Browsing by Author "Rattiya Waeonukul"

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    Characterization and high-quality draft genome sequence of Herbivorax saccincola A7, an anaerobic, alkaliphilic, thermophilic, cellulolytic, and xylanolytic bacterium
    (Elsevier GmbH, 2018) Shimpei Aikawa; Sirilak Baramee; Junjarus Sermsathanaswadi; Phakhinee Thianheng; Chakrit Tachaapaikoon; Ayumi Shikata; 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
    An anaerobic, cellulolytic-xylanolytic bacterium, designated strain A7, was isolated from a cellulose-degrading bacterial community inhabiting bovine manure compost on Ishigaki Island, Japan, by enrichment culture using unpretreated corn stover as the sole carbon source. The strain was Gram-positive, non-endospore forming, non-motile, and formed orange colonies on solid medium. Strain A7 was identified as Herbivorax saccincola by DNA-DNA hybridization, and phylogenetic analysis based on 16S rRNA gene sequences showed that it was closely related to H. saccincola GGR1 (= DSM 101079T). H. saccincola A7 (= JCM 31827 = DSM 104321) had quite similar phenotypic characteristics to those of strain GGR1. However, the optimum growth of A7 was at alkaline pH (9.0) and 55 �C, compared to pH 7.0 at 60 �C for GGR1, and the fatty acid profile of A7 contained 1.7-times more C17:0 iso than GGR1. The draft genome sequence revealed that H. saccincola A7 possessed a cellulosome-like extracellular macromolecular complex, which has also been found for Clostridium thermocellum and C. clariflavum. H. saccincola A7 contained more glycoside hydrolases (GHs) belonging to GH families-11 and -2, and more diversity of xylanolytic enzymes, than C. thermocellum and C. clariflavum. H. saccincola A7 could grow on xylan because it encoded essential genes for xylose metabolism, such as a xylose transporter, xylose isomerase, xylulokinase, and ribulose-phosphate 3-epimerase, which are absent from C. thermocellum. These results indicated that H. saccincola A7 has great potential as a microorganism that can effectively degrade lignocellulosic biomass. � 2018 Elsevier GmbH
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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 C-terminal region of xylanase domain in Xyn11A from Paenibacillus curdlanolyticus B-6 plays an important role in structural stability
    (Springer Verlag, 2014) Junjarus Sermsathanaswadi; Somsak Pianwanit; Patthra Pason; Rattiya Waeonukul; Chakrit Tachaapaikoon; Khanok Ratanakhanokchai; Krisna Septiningrum; Akihiko Kosugi
    Paenibacillus curdlanolyticus B-6 produces an extracellular multienzyme complex containing a major xylanase subunit, designated Xyn11A, which includes two functional domains belonging to glycosyl hydrolase family-11 (GH11) and carbohydrate binding module family-36 (CBM36) and possesses a glycine and asparagine-rich linker (linker). To clarify the roles of each functional domain, recombinant proteins XynXL and XynX (CBM36 deleted and CBM36 and linker deleted, respectively) were constructed. Their xylanase activities were similar toward soluble xylan, whereas XynXL showed decreased hydrolysis activity toward insoluble xylan while XynX had no xylanase activity. To determine the significance of the linker and its neighbor region, XynX was subjected to secondary structural alignments using circular dichroism (CD) spectroscopy and three-dimensional (3D) structural analysis. A seven amino acid (NTITIGG) neighbor linker sequence was highly conserved among GH11 xylanases of Paenibacillus species. Although XynX exhibited a typical GH11 xylanase structure, conformational gaps were observed in the _6- and _12-sheets and in CD spectra. Flipping of the Arg163 side chains in the subsite was also observed upon analysis of superimposed models. Docking analysis using xylohexaose indicated that flipping of the Arg163 side chains markedly affected substrate binding in the subsite. To identify the amino acids related to stabilizing the substrate binding site, XynX with an extended C-terminal region was designed. At least seven amino acids were necessary to recover substrate binding and xylanase activity. These results indicated that the seven amino acid neighbor Xyn11A linker plays an important role in the activity and conformational stability of the xylanase domain. © 2014, Springer-Verlag Berlin Heidelberg.
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    The GH67 _-glucuronidase of Paenibacillus curdlanolyticus B-6 removes hexenuronic acid groups and facilitates biodegradation of the model xylooligosaccharide hexenuronosyl xylotriose
    (Elsevier Inc., 2015) Krisna Septiningrum; Hiroshi Ohi; Rattiya Waeonukul; Patthra Pason; Chakrit Tachaapaikoon; Khanok Ratanakhanokchai; Junjarus Sermsathanaswadi; Lan Deng; Panida Prawitwong; Akihiko Kosugi
    4-O-Methylglucuronic acid (MeGlcA) side groups attached to the xylan backbone through _-1,2 linkages are converted to hexenuronic acid (HexA) during alkaline pulping. _-Glucuronidase (EC 3.2.1.139) hydrolyzes 1,2-linked MeGlcA from xylooligosaccharides. To determine whether _-glucuronidase can also hydrolyze HexA-decorated xylooligosaccharides, a gene encoding _-glucuronidase (AguA) was cloned from Paenibacillus curdlanolyticus B-6. The purified protein degraded hexenuronosyl xylotriose (_X3), a model substrate prepared from kraft pulp. AguA released xylotriose and HexA from _X3, but the Vmax and kcat values for _X3 were lower than those for MeGlcA, indicating that HexA side groups may affect the hydrolytic activity. To explore the potential for biological bleaching, _X3 degradation was performed using intracellular extract from P. curdlanolyticus B-6. The intracellular extract, with synergistic _-glucuronidase and _-xylosidase activities, degraded _X3 to xylose and HexA. These results indicate that _-glucuronidase can be used to remove HexA from _X3 derived from pulp, reducing the need for chemical treatments in the pulping process. � 2015 Elsevier Inc.