論文 - 上地　敬子

Characterization of an antifungal β-1,3-glucanase from <i>Ficus microcarpa</i> latex and comparison of plant and bacterial β-1,3-glucanases for fungal cell wall β-glucan degradation

Takashima, T; Komori, N; Uechi, K; Taira, T

PLANTA ( Planta )  258 ( 6 ) 116 - 116   2023年12月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Each β-1,3-glucanase with antifungal activity or yeast lytic activity hydrolyzes different structures of β-1,3-glucans in the fungal cell wall, respectively. Plants express several glycoside hydrolases that target chitin and β-glucan in fungal cell walls and inhibit pathogenic fungal infection. An antifungal β-1,3-glucanase was purified from gazyumaru (Ficus microcarpa) latex, designated as GlxGluA, and the corresponding gene was cloned and expressed in Escherichia coli. The sequence shows that GlxGluA belongs to glycoside hydrolase family 17 (GH17). To investigate how GlxGluA acts to degrade fungal cell wall β-glucan, it was compared with β-1,3-glucanase with different substrate specificities. We obtained recombinant β-1,3-glucanase (designated as CcGluA), which belongs to GH64, from the bacterium Cellulosimicrobium cellulans. GlxGluA inhibited the growth of the filamentous fungus Trichoderma viride but was unable to lyse the yeast Saccharomyces cerevisiae. In contrast, CcGluA lysed yeast cells but had a negligible inhibitory effect on the growth of filamentous fungi. GlxGluA degraded the cell wall of T. viride better than CcGluA, whereas CcGluA degraded the cell wall of S. cerevisiae more efficiently than GlxGluA. These results suggest that the target substrates in fungal cell walls differ between GlxGluA (GH17 class I β-1,3-glucanase) and CcGluA (GH64 β-1,3-glucanase).

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Structural Analysis and Construction of a Thermostable Antifungal Chitinase.

Kozome D, Uechi K, Taira T, Fukada H, Kubota T, Ishikawa K

Applied and environmental microbiology ( Applied and Environmental Microbiology )  88 ( 12 ) e0065222   2022年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Preparation and Crystal Structure Analysis of High-Strength Film Derived from Nigeran Ester Derivatives

Togo A.

Journal of Fiber Science and Technology ( 一般社団法人 繊維学会 )  78 ( 8 ) 126 - 132   2022年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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<p>Nigeran (<i>α</i>-1,3-<i>alt</i>-<i>α</i>-1,4-glucan) is a linear glucan with alternating <i>α</i>-1,3- and <i>α</i>-1,4-glycosidic linkages. It is extracted from the cell walls of certain species in the genera <i>Aspergillus</i> and <i>Penicillium</i>. Nigeran can be esterified and used as a film, but its strength is only approximately 4–25 MPa. However, in the present study, high-strength nigeran ester films with tensile strengths of 100 MPa were successfully prepared by thermally stretching and annealing the melt-quenched films. Two-dimensional wide-angle X-ray diffraction (2D-WAXD) analysis revealed that the highly oriented films of nigeran butyrate (NGBu), nigeran valerate (NGVa), and nigeran hexanoate (NGHx) studied herein had two-fold helix symmetry along the molecular axis. Assuming the crystal systems to be orthorhombic, the unit lattice of each ester was calculated as NGBu (<i>a</i>=28.6Å, <i>b</i>=9.07Å, <i>c</i>=16.5Å), NGVa (<i>a</i>=31.5Å, <i>b</i>=9.07Å, <i>c</i>=16.2Å), and NGHx (<i>a</i>=36.7Å, <i>b</i>=9.07Å, <i>c</i>=16.2Å). In the calculated unit lattice parameters, only the <i>a</i>-axis was extended as the number of ester carbons increased.</p>

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Identification of Genes Involved in the Synthesis of the Fungal Cell Wall Component Nigeran and Regulation of Its Polymerization in Aspergillus luchuensis

Uechi K, Yaguchi H, Tokashiki J, Taira T, Mizutani O

Applied and environmental microbiology ( Applied and Environmental Microbiology )  87 ( 21 ) e0114421   2021年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Certain Aspergillus and Penicillium spp. produce the fungal cell wall component nigeran, an unbranched D-glucan with alternating α-1,3- and α-1,4-glucoside linkages, under nitrogen starvation. The mechanism underlying nigeran biosynthesis and the physiological role of nigeran in fungal survival are not clear. We used RNA-seq to identify genes involved in nigeran synthesis in the filamentous fungus Aspergillus luchuensis when grown under nitrogen-free conditions. agsB, which encodes a putative α-1,3-glucan synthase, and two adjacent genes (agtC and gnsA) were upregulated under conditions of nitrogen starvation. Disruption of agsB in A. luchuensis (ΔagsB) resulted in the complete loss of nigeran synthesis. Furthermore, overexpression of agsB in an Aspergillus oryzae strain that cannot produce nigeran resulted in nigeran synthesis. These results indicated that agsB encodes a nigeran synthase. Therefore, we have renamed the A. luchuensis agsB as nigeran synthase gene (nisA). Nigeran synthesis in an agtC mutant (ΔagtC) increased to 121%; conversely, that in ΔgnsA and ΔagtCgnsA decreased to 64% and 63%, respectively, compared to that in the wild-type strain. Our results revealed that AgtC and GnsA play an important role in regulating not only the quantity of nigeran but also its polymerization. Collectively, our results demonstrated that nisA (agsB) is essential for nigeran synthesis in A. luchuensis, whereas agtC and gnsA contribute to the regulation of nigeran synthesis and its polymerization. This research provides insights into fungal cell wall biosynthesis, specifically the molecular evolution of fungal α-glucan synthase genes and the potential utilization of nigeran as a novel biopolymer. Importance The fungal cell wall is composed mainly of polysaccharides. Under nitrogen-free conditions, some Aspergillus and Penicillium spp. produce significant levels of nigeran, a fungal cell wall polysaccharide composed of alternating α-1,3-/1,4-glucosidic linkages. The mechanisms regulating the biosynthesis and function of nigeran are unknown. Here, we performed RNA sequencing of Aspergillus luchuensis cultured under nitrogen-free or low-nitrogen conditions. A putative α-1,3-glucan synthase gene, whose transcriptional level was upregulated under nitrogen-free conditions, was demonstrated to encode nigeran synthase. Furthermore, two genes encoding an α-glucanotransferase and a hypothetical protein were shown to be involved in controlling nigeran content and molecular weight. This study reveals genes involved in the synthesis of nigeran, a potential biopolymer, and provides a deeper understanding of fungal cell wall biosynthesis.

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Synthesis and characterization of α-1,3-<i>alt</i>-α-1,4-glucan (nigeran) ester derivatives (vol 214, 123343, 2021)

Togo, A; Uechi, K; Mizutani, O; Kimura, S; Iwata, T

POLYMER   226   2021年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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cDNA cloning, expression, and antifungal activity of chitinase from Ficus microcarpa latex: difference in antifungal action of chitinase with and without chitin-binding domain.

Takashima T, Henna H, Kozome D, Kitajima S, Uechi K, Taira T

Planta ( Planta )  253 ( 6 ) 120 - 120   2021年05月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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MAIN CONCLUSION: A chitin-binding domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions. Complementary DNA encoding the antifungal chitinase of gazyumaru (Ficus microcarpa), designated GlxChiB, was cloned and expressed in Escherichia coli cells. The results of cDNA cloning showed that the precursor of GlxChiB has an N-terminal endoplasmic reticulum targeting signal and C-terminal vacuolar targeting signal, whereas mature GlxChiB is composed of an N-terminal carbohydrate-binding module family-18 domain (CBM18) and a C-terminal glycoside hydrolase family-19 domain (GH19) with a short linker. To clarify the role of the CBM18 domain in the antifungal activity of chitinase, the recombinant GlxChiB (wild type) and its catalytic domain (CatD) were used in quantitative antifungal assays under different ionic strengths and microscopic observations against the fungus Trichoderma viride. The antifungal activity of the wild type was stronger than that of CatD under all ionic strength conditions used in this assay; however, the antifungal activity of CatD became weaker with increasing ionic strength, whereas that of the wild type was maintained. The results at high ionic strength further verified the contribution of the CBM18 domain to the antifungal ability of GlxChiB. The microscopic observations clearly showed that the wild type acted on both the tips and the lateral wall of fungal hyphae, while CatD acted only on the tips. These results suggest that the CBM18 domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions.

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Cloning, expression, and characterization of a GH 19-type chitinase with antifungal activity from Lysobacter sp. MK9-1.

Yano S, Kanno H, Tsuhako H, Ogasawara S, Suyotha W, Konno H, Makabe K, Uechi K, Taira T

Journal of bioscience and bioengineering ( Journal of Bioscience and Bioengineering )  131 ( 4 ) 348 - 355   2021年04月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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The chitin-assimilating gram-negative bacterium, Lysobacter sp. MK9-1, was isolated from soil and was the source of a glycoside hydrolase family 19-type chitinase (Chi19MK) gene that is 933-bp long and encodes a 311-residue protein. The deduced amino acid sequence of Chi19MK includes a signal peptide, an uncharacterized sequence, a carbohydrate-binding module family 12-type chitin binding domain, and a catalytic domain. The catalytic domain of Chi19MK is approximately 60% similar to those of ChiB from Burkholderia gladioli CHB101, chitinase N (ChiN) from Chitiniphilus shinanonensis SAY3T, ChiF from Streptomyces coelicolor A3(2), Chi30 from Streptomyces olivaceoviridisis, ChiA from Streptomyces cyaneus SP-27, and ChiC from Streptomyces griseus HUT6037. Chi19MK lacking the signal and uncharacterized sequences (Chi19MKΔNTerm) was expressed in Escherichia coli Rosetta-gami B(DE3), resulting in significant chitinase activity in the soluble fraction. Purified Chi19MKΔNTerm hydrolyzed colloidal chitin and released disaccharide. Furthermore, Chi19MKΔNTerm inhibited hyphal extension in Trichoderma reesei and Schizophyllum commune. Based on quantitative antifungal activity assays, Chi19MKΔNTerm inhibits the growth of Trichoderma viride with an IC50 value of 0.81 μM.

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Synthesis and characterization of α-1,3-alt-α-1,4-glucan (nigeran) ester derivatives

Azusa Togo, Keiko Uechi, Osamu Mizutani, Satoshi Kimura, Tadahisa Iwata

Polymer ( Elsevier Ltd )  214   123343   2021年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Nigeran is a linear alpha-D-glucan with alternating alpha-1,3- and alpha-1,4-glycosidic linkages. Nigeran (alpha-1,3-alt-alpha-1,4-glucan) esters with different ester chain lengths were synthesized from high molecular weight nigeran extracted from mycelium of Aspergillus oryzae over-expressing a nigeran synthase gene derived from Aspergillus luchuensis. All prepared nigeran esters were crystalline polymers with high melting point, including those with ester groups longer than octanoate. This unique crystallization behavior is not reported in other polysaccharide esters. Both solvent-cast and melt-quenched films were self-standing and high transparency. Solvent-cast films were stretched up to four times the initial length and showed well-oriented X-ray fiber diagrams. Nigeran propionate had twofold screw symmetry along the molecular axis (same as neat nigeran), suggesting that the molecular conformation remained unchanged despite esterification. This result indicates that the molecular conformation of nigeran with its alpha-1,3-alt-alpha-1,4-glycosidic linkage is very stable despite chemical modification and retains high crystallinity.

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Phenolic acid decarboxylase of Aspergillus luchuensis plays a crucial role in 4-vinylguaiacol production during awamori brewing

Mayumi Maeda, Marin Motosoko, Tatsunori Tokashiki, JikianTokashiki, Osamu Mizutani, Keiko Uechi, Masatoshi Goto, Toki Taira

Journal of Bioscience and Bioengineering   130 ( 4 ) 352 - 359   2020年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Antifungal activities of LysM-domain multimers and their fusion chitinases.

Takashima T, Sunagawa R, Uechi K, Taira T

International journal of biological macromolecules ( International Journal of Biological Macromolecules )  154   1295 - 1302   2019年11月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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PrChiA is an antifungal chitinase obtained from Pteris ryukyuensis, a fern plant. It consists of two N-terminal lysin motif (LysM) domains and a C-terminal catalytic domain of glycoside hydrolase family 18. Previous studies have shown that the deletion of LysM domains or loss of hydrolytic activity causes the loss of the antifungal activity of chitinases. In this study, we produced LysM-domain multimers (LysMn, n = 2-5) and the respective multimer fusion chitinases (LysMn-Cat, n = 1-4), and characterized their enzymatic and antifungal properties. LysMn and LysMn-Cat showed a higher affinity to insoluble chitin than single LysM domain and single catalytic domain alone, respectively. LysMn-Cat hydrolyzed insoluble chitin more efficiently than the catalytic domain alone. Surprisingly, LysMn showed antifungal activity without chitinolytic activity. Further, LysMn-Cat exhibited a stronger antifungal activity than LysMn. Microscopic observation revealed that LysMn attacked only the tips of the fungal hyphae; LysMn-Cat attacked not only the tips, but also the lateral walls around the septa of the fungal hyphae. It is suggested that the LysMn act on the growing point of the hyphal tip through their chitin-binding ability and that the LysMn-Cat act on not only the hyphal tips, but also on the lateral walls through their chitin-hydrolyzing and -binding activities.

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Characterization of D-glucoside 3-dehydrogenase from Rhizobium sp. L35 and its application for D-allose production

Yotsombat A, Uechi K., Onishi S., Mino K., Hasegawa T., Morimoto K., Takata G

Chiang Mai University Journal of Natural Sciences ( Chiang Mai University Journal of Natural Sciences )  18 ( 3 ) 267 - 284   2019年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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© 2019, CMU J. Nat. Sci. D-allose is a rare monosaccharide that possesses some interesting features for enhancing the antitumor effects of chemoradiotherapy and treatment of cancer, however, efficient production of D-allose with high percent yield has not yet been reported. In this study, we proposed a utilization of D-glucoside 3-dehydrogenase together with chemical reaction to improve the D-allose production. A D-glucoside 3-dehydrogenase, which regioselectively dehydrogenates glycosides at the C-3 position to the corresponding 3-ketoglycoside, was newly isolated as Rhizobium sp. L35 and characterized as a flavin adenine dinucleotide-dependent dehydrogenase. Its molecular weight was determined to be 67,000 by SDS-PAGE and 131,000 by size-exclusion chromatography, suggesting that it is a dimeric enzyme. Its optimum pH and temperature with respect to activity were 7.5 and 40°C, respectively. It was stable between pH 6.0 and 11.0, and below 40°C (half-life of 3 h at 40°C and 50 min at 45°C). The enzyme showed broad substrate specificity towards various glycosides, especially β-1,4-linked disaccharides such as cellobiose and lactose. Finally, D-allose production was performed by a three-step process of enzymatic-dehydrogenation, chemical reduction and acid-hydrolysis, using cellobiose as the starting material. The yield of D-allose was estimated to be 30% from cellobiose. This result indicates that D-allose can be produced by this strategy three-fold higher than the conventional method.

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Purification, cDNA cloning, and characterization of plant chitinase with a novel domain combination from lycophyte Selaginella doederleinii.

Kuba Y, Takashima T, Uechi K, Taira T

Bioscience, biotechnology, and biochemistry ( Bioscience, Biotechnology and Biochemistry )  82 ( 10 ) 1742 - 1752   2018年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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【ミニレビュー：―第6回応用糖質フレッシュシンポジウム―】　希少糖生産酵素L-リブロース3-エピメラーゼの機能と立体構造の相関性について

上地 敬子, 櫻庭 春彦, 高田 悟郎

応用糖質科学：日本応用糖質科学会誌 ( 一般社団法人 日本応用糖質科学会 )  8 ( 3 ) 224 - 228   2018年08月

掲載種別: 研究論文（学術雑誌）

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<p>希少糖とは自然界にごくわずかにしか存在しない糖類の総称であり，一部の希少糖にはがん細胞の増殖抑制効果，食後血糖値の上昇抑制効果などの生理活性が見出されている．我々は，希少糖生産酵素の1つであるケトース3-エピメラーゼについて研究を行っており，<i>Mesorhizobium loti</i>由来の新規L-リブロース3-エピメラーゼ(L-RE)を見出した．既知のケトース3-エピメラーゼがケトヘキソースに最も高い活性を示すのに対して，L-REはケトペントースに高い活性を示し，また熱安定性が高いことが明らかとなった．L-REを結晶化し，X線回折実験に供して2.7Åの分解能で構造を決定した．ホモテトラマーであるL-REのモノマーの構造は，構造既知のケトース3-エピメラーゼとよく類似していたがC末端領域のα-ヘリックスが長くなっており，隣接するサブユニットとこの部分の間に形成された特異的な相互作用が，L-REの高い熱安定性に寄与していることを明らかにした．また活性部位の構造を比較した結果，L-REの活性部位キャビティの体積が他の酵素より小さいことが，L-REのケトペントースに対する高い活性の要因であると推測された．</p>

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Characterization and induction of phenolic acid decarboxylase from Aspergillus luchuensis.

Maeda M, Tokashiki M, Tokashiki M, Uechi K, Ito S, Taira T

Journal of bioscience and bioengineering ( Journal of Bioscience and Bioengineering )  126 ( 2 ) 162 - 168   2018年08月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Amylolytic Enzymes Acquired from L-Lactic Acid Producing Enterococcus faecium K-1 and Improvement of Direct Lactic Acid Production from Cassava Starch.

Unban K, Kanpiengjai A, Takata G, Uechi K, Lee WC, Khanongnuch C

Applied biochemistry and biotechnology   183 ( 1 ) 155 - 170   2017年09月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Crystal structure of an acetyl esterase complexed with acetate ion provides insights into the catalytic mechanism.

Uechi K, Kamachi S, Akita H, Mine S, Watanabe M

Biochemical and biophysical research communications   477 ( 3 ) 383 - 7   2016年08月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Structure-Function relationship of a gellan family of polysaccharide, S-198 gum, produced by Alcaligenes ATCC31853

田幸正邦, 北島聖子, 與儀琢也, 上地敬子, 翁長正行, 玉城志博, 上地俊徳

Advances in Biological Chemistry   6 ( 3 ) 55 - 69   2016年05月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

Novel process for producing 6-deoxy monosaccharides from l-fucose by coupling and sequential enzymatic method.

Shompoosang S, Yoshihara A, Uechi K, Asada Y, Morimoto K

Journal of bioscience and bioengineering   121 ( 1 ) 1 - 6   2016年01月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Essentiality of tetramer formation of Cellulomonas parahominis L-ribose isomerase involved in novel L-ribose metabolic pathway.

Terami Y, Yoshida H, Uechi K, Morimoto K, Takata G, Kamitori S

Applied microbiology and biotechnology   99 ( 15 ) 6303 - 13   2015年08月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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Production of L-allose and D-talose from L-psicose and D-tagatose by L-ribose isomerase.

Terami Y, Uechi K, Nomura S, Okamoto N, Morimoto K, Takata G

Bioscience, biotechnology, and biochemistry   79 ( 10 ) 1725 - 9   2015年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

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