Epoxyalkyl glycosides of D-xylose and xylo-oligosaccharides are active-site markers of xylanases from glycoside hydrolase family 11, not from family 10.
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ABSTRACT:
SUBMITTER: Ntarima P
PROVIDER: S-EPMC1221026 | biostudies-other | 2000 May
REPOSITORIES: biostudies-other
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Project description:Pseudomonas cellulosa is a highly efficient xylan-degrading bacterium. Genes encoding five xylanases, and several accessory enzymes, which remove the various side chains that decorate the xylan backbone, have been isolated from the pseudomonad and characterized. The xylanase genes consist of xyn10A, xyn10B, xyn10C, xyn10D, and xyn11A, which encode Xyn10A, Xyn10B, Xyn10C, Xyn10D, and Xyn11A, respectively. In this study a sixth xylanase gene, xyn11B, was isolated which encodes a 357-residue modular enzyme, designated Xyn11B, comprising a glycoside hydrolase family 11 catalytic domain appended to a C-terminal X-14 module, a homologue of which binds to xylan. Localization studies showed that the two xylanases with glycoside hydrolase family (GH) 11 catalytic modules, Xyn11A and Xyn11B, are secreted into the culture medium, whereas Xyn10C is membrane bound. xyn10C, xyn10D, xyn11A, and xyn11B were all abundantly expressed when the bacterium was cultured on xylan or beta-glucan but not on medium containing mannan, whereas glucose repressed transcription of these genes. Although all of the xylanase genes were induced by the same polysaccharides, temporal regulation of xyn11A and xyn11B was apparent on xylan-containing media. Transcription of xyn11A occurred earlier than transcription of xyn11B, which is consistent with the predicted mode of action of the encoded enzymes. Xyn11A, but not Xyn11B, exhibits xylan esterase activity, and the removal of acetate side chains is required for xylanases to hydrolyze the xylan backbone. A transposon mutant of P. cellulosa in which xyn11A and xyn11B were inactive displayed greatly reduced extracellular but normal cell-associated xylanase activity, and its growth rate on medium containing xylan was indistinguishable from wild-type P. cellulosa. Based on the data presented here, we propose a model for xylan degradation by P. cellulosa in which the GH11 enzymes convert decorated xylans into substituted xylooligosaccharides, which are then hydrolyzed to their constituent sugars by the combined action of cell-associated GH10 xylanases and side chain-cleaving enzymes.
| S-EPMC135193 | biostudies-literature
Project description:BackgroundThe rumen harbors a complex microbial ecosystem for efficient hydrolysis of plant polysaccharides which are the main constituent of the diet. Xylanase is crucial for hemicellulose hydrolysis and plays an important role in the plant cell wall degradation. Xylanases of ruminal strains were widely studied, but few studies have focused on their diversity in rumen microenvironment.Methodology/principal findingsWe explored the genetic diversity of xylanases belonging to two major glycosyl hydrolase families (GH 10 and 11) in goat rumen contents by analyzing the amplicons generated with two degenerate primer sets. Fifty-two distinct GH 10 and 35 GH 11 xylanase gene fragments (similarity <95%) were retrieved, and most had low identities with known sequences. Based on phylogenetic analysis, all GH 10 xylanase sequences fell into seven clusters, and 88.5% of them were related to xylanases from Bacteroidetes. Five clusters of GH 11 xylanase sequences were identified. Of these, 85.7% were related to xylanases from Firmicutes, and 14.3% were related to those of rumen fungi. Two full-length xylanase genes (one for each family) were directly cloned and expressed in Escherichia coli. Both the recombinant enzymes showed substantial xylanase activity, and were purified and characterized. Combined with the results of sheep rumen, Bacteroidetes and Firmicutes are the two major phyla of xylan-degrading microorganisms in rumen, which is distinct from the representatives of other environments such as soil and termite hindgut, suggesting that xylan-degrading microorganisms are environment specific.Conclusion/significanceThe numerous new xylanase genes suggested the functional diversity of xylanase in the rumen microenvironment which may have great potential applications in industry and agriculture. The phylogenetic diversity and different distributions of xylanase genes will help us understand their roles in plant cell wall degradation in the rumen microenvironment.
| S-EPMC3033422 | biostudies-literature
Project description:Xylanases play a crucial role in the degradation of xylan in both terrestrial and marine environments. The endoxylanase XynB from the marine bacterium Glaciecola mesophila KMM 241 is a modular enzyme comprising a long N-terminal domain (NTD) (E44 to T562) with xylan-binding ability and a catalytic domain (CD) (T563 to E912) of glycoside hydrolase family 8 (GH8). In this study, the long NTD is confirmed to contain three different functional regions, which are NTD1 (E44 to D136), NTD2 (Y137 to A193), and NTD3 (L194 to T562). NTD1, mainly composed of eight ?-strands, functions as a new type of carbohydrate-binding module (CBM), which has xylan-binding ability but no sequence similarity to any known CBM. NTD2, mainly forming two ?-helices, contains one of the ?-helices of the catalytic domain's (?/?)6 barrel and therefore is essential for the activity of XynB, although it is far away from the catalytic domain in sequence. NTD3, next to the catalytic domain in sequence, is shown to be helpful in maintaining the thermostability of XynB. Thus, XynB represents a kind of xylanase with a new domain architecture. There are four other predicted glycoside hydrolase sequences with the same domain architecture and high sequence identity (?80%) with XynB, all of which are from marine bacteria. Phylogenetic analysis shows that XynB and these homologs form a new group in GH8, representing a new class of marine bacterial xylanases. Our results shed light on xylanases, especially marine xylanases.IMPORTANCE Xylanases play a crucial role in natural xylan degradation and have been extensively used in industries such as food processing, animal feed, and kraft pulp biobleaching. Some marine bacteria have been found to secrete xylanases. Characterization of novel xylanases from marine bacteria has significance for both the clarification of xylan degradation mechanisms in the sea and the development of new enzymes for industrial application. With G. mesophila XynB as a representative, this study reveals a new group of the GH8 xylanases from marine bacteria, which have a distinct domain architecture and contain a novel carbohydrate-binding module. Thus, this study offers new knowledge on marine xylanases.
| S-EPMC6238072 | biostudies-literature
Project description:Xylanase is a crucial hydrolytic enzyme that degrades plant polysaccharides in the rumen. To date, there is no information on the genetic composition and expression characteristics of ruminal xylanase during feeding cycles of ruminants. Here, the major xylanase of the glycoside hydrolase family 10 (GH 10) from the rumen of small-tail Han sheep was investigated during a feeding cycle. We identified 44 distinct GH 10 xylanase gene fragments at both the genomic and transcriptional levels. Comparison of their relative abundance showed that results from the evaluation of functional genes at the transcriptional level are more reliable indicators for understanding fluctuations in xylanase levels. The expression patterns of six xylanase genes, detected at all time points of the feeding cycle, were investigated; we observed a complex trend of gene expression over 24 h, revealing the dynamic expression of xylanases in the rumen. Further correlation analysis indicated that the rumen is a dynamic ecosystem where the transcript profiles of xylanase genes are closely related to ruminal conditions, especially rumen pH and bacterial population. Given the huge diversity and changing composition of enzymes over the entire rumen, this research provides valuable information for understanding the role of functional genes in the digestion of plant material.
| S-EPMC3568598 | biostudies-literature
Project description:Soda lakes are one of the most stable naturally occurring alkaline and saline environments, which harbor abundant microorganisms with diverse functions. In this study, culture-independent molecular methods were used to explore the genetic diversity of glycoside hydrolase (GH) family 10 and GH11 xylanases in Lake Dabusu, a soda lake with a pH value of 10.2 and salinity of 10.1%. A total of 671 xylanase gene fragments were obtained, representing 78 distinct GH10 and 28 GH11 gene fragments respectively, with most of them having low homology with known sequences. Phylogenetic analysis revealed that the GH10 xylanase sequences mainly belonged to Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes and Verrucomicrobia, while the GH11 sequences mainly consisted of Actinobacteria, Firmicutes and Fungi. A full-length GH10 xylanase gene (xynAS10-66) was directly cloned and expressed in Escherichia coli, and the recombinant enzymes showed high activity at alkaline pH. These results suggest that xylanase gene diversity within Lake Dabusu is high and that most of the identified genes might be novel, indicating great potential for applications in industry and agriculture.
| S-EPMC4231106 | biostudies-literature
Project description:BackgroundXylan is one of the most abundant biopolymers on Earth. Its degradation is mediated primarily by microbial xylanase in nature. To explore the diversity and distribution patterns of xylanase genes in soils, samples of five soil types with different physicochemical characters were analyzed.Methodology/principal findingsPartial xylanase genes of glycoside hydrolase (GH) family 10 were recovered following direct DNA extraction from soil, PCR amplification and cloning. Combined with our previous study, a total of 1084 gene fragments were obtained, representing 366 OTUs. More than half of the OTUs were novel (identities of <65% with known xylanases) and had no close relatives based on phylogenetic analyses. Xylanase genes from all the soil environments were mainly distributed in Bacteroidetes, Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria, Dictyoglomi and some fungi. Although identical sequences were found in several sites, habitat-specific patterns appeared to be important, and geochemical factors such as pH and oxygen content significantly influenced the compositions of xylan-degrading microbial communities.Conclusion/significanceThese results provide insight into the GH 10 xylanases in various soil environments and reveal that xylan-degrading microbial communities are environment specific with diverse and abundant populations.
| S-EPMC3422244 | biostudies-literature
Project description:As classified by the Carbohydrate-Active Enzymes (CAZy) database, enzymes in glycoside hydrolase (GH) family 10 (GH10) are all monospecific or bifunctional xylanases (except a tomatinase), and no endo-β-1,4-glucanase has been reported in the family. Here, we identified Arcticibacterium luteifluviistationis carboxymethyl cellulase (AlCMCase) as a GH10 endo-β-1,4-glucanase. AlCMCase originated from an Arctic marine bacterium, Arcticibacterium luteifluviistationis SM1504T It shows low identity (<35%) with other GH10 xylanases. The gene encoding AlCMCase was overexpressed in Escherichia coli Biochemical characterization showed that recombinant AlCMCase is a cold-adapted and salt-tolerant enzyme. AlCMCase hydrolyzes cello- and xylo-configured substrates via an endoaction mode. However, in comparison to its significant cellulase activity, the xylanase activity of AlCMCase is negligible. Correspondingly, AlCMCase has remarkable binding capacity for cello-oligosaccharides but no obvious binding capacity for xylo-oligosaccharides. AlCMCase and its homologs are grouped into a branch separate from other GH10 xylanases in a phylogenetic tree, and two homologs also displayed the same substrate specificity as AlCMCase. These results suggest that AlCMCase and its homologs form a novel subfamily of GH10 enzymes that have robust endo-β-1,4-glucanase activity. In addition, given the cold-adapted and salt-tolerant characters of AlCMCase, it may be a candidate biocatalyst under certain industrial conditions, such as low temperature or high salinity.IMPORTANCE Cellulase and xylanase have been widely used in the textile, pulp and paper, animal feed, and food-processing industries. Exploring novel cellulases and xylanases for biocatalysts continues to be a hot issue. Enzymes derived from the polar seas might have novel hydrolysis patterns, substrate specificities, or extremophilic properties that have great potential for both fundamental research and industrial applications. Here, we identified a novel cold-adapted and salt-tolerant endo-β-1,4-glucanase, AlCMCase, from an Arctic marine bacterium. It may be useful in certain industrial processes, such as under low temperature or high salinity. Moreover, AlCMCase is a bifunctional representative of glycoside hydrolase (GH) family 10 that preferentially hydrolyzes β-1,4-glucans. With its homologs, it represents a new subfamily in this family. Thus, this study sheds new light on the substrate specificity of GH10.
| S-EPMC6715846 | biostudies-literature
Project description:Paenibacillus sp. W-61 is capable of utilizing water-insoluble xylan for carbon and energy sources and has three xylanase genes, xyn1, xyn3, and xyn5. Xyn1, Xyn3, and Xyn5 are extracellular enzymes of the glycoside hydrolase (GH) families 11, 30, and 10, respectively. Xyn5 contains several domains including those of carbohydrate-binding modules (CBMs) similar to a surface-layer homologous (SLH) protein. This study focused on the role of Xyn5, localized on the cell surface, in water-insoluble xylan utilization. Electron microscopy using immunogold staining revealed Xyn5 clusters over the entire cell surface. Xyn5 was bound to cell wall fractions through its SLH domain. A Deltaxyn5 mutant grew poorly and produced minimal amounts of Xyn1 and Xyn3 on water-insoluble xylan. A Xyn5 mutant lacking the SLH domain (Xyn5DeltaSLH) grew poorly, secreting Xyn5DeltaSLH into the medium and producing minimal Xyn1 and Xyn3 on water-insoluble xylan. A mutant with an intact xyn5 produced Xyn5 on the cell surface, grew normally, and actively synthesized Xyn1 and Xyn3 on water-insoluble xylan. Quantitative reverse transcription-PCR showed that xylobiose, generated from water-insoluble xylan decomposition by Xyn5, is the most active inducer for xyn1 and xyn3. Luciferase assays using a Xyn5-luciferase fusion protein suggested that xylotriose is the best inducer for xyn5. The cell surface Xyn5 appears to play two essential roles in water-insoluble xylan utilization: (i) generation of the xylo-oligosaccharide inducers of all the xyn genes from water-insoluble xylan and (ii) attachment of the cells to the substrate so that the generated inducers can be immediately taken up by cells to activate expression of the xyn system.
| S-EPMC2849441 | biostudies-literature
Project description:Xylanases are crucial for lignocellulosic biomass deconstruction and generally contain noncatalytic carbohydrate-binding modules (CBMs) accessing recalcitrant polymers. Understanding how multimodular enzymes assemble can benefit protein engineering by aiming at accommodating various environmental conditions. Two multimodular xylanases, XynA and XynB, which belong to glycoside hydrolase families 11 (GH11) and GH10, respectively, have been identified from Caldicellulosiruptor sp. strain F32. In this study, both xylanases and their truncated mutants were overexpressed in Escherichia coli, purified, and characterized. GH11 XynATM1 lacking CBM exhibited a considerable improvement in specific activity (215.8 U nmol(-1) versus 94.7 U nmol(-1)) and thermal stability (half-life of 48 h versus 5.5 h at 75°C) compared with those of XynA. However, GH10 XynB showed higher enzyme activity and thermostability than its truncated mutant without CBM. Site-directed mutagenesis of N-terminal amino acids resulted in a mutant, XynATM1-M, with 50% residual activity improvement at 75°C for 48 h, revealing that the disordered region influenced protein thermostability negatively. The thermal stability of both xylanases and their truncated mutants were consistent with their melting temperature (Tm), which was determined by using differential scanning calorimetry. Through homology modeling and cross-linking analysis, we demonstrated that for XynB, the resistance against thermoinactivation generally was enhanced through improving both domain properties and interdomain interactions, whereas for XynA, no interdomain interactions were observed. Optimized intramolecular interactions can accelerate thermostability, which provided microbes a powerful evolutionary strategy to assemble catalysts that are adapted to various ecological conditions.
| S-EPMC4345376 | biostudies-literature
Project description:UnlabelledA GH8 family enzyme involved in xylan depolymerization has been characterized. The enzyme, Rex8A, is a reducing-end xylose-releasing exo-oligoxylanase (Rex) that efficiently hydrolyzes xylooligosaccharides and shows minor activity on polymeric xylan. Rex8A hydrolyzes xylooligomers of 3 to 6 xylose units to xylose and xylobiose in long-term incubations. Kinetic constants of Rex8A were determined on xylotriose, showing a Km of 1.64 ± 0.03 mM and a kcat value of 118.8 s(-1) Besides linear xylooligosaccharides, the enzyme hydrolyzed decorated xylooligomers. The catalytic activity on branched xylooligosaccharides, i.e., the release of xylose from the reducing end, is a newly described trait of xylose-releasing exo-oligoxylanases, as the exo-activity on these substrates has not been reported for the few of these enzymes characterized to date. Modeling of the three-dimensional (3D) structure of Rex8A shows an (α/α)6 barrel fold where the loops connecting the α-helices contour the active site. These loops, which show high sequence diversity among GH8 enzymes, shape a catalytic cleft with a -2 subsite that can accommodate methyl-glucuronic acid decorations. The hydrolytic ability of Rex8A on branched oligomers can be crucial for the complete depolymerization of highly substituted xylans, which is indispensable to accomplish biomass deconstruction and to generate efficient catalysts.ImportanceA GH8 family enzyme involved in xylan depolymerization has been characterized. The Rex8A enzyme from Paenibacillus barcinonensis is involved in depolymerization of glucuronoxylan, a major component of the lignocellulosic substrates. The study shows that Rex8A is a reducing-end xylose-releasing exo-oligoxylanase that efficiently hydrolyzes xylose from neutral and acidic xylooligosaccharides generated by the action of other xylanases also secreted by the strain. The activity of a Rex enzyme on branched xylooligosaccharides has not been described to date. This report provides original and useful information on the properties of a new example of the rarely studied Rex enzymes. Depolymerization of highly substituted xylans is crucial for biomass valorization as a platform for generation of biofuels, chemicals, and solvents.
| S-EPMC4988184 | biostudies-literature