Publications

Bioscience, Biotechnology, and Biochemistry. 2023 Sep, 87(12):1495-1504

• Noriaki Kitagawa, Hikaru Watanabe, Tetsuya Mori, Yuki Kawauchi, Hajime Aga, Shimpei Ushio, Koryu Yamamoto

Isomaltooligosaccharides (IMOs), including isomaltose, are valuable oligosaccharides, and the development of methods to synthesize high-purity IMOs has long been underway. We recently discovered a novel enzyme, 4-O-α-D-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase (IMM-4IH), that showed promise for improving the synthesis process. In the present study, we establish methods for synthesizing isomaltose and IMOs consisting of a variety of degrees of polymerization from starch using IMM-4IH. With 5% substrate, by combining IMM-4IH with 1,4-α-glucan 6-α-glucosyltransferase from Bacillus globisporus N75, the yield of isomaltose was 63.0%; incorporating isoamylase and cyclomaltodextrin glucanotransferase increased the yield to 75.3%. On the other hand, by combining IMM-4IH with 1,4-α-glucan 6-α-glucosyltransferase from Paenibacillus sp. PP710, IMOs were synthesized. The inclusion of isoamylase and α-amylase led to the synthesis from 10% starch of 136 mM IMOs, consisting of oligosaccharides from isomaltose to isomaltodecaose. The development of these efficient methods will be an important contribution to the industrial production of IMOs.

Bioscience, Biotechnology, and Biochemistry. 2023 Feb 24;87(3):330-337

• Noriaki Kitagawa, Hikaru Watanabe, Tetsuya Mori, Hajime Aga, Shimpei Ushio, Koryu Yamamoto

A novel enzyme, 4-O-α-D-isomaltooligosaccharylmaltooligosaccharide 1,4-α-isomaltooligosaccharohydrolase (IMM-4IH), was previously discovered from Sarocladium kiliense U4520. In order to identify the factors underlying the unique substrate specificity of IMM-4IH, we endeavored to determine the amino acid sequence of the enzyme. By comparing the partial amino acid sequence of the enzyme to whole genome sequencing data of S. kiliense U4520, the IMM-4IH gene was estimated. The putative gene was expressed in Pichia pastoris, and its activity and properties were found to be consistent with those of the native enzyme. Comparing the amino acid sequence of IMM-4IH with those in the CAZy database led to classification in the glycoside hydrolase family 49 (GH49). Several amino acids important for catalysis (Asp406, Asp425 and Asp426) and substrate recognition at subsites +1 and -3 were estimated by multiple sequence alignment analysis. These results provide important information for characterizing IMM-4IH and other GH49 enzymes.

J. Agric.FoodChem.2023,71, 5476−5482

• Izumi C. Mori*1, Takakazu Matsuura*1, Masahiro Otao, Lia Ooi, Yasuyo Nishimura, and Takashi Hirayama*1
• • *1 Institute of Plant Science and Resources, Okayama University,Kurashiki 710-0046, Japan
  • *2 Facultyof Agriculture and MarineScience, Kochi University, Nankoku 783-8502, Japan

Trehalose is a disaccharide and is often foliar applied by farmers aiming at increasing stress resistance or crop production. However, the physiological effect of exogenously applied trehalose on crops remains obscure. Here, we explored the effect of foliar trehalose application on style length of solanaceous crops, Solanum melongena and S. lycopersicum. Trehalose application promotes pistil to stamen ratio by gaining style length. Another disaccharide consisting of two glucose molecules, maltose, showed the same effect on style length of S. lycopersicum, while monosaccharide glucose did not. Trehalose is found to affect style length through uptake via roots or interaction with rhizosphere but not through absorption by shoots in S. lycopersicum. Our study suggests that yield improvement of solanaceous crops by trehalose application under stressed conditions is brought about by suppression of the occurrence of short-styled flowers. This study suggests that trehalose holds potential to act as a plant biostimulant in preventing short-styled flowers in solanaceous crops.

Journal of Community Medicine and Public Health Reports, vol.4, 2023

• Hitomi Wake, Keizo Kohno, Satomi Koya-Miyata, Kotaro Hashimoto, Hajime Kusano, Takashi Morimoto, Yosuke Shimojo, Toshio Ariyasu, Kanso Iwaki
• Pharmaceutical Ingredients Department, Personal Healthcare Division, Hayashibara Co., Ltd.

Our findings that trehalose is a suitable additive for exosomes in all of these production, purification, and preservation processes indicate that trehalose is a promising tool for the industrial application of exosomes. Furthermore, the results suggest that the separation and preservation methods presented in this study can facilitate therapeutic applications of exosomes.

Int. J. Mol. Sci. 2023, 24, 10073.

• Maria Tannous*1,2,3 , Gjylije Hoti*1 , Francesco Trotta*1, Roberta Cavalli*2, Takanobu Higashiyama, Pasquale Pagliaro*3 and Claudia Penna*3
• • *1 Department of Chemistry, University of Turin, 10125 Turin, Italy;
  • *2 Department of Drug Science and Technology, University of Turin, 10125 Turin, Italy;
  • *3 Department of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, Italy;

Nanocarriers for oxygen delivery have been the focus of extensive research to ameliorate the therapeutic effects of current anti-cancer treatments and in the organ transplant field. In the latter application, the use of oxygenated cardioplegic solution (CS) during cardiac arrest is certainly beneficial, and fully oxygenated crystalloid solutions may be excellent means of myocardial protection, albeit for a limited time. Therefore, to overcome this drawback, oxygenated nanosponges (NSs) that can store and slowly release oxygen over a controlled period have been chosen as nanocarriers to enhance the functionality of cardioplegic solutions. Different components can be used to prepare nanocarrier formulations for saturated oxygen delivery, and these include native α-cyclodextrin (αCD), αcyclodextrin-based nanosponges (αCD-NSs), native cyclic nigerosyl-nigerose (CNN), and cyclic nigerosyl-nigerose-based nanosponges (CNN-NSs). Oxygen release kinetics varied depending on the nanocarrier used, demonstrating higher oxygen release after 24 h for NSs than the native αCD and CNN. CNN-NSs presented the highest oxygen concentration (8.57 mg/L) in the National Institutes of Health (NIH) CS recorded at 37 ℃ for 12 h. The NSs retained more oxygen at 1.30 g/L than 0.13 g/L. These nanocarriers have considerable versatility and the ability to store oxygen and prolong the amount of time that the heart remains in hypothermic CS. The physicochemical characterization presents a promising oxygen-carrier formulation that can prolong the release of oxygen at low temperatures. This can make the nanocarriers suitable for the storage of hearts during the explant and transport procedure.

Carbohydrate Research 517 (2022) 108578

• Noriaki Kitagawa, Hikaru Watanabe, Tetsuya Mori, Hajime Aga, Shimpei Ushio, Koryu Yamamoto

We discovered a novel enzyme in our pursuit of an improved method for the production of isomaltose. The enzyme, 4-α-isomaltooligosylglucose 4-glucanohydrolase from Sarocladium kiliense U4520, recognizes the panose motif (α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 4)-D-Glcp) and hydrolyzes the α-1,4-glucosidic bond on the reducing end side with respect to the α-1,6-glucosidic bond. The structure on the non-reducing end of the panose motif is important for the recognition of the substrate by the enzyme, and the substrate specificity is unique and distinguished from previously reported enzymes. The enzyme catalyzes the hydrolysis of panose with a k_cat/K_m of 31.2 s^-1mM^-1, and catalysis results in anomeric inversion. These enzymatic properties suggest that this enzyme will pair well with 1,4-α-glucan 6-α-glucosyltransferase from Bacillus globisporus N75 in the efficient production of isomaltose from starch.*^-1

IFSCC Magazine 25(3), 2022, 179-186

• Kotaro Hashimoto, Masahiro Otao, Satomi Koya-Miyata, Yui Sunada, Yuki Mitsukawa, Shimpei Ushio*, Jiro Kishimoto
• • *NAGASE & Co., Ltd. Tokyo, Japan

In response to the social demand to establish a sustainable economy, biotechnology is gaining more attention as a manufacturing method to fulfill those needs. However, the productivity of bioprocesses is generally lower than conventional industrial organic chemistry. Therefore, much effort has been applied to breeding microorganisms, improving the reaction efficiencies of enzymes and developing the robustness of bioprocesses to achieve an industrial level of mass production. Since 1935, Hayashibara has been dedicated to manufacturing with a small environmental load by utilizing enzymes and microorganisms to process carbon neutral biomasses. Typical bioactive materials supplied by Hayashibara are mainly sugars and glycosides, which have remarkable biological safeties and handling abilities, and are manufactured by enzymatic synthesis (e.g. L-Ascorbic acid 2-glucoside, Glucosyl Hesperidin, Glucosyl Naringin, Trehalose and Isomalto-oligosaccharide syrup) or by microbial fermentation (e.g. Pullulan and Ergothioneine). In this review, those materials are described with respect to their physiochemical properties, their usefulness in cosmetics and personal care products, and their contribution toward sustainability. We expect that these materials will also contribute to homeostasis of the skin and the entire body systemically, and that recycling/upcycling of raw materials will become a vital factor in future material development. We believe that it is essential to pursue holistic beauty as a goal.

YAKUGAKU ZASSHI 142(5), 535-546 (2022); https://doi.org/10.1248/yakushi.21-00219

• Satomi Koya-Miyata, Keizo Kohno, Takashi Morimoto, Akira Harashima, Yuko Iwata*, Toshio Ariyasu
• * National Cerebral and Cardiovascular Center

Transient receptor potential vanilloid 2 (TRPV2) channels are expressed and play functional roles in various immune cells. Physical stimuli leading to TRPV2 activation causes mast cell degranulation. Besides their roles in immune cells, it has been shown that TRPV2 channels are pathophysiologically relevant to degenerative muscular diseases such as dilated cardiomyopathy and muscular dystrophy. Hence, development of drug candidates that inhibit human TRPV2 activation is an urgent matter. NK-4, a cryptocyanine dye, inhibited agonist-induced TRPV2 activity in mouse TRPV2-transfected HEK293 cells. However, it remains unclear whether NK-4 exerts regulatory effects on the activation of human TRPV2 channels. In this study, we show that NK-4 inhibits intracellular Ca²⁺ increase in human TRPV2-transfected HEK293 cells preactivated with a TRPV2 agonist. The inhibitory effect of NK-4 (IC50 = 0.27 mM) on human TRPV2 activation was 74-fold stronger than that on mouse TRPV2 activation (IC50 = 20 mM). NK-4 also inhibited the agonist-induced TRPV2 expression at the plasma membrane, when the human TRPV2-expressing cells were stimulated with the agonist in the presence of NK-4. These results suggest that NK-4 abrogates the agonist-induced signaling events leading to human TRPV2 activation. Furthermore, TRPV2 agonist caused degranulation of RBL-2H3 cells, which represents a phenomenon related to physical urticarias. NK-4 suppressed the release of b-hexosaminidases upon degradation with IC50 of 1.9 mM, 35-fold lower than that determined with an anti-allergic drug, Epinastine. Our results suggest that NK-4 would be a potential therapeutic strategy to resolve dilated cardiomyopathy and its associated heart failure as well as physical urticarias.

Nutrients 2022, 14(9), 1776; https://doi.org/10.3390/nu14091776

• Naomi Hamada*1,*2, Tsuyoshi Wadazumi*1, Yoko Hirata*1,*3, Hitoshi Watanabe*4, Nobuko Hongu*5 and Norie Arai
• • *1 Graduate School of Health and Well-Being, Department of Health and Well-Being, Kansai University
  • *2 Department of Applied Food Science, Higashiosaka Junior College
  • *3 Department of Food and Nutritional Science, Kobe Women’s Junior College
  • *4 Research Center for Urban Health and Sports, Osaka Metropolitan University
  • *5 Graduate School of Human Life Science, Department of Food and Human Life Science, Osaka Metropolitan University

Trehalose solution ingested during exercise induces gradual increases in blood glucose levels and the insulin response compared with glucose solution. Trehalose solution aids in the maintenance of performance in the later stages of prolonged exercise. The purpose of this study was to identify the lowest concentration at which the properties of trehalose could be exploited. After fasting for 12 h, the healthy men with recreational training performed a 60 min constant-load exercise at 40% VO₂ peak using a bicycle ergometer and ingested 500 mL of a trial drink (experiment 1: water, 8% glucose, and 6 or 8% trehalose; experiment 2: 4 or 6% trehalose). They performed four sets of the Wingate test combined with a 30 min constant-load exercise at 40% VO₂ peak. The experiment was conducted using a randomized cross-over design; trial drink experiments were conducted over intervals of 7 to 12 days. The exercise performance was evaluated based on mean power in the Wingate test. During the high-intensity 5 h intermittent exercise, no differences were found between the groups in exercise performance in the later stages with concentrations of 8, 6, and 4% trehalose solution. The results suggest that trehalose could be useful for making a new type of mixed carbohydrate solution.

Nihon Reoroji Gakkaishi　Vol.49, No.4, 241-246

• Mutsuko Taniguchi, Akira Harashima, Tatsuya Ishihara, Shimpei Ushio, Yoko Akiyama*
• • * Osaka University

When aqueous solutions of five oligosaccharides and one polyol compound were applied to the skin, tactile intensity and temporal changes, as defined by five Japanese onomatopoeic words, were observed. Moreover, the relationships between the sensations and the physical and chemical properties were investigated.
No difference in the tactile characteristics was observed among the samples during application; however, there was a difference in the characteristics among samples in the sensations of ""beta-beta (sticky)"", ""gishi-gishi (squeaky)"", and ""sara-sara (smooth)"" after application. In terms of temporal changes, the sensations of oligosaccharides were first described as ""nuru-nuru (slimy)"", which then changed to ""gishi-gishi (squeaky)"" and later to ""beta-beta (sticky)"", ""shittori (moist)"", and ""sara-sara (smooth)"". Among the samples, a difference was observed in the timing of feeling ""beta-beta (sticky)"", "" shittori (moist)"", and ""sara-sara (smooth)"".
The correlation between the ""beta-beta (sticky)"" feeling, which is a contextually undesirable quality of oligosaccharides, and the physical and chemical properties was determined. A high correlation was observed among adhesiveness, integral value of the application force for the final 20 s of application, and solubility. It was considered that the sticky oligosaccharides have these physical properties that were highly correlated described above.

International Journal of Food Science & Technology / Early View First published: 17 December 2021

• Fang Fang*1-2, Aminata Diatta*1-2, Senay Simsek*3, Pablo Torres-Aguilar*1-2, Hikaru Watanabe, Takanobu Higashiyama, Osvaldo H. Campanella*1*4, Bruce R. Hamaker*1-2
• • *1 Whistler Center for Carbohydrate Research, Purdue University
  • *2 Department of Food Science, Purdue University
  • *3 Department of Plant Sciences, North Dakota State University
  • *4 Department of Food Science and Technology, The Ohio State University

Isomaltodextrin (IMD) is a novel highly branched a-glucan ingredient that has potential applications in foods as a promising source of dietary fibre. In this study, we assessed the incorporation of IMD in baked products and its effect on dough rheology and physico-textural characteristics of wheat breads. IMD was added to wheat flour to reach the US FDA level of ‘good’ source of fibre (10% IMD). Use of the straight-dough method with IMD and regular dough mixing time (8 min) resulted in less elastic doughs and resulting breads displayed lower cohesiveness, springiness and chewiness compared to the control bread. A modified straight-dough method, with longer mixing time (36 min) and adjusted water level (70%), and a sponge-and dough method were used to allow gluten structure to more fully hydrate and develop. With IMD addition, the modified straight-dough (36 min dough mixing time) and sponge-anddough methods yielded enhanced dough elasticity compared to the original straight-dough method. Breadmaking methods were identified (improved straight dough and sponge-and-dough) whereby IMD could be added at a ’good source’ of fibre level with loaf appearance and texture similar to breads without IMD.

International Journal of Food Science and Technology, 56(4): 1631-1640 (2021) doi:10.1111/ijfs.14782

• Fang Fang*1, Anna M.R. Hayes*1, Hikaru Watanabe, Takanobu Higashiyama, Osvaldo H. Campanella*2, Bruce R. Hamaker*1
• • *1 Department of Food Science, Whistler Center for Carbohydrate Research, Purdue University
  • *2 Department of Food Science and Technology, The Ohio State University

In this study, the impact of Isomaltodextrin (IMD) on the viscoelasticity and microstructure of starch gels and starch retrogradation were investigated. IMD solutions displayed typical Newtonian behaviour. When maintained at relative humidity of 95%, IMD absorbed 40% water of its original weight. The addition of IMD to normal corn starch increased strength of gels and resulted in a denser and smaller pore structure, but there were no changes in elasticity. Upon storage for 7 days, addition of IMD at high concentration (starch: IMD = 2:1) significantly increased the degree of starch retrogradation.

Journal of Inflammation, 18:2 (2021) https://doi.org/10.1186/s12950-020-00267-z

• Keizo Kohno, Satomi Koya-Miyata, Akira Harashima, Takahiko Tsukuda, Masataka Katakami, Toshio Ariyasu, Shimpei Ushio and Kanso Iwaki

We demonstrated that NK-4 drove macrophage polarization toward an inflammatory M1-like phenotype with enhanced phagocytic activity, suggesting that NK-4 potentiates the first line of host defense against invaded pathogens during the inflammatory phase of wound healing. Furthermore, we showed that NK-4-induced M1-like macrophages undergo elevated phenotypic switching toward an anti-inflammatory M2-like phenotype upon co-culture with apoptotic cells. Our results suggest that NK-4 may provide a promising strategy for the resolution of chronic wounds.

International Journal of Oncology (2021)

• Yamamoto S*1, Lee S*1, Ariyasu T, Endo S, Miyata S, Yasuda A, Harashima A, Ohta T, Kumagai-Takei N*1, Ito T*1, Shimizu Y*2, Srinivas B*1, Sada N*1, 3, Nishimura Y*1 and Otsuki T*1.
• • *1 Kawasaki Medical School
  • *2 Dept. of Pathophysiology-Pridontal Science, Okayama University Graduate School of Medicine
  • *3 Dept of Biophysical Chemistry, Okayama University Graduate School of Medicine

These results suggest that Treh or gHesp may restore the reduced anti-tumor immunity in T helper cells caused by asbestos continuous exposure, but further detailed investigations will be needed to obtain certain confirmation for trials to give these compounds into human population with high-risk for asbestos exposure such as workers in asbestos-handling manufactures and building demolition workers.

Nutrients 2021, 13(5), 1439; https://doi.org/10.3390/nu13051439

• Naomi Hamada*1,*2, Tsuyoshi Wadazumi*2, Yoko Hirata*2,*3, Mayumi Kuriyama*4, Kanji Watanabe*5, Hitoshi Watanabe*6, Nobuko Hongu*7 and Norie Arai
• • *1 Department of Applied Food Science, Higashiosaka Junior College
  • *2 Faculty of Health and Well-Being, Kansai University
  • *3 Department of Food and Nutritional Science, Kobe Women’s Junior College
  • *4 Faculty of Nursing Sciences, Meiji University of Integrative medicine
  • *5 School of Health and Sports Sciences, Mukogawa Women’s University
  • *6 Research Center for Urban Health and Sports, Osaka City University
  • *7 Department of Food and Human Life Science, Osaka City University

Twelve healthy men first exercised with a constant load (intensity: 40% VO₂peak) for 60 min using a bicycle ergometer. They continued to exercise with a constant load (40% VO₂peak) for 30 min between four sets of the 30-second Wingate test. After the first set, each participant ingested 500 mL water (control), 8% glucose, or 8% trehalose in three trials. Blood was collected for biochemical parameters at 12 time points during the experiment. The area under the curve of adrenaline after ingestion of trehalose was significantly lower than that for water and tended to be lower than that for glucose in the later stage of the exercise. Lower secretion of adrenaline after a single dose of 8% trehalose during prolonged exercise reflected the preservation of carbohydrates in the body in the later stage of the exercise. In conclusion, a single ingestion of trehalose helped to maintain prolonged exercise performance.

Bioscience, Biotechnology, and Biochemistry, 85(3), 600-610, (2021).

• Akihiro Fujita, Akira Kawashima, Yuuki Mitsukawa, Noriaki Kitagawa, Hikaru Watanabe, Tetsuya Mori, Tomoyuki Nishimoto, Hajime Aga, Shimpei Ushio

Glucanotransferases that can synthesize cyclo-{→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→} (CI4) from dextran were purified to homogeneity from the culture supernatant of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006. The molecular mass of both enzymes was estimated to be 86 kDa by SDS-PAGE. The glucanotransferase, named CI4-forming enzyme, from Agreia sp. exhibited the highest activity at pH 6.0 and 40°C. The enzyme was stable on the pH range of 4.6-9.9 and up to 40°C. On the other hand, the enzyme from M. trichothecenolyticum exhibited the highest activity at pH 5.7 and 40°C. The enzyme was stable on the pH range of 5.0-6.9 and up to 35°C. Both enzymes catalyzed 4 reactions, namely, intramolecular α-1,6-transglycosylation (cyclization), intermolecular α-1,6-transglycosylation, hydrolysis of CI4, and coupling reaction. Furthermore, the CI4-forming enzyme produced CI4 from α-1,6-linked glucan synthesized from starch by 6-α-glucosyltransferase. These findings will enable the production of CI4 from starch.

Journal of Photopolymer Science and Technology, 34(1), 41-47, (2021).

• Yasumitsu Orii*1, Shuichi Hirose*1, Akihiro Fujita, Masakazu Kobayashi*1
• *1 NAGASE & Co., LTD.

As the use of IT increases importance with big data and AI, the issue of power consumption has been highlighted. Under these circumstances, the development of new materials is more and more important. Materials Informatics (MI) is one of the hottest technologies in the material development field, because of its potential to reduce the time and costs of discovering innovative materials. To achieve this, the key is to collect data that has been accumulated for many years at research institutions and companies, and to make information extracted from the data into knowledge. This article introduces the development of two methods based on AI: the “cognitive approach”, which reads vast amounts of literature information and digitizes data, and the “analytic approach”, which theoretically estimates the structure and physical properties of chemical substances from predictive models.

Frontiers in Cellular and Infection Microbiology,02 July 2021| https://doi.org/10.3389/fcimb.2021.670935

• Anthony M. Buckley*1, Ines B. Moura*1, Norie Arai, William Spittal*1, Emma Clark*1, Yoshihiro Nishida, Hannah C. Harris*1, Karen Bentley*1, Georgina Davis*1, Dapeng Wang*2, Suparna Mitra*1, Takanobu Higashiyama and Mark H. Wilcox*1
• • *1 Healthcare Associated Infection Research Group, Molecular Gastroenterology, Leeds Institute of Medical Research, University of Leeds, Leeds, United Kingdom
  • *2 LeedsOmics, University of Leeds, Leeds, United Kingdom

Trehalose has been reported to enhance virulence of certain Clostridioides difficile (C.difficile) ribotypes including genetic trehalose metabolism variants. However, such variants are widespread and not correlated with clinical outcomes for patients suffering from C.difficile infection (CDI). Here, we make preliminary observations on how trehalose supplementation affects the microbiota in an in vitro model and show that trehalose-induced changes can reduce the outgrowth of C.difficile, preventing simulated CDI.
Three clinically reflective human gut models instilled with glucose, trehalose or saline were exposed to C.difficile spores. The recovery of the microbiota following antibiotic treatment and CDI induction was monitored in each model. The human microbiota remodelled to utilise the bioavailable trehalose. The trehalose supplementation did not result in CDI, although limited spore germination did occur.
As a result of taxonomic and functional analysis of microbiota after antibiotic treatment, trehalose supplementation remodeled the microbiota, which prevented simulated CDI, potentially due to enhanced recovery of nutritionally competitive microbiota against C.difficile.

Bioscience, Biotechnology, and Biochemistry, 2021, Vol. 85, No. 7, 1737-1745

• Akiko Yasuda, Manabu Miyata, Osamu Sano, Tatsufumi Sogo,Seiichiro Kishishita, Takuo Yamamoto, Hajime Aga, and Koryu Yamamoto

We prepared a high-molecular-weight modified dextrin (MWS-1000) from a partial hydrolysate of waxy corn starch with a weight average molecular weight of 1 × 106 (WS-1000) using Paenibacillus alginolyticus PP710 α-glucosyltransferase. The gel permeation chromatography showed that the weight average molecular weight of MWS-1000 was almost the same as that of WS-1000. The side chain lengths of WS-1000 and MWS-1000 after isomaltodextranase digestion were also shown to be similar to each other by high-performance anion exchange chromatography with pulsed amperometric detection. Since MWS-1000 confirmed the presence of α-1,6 bonds by enzyme digestibility, methylation, and 1H-NMR analyses, it was presumed that the structure of MWS-1000 was based on the introduction of α-1,6 glucosyl residues at the nonreducing ends of the partial hydrolysate of waxy corn starch. Furthermore, the MWS-1000 solution was not retrograded even during refrigerated storage or after repeated freeze-thaw cycles.

Bioscience, Biotechnology, and Biochemistry, 2021, Vol. 85, No. 7, 1746-1752

• Rika Sumida*, Seiichiro Kishishita, Akiko Yasuda, Manabu Miyata,Akiko Mizote, Takuo Yamamoto, Hitoshi Mitsuzumi, Hajime Aga,Koryu Yamamoto, and Kiyoshi Kawai*
• * Graduate School of Integrated Sciences for Life, Hiroshima University

High-molecular-weight dextrin (WS-1000) was produced from waxy corn starch and enzymatically modified to link
glucose by α-1,6 glycosidic bond at the terminal point of the glucose chain, forming MWS-1000. In this study, the physical
properties of MWS-1000 were characterized, and the advantages of its use as a food modifier were described. From
rheological and calorimetric studies, it was found that MWS-1000 does not undergo retrogradation, but it does not
prevent the retrogradation of WS-1000, suggesting that they have no intermolecular interaction in solution. Investigation
of the effect of MWS-1000 on the viscoelasticity of gelatinized wheat starch showed that in the linear viscoelastic region,
storage modulus decreased and tan δ increased with increase in replaced MWS-1000 content. In addition, it was
confirmed that gelatinized starch containing MWS-1000 showed viscoelastic behavior similar to that of commercially
available custard cream.

JAPANESE PHAMACOLOGY & THERAPEUTICS 49(8), 1241-1252 (2021)

• Yuki Ishida, Tsuyoshi Sadakiyo, Yu Ohira, Shin Endo, Hitoshi Mitsuzumi, Nobuhiko Shioya*1, Yoshitaka Iwama*2
• • *1 KSO Corporation,
  • *2 Nihonbashi Cardiology Clinic

We examined the suppressive effect of IMD on postprandial blood glucose elevation after ingestion of cooked rice in healthy adults. The results showed that AUC of blood glucose was significantly lower with IMD than with placebo. Postprandial blood glucose levels over time were also significantly lower with IMD than with placebo. IMD intake significantly suppressed blood glucose elevation in healthy adults after ingestion of cooked rice.

Bioscience, Biotechnology, and Biochemistry. 2021 Feb 24;85(3):600-610

• Akihiro Fujita, Akira Kawashima, Yuuki Mitsukawa, Noriaki Kitagawa, Hikaru Watanabe, Tetsuya Mori, Tomoyuki Nishimoto, Hajime Aga, Shimpei Ushio

Glucanotransferases that can synthesize cyclo-{→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→} (CI4) from dextran were purified to homogeneity from the culture supernatant of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006. The molecular mass of both enzymes was estimated to be 86 kDa by SDS-PAGE. The glucanotransferase, named CI4-forming enzyme, from Agreia sp. exhibited the highest activity at pH 6.0 and 40 °C. The enzyme was stable on the pH range of 4.6-9.9 and up to 40 °C. On the other hand, the enzyme from M. trichothecenolyticum exhibited the highest activity at pH 5.7 and 40 °C. The enzyme was stable on the pH range of 5.0-6.9 and up to 35 °C. Both enzymes catalyzed 4 reactions, namely, intramolecular α-1,6-transglycosylation (cyclization), intermolecular α-1,6-transglycosylation, hydrolysis of CI4, and coupling reaction. Furthermore, the CI4-forming enzyme produced CI4 from α-1,6-linked glucan synthesized from starch by 6-α-glucosyltransferase. These findings will enable the production of CI4 from starch.

Biosci Biotechnol Biochem. 2021 Dec 22;86(1):68-77

• Fujita Akihiro, Kawashima Akira, Noguchi Yuji*, Hirose Shuichi*, Kitagawa Noriaki, Watanabe Hikaru, Mori Tetsuya, Nishimoto Tomoyuki, Aga Hajime, Ushio Shimpei, Yamamoto Koryu
• * NAGASE CO., LTD.

We performed whole genome sequence analyses of Agreia sp. D1110 and Microbacterium trichothecenolyticum D2006 that secrete enzymes to produce cyclo-{→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glcp-(1→} (CI4) from dextran. Full-length amino acid sequences of CI4-forming enzymes were identified by matching known N-terminal amino acid sequences with products of the draft genome. Domain searches revealed that the CI4-forming enzymes are composed of Glycoside Hydrolase family 66 (GH66) domain, Carbohydrate Binding Module family 35 (CBM35) domain, and CBM13 domain, categorizing the CI4-forming enzymes in the GH66. Furthermore, the amino acid sequences of the two CI4-forming enzymes were 71% similar to each other and up to 51% similar to cycloisomaltooligosaccharide glucanotransferases (CITases) categorized in GH66. Differences in sequence between the CI4-forming enzymes and the CITases suggest mechanisms to produce specific cycloisomaltooligosaccharides, and whole genome sequence analyses identified a gene cluster whose gene products likely work in concert with the CI4-forming enzymes.

Bioscience, Biotechnology, and Biochemistry. 2021 Nov 24;85(12):2450-2458

• Noriaki Kitagawa, Hikaru Watanabe, Tetsuya Mori, Tomoyuki Nishimoto, Hajime Aga, Shimpei Ushio, Koryu Yamamoto

We attempted to develop an efficient method for producing isomaltose, a disaccharide consisting of an α-(1→6)-linkage, from starch by combining enzymes of known activity. We found that the combination of 1,4-α-glucan 6-α-glucosyltransferase from Bacillus globisporus N75 and isopullulanase from Aspergillus brasiliensis ATCC 9642 led to the efficient synthesis of isomaltose. Inclusion of isoamylase and cyclomaltodextrin glucanotransferase resulted in increased efficiency, with production yields exceeding 70%. Furthermore, we considered that isomaltooligosaccharides could be synthesized from starch by combining 1,4-α-glucan 6-α-glucosyltransferase from Paenibacillus sp. PP710 and isopullulanase. In reactions that additionally utilized isoamylase and α-amylase, the total concentration of product, which included a series of isomaltooligosaccharides from isomaltose to isomaltodecaose, was 131 mM, and the ratio of 6-linked glucopyranosyl bonds to all bonds was 91.7% at a substrate concentration of 10%. The development of these manufacturing methods will accelerate the industrial production of isomaltose and isomaltooligosaccharides.

International Journal of Molecular Sciences. 2021 Apr 19;22(8):4208.

• Claudia Penna*1, Saveria Femminò*1*2, Fabrizio Caldera*3, Alberto Rubin Pedrazzo*3, Claudio Cecone*3, Edoardo Alfì*1, Stefano Comità*1, Takanobu Higashiyama, Francesco Trotta*3, Pasquale Pagliaro*1, Roberta Cavalli*4
• • *1 Department of Clinical and Biological Sciences, University of Turin, 10043 Turin, Italy.
  • *2 Department of Medical Sciences, University of Turin, 10126 Turin, Italy.
  • *3 Department of Chemistry, University of Turin, 10125 Turin, Italy.
  • *4 Department of Drug Science and Technology, University of Turin, 10125 Turin, Italy.

Heart failure (HF) prevalence is increasing among the aging population, and the mortality
rate remains unacceptably high despite improvements in therapy. Myocardial ischemia (MI) and, consequently, ischemia/reperfusion injury (IRI), are frequently the basis of HF development. Therefore, cardioprotective strategies to limit IRI are mandatory. Nanocarriers have been proposed as alternative therapy for cardiovascular disease. Controlled reoxygenation may be a promising strategy. Novel nanocarriers, such as cyclic nigerosyl-nigerose (CNN), can be innovative tools for oxygen delivery in a controlled manner. In this study we analyzed new CNN-based formulations as oxygen nanocarriers (O₂-CNN), and compared them with nitrogen CNN (N₂-CNN). These different CNN-based formulations were tested using two cellular models, namely, cardiomyoblasts (H9c₂), and endothelial (HMEC) cell lines, at different concentrations. The effects on the growth curve during normoxia (21% O₂, 5% CO₂ and 74% N₂) and their protective effects during hypoxia (1% O₂, 5% CO₂ and 94% N₂) and reoxygenation (21% O₂, 5% CO₂ and 74% N₂) were studied. Neither O₂-CNN nor N₂-CNN has any effect on the growth curve during normoxia. However, O₂-CNN applied before hypoxia induces a 15-30% reduction in cell mortality after hypoxia/re-oxygenation when compared to N₂-CNN. O₂-CNN showed a marked efficacy in controlled oxygenation, which suggests an interesting potential for the future medical application of soluble nanocarrier systems for MI treatment.

KDD '20: Proceedings of the 26th ACM SIGKDD International Conference on Knowledge Discovery & Data MiningAugust 2020 Pages 2961-2969. https://doi.org/10.1145/3394486.3403346

• Seiji Takeda*1, Toshiyuki Hama*1, Hsiang-Han Hsu*1, Victoria A. Piunova*2, Dmitry Zubarev*2, Daniel P. Sanders*2, Jed W. Pitera*2, Makoto Kogoh*3, Takumi Hongo*3, Yenwei Cheng*3, Wolf Bocanett*3, Hideaki Nakashika*3, Akihiro Fujita, Yuta Tsuchiya, Katsuhiko Hino, Kentaro Yano, Shuichi Hirose*4, Hiroki Toda*4, Yasumitsu Orii*4, Daiju Nakano*1
• • *1 IBM Research - Tokyo
  • *2 IBM Almaden Research Center
  • *3 IBM Garage, Tokyo Laboratory
  • *4 NAGASE Co., Ltd.

The discovery of new materials is essential for the sustainable development of industry. However, it is realistically difficult for human professionals to examine all possibilities for new materials. In this paper, we constructed a molecular inverse design platform using artificial intelligence as a web service. In this platform, it becomes possible to output the chemical structure with the desired property to the artificial intelligence by allowing the relationship between the structure and the property of the compound to be learned to the artificial intelligence. This time, we developed a system especially for dyes and carbohydrates to obtain the target structure. The development process and results of the system are reported in this paper.

Carbohydrate ResearchVolume 496, October 2020, 108104

• Akihiro Fujita, Akira Kawashima, Hiromi Ota*, Hikaru Watanabe, Tetsuya Mori,Tomoyuki Nishimoto, Hajime Aga, Shimpei Ushio
• * Okayama University

Two strains isolated from soils, Agreia sp. D1110 and Microbacterium trichocethenolyticum D2006, were revealed to enzymatically produce a novel cyclic tetrasaccharide cycloisomaltotetraose (CI4). Single crystals of CI4 were also obtained to clarify the stereostructure of CI4. CI4 in CI4 single crystals revealed that the two glucosyl residues each exhibited a unique 3D structure facing each other, which was greatly different from the known cyclic tetrasaccharide. This is the first report on CI4 molecules and their unique crystallographic structure.

Nutrition Journal (2020) 19:68

• Chiyo Yoshizane, Akiko Mizote, Chikako Arai, Norie Arai, Rieko Ogawa, Shin Endo, Hitoshi Mitsuzumi, Shimpei Ushio

One teaspoon (3.3g) of trehalose per day was consumed for 12 weeks, and a glucose tolerance test was performed before and after ingestion to examine the ease of returning postprandial blood glucose levels. As the result, the blood glucose level after meals was easily returned by the trehalose intake as well as the previously reported trehalose intake of 10g. Daily intake of one teaspoon of trehalose is easily incorporated into a meal, and may therefore contribute to the prevention of pathologies that are predisposed to by postprandial hyperglycemia.

PLoS ONE 15(11): e0241912　(2020)

• Masaki Kohno*1, Takatoshi Arakawa*1,2, Naoki Sunagawa*3, Tetsuya Mori, Kiyohiko Igarashi*3,4, Tomoyuki Nishimoto, Shinya Fushinobu*1,2
• • *1 Department of Biotechnology, The University of Tokyo, Tokyo, Japan,
  • *2 Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo, Japan
  • *3 Department of Biomaterial Sciences, The University of Tokyo, Tokyo, Japan
  • *4 VTT Technical Research Centre of Finland Ltd., Espoo, Finland

Cyclic α-maltosyl-(1→6)-maltose (CMM) is a cyclic glucotetrasaccharide with alternating α-1,4 and α-1,6 linkages. Here, we report functional and structural analyses on CMM-binding protein (CMMBP), which is a substrate-binding protein (SBP) of an ABC importer system of the bacteria Arthrobacter globiformis. Isothermal titration calorimetry analysis revealed that CMMBP specifically bound to CMM with a Kd value of 9.6 nM. The crystal structure of CMMBP was determined at a resolution of 1.47 Å, and a panose molecule was bound in a cleft between two domains. To delineate its structural features, the crystal structure of CMMBP was compared with other SBPs specific for carbohydrates, such as cyclic α-nigerosyl-(1→6)-nigerose and cyclodextrins. These results indicate that A. globiformis has a unique metabolic pathway specialized for CMM.

Medical Consultation ＆ New Remedies 57(2), 129-134 (2020)

• Naoki Morishita, Saori Ogihara, Shin Endo, Hitoshi Mitsuzumi, Shimpei Ushio

Twenty-four healthy adult men and women took a single dose of 100 mg of glucosyl hesperidin and examined blood flow and the temperature of the skin after cold water loading. The results showed that intake of 100 mg of glycosylated hesperidin, as well as 340 mg previously reported, promoted the recovery of blood flow and the temperature of the skin after cold water loading.

JAPANESE PHAMACOLOGY & THERAPEUTICS 48(8), 1437-1446 (2020)

• Yuki Ishida, Tsuyoshi Sadakiyo, Shin Endo, Hitoshi Mitsuzumi, Shimpei Ushio, Nobuhiko Shioya*1, Yoshitaka Iwama*2
• • *1 KSO Corporation
  • *2 Nihonbashi Cardiology Clinic

We examined the inhibitory effect of IMD on postprandial triglycerides (TG) elevation in the subjects with high-normal to slightly-high fasting TG levels. The results showed that AUC of TG and Remnant-like Lipoprotein particle cholesterol (RLP-C) after intake of the high-fat diet with IMD was significantly lower than the placebo. Similar results were seen in a sub-group analysis of subjects with normal fasting TG levels.

Natural Product Communications 15(11): 1-6 (2020)

• Yoshio Suzuki*1, 2, Kotaro Sato*2, Norie Arai, Shin Endo
• • *1 Juntendo University, Graduate School of Health and Sports Science
  • *2 Juntendo University, Faculty of Health and Sports Science

Pre-exercise trehalose ingestion enhances exercise performance within 30 minutes. To examine whether the enhanced effect was due to a mouth rinse effect, ten healthy male collegiate distance runners performed an exercise assessment　composed of 6-second peak power and endurance tests. Before each test, they rinsed their mouths with either trehalose (6% w/v), maltose (6% w/v) or acesulfame potassium (0.04 mg/mL) for 5 seconds. Trehalose induced the highest mean power output in peak power tests. In the endurance test, trehalose consistently showed higher mean power output than maltose.
Trehalose activates taste receptors T1R1-T1R3, T1R3-T1R3 homodimer, and T1R2-T1R3, whereas sucrose activates only T1R2-T1R3. Therefore, a difference in mouth rinse effect might be due to a specific receptor in the oral cavity that recognizes differences between trehalose and maltose.

Gastroenterology, 158:1402-1416 (2020)

• Yiming Zhang*1, Nurmohammad Shaikh*1, Jeremie L. Ferey*2, Umesh D. Wankhade*3, Sree V. Chintapalli*3, Cassandra B. Higgins*1, Jan R. Crowley*4, Monique R. Heitmeier*1, Alicyn I. Stothard*5, Belgacem Mihi*1, Misty Good*1, Takanobu Higashiyama, Benjamin M. Swarts*5, Paul W. Hruz*1, Kartik Shankar*3, Phillip I. Tarr*1,6, Brian J. DeBosch*1,7
• • *1 Department of Pediatrics, Washington University School of Medicine
  • *2 Department of Obstetrics and Gynecology, Washington University School of Medicine
  • *3 Department of Pediatrics, University of Arkansas Medical School
  • *4 Department of Medicine, Washington University School of Medicine
  • *5 Department of Chemistry and Biochemistry, Central Michigan University
  • *6 Department of Molecular Microbiology, Washington University School of Medicine
  • *7 Department of Cell Biology and Physiology, Washington University School of Medicine

Lactotrehalose is a trehalaseresistant analogue that increases metabolic parameters, compared with trehalose, without increasing the abundance or virulence of C. difficile strain CD027. Trehalase-resistant trehalose analogues might be developed as next-generation fastingmimetics for the treatment of diabetes and nonalcoholic fatty liver disease.

Medical Consultation ＆ New Remedies 56(5), 358-364 (2019)

• Takeo Sakurai, Yuki Ishida, Shin-ichiro Inoue, Mayumi Kurose, Yukinobu Uchida, Nariaki Yoshida, Tsuyoshi Sadakiyo, Hikaru Watanabe, Hitoshi Mitsuzumi, Shimpei Ushio, Junichi Hiramatsu*
• * Health Research Institute, National Institute of Advanced Industrial Science and Technology

Overnight-fasted subjects consumed bread (containing 2.93 g IMD or not containing IMD) with breakfast, and blood glucose levels were determined over time from immediately before to 120 minutes after meal intake. A stratified analysis of subjects with a tendency for increased postprandial blood glucose levels showed a significant higher attenuating effect of IMD intake on blood glucose elevation compared with non-IMD intake in terms of blood glucose levels over time and area under the curve.

Sports 2019, 7, 100; doi:10.3390/sports7050100

• Tsuyoshi Wadazumi*1, Kanji Watanabe*2, Hitoshi Watanabe*3, Hisayo Yokoyama*3, Nobuko Hongu*4, Norie Arai
• • *1 Kansai University,
  • *2 Mukogawa Womes’s University
  • *3 Osaka City University
  • *4 University of Arizona

In the prolonged exercise test, the effect of trehalose single ingestion on exercise performance and energy utilization was investigated, and the results showed that it was effective in improving the performance at the later stages of exercise through effective utilization of glucose and lipids.

In Proceedings of KDD 2019: Workshop on Applied Data Science for Healthcare (KDD 2019 Workshop). ACM, New York, NY, USA, 4 pages.

• Matteo Manica*1, Christoph Auer*1, Valery Weber*1, Federico Zipoli*1, Michele Dolfi*1, Peter Staar*1, Teodoro Laino*1, Costas Bekas*1, Akihiro Fujita, Hiroki Toda*2, Shuichi Hirose*2, Yasumitsu Orii*2
• • *1 IBM Zurich
  • *2 NAGASE CO., LTD.

In human exploration, there are discoveries that are unnoticed due to the preconceptions of the researchers and the limited amount of information that can be processed. In this paper, we have created a knowledge graph on biochemistry by extracting information from articles and public databases in the field. It is expected that this knowledge graph will lead to discoveries that have not been noticed by humans. It is also expected to significantly reduce the search time.

Medical Consultation ＆ New Remedies 56(12), 907-916 (2019)

• Yuki Ishida, Tsuyoshi Sasakiyo, Hikaru Watanabe, Hitoshi Mitsuzumi, Shimpei Ushio, Shuji Nakada*
• * Medical Corporation Bokushinkai CLINITEXE Clinic

Healthy men and women with awareness of dry skin symptoms and a tendency toward constipation constipation consumed IMD for 8 weeks. The results showed that IMD had an improvement in skin condition with evaluation by a dermatologist. Furthermore, in those who had a relatively high serum phenol concentration, which is an indicator of the composition of the intestinal environment, the serum phenol concentration was significantly lower after intake of IMD versus placebo, and a significantly higher moisture content of the skin on the foot.

Applied Physics Express 12(9), 097003.1-097003.5 (2019)

• Haruka Omachi*1-2, Tomohiko Komuro*1, Kaisei Matsumoto*1, Minako Nakajima*2, Hikaru Watanabe, Jun Hirotani*3, Yutaka Ohno*3-4, Hisanori Shinohara*1
• • *1 Department of Chemistry, Graduate School of Science, Nagoya University
  • *2 Research Center for Materials Science, Nagoya University
  • *3 Department of Electronics, Graduate School of Engineering, Nagoya University
  • *4 Institute of Materials and Systems for Sustainability, Nagoya University

We developed a rapid single-step aqueous two-phase extraction of high-purity semiconducting single-wall carbon nanotubes (s-SWCNTs) using isomaltodextrin, which is a cost-effective polysaccharide that contains a large amount of α-1,6-glucosidic linkages. We also succeeded in fabricating high-density uniform films directly from the high-purity s-SWCNT extract. Fabricated thin-film transistors (TFTs) exhibited an excellent on/off ratio, carrier mobility, and on-current density.