Characterization of a new bifunctional endo-1,4-β-xylanase/esterase found in the rumen metagenome.
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ABSTRACT:
SUBMITTER: Pavarina GC
PROVIDER: S-EPMC8128909 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundThe utilization of raw biomass components such as cellulose or hemicellulose for the production of valuable chemicals has attracted considerable research interest in recent years. One promising approach is the application of microorganisms that naturally convert biomass constituents into value added chemicals. One of these organisms--Ustilago maydis--can grow on xylan, the second most abundant polysaccharide in nature, while at the same time it produces chemicals of biotechnological interest.ResultsIn this study, we present the identification of an endo-1,4-beta xylanase responsible for xylan degradation. Xylanase activity of U. maydis cells was indirectly detected by the quantification of released reducing sugars and could be confirmed by visualizing oligosaccharides as degradation products of xylan by thin layer chromatography. A putative endo-1,4-beta-xylanase, encoded by um06350.1, was identified in the supernatant of xylan-grown cells. To confirm the activity, we displayed the putative xylanase on the surface of the xylanase negative Saccharomyces cerevisiae EBY100. The presented enzyme converted xylan to xylotriose, similar to the source organism U. maydis.ConclusionsThe xylan degradation ability together with its unicellular and yeast-like growth makes U. maydis MB215 a promising candidate for the production of valuable chemicals such as itaconic acid or glycolipids from lignocellulosic biomass. Therefore, the characterization of the endo-1,4-beta-xylanase, encoded by um06350.1, is a further step towards the biotechnological application of U. maydis and its enzymes.
| S-EPMC3737115 | biostudies-literature
Project description:The field of computational protein design has experienced important recent success. However, the de novo computational design of high-affinity protein-ligand interfaces is still largely an open challenge. Using the Rosetta program, we attempted the in silico design of a high-affinity protein interface to a small peptide ligand. We chose the thermophilic endo-1,4-?-xylanase from Nonomuraea flexuosa as the protein scaffold on which to perform our designs. Over the course of the study, 12 proteins derived from this scaffold were produced and assayed for binding to the target ligand. Unfortunately, none of the designed proteins displayed evidence of high-affinity binding. Structural characterization of four designed proteins revealed that although the predicted structure of the protein model was highly accurate, this structural accuracy did not translate into accurate prediction of binding affinity. Crystallographic analyses indicate that the lack of binding affinity is possibly due to unaccounted for protein dynamics in the 'thumb' region of our design scaffold intrinsic to the family 11 ?-xylanase fold. Further computational analysis revealed two specific, single amino acid substitutions responsible for an observed change in backbone conformation, and decreased dynamic stability of the catalytic cleft. These findings offer new insight into the dynamic and structural determinants of the ?-xylanase proteins.
| S-EPMC3092692 | biostudies-literature
Project description:This study describes the catalytic properties of a GH30_7 xylanase produced by the fungus Talaromyces leycettanus. The enzyme is an ando-β-1,4-xylanase, showing similar specific activity towards glucuronoxylan, arabinoxylan, and rhodymenan (linear β-1,3-β-1,4-xylan). The heteroxylans are hydrolyzed to a mixture of linear as well as branched β-1,4-xylooligosaccharides that are shorter than the products generated by GH10 and GH11 xylanases. In the rhodymenan hydrolyzate, the linear β-1,4-xylooligosaccharides are accompanied with a series of mixed linkage homologues. Initial hydrolysis of glucuronoxylan resembles the action of other GH30_7 and GH30_8 glucuronoxylanases, resulting in a series of aldouronic acids of a general formula MeGlcA2Xyln. Due to the significant non-specific endoxylanase activity of the enzyme, these acidic products are further attacked in the unbranched regions, finally yielding MeGlcA2Xyl2-3. The accommodation of a substituted xylosyl residue in the -2 subsite also applies in arabinoxylan depolymerization. Moreover, the xylose residue may be arabinosylated at both positions 2 and 3, without negatively affecting the main chain cleavage. The catalytic properties of the enzyme, particularly the great tolerance of the side-chain substituents, make the enzyme attractive for biotechnological applications. The enzyme is also another example of extraordinarily great catalytic diversity among eukaryotic GH30_7 xylanases.
| S-EPMC8347862 | biostudies-literature
Project description:In this study, an endo-1,4-?-xylanase was purified from wheat malt following the procedures of ammonium sulfate precipitation, cation-exchange chromatography, and two-step anion-exchange chromatography. The purified endo-1,4-?-xylanase had a specific activity of 3.94 u/mg, demonstrating a weight average molecular weight (Mw) of approximately 58,000 Da. After LC-MS/MS (Liquid chromatography-tandem mass spectrometry) identification, the purified enzyme had the highest matching degree with a GH10 (Glycoside Hydrolase 10) domain-containing protein from wheat, there were 23 match peptides with a score above the threshold and the prot-cover was 45.5%. The resulting purified enzyme was used to investigate its degradation ability on high viscosity wheat-derived water-extractable arabinoxylan (WEAX). Degradation experiments confirmed that the purified enzyme was a true endo-acting enzyme, which could degrade large WEAX into smaller WEAX. The average degree of polymerization (avDP) and the viscosity of WEAX decreased with the increasing reaction time. The enzyme could degrade a small amount of WEAX into arabinoxylan-oligosaccharides (AXOS) with a degree of polymerization of 2-6, but no monosaccharide was produced. The degradation occurred rapidly in the first 3.5 h and decreased with the further prolongation of reaction time.
| S-EPMC7180899 | biostudies-literature
Project description:The food enzyme is an endo-1,4-β-xylanase (4-β-d-xylan xylanohydrolase; EC 3.2.1.8) produced with the genetically modified Bacillus subtilis strain XAS. Antibiotic resistance genes are present in the production organism on a self-replicative vector. The endo-1,4-β-xylanase is intended to be used in baking processes. Based on the maximum use levels, dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.014 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no-observed-adverse-effect level (NOAEL) of 55 mg TOS/kg bw per day that, compared with the estimated dietary exposure, results in a sufficiently high margin of exposure (MOE) (of at least 3,600). Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood to occur is considered to be low. Since the absence of viable cells in the food enzyme has not been adequately demonstrated, the Panel cannot conclude on the risks associated with the possible spread of a genetically modified bacterial strain carrying antimicrobial resistance determinants.
| S-EPMC7009094 | biostudies-literature
Project description:A series of strategies were applied to improve expression level of recombinant endo-β-1,4-xylanase from Aspergillus usamii (A. usamii) in Pichia pastoris (P. pastoris). Firstly, the endo-β-1,4-xylanase (xynB) gene from A. usamii was optimized for P. pastoris and expressed in P. pastoris. The maximum xylanase activity of optimized (xynB-opt) gene was 33500 U/mL after methanol induction for 144 h in 50 L bioreactor, which was 59% higher than that by wild-type (xynB) gene. To further increase the expression of xynB-opt, the Vitreoscilla hemoglobin (VHb) gene was transformed to the recombinant strain containing xynB-opt. The results showed that recombinant strain harboring the xynB-opt and VHb (named X33/xynB-opt-VHb) displayed higher biomass, cell viability, and xylanase activity. The maximum xylanase activity of X33/xynB-opt-VHb in 50 L bioreactor was 45225 U/mL, which was 35% and 115% higher than that by optimized (xynB-opt) gene and wild-type (xynB) gene. Finally, the induction temperature of X33/xynB-opt-VHb was optimized in 50 L bioreactor. The maximum xylanase activity of X33/xynB-opt-VHb reached 58792 U/mL when the induction temperature was 22°C. The results presented here will greatly contribute to improving the production of recombinant proteins in P. pastoris.
| S-EPMC4811622 | biostudies-literature
Project description:The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-β-1,4-glucanase. The recombinant KG35 endo-β-1,4-glucanase showed optimal activity within the range of 30-50°C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50°C at a pH of 5-7.
| S-EPMC5628298 | biostudies-literature
Project description:Endo-1,4-β-xylanase (EC 3.2.1.8) is the enzyme from Ruminococcus albus 8 (R. albus 8) (Xyn10A), and catalyzes the degradation of arabinoxylan, which is a major cell wall non-starch polysaccharide of cereals. The crystallographic structure of Xyn10A is still unknown. For this reason, we report a computer-assisted homology study conducted to build its three-dimensional structure based on the known sequence of amino acids of this enzyme. In this study, the best similarity was found with the Clostridium thermocellum (C. thermocellum) N-terminal endo-1,4-β-D-xylanase 10 b. Following the 100 ns molecular dynamics (MD) simulation, a reliable model was obtained for further studies. Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) methods were used for the substrate xylotetraose having the reactive sugar, which was bound in the -1 subsite of Xyn10A in the 4C1 (chair) and 2SO (skew boat) ground state conformations. According to the simulations and free energy analysis, Xyn10A binds the substrate with the -1 sugar in the 2SO conformation 39.27 kcal·mol(-1) tighter than the substrate with the sugar in the 4C1 conformation. According to the Xyn10A-2SO Xylotetraose (X4(sb) interaction energies, the most important subsite for the substrate binding is subsite -1. The results of this study indicate that the substrate is bound in a skew boat conformation with Xyn10A and the -1 sugar subsite proceeds from the 4C1 conformation through 2SO to the transition state. MM-PBSA free energy analysis indicates that Asn187 and Trp344 in subsite -1 may an important residue for substrate binding. Our findings provide fundamental knowledge that may contribute to further enhancement of enzyme performance through molecular engineering.
| S-EPMC4227162 | biostudies-literature
Project description:BackgroundReplacing fossil fuel with renewable sources such as lignocellulosic biomass is currently a promising alternative for obtaining biofuel and for fighting against the consequences of climate change. However, the recalcitrant structure of lignocellulosic biomass residues constitutes a major limitation for its widespread use in industry. The efficient hydrolysis of lignocellulosic materials requires the complementary action of multiple enzymes including xylanases and β-xylosidases, which are responsible for cleaving exo- and endoxylan linkages, that release oligocarbohydrates that can be further processed by other enzymes.ResultsWe have identified the endo-β-1,4-xylanase Xyl2 from Fusarium oxysporum as a promising glycoside hydrolase family 11 enzyme for the industrial degradation of xylan. To characterize Xyl2, we have cloned the synthetic optimized gene and expressed and purified recombinant Xyl2 to homogeneity, finally obtaining 10 mg pure Xyl2 per liter of culture. The crystal structure of Xyl2 at 1.56 Å resolution and the structure of a methyl-xylopyranoside Xyl2 complex at 2.84 Å resolution cast a highly detailed view of the active site of the enzyme, revealing the molecular basis for the high catalytic efficiency of Xyl2. The kinetic analysis of Xyl2 demonstrates high xylanase activity and non-negligible β-xylosidase activity under a variety of experimental conditions including alkaline pH and elevated temperature. Immobilizing Xyl2 on a variety of solid supports enhances the enzymatic properties that render Xyl2 a promising industrial biocatalyst, which, together with the detailed structural data, may establish Xyl2 as a platform for future developments of industrially relevant xylanases.ConclusionsF. oxysporum Xyl2 is a GH11 xylanase which is highly active in free form and immobilized onto a variety of solid supports in a wide pH range. Furthermore, immobilization of Xyl2 on certain supports significantly increases its thermal stability. A mechanistic rationale for Xyl2's remarkable catalytic efficiency at alkaline pH is proposed on the basis of two crystallographic structures. Together, these properties render Xyl2 an attractive biocatalyst for the sustainable industrial degradation of xylan.
| S-EPMC5011838 | 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