repressed under red-light illumination and increased over 10-fold by illuminating with green light. Control vectors missing either the ccaS sensor histidine kinase gene or the ccaR response regulator gene showed no detectable induction of GFPuv expression. Green-light induction of gfp uv expression was further confirmed by quantitative reverse transcription PCR. The constructed system was effective at regulating the recombinant expression of a target gene using green light in a marine cyanobacterial strain that does not naturally possess such a green-light regulation system. Thus, constructed green-light-regulated gene expression system may be used as a core platform technology for the development of marine cyanobacterial strains in which bioprocesses will be regulated by light.
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http://dx.doi.org/10.1007/s10126-015-9616-1DOI Listing
June 2015

Evaluation of the possibility to use the plant-microbe interaction to stimulate radioactive 137Cs accumulation by plants in a contaminated farm field in Fukushima, Japan.

J Plant Res 2015 Jan 15;128(1):147-59. Epub 2014 Nov 15.

Department of Biological Production Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-chou, Fuchu, Tokyo, 183-8509, Japan.

Field experiments in a contaminated farmland in Nihonmatsu city, Fukushima were conducted to assess the effectiveness of the plant-microbe interaction on removal of radiocesium. Before plowing, 93.3% of radiocesium was found in the top 5 cm layer (5,718 Bq kg DW(-1)). After plowing, Cs radioactivity in the 0-15 cm layer ranged from 2,037 to 3,277 Bq kg DW(-1). Based on sequential extraction, the percentage of available radiocesium (water soluble + exchangeable) was fewer than 10% of the total radioactive Cs. The transfer of (137)Cs was investigated in three agricultural crops; komatsuna (four cultivars), Indian mustard and buckwheat, inoculated with a Bacillus or an Azospirillum strains. Except for komatsuna Nikko and Indian mustard, inoculation with both strains resulted in an increase of biomass production by the tested plants. The highest (137)Cs radioactivity concentration in above-ground parts was found in Bacillus-inoculated komatsuna Nikko (121 Bq kg DW(-1)), accompanied with the highest (137)Cs TF (0.092). Furthermore, komatsuna Nikko-Bacillus and Indian mustard-Azospirillum associations gave the highest (137)Cs removal, 131.5 and 113.8 Bq m(-2), respectively. Despite the beneficial effect of inoculation, concentrations of (137)Cs and its transfer to the tested plants were not very high; consequently, removal of (137)Cs from soil would be very slow.
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http://dx.doi.org/10.1007/s10265-014-0678-3DOI Listing
January 2015

Stable cesium uptake and accumulation capacities of five plant species as influenced by bacterial inoculation and cesium distribution in the soil.

J Plant Res 2014 Sep 8;127(5):585-97. Epub 2014 Jul 8.

Department of Biological Production Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-chou, Fuchu, Tokyo, 183-8509, Japan.

The effects of inoculation with Bacillus and Azospirillum strains on growth and cesium accumulation of five plant species, Komatsuna, Amaranth, sorghum, common millet and buckwheat, grown on cesium-spiked soil were assessed for potential use in cesium remediation. Pot experiments were performed using "artificially" Cs-contaminated soil. Three treatments were applied based on Cs location in the soil. For a soil height of 15 cm in the pots, Cs was added as follows: in the top five cm to imitate no ploughing condition; in the bottom five cm simulating inverted ploughing; and uniformly distributed Cs reproducing normal plowing. Generally, inoculation of Cs-exposed plants significantly enhanced growth and tolerance to this element. Transfer factor (ratio of Cs concentration in the plant tissues to that in surrounding soil) was strongly influenced by Cs distribution, with higher values in the top-Cs treatment. Within this treatment, inoculation of Komatsuna with Bacillus and Azospirillum strains resulted in the greatest transfer factors of 6.55 and 6.68, respectively. Cesium content in the shoots was high in the Azospirillum-inoculated Komatsuna, Amaranth, and buckwheat, i.e., 1,830, 1,220, and 1,030 µg per pot, respectively (five plants were grown in each pot). Therefore, inoculation of Komatsuna and Amaranth with the strains tested here could be effective in enhancing Cs accumulation. The decrease of Cs transfer under uniform- and bottom-Cs treatments would suggest that countermeasures aiming at decreasing the transfer of Cs could rely on ploughing practices.
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http://dx.doi.org/10.1007/s10265-014-0647-xDOI Listing
September 2014

A green-light inducible lytic system for cyanobacterial cells.

Biotechnol Biofuels 2014 9;7:56. Epub 2014 Apr 9.

Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture & Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan ; JST, CREST, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.

Background: Cyanobacteria are an attractive candidate for the production of biofuel because of their ability to capture carbon dioxide by photosynthesis and grow on non-arable land. However, because huge quantities of water are required for cultivation, strict water management is one of the greatest issues in algae- and cyanobacteria-based biofuel production. In this study, we aim to construct a lytic cyanobacterium that can be regulated by a physical signal (green-light illumination) for future use in the recovery of biofuel related compounds.

Results: We introduced T4 bacteriophage-derived lysis genes encoding holin and endolysin under the control of the green-light regulated cpcG2 promoter in Synechocystis sp. PCC 6803. When cells harboring the lysis genes were illuminated with both red and green light, we observed a considerable decrease in growth rate, a significant increase in cellular phycocyanin released in the medium, and a considerable fraction of dead cells. These effects were not observed when these cells were illuminated with only red light, or when cells not containing the lysis genes were grown under either red light or red and green light. These results indicate that our constructed green-light inducible lytic system was clearly induced by green-light illumination, resulting in lytic cells that released intracellular phycocyanin into the culture supernatant. This property suggests a future possibility to construct photosynthetic genetically modified organisms that are unable to survive under sunlight exposure. Expression of the self-lysis system with green-light illumination was also found to greatly increase the fragility of the cell membrane, as determined by subjecting the induced cells to detergent, osmotic-shock, and freeze-thaw treatments.

Conclusions: A green-light inducible lytic system was constructed in Synechocystis sp. PCC 6803. The engineered lytic cyanobacterial cells should be beneficial for the recovery of biofuels and related compounds from cells with minimal effort and energy, due to the fragile nature of the induced cells. Furthermore, the use of light-sensing two-component systems to regulate the expression of exogenous genes in cyanobacteria promises to replace conventional chemical inducers in many bioprocess applications, impacting the limiting water management issues.
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http://dx.doi.org/10.1186/1754-6834-7-56DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4021604PMC
May 2014

Engineering of a green-light inducible gene expression system in Synechocystis sp. PCC6803.

Microb Biotechnol 2014 Mar 12;7(2):177-83. Epub 2013 Dec 12.

Department of Biotechnology and Life Science, Tokyo University of Agriculture & Technology, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan; JST, CREST, 2-24-16 Naka-cho, Koganei, Tokyo, 184-8588, Japan.

In order to construct a green-light-regulated gene expression system for cyanobacteria, we characterized a green-light sensing system derived from Synechocystis sp. PCC6803, consisting of the green-light sensing histidine kinase CcaS, the cognate response regulator CcaR, and the promoter of cpcG2 (PcpcG 2 ). CcaS and CcaR act as a genetic controller and activate gene expression from PcpcG 2 with green-light illumination. The green-light induction level of the native PcpcG 2 was investigated using GFPuv as a reporter gene inserted in a broad-host-range vector. A clear induction of protein expression from native PcpcG 2 under green-light illumination was observed; however, the expression level was very low compared with Ptrc , which was reported to act as a constitutive promoter in cyanobacteria. Therefore, a Shine-Dalgarno-like sequence derived from the cpcB gene was inserted in the 5' untranslated region of the cpcG2 gene, and the expression level of CcaR was increased. Thus, constructed engineered green-light sensing system resulted in about 40-fold higher protein expression than with the wild-type promoter with a high ON/OFF ratio under green-light illumination. The engineered green-light gene expression system would be a useful genetic tool for controlling gene expression in the emergent cyanobacterial bioprocesses.
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http://dx.doi.org/10.1111/1751-7915.12098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937721PMC
March 2014

Construction of mutant glucose oxidases with increased dye-mediated dehydrogenase activity.

Int J Mol Sci 2012 Nov 2;13(11):14149-57. Epub 2012 Nov 2.

Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei, Tokyo 184-8588, Japan.

Mutagenesis studies on glucose oxidases (GOxs) were conducted to construct GOxs with reduced oxidase activity and increased dehydrogenase activity. We focused on two representative GOxs, of which crystal structures have already been reported—Penicillium amagasakiense GOx (PDB ID; 1gpe) and Aspergillus niger GOx (PDB ID; 1cf3). We constructed oxygen-interacting structural models for GOxs, and predicted the residues responsible for oxidative half reaction with oxygen on the basis of the crystal structure of cholesterol oxidase as well as on the fact that both enzymes are members of the glucose/methanol/choline (GMC) oxidoreductase family. Rational amino acid substitution resulted in the construction of an engineered GOx with drastically decreased oxidase activity and increased dehydrogenase activity, which was higher than that of the wild-type enzyme. As a result, the dehydrogenase/oxidase ratio of the engineered enzyme was more than 11-fold greater than that of the wild-type enzyme. These results indicate that alteration of the dehydrogenase/oxidase activity ratio of GOxs is possible by introducing a mutation into the putative functional residues responsible for oxidative half reaction with oxygen of these enzymes, resulting in a further increased dehydrogenase activity. This is the first study reporting the alteration of GOx electron acceptor preference from oxygen to an artificial electron acceptor.
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http://dx.doi.org/10.3390/ijms131114149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3509572PMC
November 2012

Substrate specificity engineering of Escherichia coli derived fructosamine 6-kinase.

Biotechnol Lett 2013 Feb 18;35(2):253-8. Epub 2012 Oct 18.

Ultizyme International Ltd, 1-13-16, Minami, Meguro, Tokyo, Japan.

A three-dimensional structural model of Escherichia coli fructosamine 6-kinase (FN6K), an enzyme that phosphorylates fructosamines at C6 and catalyzes the production of the fructosamine 6-phosphate stable intermediate, was generated using the crystal structure of 2-keto-3-deoxygluconate kinase isolated from Thermus thermophilus as template. The putative active site region was then investigated by site-directed mutagenesis to reveal several amino acid residues that likely play important roles in the enzyme reaction. Met220 was identified as a residue that plays a role in substrate recognition when compared to Bacillus subtilis derived FN6K, which shows different substrate specificity from the E. coli FN6K. Among the various Met220-substituted mutant enzymes, Met220Leu, which corresponded to the B. subtilis residue, resulted in an increased activity of fructosyl-valine and decreased activity of fructosyl-lysine, thus increasing the specificity for fructosyl-valine by 40-fold.
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http://dx.doi.org/10.1007/s10529-012-1062-9DOI Listing
February 2013
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