Characterization of a Broad-Specificity β-Glucanase Acting on β-(1,3)-, β-(1,4)-, and β-(1,6)-Glucans That Defines a New Glycoside Hydrolase Family

ABSTRACTHere we report the cloning of thePa_3_10940gene from the coprophilic fungusPodospora anserina, which encodes a C-terminal family 1 carbohydrate binding module (CBM1) linked to a domain of unknown function. The function of the gene was investigated by expression of the full-length protein and a truncated derivative without the CBM1 domain in the yeastPichia pastoris. Using a library of polysaccharides of different origins, we demonstrated that the full-length enzyme displays activity toward a broad range of β-glucan polysaccharides, including laminarin, curdlan, pachyman, lichenan, pustulan, and cellulosic derivatives. Analysis of the products released from polysaccharides revealed that this β-glucanase is an exo-acting enzyme on β-(1,3)- and β-(1,6)-linked glucan substrates and an endo-acting enzyme on β-(1,4)-linked glucan substrates. Hydrolysis of short β-(1,3), β-(1,4), and β-(1,3)/β-(1,4) gluco-oligosaccharides confirmed this striking feature and revealed that the enzyme performs in an exo-type mode on the nonreducing end of gluco-oligosaccharides. Excision of the CBM1 domain resulted in an inactive enzyme on all substrates tested. To our knowledge, this is the first report of an enzyme that displays bifunctional exo-β-(1,3)/(1,6) and endo-β-(1,4) activities toward beta-glucans and therefore cannot readily be assigned to existing Enzyme Commission groups. The amino acid sequence has high sequence identity to hypothetical proteins within the fungal taxa and thus defines a new family of glycoside hydrolases, the GH131 family.

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Characterization of a galactosyl-binding protein module from a Cellvibrio japonicus endo-xyloglucanase defines a new family of Carbohydrate Binding Modules

Applied and Environmental Microbiology ◽

10.1128/aem.02634-20 ◽

2020 ◽

pp. AEM.02634-20

Author(s):

Mohamed A. Attia ◽

Harry Brumer

Keyword(s):

Binding Protein ◽

Glycoside Hydrolases ◽

Glycoside Hydrolase Family ◽

Substrate Affinity ◽

Carbohydrate Binding ◽

Carbohydrate Active Enzymes ◽

Plant Polysaccharides ◽

New Family ◽

Carbohydrate Binding Modules ◽

Cellvibrio Japonicus

Carbohydrate-binding modules (CBMs) are usually appended to carbohydrate-active enzymes (CAZymes) and serve to potentiate catalytic activity, e.g. by increasing substrate affinity. The Gram-negative soil saprophyte Cellvibrio japonicus is valuable source for CAZyme and CBM discovery and characterization, due to its innate ability to degrade a wide array of plant polysaccharides. Bioinformatic analysis of the CJA_2959 gene product from C. japonicus revealed a modular architecture consisting of a fibronectin type III (Fn3) module, a cryptic module of unknown function (“X181”), and a Glycoside Hydrolase Family 5 subfamily 4 (GH5_4) catalytic module. We previously demonstrated that the last of these, CjGH5F, is an efficient and specific endo-xyloglucanase [Attia et al. 2018. Biotechnol. Biofuels, 11: 45]. In the present study, C-terminal fusion of superfolder green fluorescent protein in tandem with the Fn3-X181 modules enabled recombinant production and purification from Escherichia coli. Native affinity gel electrophoresis revealed binding specificity for the terminal galactose-containing plant polysaccharides galactoxyloglucan and galactomannan. Isothermal titration calorimetry further evidenced a preference for galactoxyloglucan polysaccharide over short oligosaccharides comprising the limit-digest product of CjGH5F. Thus, our results identify the X181 module as the defining member of a new CBM family, CBM88. In addition to directly revealing the function of this CBM in the context of xyloglucan metabolism by C. japonicus, this study will guide future bioinformatic and functional analyses across microbial (meta)genomes.Importance This study reveals Carbohydrate Binding Module Family 88 (CBM88) as a new family of galactose-binding protein modules, which are found in series with diverse microbial glycoside hydrolases, polysaccharide lyases, and carbohydrate esterases. The definition of CBM88 in the Carbohydrate-Active Enzymes classification (http://www.cazy.org/CBM88.html) will significantly enable future microbial (meta)genome analysis and functional studies.

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Cello-Oligosaccharide Oxidation Reveals Differences between Two Lytic Polysaccharide Monooxygenases (Family GH61) from Podospora anserina

Applied and Environmental Microbiology ◽

10.1128/aem.02942-12 ◽

2012 ◽

Vol 79 (2) ◽

pp. 488-496 ◽

Cited By ~ 111

Author(s):

Mathieu Bey ◽

Simeng Zhou ◽

Laetitia Poidevin ◽

Bernard Henrissat ◽

Pedro M. Coutinho ◽

...

Keyword(s):

Cellulose Hydrolysis ◽

Podospora Anserina ◽

Striking Difference ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Pycnoporus Cinnabarinus ◽

Content Type ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases

ABSTRACTThe genome of the coprophilic ascomycetePodospora anserinaencodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserinaGH61A [PaGH61A] andPaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced inPichia pastoris. Synergistic cooperation betweenPaGH61A orPaGH61B with the cellobiose dehydrogenase (CDH) ofPycnoporus cinnabarinuson cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference betweenPaGH61A andPaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination ofPaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties ofPaGH61A andPaGH61B and their effect on the interaction with CDH are discussed in regard to the proposedin vivofunction of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

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Multiple Transporters and Glycoside Hydrolases Are Involved in Arabinoxylan-Derived Oligosaccharide Utilization in Bifidobacterium pseudocatenulatum

Applied and Environmental Microbiology ◽

10.1128/aem.01782-20 ◽

2020 ◽

Vol 86 (24) ◽

Author(s):

Yuki Saito ◽

Akira Shigehisa ◽

Yohei Watanabe ◽

Naoki Tsukuda ◽

Kaoru Moriyama-Ohara ◽

...

Keyword(s):

Abc Transporters ◽

Binding Proteins ◽

Molecular Mechanisms ◽

Bifidobacterium Longum ◽

Gene Clusters ◽

Glycoside Hydrolases ◽

Glycoside Hydrolase Family ◽

Carbohydrate Utilization ◽

Bifidobacterium Adolescentis ◽

Content Type

ABSTRACT Arabinoxylan hydrolysates (AXH) are the hydrolyzed products of the major components of the dietary fiber arabinoxylan. AXH include diverse oligosaccharides varying in xylose polymerization and side residue modifications with arabinose at the O-2 and/or O-3 position of the xylose unit. Previous studies have reported that AXH exhibit prebiotic properties on gut bifidobacteria; moreover, several adult-associated bifidobacterial species (e.g., Bifidobacterium adolescentis and Bifidobacterium longum subsp. longum) are known to utilize AXH. In this study, we tried to elucidate the molecular mechanisms of AXH utilization by Bifidobacterium pseudocatenulatum, which is a common bifidobacterial species found in adult feces. We performed transcriptomic analysis of B. pseudocatenulatum YIT 4072T, which identified three upregulated gene clusters during AXH utilization. The gene clusters encoded three sets of ATP-binding cassette (ABC) transporters and five enzymes belonging to glycoside hydrolase family 43 (GH43). By characterizing the recombinant proteins, we found that three solute-binding proteins of ABC transporters showed either broad or narrow specificity, two arabinofuranosidases hydrolyzed either single- or double-decorated arabinoxylooligosaccharides, and three xylosidases exhibited functionally identical activity. These data collectively suggest that the transporters and glycoside hydrolases, encoded in the three gene clusters, work together to utilize AXH of different sizes and with different side residue modifications. Thus, our study sheds light on the overall picture of how these proteins collaborate for the utilization of AXH in B. pseudocatenulatum and may explain the predominance of this symbiont species in the adult human gut. IMPORTANCE Bifidobacteria commonly reside in the human intestine and possess abundant genes involved in carbohydrate utilization. Arabinoxylan hydrolysates (AXH) are hydrolyzed products of arabinoxylan, one of the most abundant dietary fibers, and they include xylooligosaccharides and those decorated with arabinofuranosyl residues. The molecular mechanism by which B. pseudocatenulatum, a common bifidobacterial species found in adult feces, utilizes structurally and compositionally variable AXH has yet to be extensively investigated. In this study, we identified three gene clusters (encoding five GH43 enzymes and three solute-binding proteins of ABC transporters) that were upregulated in B. pseudocatenulatum YIT 4072T during AXH utilization. By investigating their substrate specificities, we revealed how these proteins are involved in the uptake and degradation of AXH. These molecular insights may provide a better understanding of how resident bifidobacteria colonize the colon.

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Crystal structure of GH43 exo-β-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium provides insights into the mechanism of bypassing side chains

10.1101/2020.09.23.310037 ◽

2020 ◽

Author(s):

Kaori Matsuyama ◽

Naomi Kishine ◽

Zui Fujimoto ◽

Naoki Sunagawa ◽

Toshihisa Kotake ◽

...

Keyword(s):

Crystal Structure ◽

Phanerochaete Chrysosporium ◽

Catalytic Domain ◽

Interaction Mechanism ◽

Binding Domain ◽

Glycoside Hydrolases ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Side Chains ◽

Ligand Interaction

AbstractArabinogalactan proteins (AGPs) are functional plant proteoglycans, but their functions are largely unexplored, mainly because of the complexity of the sugar moieties, which are generally analyzed with the aid of glycoside hydrolases. In this study, we solved the apo and liganded structures of exo-β-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. It is composed of a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family (CBM) 35 binding domain. GH43_sub24 lacks the catalytic base Asp that is conserved among other GH43 subfamilies. Crystal structure and kinetic analyses indicated that the tautomerized imidic acid function of Gln263 serves instead as the catalytic base residue. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite -1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6-linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s. Some of the residues involved in ligand recognition differ from those of galactomannan-binding CBM35, including substitution of Trp for Gly, which affects pyranose stacking, and substitution of Asn for Asp in the lower part of the binding pocket. Pc1,3Gal43A WT and its mutants at residues involved in substrate recognition are expected to be useful tools for structural analysis of AGPs. Our findings should also be helpful in engineering designer enzymes for efficient utilization of various types of biomass.

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S-Layer Homology Domain Proteins Csac_0678 and Csac_2722 Are Implicated in Plant Polysaccharide Deconstruction by the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus

Applied and Environmental Microbiology ◽

10.1128/aem.07031-11 ◽

2011 ◽

Vol 78 (3) ◽

pp. 768-777 ◽

Cited By ~ 43

Author(s):

Inci Ozdemir ◽

Sara E. Blumer-Schuette ◽

Robert M. Kelly

Keyword(s):

Cellular Localization ◽

Catalytic Domain ◽

Plant Biomass ◽

Thermophilic Bacteria ◽

Biochemical Properties ◽

Crystalline Cellulose ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Caldicellulosiruptor Saccharolyticus ◽

Content Type

ABSTRACTThe genusCaldicellulosiruptorcontains extremely thermophilic bacteria that grow on plant polysaccharides. The genomes ofCaldicellulosiruptorspecies reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins inCaldicellulosiruptor saccharolyticus(Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequencedCaldicellulosiruptorspecies. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in theC. saccharolyticusgenome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to theC. saccharolyticusS-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in otherCaldicellulosiruptorgenomes may also be important contributors to plant biomass utilization.

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Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

Applied and Environmental Microbiology ◽

10.1128/aem.06814-11 ◽

2012 ◽

Vol 78 (7) ◽

pp. 2230-2240 ◽

Cited By ~ 40

Author(s):

Xiaoyun Su ◽

Roderick I. Mackie ◽

Isaac K. O. Cann

Keyword(s):

Catalytic Efficiency ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Wild Type ◽

Ph Optimum ◽

Content Type ◽

Caldicellulosiruptor Bescii ◽

Type Protein ◽

Wild Type Protein ◽

Carbohydrate Binding Modules

ABSTRACTThermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses ofCaldicellulosiruptor besciiCel9B/Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins ofC. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydrate-binding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/Man5A were assigned to the N- and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ∼5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s−1ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparentkcatof the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in theKmled to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.

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Expression and Characterization of a Bifidobacterium adolescentis Beta-Mannanase Carrying Mannan-Binding and Cell Association Motifs

Applied and Environmental Microbiology ◽

10.1128/aem.02118-12 ◽

2012 ◽

Vol 79 (1) ◽

pp. 133-140 ◽

Cited By ~ 22

Author(s):

Evelina Kulcinskaja ◽

Anna Rosengren ◽

Romany Ibrahim ◽

Katarína Kolenová ◽

Henrik Stålbrand

Keyword(s):

Signal Peptide ◽

High Performance ◽

Guar Gum ◽

Transmembrane Helix ◽

Exchange Chromatography ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Bifidobacterium Adolescentis ◽

Content Type ◽

Carbohydrate Binding Modules

ABSTRACTThe gene encoding β-mannanase (EC 3.2.1.78) BaMan26A from the bacteriumBifidobacterium adolescentis(living in the human gut) was cloned and the gene product characterized. The enzyme was found to be modular and to contain a putative signal peptide. It possesses a catalytic module of the glycoside hydrolase family 26, a predicted immunoglobulin-like module, and two putative carbohydrate-binding modules (CBMs) of family 23. The enzyme is likely cell attached either by the sortase mechanism (LPXTG motif) or via a C-terminal transmembrane helix. The gene was expressed inEscherichia coliwithout the native signal peptide or the cell anchor. Two variants were made: one containing all four modules, designated BaMan26A-101K, and one truncated before the CBMs, designated BaMan26A-53K. BaMan26A-101K, which contains the CBMs, showed an affinity to carob galactomannan having a dissociation constant of 0.34 μM (8.8 mg/liter), whereas BaMan26A-53K did not bind, showing that at least one of the putative CBMs of family 23 is mannan binding. For BaMan26A-53K,kcatwas determined to be 444 s−1andKm21.3 g/liter using carob galactomannan as the substrate at the optimal pH of 5.3. Both of the enzyme variants hydrolyzed konjac glucomannan, as well as carob and guar gum galactomannans to a mixture of oligosaccharides. The dominant product from ivory nut mannan was found to be mannotriose. Mannobiose and mannotetraose were produced to a lesser extent, as shown by high-performance anion-exchange chromatography. Mannobiose was not hydrolyzed, and mannotriose was hydrolyzed at a significantly lower rate than the longer oligosaccharides.

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Novel Trifunctional Xylanolytic Enzyme Axy43A from Paenibacillus curdlanolyticus Strain B-6 Exhibiting Endo-Xylanase, β-d-Xylosidase, and Arabinoxylan Arabinofuranohydrolase Activities

Applied and Environmental Microbiology ◽

10.1128/aem.02256-16 ◽

2016 ◽

Vol 82 (23) ◽

pp. 6942-6951 ◽

Cited By ~ 9

Author(s):

Thitiporn Teeravivattanakit ◽

Sirilak Baramee ◽

Paripok Phitsuwan ◽

Rattiya Waeonukul ◽

Patthra Pason ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Xylanase Activity ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Metabolic Potential ◽

Gene Encoding ◽

Content Type ◽

Arabinoxylan Arabinofuranohydrolase ◽

Paenibacillus Curdlanolyticus ◽

Xylanolytic Enzyme

ABSTRACTTheaxy43Agene encoding the intracellular trifunctional xylanolytic enzyme fromPaenibacillus curdlanolyticusB-6 was cloned and expressed inEscherichia coli. Recombinant PcAxy43A consisting of a glycoside hydrolase family 43 and a family 6 carbohydrate-binding module exhibited endo-xylanase, β-xylosidase, and arabinoxylan arabinofuranohydrolase activities. PcAxy43A hydrolyzed xylohexaose and birch wood xylan to release a series of xylooligosaccharides, indicating that PcAxy43A contained endo-xylanase activity. PcAxy43A exhibited β-xylosidase activity toward a chromogenic substrate,p-nitrophenyl-β-d-xylopyranoside, and xylobiose, while it preferred to hydrolyze long-chain xylooligosaccharides rather than xylobiose. In addition, surprisingly, PcAxy43A showed arabinoxylan arabinofuranohydrolase activity; that is, it released arabinose from both singly and doubly arabinosylated xylose, α-l-Araf-(1→2)-d-Xylpor α-l-Araf-(1→3)-d-Xylpand α-l-Araf-(1→2)-[α-l-Araf-(1→3)]-β-d-Xylp. Moreover, the combination of PcAxy43A andP. curdlanolyticusB-6 endo-xylanase Xyn10C greatly improved the efficiency of xylose and arabinose production from the highly substituted rye arabinoxylan, suggesting that these two enzymes function synergistically to depolymerize arabinoxylan. Therefore, PcAxy43A has the potential for the saccharification of arabinoxylan into simple sugars for many applications.IMPORTANCEIn this study, the glycoside hydrolase 43 (GH43) intracellular multifunctional endo-xylanase, β-xylosidase, and arabinoxylan arabinofuranohydrolase (AXH) fromP. curdlanolyticusB-6 were characterized. Interestingly, PcAxy43A AXH showed a new property that acted on both the C(O)-2 and C(O)-3 positions of xylose residues doubly substituted with arabinosyl, which usually obstruct the action of xylanolytic enzymes. Furthermore, the studies here show interesting properties for the processing of xylans from cereal grains, particularly rye arabinoxylan, and show a novel relationship between PcAxy43A and endo-xylanase Xyn10C from strain B-6, providing novel metabolic potential for processing arabinoxylans into xylose and arabinose.

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Transcriptomic Analysis of Xylan Utilization Systems in Paenibacillus sp. Strain JDR-2

Applied and Environmental Microbiology ◽

10.1128/aem.03523-14 ◽

2014 ◽

Vol 81 (4) ◽

pp. 1490-1501 ◽

Cited By ~ 10

Author(s):

Neha Sawhney ◽

Casey Crooks ◽

Franz St. John ◽

James F. Preston

Keyword(s):

Consolidated Bioprocessing ◽

Direct Conversion ◽

Glycoside Hydrolases ◽

Glycoside Hydrolase Family ◽

Content Type ◽

Expression Of Genes ◽

Paenibacillus Sp ◽

Genes Encoding ◽

Elevated Expression ◽

A Cell

ABSTRACTXylans, including methylglucuronoxylans (MeGXn) and methylglucuronoarabinoxylans (MeGAXn), are the predominant polysaccharides in hemicellulose fractions of dicots and monocots available for conversion to biofuels and chemicals.Paenibacillussp. strain JDR-2 (Pjdr2) efficiently depolymerizes MeGXnand MeGAXnand assimilates the generated oligosaccharides, resulting in efficient saccharification and subsequent metabolism of these polysaccharides. A xylan utilization regulon encoding a cell-associated GH10 (glycoside hydrolase family 10) endoxylanase, transcriptional regulators, ABC (ATP binding cassette) transporters, an intracellular GH67 α-glucuronidase, and other glycoside hydrolases contributes to complete metabolism. This GH10/GH67 system has been proposed to account for preferential utilization of xylans compared to free oligo- and monosaccharides. To identify additional genes contributing to MeGXnand MeGAXnutilization, the transcriptome of Pjdr2 has been sequenced following growth on each of these substrates as well as xylose and arabinose. Increased expression of genes with different substrates identified pathways common or unique to the utilization of MeGXnor MeGAXn. Coordinate upregulation of genes comprising the GH10/GH67 xylan utilization regulon is accompanied with upregulation of genes encoding a GH11 endoxylanase and a GH115 α-glucuronidase, providing evidence for a novel complementary pathway for processing xylans. Elevated expression of genes encoding a GH43 arabinoxylan arabinofuranohydrolase and an arabinose ABC transporter on MeGAXnbut not on MeGXnsupports a process in which arabinose may be removed extracellularly followed by its rapid assimilation. Further development of Pjdr2 for direct conversion of xylans to targeted products or introduction of these systems into fermentative strains of related bacteria may lead to biocatalysts for consolidated bioprocessing of hemicelluloses released from lignocellulose.

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A Polysaccharide Utilization Locus from an Uncultured Bacteroidetes Phylotype Suggests Ecological Adaptation and Substrate Versatility

Applied and Environmental Microbiology ◽

10.1128/aem.02858-14 ◽

2014 ◽

Vol 81 (1) ◽

pp. 187-195 ◽

Cited By ~ 25

Author(s):

A. K. Mackenzie ◽

A. E. Naas ◽

S. K. Kracun ◽

J. Schückel ◽

J. U. Fangel ◽

...

Keyword(s):

Anion Exchange Chromatography ◽

Gene Organization ◽

Exchange Chromatography ◽

Glycoside Hydrolases ◽

Glycoside Hydrolase Family ◽

Content Type ◽

Svalbard Reindeer ◽

Uncultured Bacteria ◽

Rumen Microbiome ◽

Polysaccharide Utilization Locus

ABSTRACTRecent metagenomic analyses have identified uncultured bacteria that are abundant in the rumen of herbivores and that possess putative biomass-converting enzyme systems. Here we investigate the saccharolytic capabilities of a polysaccharide utilization locus (PUL) that has been reconstructed from an unculturedBacteroidetesphylotype (SRM-1) that dominates the rumen microbiome of Arctic reindeer. Characterization of the three PUL-encoded outer membrane glycoside hydrolases was performed using chromogenic substrates for initial screening, followed by detailed analyses of products generated from selected substrates, using high-pressure anion-exchange chromatography with electrochemical detection. Two glycoside hydrolase family 5 (GH5) endoglucanases (GH5_g and GH5_h) demonstrated activity against β-glucans, xylans, and xyloglucan, whereas GH5_h and the third enzyme, GH26_i, were active on several mannan substrates. Synergy experiments examining different combinations of the three enzymes demonstrated limited activity enhancement on individual substrates. Binding analysis of a SusE-positioned lipoprotein revealed an affinity toward β-glucans and, to a lesser extent, mannan, but unlike the two SusD-like lipoproteins previously characterized from the same PUL, binding to cellulose was not observed. Overall, these activities and binding specificities correlated well with the glycan content of the reindeer rumen, which was determined using comprehensive microarray polymer profiling and showed an abundance of various hemicellulose glycans. The substrate versatility of this single PUL putatively expands our perceptions regarding PUL machineries, which so far have demonstrated gene organization that suggests one cognate PUL for each substrate type. The presence of a PUL that possesses saccharolytic activity against a mixture of abundantly available polysaccharides supports the dominance of SRM-1 in the Svalbard reindeer rumen microbiome.

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