Main Components Of Hemicellulose Research Articles

The Discovery of a Multidomain Mannanase Containing Dual-Catalytic Domain of the Same Activity: Biochemical Properties and Synergistic Effect.

In recent years, the wide application of mannan has driven the demand for the exploration of mannanase. As one of the main components of hemicellulose, mannan is an important polysaccharide that ruminants need to degrade and utilize, making rumen a rich source of mannanases. In this study, gene mining of mannanases was performed using bioinformatics, and potential dual-catalytic domain mannanases were heterologously expressed to analyze their properties. The hydrolysis pattern and enzymatic products were identified by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). A dual-catalytic domain mannanase Man26/5 with the same function as the substrate was successfully mined from the genome of cattle rumen microbiota. Compared to the single-catalytic domain, its higher thermal stability (≤50 °C) and catalytic efficiency confirm the synergistic effect between the two catalytic domains. It exhibited a unique "crab-like" structure where the CBM located in the middle is responsible for binding, and the catalytic domains at both ends are responsible for cutting. The exploration of its multidomain structure and synergistic patterns could provide a reference for the artificial construction and molecular modification of enzymes.

Critical Roles of Acidic Residues in Loop Regions of the Structural Surface for the Salt Tolerance of a GH39 β-d-Xylosidase.

Xylan is the main component of hemicellulose. Complete hydrolysis of xylan requires synergistically acting xylanases, such as β-d-xylosidases. Salt-tolerant β-d-xylosidases have significant application benefits, but few reports have explored the critical amino acids affecting the salt tolerance of xylosidases. Herein, the site-directed mutation was used to demonstrate that negative electrostatic potentials generated by 19 acidic residues in the loop regions of the structural surface positively correlated with the improved salt tolerance of GH39 β-d-xylosidase JB13GH39P28. These mutants showed reduced negative potentials on structural surfaces as well as a 13-43% decrease in stability in 3.0-30.0% (w/v) NaCl. Six key residue sites, D201, D259, D297, D377, D395, and D474, were confirmed to influence both the stability and activity of GH39 β-d-xylosidase. The activity of the GH39 β-d-xylosidase was found promoting by SO42- and inhibiting by NO3-. Values of Km and Kcat/Km decreased aggravatedly in 30.0% (w/v) NaCl when mutation operated on residues E179 and D182 in the loop regions of the catalytic domain. Taken together, mutation on acidic residues in loop regions from catalytic and noncatalytic domains may cause the deformation of catalytic pocket and aggregation of protein particles then decrease the stability, binding affinity, and catalytic efficiency of the β-d-xylosidase.

Effect of Steam Explosion on the Structure, Chemical Composition and Pyrolytic Volatile Composition of Tobacco Waste

Tobacco stems are an important part of the tobacco leaf, containing a lot of cellulose, result in the low utilization of tobacco leaves in cigarettes. The pre-treatment technology of tobacco stems is a key to increase the utilization of tobacco stems. In this work, steam explosion treatment was carried out on tobacco stems with different moisture contents containing 2%, 5% and 10%, respectively. The steam explosion is carried out under different pressure and pressure holding time conditions, the changes in physical structure, chemical composition and pyrolysis volatiles of tobacco stem was investigated. The results indicate that steam explosion has a negligible impact on the crystalline structure of cellulose and low-order polysaccharide crystals, while it has an obvious effect on KCl. Steam explosion increases the specific surface area by destroying the amorphous structure on tobacco stems. The contents of chemical constituents and pyrolytic volatiles indicate that there is a significant leaching effect of lignin, but no change in cellulose content. Soluble polysaccharides, the main component of hemicellulose, were converted into pyrolytic volatiles e.g., furfural and furfuryl alcohol. In conclusion, the technology of steam explosion promotes the leaching, decomposition and chemical transformation of amorphous structures on tobacco stems, thus destroying the structure of the cell walls.

Synergistic Effects of Stirring and Aeration Rate on Carotenoid Production in Yeast Rhodotorula toruloides CCT 7815 Envisioning Their Application as Soap Additives

The production of carotenoids by microbial organisms has gained significant interest due to the growing demand for natural products. Among the non-model oleaginous red yeasts, Rhodotorula toruloides stands out as an appealing host for natural carotenoid production. R. toruloides possesses the natural ability to metabolize a wide range of substrates, including lignocellulosic hydrolysates, and convert them into lipids and carotenoids. In this study, we focused on utilizing xylose, the main component of hemicellulose, as the major substrate for R. toruloides. We conducted a comprehensive kinetic evaluation to examine the impact of aeration and agitation on carotenoid production. Results in stirred-tank reactor demonstrated that under milder conditions (300 rpm and 0.5 vvm), R. toruloides accumulated over 70% of its cell mass as lipids. Furthermore, the highest carotenoid yields were achieved at high agitation rates (700 rpm), with carotenoid levels reaching nearly 120 µg/mL. Several carotenoids were identified, including β-carotene, γ-carotene, torularhodin, and torulene, with β-carotene being the major carotenoid, accounting for up to 70% of the total carotenoid content. The carotenoid-rich extract produced by R. toruloides under evaluated conditions was successfully incorporated into soap formulations, demonstrating the addition of antioxidant properties. This work provides a comprehensive understanding of xylose conversion into natural carotenoids by R. toruloides, presenting a promising avenue for their application in cosmetics. Furthermore, this study highlights the potential of a renewable and cost-effective approach for carotenoid production in the soap industry.

Production and Functional Characterization of a Novel Mannanase from Alteromonadaceae Bacterium Bs31.

Mannans are the main components of hemicellulose in nature and serve as the major storage polysaccharide in legume seeds. To mine new mannanase genes and identify their functional characteristics are an important basis for mannan biotechnological applications. In this study, a putative mannanase gene (ManBs31) from the genome of the marine bacterium Alteromonadaceae Bs31 was characterized. Amino acid sequence analysis and protein structural modeling were used to reveal the molecular features of ManBs31. The catalytic domain of ManBs31 was recombinantly produced using Escherichia coli and Pichia pastoris expression systems. The biochemical properties of the enzymes were determined by reducing sugar assay and thin-layer chromatography. Sequence analysis revealed that ManBs31 was a multidomain protein, consisting of a catalytic domain belonging to glycoside hydrolase family 5 (GH5) and two cellulose-binding domains. Recombinant ManBs31-GH5 exhibited the maximum hydrolytic performance at 70 ºC and pH 6. It showed the best hydrolysis capacity toward konjac glucomannan (specific enzyme activity up to 1070.84 U/mg) and poor hydrolysis ability toward galactomannan with high side-chain modifications (with a specific activity of 344.97 U/mg and 93.84 U/mg to locust bean gum and ivory nut mannan, respectively). The hydrolysis products of ManBs31-GH5 were mannooligosaccharides, and no monosaccharide was generated. Structural analysis suggested that ManBs31-GH5 had a noncanonical +2 subsite compared with other GH5 mannanases. ManBs31 was a novel thermophilic endo-mannanase and it provided a new alternative for the biodegradation of mannans, especially for preparation of probiotic mannooligosaccharides.

Structural and biophysical characterization of the multidomain xylanase Xyl.

The depletion of fossil fuels, associated pollution, and resulting health hazards are of concern worldwide. Woody biomass constitutes an alternative source of cleaner and renewable energy. The efficient use of woody biomass depends on xylan depolymerisation as the endo-β-1,4-xylopyranosyl homopolymer is the main component of hemicellulose, the second most abundant component of wood. Xylan depolymerisation is achieved by hemicellulolytic xylanases of glycoside hydrolase (GH) families 5, 8, 10, 11, 30 and 43 of the CAZY database. We analysed a multidomain xylanase (Xyl) from the hindgut metagenome of the snouted harvester termite Trinervitermes trinervoides that releases xylobiose and xylotriose from beech and birch xylan and wheat arabinoxylan. The four domains of Xyl include an N-terminal GH11 xylanase domain, two family 36-like carbohydrate-binding domains CBM36-1 and 2, and a C-terminal CE4 esterase domain. Previous analyses indicated that CBM36-1 deletion slightly increased GH11 catalysis at low pH whereas removal of both CBMs decreased xylanase activity at 60°C from 90 to 56%. Possible cooperativity between the domains suggested by these observations was explored. A crystal structure of the two-domain construct, GH11-CBM36-1, confirmed the structure of the GH11 domain whereas the CBM36-1 domain lacked electron density, possibly indicating a random orientation of the CBM36-1 domain around the GH11 domain. Isothermal titration calorimetry (ITC) experiments similarly did not indicate specific interactions between the individual domains of Xyl supporting a “beads-on-a-string” model for Xyl domains.

Purification of Xylooligosaccharide From Casavva Pulp by Ultrafiltration Method

Xylan is the main component of hemicellulose. Xylan can be extracted from agricultural waste, such as cassava pulp. Xylan is used as an endo-β-1,4-D-xylanase substrate to produce impure xylooligosaccharides (XOS)This study aims to purify XOS from cassava pulp using the ultrafiltration method. The components of XOS obtained from the enzymatic hydrolysis were analyzed using thin layer chromatography (TLC) and densitometry methods. In addition, the XOS was purified by the ultrafiltration method using a cellulose membrane with a Molecular Weight Cut Off (MWCO) of 12 kDa. The permeate obtained from the purification results was also analyzed using TLC and densitometry. The results of this study indicated that the components in XOS cassava pulp before and after purification by the TLC method were X5 and X6, while the XOS components before and after purification by the densitometric method were X3, X4 and X5.

Xylose Metabolism and the Effect of Oxidative Stress on Lipid and Carotenoid Production in Rhodotorula toruloides: Insights for Future Biorefinery.

The use of cell factories to convert sugars from lignocellulosic biomass into chemicals in which oleochemicals and food additives, such as carotenoids, is essential for the shift toward sustainable processes. Rhodotorula toruloides is a yeast that naturally metabolises a wide range of substrates, including lignocellulosic hydrolysates, and converts them into lipids and carotenoids. In this study, xylose, the main component of hemicellulose, was used as the sole substrate for R. toruloides, and a detailed physiology characterisation combined with absolute proteomics and genome-scale metabolic models was carried out to understand the regulation of lipid and carotenoid production. To improve these productions, oxidative stress was induced by hydrogen peroxide and light irradiation and further enhanced by adaptive laboratory evolution. Based on the online measurements of growth and CO2 excretion, three distinct growth phases were identified during batch cultivations. Majority of the intracellular flux estimations showed similar trends with the measured protein levels and demonstrated improved NADPH regeneration, phosphoketolase activity and reduced β-oxidation, correlating with increasing lipid yields. Light irradiation resulted in 70% higher carotenoid and 40% higher lipid content compared to the optimal growth conditions. The presence of hydrogen peroxide did not affect the carotenoid production but culminated in the highest lipid content of 0.65 g/gDCW. The adapted strain showed improved fitness and 2.3-fold higher carotenoid content than the parental strain. This work presents a holistic view of xylose conversion into microbial oil and carotenoids by R. toruloides, in a process toward renewable and cost-effective production of these molecules.

The Xylanase Inhibitor TAXI-I Increases Plant Resistance to Botrytis cinerea by Inhibiting the BcXyn11a Xylanase Necrotizing Activity.

During host plant infection, pathogens produce a wide array of cell wall degrading enzymes (CWDEs) to break the plant cell wall. Among CWDEs, xylanases are key enzymes in the degradation of xylan, the main component of hemicellulose. Targeted deletion experiments support the direct involvement of the xylanase BcXyn11a in the pathogenesis of Botrytis cinerea. Since the Triticum aestivum xylanase inhibitor-I (TAXI-I) has been shown to inhibit BcXyn11a, we verified if TAXI-I could be exploited to counteract B. cinerea infections. With this aim, we first produced Nicotiana tabacum plants transiently expressing TAXI-I, observing increased resistance to B. cinerea. Subsequently, we transformed Arabidopsis thaliana to express TAXI-I constitutively, and we obtained three transgenic lines exhibiting a variable amount of TAXI-I. The line with the higher level of TAXI-I showed increased resistance to B. cinerea and the absence of necrotic lesions when infiltrated with BcXyn11a. Finally, in a droplet application experiment on wild-type Arabidopsis leaves, TAXI-I prevented the necrotizing activity of BcXyn11a. These results would confirm that the contribution of BcXyn11a to virulence is due to its necrotizing rather than enzymatic activity. In conclusion, our experiments highlight the ability of the TAXI-I xylanase inhibitor to counteract B. cinerea infection presumably by preventing the necrotizing activity of BcXyn11a.

APPLICATION OF COFFEE PEEL WASTE AS RAW MATERRIAL FOR XYLOOLIGOSACCHARIDE PRODUCTION

<p>Coffee is the second most common trade commodity in the world after petroleum. The coffee industry generates large amounts of waste in the form of coffee peels. Coffee peel waste consist of lignocellulose containing hemicellulose and other chemical compounds. The objective of this research was to extract xylan, the main component of hemicellulose from coffee waste, and to utilize the xylan in the production of xylooligosaccharide (XOS). Xylan was extracted from coffee waste using NaOH solution and neutralized by HCl 6 M. Afterward, xylan was precipitated using ethanol. Various NaOH concentrations (4, 8, 12 and 16 % w/v) were used to obtain the xylan. NaOH solution with a concentration of 12 % results 43 % extraction of xylan from coffee waste. Xylan obtained from the extraction was hydrolyzed using endo-β-1,4-xylanase from Bacillus sp. to produce XOS. The incubation time of enzyme-substrate was observed at 40 oC, pH 5 and enzyme dose of 23.6 U. Thin layer chromatography results showed that the hydrolyzed products of xylan are XOS with composition xylobiose (X2), xylotriose (X3), xylotetraose (X4) and xylopentaose (X5). LC-MS studies revealed that X2 is the dominant product with the concentration of 6.00 ppm. This research demonstrates the potential to utilizate coffee peel waste as a source of xylan for the production of XOS.</p>

Hemicellulose modification promotes cadmium hyperaccumulation by decreasing its retention on roots in Sedum alfredii

As a vital polysaccharide related to mechanisms of plant resistance to trace metal in the root cell wall, the role of hemicellulose in cadmium (Cd) accumulation in hyperaccumulators is still unknown. We investigated hemicellulose modification in response to Cd in two populations of Sedum alfredii. Nonhyperaccumulating population (NHP) and hyperaccumulating population (HP) of S. alfredii were grown in nutrient solutions with or without 25 μM Cd for 15 d. Monosaccharide composition of root cell wall hemicellulose and its remolding mechanisms (e.g. enzyme activity and gene expression) were analyzed by using gas chromatography-mass spectrometer (GC-MS), fourier transform infrared spectrometer (FTIR), nuclear magnetic resonance (NMR) and quantitative real-time PCR techniques. In 25 μM Cd treatment, root cell wall hemicellulose in the NHP significantly (P < 0.05) increased and its hemicellulose-bound Cd was nearly 2.5-fold higher than that of HP. In the presence of Cd, xylose and glucose, proved to be the main component of hemicellulose, were higher in the NHP than in the HP owing to the up-regulation of XET/XEH and encoding gene (XTH 31). 113Cd-NMR and FTIR results indicated that hemicellulose with hydroxyl and carboxyl groups of HP retained less Cd than that of NHP. Hemicellulose modification decreased the Cd-binding capacity of the root cell wall and increased the entry of Cd in the shoot of HP S. alfredii.

Characterization of hemicelluloses from leaves and tops of the CC 8475, CC 8592, and V 7151 varieties of sugarcane (Saccharum officinarum L

In this research, the types of hemicellulose that predominate in the leaves and tops of the three most cultivated varieties (CC 8475, CC 8592, V 7151) of sugarcane (Saccharum officinarum L.) in Valle del Cauca, Colombia were determined. Hemicellulose analyses were performed after delignification with sodium chlorite and extraction with 18% NaOH and 24% KOH containing 0.26 M NaBH4. The main components of hemicellulose were identified via FTIR and NMR spectroscopy, and monomeric sugars were identified via HPLC. Hemicellulose A composed of arabinoxylans, glucomannans and arabinogalactactans and hemicellulose B primarily composed of arabinoxylans were extracted. The hemicelluloses of the Colombian varieties were more stable against heat than those of the Venezuelan variety. The results of this project allowed discovery of the potential use of agricultural sugarcane residues for bioethanol production because they have a holocellulose content of more than 60%.

The integration of dilute acid hydrolysis of xylan and fast pyrolysis of glucan to obtain fermentable sugars.

BackgroundFermentable sugars are important intermediates in the biological conversion of biomass. Hemicellulose and amorphous cellulose are easily hydrolyzed to fermentable sugars in dilute acid, whereas crystalline cellulose is more difficult to be hydrolyzed. Cellulose fast pyrolysis is an alternative method to liberate valuable fermentable sugars from biomass. The amount of levoglucosan generated from lignocellulose by fast pyrolysis is usually lower than the theoretical yield based on the cellulose fraction. Pretreatment is a promising route to improve the yield of levoglucosan from lignocellulose.ResultsThe integration of dilute sulfuric acid hydrolysis and fast pyrolysis to obtain fermentable sugars was evaluated in this study. Dilute sulfuric acid hydrolysis could remove more than 95.1 and 93.4 % of xylan (the main component of hemicellulose) from sugarcane bagasse and corncob with high yield of xylose. On the other hand, dilute sulfuric acid hydrolysis was also an effective pretreatment to enhance levoglucosan yield from lignocellulose. Dilute acid hydrolysis could accumulate glucan (the component of cellulose) and remove most of the alkali and alkaline earth metals which were powerful catalysts during fast pyrolysis. Further increase in dilute acid concentration (from 0 to 2 %) in pretreatment could promote the yield of levoglucosan in fast pyrolysis. The acid pretreated sugarcane bagasse and corncob gave levoglucosan yields of 43.8 and 35.2 % which were obvious higher than those of raw sugarcane bagasse (12.0 %) and corncob (7.0 %).ConclusionsObtaining fermentable sugars by combination dilute acid hydrolysis of xylan and fast pyrolysis of glucan could make full utilization of biomass, and get fermentable sugars economically from biomass for bio-refinery.

High production of fumaric acid from xylose by newly selected strain Rhizopus arrhizus RH 7-13-9#

Fumaric acid, as an important material for polymerization, is highly expected to be produced by fermentation of lignocellulosic biomass which is composed of cellulose, hemicellulose and lignin. Xylose as the main component of hemicellulose cannot be efficiently utilized by most of the common fermentation. In this study, a new strain Rhizopus arrhizus RH 7-13-9# was selected from the R. arrhizus RH 7-13 through a novel convenient and efficient selection method. Efficient production of fumaric acid (45.31g/L) from xylose was achieved by the new strain, and the volumetric productivity was still 0.472g/Lh. Moreover, the conversion of xylose reached 73% which is close to the theoretic yield (77%). The production of fumaric acid was increased approximate by 172%, compared with the initial strain counterpart. These results indicated that xylose, as the main component of hemicellulose, has a promising application for the production of fumaric acid on an industrial-scale.

Obtaining new cultures of microorganisms that produces cellulases and xylanases from the sugarcane bagasse

Background The international energetic system strongly depends on fossil fuels, which causes negative effects in the environment, such as the global warming. Biofuels appear as an environmental and economic alternative for the energetic industry because of their potential source of renewable energy. Several studies are based on sugarcane culture and its derivatives, as bagasse, the sugarcane residue. Bioethanol can be produced by the fermentation of sugar or by the hydrolysis of cellulosic biomass [1]. The plant cell wall is constituted of cellulose (40-50%), hemicellulose (15-30%) and lignin (1030%), forming the vegetal biomass. Cellulases are enzymes that form a complex that hydrolyses cellulosic materials, releasing sugars [2]. The main component of hemicellulose is the xylan, which is hydrolyzed by xylanases [3]. Cellulases as xylanases have a great biotechnological potential, they can be used in a variety of field: food, animal feed, textile and paper recycling industries. The sugarcane bagasse (SCB) is the most studied lignocellulosic waste for bioethanol production, because it is a by-product of conventional ethanol and can be find in large amount in Brazil [4]. Nowadays, the process of bioconversion of biomass has high cost and low specific activity of the enzymes that are necessary for the cellulose saccharification [5]. The aim of this research is to obtain microorganisms that hydrolyze the sugarcane bagasse and to quantify the sugar production.

First Structural Insights into α-l-Arabinofuranosidases from the Two GH62 Glycoside Hydrolase Subfamilies

α-L-arabinofuranosidases are glycoside hydrolases that specifically hydrolyze non-reducing residues from arabinose-containing polysaccharides. In the case of arabinoxylans, which are the main components of hemicellulose, they are part of microbial xylanolytic systems and are necessary for complete breakdown of arabinoxylans. Glycoside hydrolase family 62 (GH62) is currently a small family of α-L-arabinofuranosidases that contains only bacterial and fungal members. Little is known about the GH62 mechanism of action, because only a few members have been biochemically characterized and no three-dimensional structure is available. Here, we present the first crystal structures of two fungal GH62 α-L-arabinofuranosidases from the basidiomycete Ustilago maydis (UmAbf62A) and ascomycete Podospora anserina (PaAbf62A). Both enzymes are able to efficiently remove the α-L-arabinosyl substituents from arabinoxylan. The overall three-dimensional structure of UmAbf62A and PaAbf62A reveals a five-bladed β-propeller fold that confirms their predicted classification into clan GH-F together with GH43 α-L-arabinofuranosidases. Crystallographic structures of the complexes with arabinose and cellotriose reveal the important role of subsites +1 and +2 for sugar binding. Intriguingly, we observed that PaAbf62A was inhibited by cello-oligosaccharides and displayed binding affinity to cellulose although no activity was observed on a range of cellulosic substrates. Bioinformatic analyses showed that UmAbf62A and PaAbf62A belong to two distinct subfamilies within the GH62 family. The results presented here provide a framework to better investigate the structure-function relationships within the GH62 family.

Phenotypic manifestation of gene expression encoding xyloglucanase from Penicillium canescens in transgenic aspen plants

Plant xyloglucans play an important role in the processes of cell wall extension, determine their mechanical properties, thus affecting growth and morphology of individual cells and whole organs. Being one of the main components of hemicellulose, xyloglucans play a particular physiological role in woody plants. To study xyloglucan physiological role, transgenic aspen (Populus tremula L.) plants with a recombinant sp-Xeg gene from the fungus Penicillium canescens were produced. Constitutive expression of this gene in the heterologous surrounding was confirmed by RT-PCR method. The analysis of protein extracts from the leaves of greenhouse-grown plants and microshoots grown in vitro showed activation of xylogluconase in transgenic lines. The strongest activation (1.6-fold) was observed in the leaf extracts (clone PtXVXeg1b) and in vitro microshoots (clone PtXVXeg1c). In transgenic plants, the relative content of pentosans in the wood was declined. In control plants (Pt genotype), it was equal to 148 mg/g dry wt, whereas in tested clones (PtXVXeg1a, PtXVXeg1b, and PtXVXeg1c), it varied from 100 to 140 mg/g dry wt. The strongest decrease (by 31%) in the content of pentosans was observed for the line PtXVXeg1c; the content was equal to 102.1 ± 1.5 mg/g dry wt. A comparative analysis of leaf morphology revealed an increase in the length of petiole and a decrease in the length of the main vein in transgenic lines. In control plants, the ratio of the petiole length to the length of the main vein was equal to 0.49, whereas in transgenic plants, it varied from 0.51 to 0.66. A significant increase of this index was observed in 12 from 14 transgenic lines.

Cloning and Characterization of the Xyn11A Gene from Lentinula edodes

Hemicellulose represents a rich source of biomass that can be converted into useful chemical feedstocks. One of the main components of hemicellulose is xylan, a polymer of xylose residues. Xylanase enzymes that hydrolyze xylan are therefore of great commercial interest. We have cloned a gene (xyn11A) that encodes a 283-amino acid xylanase enzyme from the fungus Lentinula edodes. The enzyme has a pI of 4.6 and belongs to the highly conserved glycosyl hydrolase family 11. The xylanase gene was cloned into a Pichia pastoris expression vector that secretes active enzyme into both solid and liquid media. The optimal reaction conditions were at pH 4.5 and 50 degrees C. The enzyme had a Km of 1.5 mg/ml and a Vmax of 2.1 mmol/min/mg. Xyn11A produced primarily xylobiose, xylotriose, and xylotetraose from a birchwood xylan substrate. This is the first report on the cloning of a hemicellulase gene from L. edodes.