Browsing by Author "A. Kosugi; Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, 1-1 Ohwashi, 305-8686, Japan; email: akosugi@affrc.go.jp"

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    Characterization of an Anaerobic, Thermophilic, Alkaliphilic, High Lignocellulosic Biomass-Degrading Bacterial Community, ISHI-3, Isolated from Biocompost
    (Elsevier Inc., 2018) Ayumi Shikata; Junjarus Sermsathanaswadi; Phakhinee Thianheng; Sirilak Baramee; Chakrit Tachaapaikoon; Rattiya Waeonukul; Patthra Pason; Khanok Ratanakhanokchai; Akihiko Kosugi; A. Kosugi; Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, 1-1 Ohwashi, 305-8686, Japan; email: akosugi@affrc.go.jp
    The generation of a complex microbial consortium is a promising approach for efficient biomass decomposition. An anaerobic thermophilic alkaliphilic microbial consortium with efficient degradation ability was screened from bovine manure compost using non-pretreated milling corn stover (CS) and rice straw (RS). A stable microbial consortium ISHI-3 with high degradation ability for CS and RS was isolated by the roll tube technique. ISHI-3 comprised Herbivorax saccincola and bacteria belonging to the classes Pelotomaculum, Tepidanaerobacter, and Tepidimicrobium, as determined by DGGE of the PCR-generated 16S rRNA genes. Furthermore, metagenomics analysis using a 16S rRNA library was carried out to determine the bacterial distribution during degradation of CS and RS. H. saccincola and bacteria belonging to Pelotomaculum were relatively abundant in the beginning to middle periods of culture with CS and RS whereas bacteria belonging to Tepidanaerobacter and Tepidimicrobium gradually increased in the population during the later stages. To understand the role of non-cellulolytic bacteria in the consortium, novel strains ET1 and GL4, which were most closely related to Tepidimicrobium ferriphilum and Tepidanaerobacter acetatoxydans, were isolated from ISHI-3. Based on their carbon source usage, morphology, and phylogenetic analysis, we propose that strains ET1 and GL4 should be classified as a novel genus or species. Bacteria ET1 and GL4 can utilize different organic compounds as carbon and energy sources such as organic acids, alcohols, sugars, and amino acids, showing a preference for organic acids and alcohols rather than sugars such as glucose and cellobiose. These results indicated that ET1 and GL4 help to accelerate efficient lignocellulose degradation of H. saccincola. � 2018 Elsevier Inc.
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    Molecular characterization of hypothetical scaffolding-like protein S1 in multienzyme complex produced by Paenibacillus curdlanolyticus B-6
    (Springer Verlag, 2019) Patthra Pason; Junjarus Sermsathanaswadi; Rattiya Waeonukul; Chakrit Tachaapaikoon; Sirilak Baramee; Khanok Ratanakhanokchai; Akihiko Kosugi; A. Kosugi; Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, 1-1 Ohwashi, 305-8686, Japan; email: akosugi@affrc.go.jp
    Paenibacillus curdlanolyticus B-6 produces an extracellular multienzyme complex containing a hypothetical scaffolding-like protein and several xylanases and cellulases. The largest (280-kDa) component protein, called S1, has cellulose-binding ability and xylanase activity, thus was considered to function like the scaffolding proteins found in cellulosomes. S1 consists of 863 amino acid residues with predicted molecular mass 91,029�Da and includes two N-terminal surface layer homology (SLH) domains, but most of its sequence shows no homology with proteins of known function. Native S1 (nS1) was highly glycosylated. Purified nS1 and recombinant Xyn11A (rXyn11A) as a major xylanase subunit could assemble in a complex, but recombinant S1 (rS1) could not interact with rXyn11A, indicating that S1 glycosylation is necessary for assembly of the multienzyme complex. nS1 and rS1 showed weak, typical endo-xylanase activity, even though they have no homology with known glycosyl hydrolase family enzymes. S1 and its SLH domains bound tightly to the peptide-glycan layer of P. curdlanolyticus B-6, microcrystalline cellulose, and insoluble xylan, indicating that the SLHs of S1 bind to carbohydrate polymers and the cell surface. When nS1 and rXyn11A were co-incubated with birchwood xylan, the degradation ability was synergistically increased compared with that for each protein; however synergy was not observed for rS1 and rXynA. These results indicate that S1 may have a scaffolding protein-like function by interaction with enzyme subunits and polysaccharides through its glycosylated sites and SLH domains. � 2019, The Author(s).
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    The family 22 carbohydrate-binding module of bifunctional xylanase/_-glucanase Xyn10E from Paenibacillus curdlanolyticus B-6 has an important role in lignocellulose degradation
    (Elsevier Inc., 2017) Junjarus Sermsathanaswadi; Sirilak Baramee; Chakrit Tachaapaikoon; Patthra Pason; Khanok Ratanakhanokchai; Akihiko Kosugi; A. Kosugi; Biological Resources and Post-harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba, 1-1 Ohwashi, 305-8686, Japan; email: akosugi@affrc.go.jp
    A newly isolated endo-_-1,4-xylanase (Xyn10E) from Paenibacillus curdlanolyticus B-6 has a modular structure consisting of a family 22 carbohydrate-binding module (CBM), a glycoside hydrolase (GH) family 10 catalytic domain, two fibronectin type III (Fn3) domains, and a family 3 CBM at the C-terminus. Intact Xyn10E (rXyn10E), CBM22-deleted Xyn10E (X-CBM3), CBM3-deleted Xyn10E (X-CBM22), and GH10 catalytic domain only (X-GH10) were expressed in Escherichia coli. rXyn10E showed bifunctional degradation activity toward xylan and _-glucan and also degraded microcrystalline cellulose. Although X-CBM3 and X-GH10 had drastically reduced xylanase and _-glucanase activities, X-CBM22 mostly retained these activities. Similar Km values were obtained for rXyn10E and X-CBM3, but kcat and kcat/Km values for X-CBM3 and X-GH10 were lower than those for rXyn10E, suggesting that CBM22 of Xyn10E may contribute to catalytic efficiency. In binding assays, X-CBM3 was still able to bind to _-glucan, soluble xylan, insoluble xylan, and cellulose through GH10 and CBM3. These results indicate that CBM22 has an important role not only in binding to xylan and _-glucan but also in feeding both polysaccharides into the neighboring GH10 catalytic domain. rXyn10E showed remarkable synergism with rXyn11A, a major xylanase subunit of P. curdlanolyticus B-6, in the degradation of untreated corn stover and sugarcane bagasse; however, the combination of X-CBM3 and rXyn11A was not synergistic. These results indicate that Xyn10E and Xyn11A act synergistically on lignocellulosic biomass, and CBM22 is essential for efficient degradation of lignocellulosic materials. � 2016 Elsevier Inc.