Insoluble Xylan Research Articles

Cross-linked enzyme aggregates of xylanase, XynR8(N58D), for effective degradation of untreated lignocellulosic biomass

Enzymatic degradation of biomass is preferred over chemical methods for environmental protection. However, in most cases, a chemical pretreatment is still required to help improve enzyme accessibility of the biomass. Therefore, it is necessary to develop highly efficient enzymes for those reasons and to save costs accordingly. Costs can be further reduced if the enzymes are recycled. XynR8(N58D) is an engineered and highly active xylanase originating from the rumen fungus Neocallimastix patriciarum. In this study, XynR8(N58D) was extracellularly over-expressed by Pichia pastoris. The resulting crude enzyme solution, with a purity of XynR8(N58D) of over 80%, was used to prepare the cross-linked enzyme aggregates (CLEA) of XynR8(N58D). Specific activity of the crude enzyme solution was 11,599.39 IU/mg, and that of CLEA was 6129.42 IU/mg. The immobilization yield and the immobilization efficiency were 62.68 ± 16.20% and 84.31 ± 3.11%, respectively. The thermostability, stress tolerance, and shelf life of CLEA were significantly better than those of the free enzyme. The free enzyme and CLEA both effectively decomposed insoluble xylan and autoclaved corncob powders. Therefore, XynR8(N58D) made into CLEA not only maintained high activity, but also improved its stability and permitted its reuse or recyclability. Both forms of the enzyme can effectively degrade lignocellulosic biomass without relying on chemical pretreatment to reduce recalcitrance of the biomass structure. The application of XynR8(N58D) and its CLEA to biomass degradation can help establish an eco-friendly and more cost-effective process.

Size-controlled synthesis of xylan micro / nanoparticles by self-assembly of alkali-extracted xylan

Valorization of underutilized biobased feedstocks like hetero-polysaccharides is critical for the development of the biorefinery concept. Towards this goal, highly uniform xylan micro/nanoparticles with a particle size ranging from 400 nm to 2.5 μm in diameter were synthesized by a facile self-assembly method in aqueous solutions. Initial concentration of the insoluble xylan suspension was utilized to control the particle size. The method utilized supersaturated aqueous suspensions formed at standard autoclaving conditions without any other chemical treatments to create the resulting particles as solutions cooled to room temperature. Processing parameters of the xylan micro/nanoparticles were systematically studied and correlated with both the morphology and size of xylan particles. By adjusting the crowding of the supersaturated solutions, highly uniform dispersions of xylan particles were synthesized of defined size. The xylan micro/nanoparticles prepared by self-assembly have a quasi-hexagonal shape, like a tile, and depending upon solution concentrations xylan nanoparticles with a thickness of <100 nm were achieved at high concentrations. Based on the usefulness of polysaccharide nanoparticles, like cellulose nanocrystals, these particles have potential for unique structures for hydrogels, aerogels, drug delivery, and photonic materials. This study highlights the formation of a diffraction grating film for visible light with these size-controlled particles.

Characterization of a GH Family 43 β-Xylosidase Having a Novel Carbohydrate-binding Module from Paenibacillus xylaniclasticus Strain TW1.

Paenibacillus xylaniclasticus strain TW1, a gram-positive facultative anaerobic bacterium, was isolated as a xylanolytic microorganism from the wastes of a pineapple processing factory. A gene encoding one of its xylanolytic enzymes, a β-xylosidase, was cloned and sequenced. Sequence analysis revealed that this β-xylosidase, named PxXyl43A, was composed of a glycoside hydrolase (GH) family 43 subfamily 12 catalytic module and an unknown function module (UM). The full-length PxXyl43A (PxXyl43A) was heterologously expressed in Escherichia coli and purified. Recombinant PxXyl43A exhibited hydrolysis activity against both p-nitrophenyl-β-D-xylopyranoside (pNPX) and p-nitrophenyl-α-L-arabinofuranoside at specific activities of 250 and 310 mU/mg, respectively. The optimal reaction pH and temperature for pNPX hydrolysis were 7.1 and 54 ˚C, respectively. At pH 7.0 and 54 ˚C, the Km and kcat for pNPX were 1.2 mM and 2.8 ± 0.15 s-1, respectively. It was also discovered that the recombinant unknown function module of PxXyl43A (PxXyl43A-UM) could bind to insoluble xylans like birchwood xylan and oat spelt xylan, whereas it did not bind to cellulosic substrates such as ball-milled cellulose, carboxymethyl cellulose or lichenan. The PxXyl43A-UM's binding constant value Ka for oat spelt xylan was 2.0 × 10-5 M-1. These results suggest that PxXyl43A possesses a novel carbohydrate-binding module, named as CBM91, specific for xylan-containing polysaccharides.

Characterization of a New Xylanase Found in the Rumen Metagenome and Its Effects on the Hydrolysis of Wheat.

Wheat is the main ingredient of poultry diet, but its xylan has an adverse impact on poultry production. A novel xylanase from beef cattle rumen metagenome (RuXyn) and its effect on the wheat hydrolysis were investigated in the present study. The RuXyn coded for 377 amino acids and exhibited low identity (<40%) to previously reported proteins. The RuXyn was heterologously expressed in Escherichia coli and showed maximum activity at pH 6.0 and 40 °C. The activity of RuXyn could be increased by 79.8 and 36.0% in the presence of Ca2+ and Tween 20, respectively. The soluble xylan and insoluble xylan in wheat could be effectively degraded by RuXyn and xylooligosaccharides produced accounting for more than 80% of the products. This study demonstrates that RuXyn has substantial potential to improve the application of wheat in poultry production by degrading wheat xylan and the accompanying xylooligosaccharides produced.

In vitro study of the effect of xylooligosaccharides obtained from banana pseudostem xylan by enzymatic hydrolysis on probiotic bacteria

Xylooligosaccharides (XOS) are xylose oligomers, recognized for their prebiotic effect, with the ability to impact health by influencing the gut microbiota . This study aimed to produce XOS by xylan enzymatic hydrolysis and evaluate the prebiotic effect. Endoxylanase was produced by Aspergillus versicolor and purified, and the xylan from banana pseudostem was alkali extracted. XOS production was performed through enzymatic hydrolysis by varying the parameters of xylanase load, substrate mass load, and reaction time. Lactobacillus plantarum and Lactobacillus fermentum were grown using XOS as a carbon source. Using an enzymatic load of 30 IU. g −1 , it was possible to obtain a good amount of XOS and good yield, around 11 g.L −1 , and 61%, respectively. Regarding the substrate concentration , the increase of concentration also increased XOS production, however, decreased the yield. There was not significant XOS production until 16 h and production was similar after 24 h. XOS with a higher degree of polymerization were mainly produced. Both probiotic bacteria were able to grow on XOS containing medium. Up to 48 h, the growth was similar, of 10 10 CFU/mL, with glucose and XOS as the carbon source. The growth with XOS increased after 48 h, while the growth with glucose remained stationary. This study demonstrates that XOS from banana pseudostem xylan presents prebiotic properties by increasing probiotic bacteria growth. • Xylooligosaccharides (XOS) with higher degree of polymerization were mainly produced. • Different degree of polymerization XOS were produced by enzymatic hydrolysis of soluble and insoluble xylan. • Probiotic bacterias grew on media containing high degree of polymerization XOS, evidencing their prebiotic property.

In vitro versus in situ evaluation of xylan hydrolysis into xylo-oligosaccharides in broiler chicken gastrointestinal tract

Xylan hydrolysis into xylo-oligosaccharides (XOS) was evaluated both in the gizzard and ileum of broiler chickens, and by a 2-step in vitro digestion assay that simulated the pH, temperature and time period of the gastric and small intestine (SI) phases. Twelve dietary treatments with varying soluble and insoluble xylan levels, either with or without supplemental xylanase, were fed to broiler chickens (n = 576) for the in situ analysis, and were exposed to the in vitro assay. Relatedness of the two methods was strong for determination of XOS production in all dietary treatments for X5, X4, X3, X2 and X1, respectively, in both the gastric (r = 0.980, 0.853, 0.894, 0.870 and 0.951) and small intestine phase (r = 0.957, 0.923, 0.940, 0.970, 0.969) (P < 0.05). Consequently, the in vitro assay was used to illustrated the diversity of XOS production across different batches of wheat and barley in the presence of xylanase.

A mini review of xylanolytic enzymes with regards to their synergistic interactions during hetero-xylan degradation.

This review examines the recent models describing the mode of action of various xylanolytic enzymes and how these enzymes can be applied (sequentially or simultaneously) with their distinctive roles in mind to achieve efficient xylan degradation. With respect to homeosynergy, synergism appears to be as a result of β-xylanase and/or oligosaccharide reducing-end β-xylanase liberating xylo-oligomers (XOS) that are preferred substrates of the processive β-xylosidase. With regards to hetero-synergism, two cross relationships appear to exist and seem to be the reason for synergism between the enzymes during xylan degradation. These cross relations are the debranching enzymes such as α-glucuronidase or side-chain cleaving enzymes such as carbohydrate esterases (CE) removing decorations that would have hindered back-bone-cleaving enzymes, while backbone-cleaving-enzymes liberate XOS that are preferred substrates of the debranching and side-chain-cleaving enzymes. This interaction is demonstrated by high yields in co-production of xylan substituents such as arabinose, glucuronic acid and ferulic acid, and XOS. Finally, lytic polysaccharide monooxygenases (LPMO) have also been implicated in boosting whole lignocellulosic biomass or insoluble xylan degradation by glycoside hydrolases (GH) by possibly disrupting entangled xylan residues. Since it has been observed that the same enzyme (same Enzyme Commission, EC, classification) from different GH or CE and/or AA families can display different synergistic interactions with other enzymes due to different substrate specificities and properties, in this review, we propose an approach of enzyme selection (and mode of application thereof) during xylan degradation, as this can improve the economic viability of the degradation of xylan for producing precursors of value added products.

Molecular characterization of hypothetical scaffolding-like protein S1 in multienzyme complex produced by Paenibacillus curdlanolyticus B-6

Paenibacillus curdlanolyticus B-6 produces an extracellular multienzyme complex containing a hypothetical scaffolding-like protein and several xylanases and cellulases. The largest (280-kDa) component protein, called S1, has cellulose-binding ability and xylanase activity, thus was considered to function like the scaffolding proteins found in cellulosomes. S1 consists of 863 amino acid residues with predicted molecular mass 91,029 Da and includes two N-terminal surface layer homology (SLH) domains, but most of its sequence shows no homology with proteins of known function. Native S1 (nS1) was highly glycosylated. Purified nS1 and recombinant Xyn11A (rXyn11A) as a major xylanase subunit could assemble in a complex, but recombinant S1 (rS1) could not interact with rXyn11A, indicating that S1 glycosylation is necessary for assembly of the multienzyme complex. nS1 and rS1 showed weak, typical endo-xylanase activity, even though they have no homology with known glycosyl hydrolase family enzymes. S1 and its SLH domains bound tightly to the peptide-glycan layer of P. curdlanolyticus B-6, microcrystalline cellulose, and insoluble xylan, indicating that the SLHs of S1 bind to carbohydrate polymers and the cell surface. When nS1 and rXyn11A were co-incubated with birchwood xylan, the degradation ability was synergistically increased compared with that for each protein; however synergy was not observed for rS1 and rXynA. These results indicate that S1 may have a scaffolding protein-like function by interaction with enzyme subunits and polysaccharides through its glycosylated sites and SLH domains.

The extracellular endo-\u03b2-1,4-xylanase with multidomain from the extreme thermophile Caldicellulosiruptor lactoaceticus is specific for insoluble xylan degradation

BackgroundThe extremely thermophilic bacterium Caldicellulosiruptor lactoaceticus can degrade and metabolize untreated lignocellulosic biomass containing xylan. The mechanism of the bacterium for degradation of insoluble xylan in untreated biomass has not been revealed.ResultsIn the present study, the only annotated extracellular endo-β-1,4-xylanase (Xyn10B) with multidomain structures in C. lactoaceticus genome was biochemically characterized. Xyn10B contains three N-terminal consecutive family 22 carbohydrate-binding modules (CBMs), one GH10 catalytic domain (CD), two family 9 CBMs and two S-layer homology (SLH) modules in the C-terminal. CBM22a shares 27.1% and 27.2% sequence homology with CBM22b and CBM22c, respectively. The sequence homology between two CBM9 s and two SLHs is 26.8% and 25.6%, respectively. To elucidate the effect of multiple domains on the enzymatic properties of Xyn10B, the truncated variants of which (Xyn10B-TM1: CBM22a-CBM22b-CBM22c-CD10; Xyn10B-TM2: CBM22c-CD10; Xyn10B-TM3: CBM22c-CD10-CBM9a; and Xyn10B-TM4: CD10-CBM9a) were separately reconstructed, recombinantly expressed and biochemically characterized. Enzymatic properties studies showed that the optimal temperature for all four Xyn10B truncations was 65 °C. Compared to Xyn10B-TM3 and Xyn10B-TM4, Xyn10B-TM1 and Xyn10B-TM2 had higher hydrolytic activity, thermostability and affinity on insoluble substrates. It is noteworthy that Xyn10B-TM1 and Xyn10B-TM2 have higher enzymatic activity on insoluble xylan than the soluble counterparts, whereas Xyn10B-TM3 and Xyn10B-TM4 showed opposite characteristics. The kinetic parameters analysis of Xyn10B-TM1 on xylan showed Vmax was 5740, 1300, 1033, and 3925 U/μmol on insoluble oat spelt xylan (OSX), soluble beechwood xylan (BWX), soluble sugar cane xylan (SCX), and soluble corncob xylan (CCX), respectively. The results indicated that CBM22s especially CBM22c promoted the hydrolytic activity, thermostability and affinity on insoluble substrates of the Xyn10B truncations. The functions of CBM22, CBM9, CD and SLH are different, while contribute synergetically to the thermostability, protein structure integrity, substrate binding, and high hydrolytic activity on insoluble xylan of untreated lignocellulosic biomass. The domains of CBM22, CBM9, CD and SLH have different characteristics, which synergistically promote the thermostability, protein structure integrity, affinity on insoluble substrates and enzymatic activity properties of Xyn10B.ConclusionsThe extracellular endo-β-1,4-xylanase with multidomain structures of CBM, CD and SLH promote the biodegradation of insoluble xylan in untreated lignocellulosic biomass by thermophilic C. lactoaceticus.

Overexpression and characterization of alkaliphilic Bacillus firmus strain K-1 xylanase

The alkaliphilic Bacillus firmus strain K-1 produces an alkaliphilic xylanase (Xyn11A) which can be stable across a wide pH range and active at high temperatures. However, the enzyme suffers from low activity when isolated directly from the culture broth using corn husk. A method was developed which employed recombinant DNA technology to produce recombinant Xyn11A (rXyn11A) in an Escherichia coli (E. coli) expression system. The thioredoxin-fused Xyn11A (Thio-rXyn11A) could be expressed as a soluble form in BL21 (DE3). The expressed protein was tobacco etch virus (TEV) cleavaged to remove the thioredoxin tag and subjected to further purification using Ni2+-NTA affinity chromatography followed by gel-filtration chromatography. Activity of rXyn11A was shown to be under the same conditions as the native enzyme isolated directly from the K-1, having a broad range of active pH (5.0–12.0), with the maximum activity obtained from pH 5.0 at 60 °C. Interestingly, the obtained rXyn11A exhibited a very large increase in specific activity (3034 U/mg), which was 84-times higher than that reported in the native enzyme when observed under the same conditions. Also seen in the xylan hydrozation assay, the rXyn11A hydrolyzed insoluble xylans around 100-times more effectively than the native enzyme. The results from this study demonstrated a successful method for generating the enzyme rXyn11A with much improved activity, making it feasible for industrial applications.

A novel trifunctional, family GH10 enzyme from Acidothermus cellulolyticus 11B, exhibiting endo-xylanase, arabinofuranosidase and acetyl xylan esterase activities.

A novel, family GH10 enzyme, Xyn10B from Acidothermus cellulolyticus 11B was cloned and expressed in Escherichia coli. This enzyme was purified to homogeneity by binding to regenerated amorphous cellulose. It had higher binding on Avicel as compared to insoluble xylan due to the presence of cellulose-binding domains, CBM3 and CBM2. This enzyme was optimally active at 70°C and pH 6.0. It was stable up to 70°C while the CD spectroscopy analysis showed thermal unfolding at 80°C. Xyn10B was found to be a trifunctional enzyme having endo-xylanase, arabinofuranosidase and acetyl xylan esterase activities. Its activities against beechwood xylan, p-Nitrophenyl arabinofuranoside and p-Nitrophenyl acetate were found to be 126,480, 10,350 and 17,250Uμmol-1, respectively. Xyn10B was highly active producing xylobiose and xylose as the major end products, as well as debranching the substrates by removing arabinose and acetyl side chains. Due to its specific characteristics, this enzyme seems to be of importance for industrial applications such as pretreatment of poultry cereals, bio-bleaching of wood pulp and degradation of plant biomass.

Recombinant Bacillus amyloliquefaciens xylanase A expressed in Pichia pastoris and generation of xylooligosaccharides from xylans and wheat bran

In this study, BaxA (GenBank: KM624029), which encodes the Bacillus amyloliquefaciens xylanase A (BaxA), was highly expressed in Pichia pastoris GS115 under the control of the AOX1 promoter. The recombinant xylanase, namely rePBaxA, was purified to homogeneity by using Ni-affinity resin and its molecular weight was 35.0kDa. The optimum temperature and pH of rePBaxA were 50°C and 5.0, respectively. The kinetic parameters Michaelis–Menten constant (Km) and maximum reaction rate (Vmax) of rePBaxA were 5.41mg/mL and 22.42μmol/min/mL, respectively. High–performance liquid chromatography results showed that after 6h of hydrolysis, rePBaxA released xylose–xylohexaose (X1–X6) mixture from beechwood and birchwood xylan, with xylobiose (X2) and xylotriose (X3) as the major products, respectively. The hydrolyates from oat spelt, wheat bran insoluble xylan and pretreated wheat bran by rePBaxA included X2–X6, with X6 having the highest concentration. The mode of action analysis revealed that rePBaxA was an endo-acting xylanase with transglycosylation activity. X2 might be the minimum oligomer hydrolyzed by rePBaxA. The pretreated wheat bran and wheat bran insoluble xylan could be directly hydrolyzed by rePBaxA. This study provided a basis for using agricultural waste by-products as substrates for manufacting value-added probiotics, namely, xylooligosaccharides.

Recombinant Thermostable Thermomonospora fusca TF Endo-xylanase A and Its Immobilization on Modified Mesoporous SiO2 Microspheres for Manufacturing Xylooligosaccharides

tfxa (GenBank: U01242), which encoded the mature peptide of Thermomonospora fusca TF xylanase A (TfxA), was high-level expressed in Escherichia coli BL21 (DE3). The recombinant xylanase, reETfxA, was both secreted into culture medium and in the cytoplasm. reETfxA showed high xylanase activity and was purified to homogeneity by Ni-affinity resin (182.8 and 681.4 U mg−1 for crude and purified enzyme, respectively). Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and Western blot analysis revealed that the molecular mass of reETfxA was approximately 42.7 kDa. The reETfxA was immobilized on a novel mesoporous SiO2 microsphere (MSM)-coated chitosan via covalent bonds, which formed from Schiff base reaction between supports and enzyme. The binding capacity of the prepared MSM-coated chitosan particles to reETfxA was approximately 272.6 mg g−1-particles. The optimum temperature values of the free and immobilized reETfxA were 65 and 70 °C, respectively. Moreover, the optimum pH values of the free and immobilized reETfxA were pH 6.0 and 5.0, respectively. The reETfxA showed relatively high thermostability. When treated at 70 °C and pH 6.0 for 15 min, the residual activities of free and immobilized reETfxA were 84.2 and 100.2%, respectively. The results obtained from the HPLC analysis showed that immobilized reETfxA released xylooligosaccharides from oat spelt, beechwood and birchwood xylans, with xylotetraose, xylotriose, and xylopentaose as the major products, respectively. Additionally, the immobilized reETfxA could directly hydrolyze the wheat bran insoluble xylan.

High stability and low competitive inhibition of thermophilic Thermopolyspora flexuosa GH10 xylanase in biomass-dissolving ionic liquids.

Thermophilic Thermopolyspora flexuosa GH10 xylanase (TfXYN10A) was studied in the presence of biomass-dissolving hydrophilic ionic liquids (ILs) [EMIM]OAc, [EMIM]DMP and [DBNH]OAc. The temperature optimum of TfXYN10A with insoluble xylan in the pulp was at 65-70 °C, with solubilised 1% xylan at 70-75 °C and with 3% xylan at 75-80 °C. Therefore, the amount of soluble substrate affects the enzyme activity at high temperatures. The experiments with ILs were done with 1% substrate. TfXYN10A can partially hydrolyse soluble xylan even in the presence of 40% (v/v) ILs. Although ILs decrease the apparent temperature optimum, a surprising finding was that at the inactivating temperatures (80-90 °C), especially [EMIM]OAc increases the stability of TfXYN10A indicating that the binding of IL molecules strengthens the protein structure. Earlier kinetic studies showed an increased K m with ILs, indicating that ILs function as competitive inhibitors. TfXYN10A showed low increase of K m, which was 2-, 3- and 4-fold with 15% [EMIM]OAc, [DBNH]OAc and [EMIM]DMP, respectively. One reason for the low competitive inhibition could be the high affinity to the substrate (low K m). Xylanases with low K m (~1mg/mL) appear to show higher tolerance to ILs than xylanases with higher K m (~2mg/mL). Capillary electrophoresis showed that TfXYN10A hydrolyses xylan to the end-products in 15-35% ILs practically as completely as without IL, also indicating good binding of the short substrate molecules by TfXYN10A despite of major apparent IL binding sites above the catalytic residues. Substrate binding interactions in the active site appear to explain the high tolerance of TfXYN10A to ILs.

The family 22 carbohydrate-binding module of bifunctional xylanase/β-glucanase Xyn10E from Paenibacillus curdlanolyticus B-6 has an important role in lignocellulose degradation.

A newly isolated endo-β-1,4-xylanase (Xyn10E) from Paenibacillus curdlanolyticus B-6 has a modular structure consisting of a family 22 carbohydrate-binding module (CBM), a glycoside hydrolase (GH) family 10 catalytic domain, two fibronectin type III (Fn3) domains, and a family 3 CBM at the C-terminus. Intact Xyn10E (rXyn10E), CBM22-deleted Xyn10E (X-CBM3), CBM3-deleted Xyn10E (X-CBM22), and GH10 catalytic domain only (X-GH10) were expressed in Escherichia coli. rXyn10E showed bifunctional degradation activity toward xylan and β-glucan and also degraded microcrystalline cellulose. Although X-CBM3 and X-GH10 had drastically reduced xylanase and β-glucanase activities, X-CBM22 mostly retained these activities. Similar Km values were obtained for rXyn10E and X-CBM3, but kcat and kcat/Km values for X-CBM3 and X-GH10 were lower than those for rXyn10E, suggesting that CBM22 of Xyn10E may contribute to catalytic efficiency. In binding assays, X-CBM3 was still able to bind to β-glucan, soluble xylan, insoluble xylan, and cellulose through GH10 and CBM3. These results indicate that CBM22 has an important role not only in binding to xylan and β-glucan but also in feeding both polysaccharides into the neighboring GH10 catalytic domain. rXyn10E showed remarkable synergism with rXyn11A, a major xylanase subunit of P. curdlanolyticus B-6, in the degradation of untreated corn stover and sugarcane bagasse; however, the combination of X-CBM3 and rXyn11A was not synergistic. These results indicate that Xyn10E and Xyn11A act synergistically on lignocellulosic biomass, and CBM22 is essential for efficient degradation of lignocellulosic materials.

Metagenomic mining of glycoside hydrolases from the hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) and the characterization of a multimodular β-1,4-xylanase (GH11).

In recent years, there have been particular emphases worldwide on the development and optimization of bioprocesses for the utilization of biomass. An essential component of the biomass processing conduit has been the need for robust biocatalysts as high-performance tools for both the depolymerization of lignocellulosic biomass and synthesis of new high-value bio-based chemical entities. Through functional screening of the metagenome of the hindgut bacterial symbionts of a termite, Trinervitermes trinervoides, we discovered open reading frames for 25 cellulases and hemicellulases. These were classified into 14 different glycoside hydrolase (GH) families: eight GH family 5; four GH9, two GH13, and one each in GH2, GH10, GH11, GH26, GH29, GH43, GH44, GH45, GH67, and GH94 families. Of these, eight were overexpressed and partially characterized to be shown to be endocellulases (GH5C, GH5E, GH5F, and GH5G), an exocellulase (GH5D), endoxylanases (GH5H and GH11), and an α-fucosidase (GH29). The GH11 (Xyl1) was of particular interest as it was discovered to be a multimodular β-1,4-xylanase, consisting of a catalytic domain and two carbohydrate-binding modules (CBMs). The CBM functions to selectively bind insoluble xylan and increases the rate of hydrolysis. The primary structure of GH11 showed a classical catalytic dyad of glutamic acid residues that generally forms part of the active site in GH11 enzyme family. This endoxylanase was optimal at pH 6 and 50°C, and generated xylobiose and xylotriose from various xylan sources, including beechwood, birchwood, and wheat arabinoxylan. The catalytic ability of GH11 against natural substrate (e.g., wheat arabinoxylan) renders GH11 as a potential useful biocatalyst in the effective dismantling of complex plant biomass architecture.

Reducing the heterogeneity of xylan through processing

Glycerol thermal processing (GTP) of hardwood biomass at temperatures between 200 and 240°C facilitated stepwise biopolymer fractionation, while limiting significant degradation of the major hemicellulose, glucuronoxylan, into water-extractable oligosaccharides. After GTP pretreatment and sequential water and organic solvent extraction, up to 80% of the initial xylan remained in the pretreated biomass. The majority of the xylan from GTP pretreated and water/solvent extracted biomass was removed using a mild alkali extraction and the composition was compared to xylan directly isolated from untreated hardwood. The precipitated xylan from the neutralized alkaline filtrate was isolated as a water insoluble xylan portion (WIX). The residual xylan dissolved in the neutralized filtrate was precipitated in cold methanol and recovered as the water soluble xylan portion (WSX). Results showed that xylan in WIX was in a polymeric form with a number average degree of polymerization (DP) over 100, whereas the WSX had a much lower average DP of 27 (ca) and contained more substitution. As the processing severity increased during GTP pretreatment, the proportion of WIX increased and the purity of the xylan within the WIX sample reached 84% based on compositional analysis. FT-IR analysis of WIX revealed that xylan isolated after GTP contained peaks related to a reduced carbonyl signal compared to the control. Furthermore, crude WSX contained less xylan with more lignin contamination at severe GTP conditions. The recovery of the xylan in two portions facilitated a preferential purification strategy resulting in WIX with an extremely narrow polydispersity index between 1.1 and 1.25, dependent upon the GTP severity. This study provided insight into fractionating higher molecular weight xylan that may serve value-added applications such as healthcare materials and advanced packaging.

Improvement of thermostability and activity of Trichoderma reesei endo-xylanase Xyn III on insoluble substrates.

Trichoderma reesei Xyn III, an endo-β-1,4-xylanase belonging to glycoside hydrolase family 10 (GH10), is vital for the saccharification of xylans in plant biomass. However, its enzymatic thermostability and hydrolytic activity on insoluble substrates are low. To overcome these difficulties, the thermostability of Xyn III was improved using random mutagenesis and directed evolution, and its hydrolytic activity on insoluble substrates was improved by creating a chimeric protein. In the screening of thermostable Xyn III mutants from a random mutagenesis library, we identified two amino acid residues, Gln286 and Asn340, which are important for the thermostability of Xyn III. The Xyn III Gln286Ala/Asn340Tyr mutant showed xylanase activity even after heat treatment at 60°C for 30min or 50°C for 96h, indicating a dramatic enhancement in thermostability. In addition, we found that the addition of a xylan-binding domain (XBD) to the C-terminal of Xyn III improved its hydrolytic activity on insoluble xylan.

An immobilized bifunctional xylanase on carbon-coated chitosan nanoparticles with a potential application in xylan-rich biomass bioconversion

Immobilization technology offers many enzymatic advantages and overcomes the limitations of free enzymes. Bi- or multifunctional enzymes for industrial use have elicited much interest in recent years. The present work reported that a novel carbon nanoparticle-based supports was prepared by layer-by-layer self-assemble approach. The constructed bifunctional enzyme (ATXX) was successfully immobilized on the supports by covalent bonds. The prepared carbon-coated chitosan nanoparticles showed high binding capacity of about 289.9mgg−1-particles for ATXX. The Michaelis–Menten constants (Km) and maximal activity (Vmax) of immobilized ATXX were 4.83mgml−1 and 67.42μmolmin−1mg−1-particles (xylanase activity), as well as 6.13mgml−1 and 17.92μmolmin−1mg−1-particles (cellulase activity), respectively. The immobilized ATXX showed improved thermostability and storage stability compared with the free enzyme. The immobilized ATXX retained 82.6% xylanase activity after seven successive reactions. High-performance liquid chromatography (HPLC) analysis revealed that xylobiose (X2) was the main hydrolysis product released from beechwood xylan, birchwood xylan, and oat spelt xylan by immobilized ATXX. Wheat bran and wheat bran insoluble xylan could be directly hydrolyzed by immobilized ATXX, which demonstrated a potential use for xylan bioconversion to xylooligosaccharides by the immobilized ATXX.

Contribution of a family 1 carbohydrate-binding module in thermostable glycoside hydrolase 10 xylanase from Talaromyces cellulolyticus toward synergistic enzymatic hydrolysis of lignocellulose.

BackgroundEnzymatic removal of hemicellulose components such as xylan is an important factor for maintaining high glucose conversion from lignocelluloses subjected to low-severity pretreatment. Supplementation of xylanase in the cellulase mixture enhances glucose release from pretreated lignocellulose. Filamentous fungi produce multiple xylanases in their cellulase system, and some of them have modular structures consisting of a catalytic domain and a family 1 carbohydrate-binding module (CBM1). However, the role of CBM1 in xylanase in the synergistic hydrolysis of lignocellulose has not been investigated in depth.ResultsThermostable endo-β-1,4-xylanase (Xyl10A) from Talaromyces cellulolyticus, which is recognized as one of the core enzymes in the fungal cellulase system, has a modular structure consisting of a glycoside hydrolase family 10 catalytic domain and CBM1 at the C-terminus separated by a linker region. Three recombinant Xyl10A variants, that is, intact Xyl10A (Xyl10Awt), CBM1-deleted Xyl10A (Xyl10AdC), and CBM1 and linker region-deleted Xyl10A (Xyl10AdLC), were constructed and overexpressed in T. cellulolyticus. Cellulose-binding ability of Xyl10A CBM1 was demonstrated using quartz crystal microbalance with dissipation monitoring. Xyl10AdC and Xyl10AdLC showed relatively high catalytic activities for soluble and insoluble xylan substrates, whereas Xyl10Awt was more effective in xylan hydrolysis of wet disc-mill treated rice straw (WDM-RS). The enzyme mixture of cellulase monocomponents and intact or mutant Xyl10A enhanced the hydrolysis of WDM-RS glucan, with the most efficient synergism found in the interactions with Xyl10Awt. The increased glucan hydrolysis yield exhibited a linear relationship with the xylan hydrolysis yield by each enzyme. This relationship revealed significant hydrolysis of WDM-RS glucan with lower supplementation of Xyl10Awt.ConclusionsOur results suggest that Xyl10A CBM1 has the following two roles in synergistic hydrolysis of lignocellulose by Xyl10A and cellulases: enhancement of lignocellulosic xylan hydrolysis by binding to cellulose, and the efficient removal of xylan obstacles that interrupt the cellulase activity (because of similar binding target of CBM1). The combination of CBM-containing cellulases and xylanases in a fugal cellulase system could contribute to reduction of the enzyme loading in the hydrolysis of pretreated lignocelluloses.