Characterization of a novel GH10 alkali-thermostable xylanase from a termite microbiome
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SUBMITTER: Mon M
PROVIDER: S-EPMC10992782 | biostudies-literature | 2022 Aug
REPOSITORIES: biostudies-literature
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Project description:Xylanolytic enzymes have a broad range of applications in industrial biotechnology as biocatalytic components of various processes and products, such as food additives, bakery products, coffee extraction, agricultural silage and functional foods. An increasing market demand has driven the growing interest for the discovery of xylanases with specific industrially relevant characteristics, such as stability at elevated temperatures and in the presence of other denaturing factors, which will facilitate their incorporation into industrial processes. In this work, we report the discovery and biochemical characterization of a new thermostable GH10 xylanase, termed XynDZ5, exhibiting only 26% amino acid sequence identity to the closest characterized xylanolytic enzyme. This new enzyme was discovered in an Icelandic hot spring enrichment culture of a Thermoanaerobacterium species using a recently developed bioinformatic analysis platform. XynDZ5 was produced recombinantly in Escherichia coli, purified and characterized biochemically. This analysis revealed that it acts as an endo-1,4-β-xylanase that performs optimally at 65-75°C and pH 7.5. The enzyme is capable of retaining high levels of catalytic efficiency after several hours of incubation at high temperatures, as well as in the presence of significant concentrations of a range of metal ions and denaturing agents. Interestingly, the XynDZ5 biochemical profile was found to be atypical, as it also exhibits significant exo-activity. Computational modeling of its three-dimensional structure predicted a (β/α)8 TIM barrel fold, which is very frequently encountered among family GH10 enzymes. This modeled structure has provided clues about structural features that may explain aspects of its catalytic performance. Our results suggest that XynDZ5 represents a promising new candidate biocatalyst appropriate for several high-temperature biotechnological applications in the pulp, paper, baking, animal-feed and biofuel industries.
| S-EPMC7193231 | biostudies-literature
Project description:The functional screening of a Pseudacanthotermes militaris termite gut metagenomic library revealed an array of xylan-degrading enzymes, including P. militaris 25 (Pm25), a multimodular glycoside hydrolase family 10 (GH10). Sequence analysis showed details of the unusual domain organization of this enzyme. It consists of one catalytic domain, which is intercalated by two carbohydrate binding modules (CBMs) from family 4. The genes upstream of the genes encoding Pm25 are susC-susD-unk, suggesting Pm25 is a Xyn10C-like enzyme belonging to a polysaccharide utilization locus. The majority of Xyn10C-like enzymes shared the same interrupted domain architecture and were vastly distributed in different xylan utilization loci found in gut Bacteroidetes, indicating the importance of this enzyme in glycan acquisition for gut microbiota. To understand its unusual multimodularity and the possible role of the CBMs, a detailed characterization of the full-length Pm25 and truncated variants was performed. Results revealed that the GH10 catalytic module is specific toward the hydrolysis of xylan. Ligand binding results indicate that the GH10 module and the CBMs act independently, whereas the tandem CBM4s act synergistically with each other and improve enzymatic activity when assayed on insoluble polysaccharides. In addition, we show that the UNK protein upstream of Pm25 is able to bind arabinoxylan. Altogether, these findings contribute to a better understanding of the potential role of Xyn10C-like proteins in xylan utilization systems of gut bacteria.IMPORTANCE Xylan is the major hemicellulosic polysaccharide in cereals and contributes to the recalcitrance of the plant cell wall toward degradation. Members of the Bacteroidetes, one of the main phyla in rumen and human gut microbiota, have been shown to encode polysaccharide utilization loci dedicated to the degradation of xylan. Here, we present the biochemical characterization of a xylanase encoded by a Bacteroidetes strain isolated from the termite gut metagenome. This xylanase is a multimodular enzyme, the sequence of which is interrupted by the insertion of two CBMs from family 4. Our results show that this enzyme resembles homologues that were shown to be important for xylan degradation in rumen or human diet and show that the CBM insertion in the middle of the sequence seems to be a common feature in xylan utilization systems. This study shed light on our understanding of xylan degradation and plant cell wall deconstruction, which can be applied to several applications in food, feed, and bioeconomy.
| S-EPMC7848910 | biostudies-literature
Project description:BackgroundThe search for novel thermostable xylanases for industrial use has intensified in recent years, and thermophilic fungi are a promising source of useful enzymes. The present work reports the heterologous expression and biochemical characterization of a novel thermostable xylanase (GH10) from the thermophilic fungus Malbranchea pulchella, the influence of glycosylation on its stability, and a potential application in sugarcane bagasse hydrolysis.ResultsXylanase MpXyn10A was overexpressed in Aspergillus nidulans and was active against birchwood xylan, presenting an optimum activity at pH 5.8 and 80°C. MpXyn10A was 16% glycosylated and thermostable, preserving 85% activity after 24 hours at 65°C, and deglycosylation did not affect thermostability. Circular dichroism confirmed the high alpha-helical content consistent with the canonical GH10 family (β/α)8 barrel fold observed in molecular modeling. Primary structure analysis revealed the existence of eight cysteine residues which could be involved in four disulfide bonds, and this could explain the high thermostability of this enzyme even in the deglycosylated form. MpXyn10A showed promising results in biomass degradation, increasing the amount of reducing sugars in bagasse in natura and in three pretreated sugarcane bagasses.ConclusionsMpXyn10A was successfully secreted in Aspergillus nidulans, and a potential use for sugarcane bagasse biomass degradation was demonstrated.
| S-EPMC4364333 | biostudies-literature
Project description:An alkalitolerant fungus, Aspergillus fumigatus strain MA28 produced significant amounts of cellulase-free xylanase when grown on a variety of agro-wastes. Wheat bran as the sole carbon source supported higher xylanase production (8,450 U/L) than xylan (7,500 U/L). Soybean meal was observed to be the best nitrogen source for xylanase production (9,000 U/L). Optimum medium pH for xylanase production was 8 (9,800 U/L), though, significant quantities of the enzyme was also produced at pH 7 (8,500 U/L), 9 (8,200 U/L) and 10 (4,600 U/L). The xylanase was purified by ammonium sulphate precipitation and carboxymethyl cellulose chromatography, and was found to have a molecular weight of 14.4 kDa with a V(max) of 980 μmol/min/mg of protein and a K(m) of approximately 4.9 mg/mL. The optimum temperature and pH for enzyme activity was 50 °C and pH 8, respectively. However, the enzyme also showed substantial residual activity at 60-70 °C (53-75%) and at alkaline pH 8-9 (56-88%).
| S-EPMC3339620 | biostudies-literature
Project description:Xylanases have the potential to be used as bio-deinking and bio-bleaching materials and their application will decrease the consumption of the chlorine-based chemicals currently used for this purpose. However, xylanases with specific properties could act effectively, such as having significant thermostability and alkali resistance, etc. In this study, we found that TfXyl10A, a xylanase from Thermobifida fusca, was greatly induced to transcript by microcrystalline cellulose (MCC) substrate. Biochemical characterization showed that TfXyl10A is optimally effective at temperature of 80 °C and pH of 9.0. After removing the carbohydrate-binding module (CBM) and linker regions, the optimum temperature of TfXyl10A-CD was reduced by 10°C (to 70°C), at which the enzyme's temperature tolerance was also weakened. While truncating only the CBM domain (TfXyl10AdC) had no significant effect on its thermostability. Importantly, polysaccharide-binding experiment showed that the auxiliary domain CBM2 could specifically bind to cellulose substrates, which endowed xylanase TfXyl10A with the ability to degrade xylan surrounding cellulose. These results indicated that TfXyl10A might be an excellent candidate in bio-bleaching processes of paper industry. In addition, the features of active-site architecture of TfXyl10A in GH10 family were further analyzed. By mutating each residue at the -2 and -1 subsites to alanine, the binding force and enzyme activity of mutants were observably decreased. Interestingly, the mutant E51A, locating at the distal -3 subsite, exhibited 90% increase in relative activity compared with wild-type (WT) enzyme TfXyl10A-CD (the catalytic domain of TfXyl110A). This study explored the function of a GH10 xylanase containing a CBM2 domain and the contribution of amino acids in active-site architecture to catalytic activity. The results obtained provide guidance for the rational design of xylanases for industrial applications under high heat and alkali-based operating conditions, such as paper bleaching.
| S-EPMC9459120 | biostudies-literature
Project description:Xylanase with a high thermostability will satisfy the needs of raising the temperature of hydrolysis to improve the rheology of the broth in industry of biomass conversion. In this study, a xylanase gene (xyn10A), predicted to encode a hydrolase domain of GH10, a linker region and a CBM1 domain, was cloned from a superior lignocellulose degrading strain Aspergillus fumigatus Z5 and successfully expressed in Pichia pastoris X33. Xyn10A has a specific xylanase activity of 34.4 U mg-1, and is optimally active at 90 °C and pH 6.0. Xyn10A shows quite stable at pHs ranging from 3.0 to 11.0, and keeps over 40% of xylanase activity after incubation at 70 °C for 1 h. Removal of CBM1 domain has a slight negative effect on its thermostability, but the further cleavage of linker region significantly decreased its stability at high temperature. The transfer of CBM1 and linker region to another GH10 xylanase can help to increase the thermostability. In addition, hydrolase domains between the two Xyn10A proteins naturally formed a dimer structure, which became more thermostable after removing the CBM1 or/and linker region. This thermostable Xyn10A is a suitable candidate for the highly efficient fungal enzyme cocktails for biomass conversion.
| S-EPMC5862715 | biostudies-literature
Project description:A two-domain GH10 xylanase-encoding gene (amor_gh10a) was discovered from a metagenomic data set, generated after in situ incubation of a lignocellulosic substrate in hot sediments on the sea floor of the Arctic Mid-Ocean Ridge (AMOR). AMOR_GH10A comprises a signal peptide, a carbohydrate-binding module belonging to a previously uncharacterized family, and a catalytic glycosyl hydrolase (GH10) domain. The enzyme shares the highest sequence identity (42%) with a hypothetical protein from a Verrucomicrobia bacterium, and its GH10 domain shares low identity (24 to 28%) with functionally characterized xylanases. Purified AMOR_GH10A showed thermophilic and halophilic properties and was active toward various xylans. Uniquely, the enzyme showed high activity toward amorphous cellulose, glucomannan, and xyloglucan and was more active toward cellopentaose than toward xylopentaose. Binding assays showed that the N-terminal domain of this broad-specificity GH10 binds strongly to amorphous cellulose, as well as to microcrystalline cellulose, birchwood glucuronoxylan, barley β-glucan, and konjac glucomannan, confirming its classification as a novel CBM (CBM85).IMPORTANCE Hot springs at the sea bottom harbor unique biodiversity and are a promising source of enzymes with interesting properties. We describe the functional characterization of a thermophilic and halophilic multidomain xylanase originating from the Arctic Mid-Ocean Ridge vent system, belonging to the well-studied family 10 of glycosyl hydrolases (GH10). This xylanase, AMOR_GH10A, has a surprisingly wide substrate range and is more active toward cellopentaose than toward xylopentaose. This substrate promiscuity is unique for the GH10 family and could prove useful in industrial applications. Emphasizing the versatility of AMOR_GH10A, its N-terminal domain binds to both xylans and glycans, while not showing significant sequence similarities to any known carbohydrate-binding module (CBM) in the CAZy database. Thus, this N-terminal domain lays the foundation for the new CBM85 family.
| S-EPMC6414390 | biostudies-literature
Project description:Glycoside hydrolase family 10 (GH10) xylanases are responsible for enzymatic cleavage of the internal glycosidic linkages of the xylan backbone, to generate xylooligosaccharides (XOS) and xyloses. The topologies of active-site cleft determine the substrate preferences and product profiles of xylanases. In this study, positional bindings and substrate interactions of TmxB, one of the most thermostable xylanases characterized from Thermotoga maritima to date, was investigated by docking simulations. XOS with backbone lengths of two to five (X2?X5) were docked into the active-site cleft of TmxB by AutoDock The modeled complex structures provided a series of snapshots of the interactions between XOS and TmxB. Changes in binding energy with the length of the XOS backbone indicated the existence of four effective subsites in TmxB. The interaction patterns at subsites -2 to +1 in TmxB were conserved among GH10 xylanases whereas those at distal aglycone subsite +2, consisting of the hydrogen bond network, was unique for TmxB. This work helps in obtaining an in-depth understanding of the substrate-binding property of TmxB and provides a basis for rational design of mutants with desired product profiles.
| S-EPMC6163442 | biostudies-literature
Project description:BACKGROUND:Xylanase is an important component of hemicellulase enzyme system. Since it plays an important role in the hydrolysis of hemicellulose into xylooligosaccharides (XOs), high thermostable xylanase has been the focus of much recent attention as powerful enzyme as well as in the field of biomass utilization. RESULTS:A xylanase gene (xyn10A) with 3,474 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum that encodes a protein containing 1,158 amino acid residues. Based on amino acid sequence homology, hydrophobic cluster and three dimensional structure analyses, it was attested that the xylanase belongs to the glycoside hydrolase (GH) families 10 with five carbohydrate binding domains. When the xylanase gene was cloned and expressed in Escherichia coli BL21 (DE3), the specific enzyme activity of xylanase produced by the recombinant strain was up to 145.8 U mg-1. The xylanase was optimally active at 95°C, pH 7.0. In addition, it exhibited high thermostability over broad range of pH 4.0-8.5 and temperature 55-90°C upon the addition of 5 mM Ca2+. Confirmed by Ion Chromatography System (ICS) analysis, the end products of the hydrolysis of beechwood xylan were xylose, xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose. CONCLUSIONS:The xylanase from T. thermarum is one of the hyperthermophilic xylanases that exhibits high thermostability, and thus, is a suitable candidate for generating XOs from cellulosic materials such as agricultural and forestry residues for the uses as prebiotics and precursors for further preparation of furfural and other chemicals.
| S-EPMC3598563 | biostudies-literature
Project description:Wheat (Triticum aestivum L.) is an important staple crop. Rhizoctonia cerealis is the causal agent of diseases that are devastating to cereal crops, including wheat. Xylanases play an important role in pathogenic infection, but little is known about xylanases in R. cerealis. Herein, we identified nine xylanase-encoding genes from the R. cerealis genome, named RcXYN1-RcXYN9, examined their expression patterns, and investigated the pathogenicity role of RcXYN1. RcXYN1-RcXYN9 proteins contain two conserved glutamate residues within the active motif in the glycoside hydrolase 10 (GH10) domain. Of them, RcXYN1-RcXYN4 are predicted to be secreted proteins. RcXYN1-RcXYN9 displayed different expression patterns during the infection process of wheat, and RcXYN1, RcXYN2, RcXYN5, and RcXYN9 were expressed highly across all the tested inoculation points. Functional dissection indicated that the RcXYN1 protein was able to induce necrosis/cell-death and H2O2 generation when infiltrated into wheat and Nicotiana benthamiana leaves. Furthermore, application of RcXYN1 protein followed by R. cerealis led to significantly higher levels of the disease in wheat leaves than application of the fungus alone. These results demonstrate that RcXYN1 acts as a pathogenicity factor during R. cerealis infection in wheat. This is the first investigation of xylanase genes in R. cerealis, providing novel insights into the pathogenesis mechanisms of R. cerealis.
| S-EPMC7084588 | biostudies-literature