Characterization of a new bifunctional endo-1,4-β-xylanase/esterase found in the rumen metagenome.
Ontology highlight
ABSTRACT:
SUBMITTER: Pavarina GC
PROVIDER: S-EPMC8128909 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
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: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: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-β-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 β-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 β-xylanase proteins.
| S-EPMC3092692 | 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 enzymatic degradation of semi-cellulosic substrates has recently received immense attention. The enzyme endo-1,4-β-xylanase is essential for the complete digestion of complex and heterogeneous hemicellulose. Here, the purification, crystallization and preliminary X-ray free-electron laser (XFEL) diffraction analysis of endo-1,4-β-xylanase from the fungus Hypocrea virens (HviGH11) are reported. Codon-optimized HviGH11 was overexpressed in Escherichia coli and spontaneously crystallized after His-tag purification and concentration. Preliminary XFEL diffraction data were collected at the Pohang Accelerator Laboratory XFEL (PAL-XFEL). A total of 1021 images containing Bragg peaks were obtained and indexed. The HviGH11 crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 43.80, b = 51.90, c = 94.90 Å. Using 956 diffraction patterns, the phasing problem was solved and an initial model structure of HviGH11 was obtained.
| S-EPMC9158662 | 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 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: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:Xylanases are important for the enzymatic breakdown of lignocellulose-based biomass to produce biofuels and other value-added products. We report functional and structural analyses of TsaGH11, an endo-1,4-β-xylanase from the hemicellulose-degrading bacterium, Thermoanaerobacterium saccharolyticum. TsaGH11 was shown to be a thermophilic enzyme that favors acidic conditions with maximum activity at pH 5.0 and 70 °C. It decomposes xylans from beechwood and oat spelts to xylose-containing oligosaccharides with specific activities of 5622.0 and 3959.3 U mg-1, respectively. The kinetic parameters, Km and kcat towards beechwood xylan, are 12.9 mg mL-1 and 34,015.3 s-1, respectively, resulting in kcat/Km value of 2658.7 mL mg-1 s-1, higher by 102-103 orders of magnitude compared to other reported GH11s investigated with the same substrate, demonstrating its superior catalytic performance. Crystal structures of TsaGH11 revealed a β-jelly roll fold, exhibiting open and close conformations of the substrate-binding site by distinct conformational flexibility to the thumb region of TsaGH11. In the room-temperature structure of TsaGH11 determined by serial synchrotron crystallography, the electron density map of the thumb domain of the TsaGH11 molecule, which does not affect crystal packing, is disordered, indicating that the thumb domain of TsaGH11 has high structural flexibility at room temperature, with the water molecules in the substrate-binding cleft being more disordered than those in the cryogenic structure. These results expand our knowledge of GH11 structural flexibility at room temperature and pave the way for its application in industrial biomass degradation.
| S-EPMC10576002 | biostudies-literature
Project description:BackgroundThe coffee berry borer, Hypothenemus hampei, reproduces and feeds exclusively on the mature endosperm of the coffee seed, which has a cell wall composed mainly of a heterogeneous mixture of hemicellulose polysaccharides, including arabinoxylans. Xylanases are digestive enzymes responsible for the degradation of xylan based polymers, hydrolyzing them into smaller molecules that are easier to assimilate by insects. We report the cloning, expression and enzymatic characterization of a xylanase gene that was identified in the digestive tract of the coffee berry borer.MethodsThe complete DNA sequence encoding a H. hampei xylanase (HhXyl) was obtained using a genome walking technique in a cDNA library derived from the borer digestive tract. The XIP-I gene was amplified from wheat (Triticum aestivum variety Soisson). A Pichia pastoris expression system was used to express the recombinant form of these enzymes. The xylanase activity and XIP-I inhibitory activity was quantified by the 3,5-dinitrosalicylic (DNS). The biological effects of XIP-I on borer individuals were evaluated by providing an artificial diet enriched with the recombinant XIP-I protein to the insects.ResultsThe borer xylanase sequence contains a 951 bp open reading frame that is predicted to encode a 317-amino acid protein, with an estimated molecular weight of 34.92 kDa and a pI of 4.84. Bioinformatic analysis revealed that HhXyl exhibits high sequence homology with endo-β-D-xylanases of Streptomyces bingchenggensis from glycosyl hydrolase 10 (GH10). The recombinant xylanase showed maximal activity at pH 5.5 and 37°C. XIP-I expressed as a recombinant protein inhibited HhXyl activity in vitro and caused individual H. hampei mortality in bioassays when included as a supplement in artificial diets.ConclusionA xylanase from the digestive tract of the coffee berry borer was identified and functionally characterized. A xylanase inhibitor protein, XIP-I, from wheat was shown to be a potent inhibitor of this xylanase, suggesting that its deployment has potential as a strategy to control coffee berry borer colonization of coffee plants.
| S-EPMC3283504 | biostudies-literature