Beechwood Xylan Research Articles

Optimization of Cellulase Production from Agri-Industrial Residues by Aspergillus terreus NIH2624

The objective of this study was to assess the cellulase production of four fungi: Aspergillus terreus NIH2624, Aspergillus clavatus NRRL1, Aspergillus versicolor CBS583.65 and Aspergillus phoenicis ATCC3157, under submerged cultivation conditions. When these fungi were cultured in shake flasks using Mandels and Weber’s minimal medium with 1% sugarcane bagasse as a carbon source and 1.8 g/L of rice bran extract as a nitrogen source, A. terreus showed maximum cellulase production (filter paper activity (FPase) 3.35 U/mL; carboxymethyl cellulase activity (CMCase) 1.69 U/mL). Consequently, A. terreus was selected for the optimization study for cellulase production. Among the different tested carbon sources, A. terreus showed higher CMCase activity when it was cultivated on delignified sugarcane bagasse (1.64 U/mL) and higher FPase activity on sugarcane straw (7.95 U/mL). Regarding the nitrogen sources, the maximum FPase activity was observed when using rice bran (FPase, 8.90 U/mL) and soybean meal (FPase, 9.63 U/mL). The optimized fermentation medium (minimal medium with delignified sugarcane bagasse and rice bran as carbon and nitrogen sources, respectively) resulted in an enzymatic cocktail mainly composed of xylanases, with a maximum activity of 1701.85 U/mL for beechwood xylan, 77.12 U/mL for endoglucanase and 21.02 U/mL for cellobiohydrolase. Additionally, the enzymatic cocktail showed efficient activities for β-glucosidase, β-xylanase, arabinofuranosidase and lytic polysaccharide monoxygenases (LPMOs). This cellulase enzyme solution has the potential to efficiently hydrolyze lignocellulosic biomass, producing second-generation sugars in biorefineries.

Exploring the composition and function profiles of bacteria from wood- and soil-feeding termites for effective degradation of lignin-based aromatics

Lignocellulosic biomass (LCB) in the form of agricultural, forestry, and agro-industrial wastes is globally generated in large volumes every year. The chemical components of lignocellulosic biomass render them a substrate valuable for biofuel production. However, the rigid association of lignin, cellulose, and hemicellulose component of LCB forms a complex, hierarchical, and recalcitrant structure, which inhibits the solubilization of LCB resources for biofuel production. The learning from termites (wood-feeding and soil-feeding) and further application of their gut bacteria for lignin degradation and bioconversion remain unexplored or at its early stage. With reference to this scientific knowledge gap, this study seeks to highlight the culturable gut bacterial community in soil- and wood-feeding termites, namely Pericapritermes nitobei and Microcerotermes sp., respectively to design a future biorefinery and bioremediation technologies. In this study, a total of 40 and 67 bacterial isolates were indeed identified from Pericapritermes nitobei and Microcerotermes sp., respectively. The identified isolates from both termite species were actually classified into four different phyla: Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes, where the phylum Actinobacteria dominated gut bacteria isolates identified from P. nitobei at 47.5 % while Proteobacteria were dominant in wood-feeding termite, Microcerotermes sp. at 46.27 %. At the genus level, the bacterial isolates enriched from the gut of Microcerotermes sp. belonged to 17 different genera, among which the bacterial genus Streptomyces (28 %) was most prevalent, followed by Enterobacter (11 %). Meanwhile, 9 genera were recorded for gut bacterial isolates from P. nitobei and were dominated by Streptomyces (37.5 %), followed by Acinetobacter (25 %). In general, the gut bacterial symbionts from both termites showed a congruency at the phyla level but were more diverged at a lower classification, inferring that different termite species evolved a unique repertoire of gut bacteria. Moreover, 61 % and 55 % of the gut bacterial isolates from the wood- and soil-feeding termites demonstrated a significant and multifunctional role in the hydrolysis of three lignocellulolytic substrates, including carboxymethyl cellulose, beechwood xylan, and aniline blue dye, indicating their unique functions and assistance to the host for the degradation of lignocellulose or other xenobiotic compounds from soil.

Molecular Identification and Engineering a Salt-Tolerant GH11 Xylanase for Efficient Xylooligosaccharides Production.

This study identified a salt-tolerant GH11 xylanase, Xynst, which was isolated from a soil bacterium Bacillus sp. SC1 and can resist as high as 4 M NaCl. After rational design and high-throughput screening of site-directed mutant libraries, a double mutant W6F/Q7H with a 244% increase in catalytic activity and a 10 °C increment in optimal temperature was obtained. Both Xynst and W6F/Q7H xylanases were stimulated by high concentrations of salts. In particular, the activity of W6F/Q7H was more than eight times that of Xynst in the presence of 2 M NaCl at 65 °C. Kinetic parameters indicated they have the highest affinity for beechwood xylan (Km = 0.30 mg mL-1 for Xynst and 0.18 mg mL-1 for W6F/Q7H), and W6F/Q7H has very high catalytic efficiency (Kcat/Km = 15483.33 mL mg-1 s-1). Molecular dynamic simulation suggested that W6F/Q7H has a more compact overall structure, improved rigidity of the active pocket edge, and a flexible upper-end alpha helix. Hydrolysis of different xylans by W6F/Q7H released more xylooligosaccharides and yielded higher proportions of xylobiose and xylotriose than Xynst did. The conversion efficiencies of Xynst and W6F/Q7H on all tested xylans exceeded 20%, suggesting potential applications in the agricultural and food industries.

Synergistic xylan decomposition by a reducing-end xylose-releasing exo-oligoxylanase with other xylanolytic enzymes derived from Paenibacillus xylaniclasticus strain TW1.

Paenibacillus xylaniclasticus strain TW1 is a promising tool for decomposing xylan-containing lignocellulosic biomass, since this strain possesses various genes encoding cellulolytic/hemicellulolytic enzymes. In this study, PxRex8A from the TW1 strain was found to be a reducing-end xylose-releasing exo-oligoxylanase of glycoside hydrolase family 8, which cleaves xylose from xylooligosacchrides of corn core xylan. In synergistic assay, the efficient decomposition of oat spelt xylan (OSX) and beech wood xylan was exemplified in the combination of endo β-1,4-xylanase (PxXyn11A) and PxRex8A from the TW1 strain in a molar ratio of 4:1. Furthermore, it was found that the addition of β-d-xylosidase/α-l-arabinofuranosidase (PxXyl43A) from this strain with PxXyn11A and PxRex8A achieved twice the amount of reducing sugars (1.1 mg/mL) against OSX after 24 hours compared to PxXyn11A alone (0.5 mg/mL). These results present that synergy effect of PxRex8A and PxXyl43A with PxXyn11A promotes xylan degradation into xylose.

Expression in Pichia pastoris of Thermostable Endo-1,4-β-xylanase from the Actinobacterium Nocardiopsis halotolerans: Properties and Use for Saccharification of Xylan-Containing Products.

A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-β-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-β-xylanase NhX1 showed maximum activity and stability at pH 6.0-7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed Km 2.16 mg/mL and Vmax 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-β-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria.

Isolation and Identification of Xylanase Producing Bacteria from Soil Samples of Kerala

Microorganisms play a very vital role in the biological recycling of plant bio-matter. Xylan is the second most plentiful polysaccharide and is found as an interface between lignin and other polysaccharide in the secondary cell wall. When these polysaccharides are hydrolyzed by a suitable strain of bacteria it will become an end to many environmental issues and had many useful applications such as in paper and pulp industry, food and feed industry, bread quality improvement, agro waste treatment, seed germination, generation of bio-fuels etc. To isolate a suitable strain of bacteria which possess good xylanase activity several samples of soils were collected from various regions of Kerala and were undergone a three step enrichment process. In the primary screening 85 bacterial strains were obtained. 22 isolates produces clear zone during plate assay using beech wood xylan. The isolates which possess more than 0.5cm clear zone were undergone final screening process and the strain PSS 1 showed the maximum enzyme activity of 112.38 U/ml.

Identification of a novel glycoside hydrolase family 8 xylanase from Deinococcus geothermalis and its application at low temperatures.

Xylanase is the most important hydrolase in the xylan hydrolase system, the main function of which is β-1,4-endo-xylanase, which randomly cleaves xylans to xylo-oligosaccharides and xylose. Xylanase has wide ranging of applications, but there remains little research on the cold-adapted enzymes required in some low-temperature industries. Glycoside hydrolase family 8 (GH8) xylanases have been reported to have cold-adapted enzyme activity. In this study, the xylanase gene dgeoxyn was excavated from Deinococcus geothermalis through sequence alignment. The recombinant xylanase DgeoXyn encodes 403 amino acids with a theoretical molecular weight of 45.39kDa. Structural analysis showed that DgeoXyn has a (α/α)6-barrel fold structure typical of GH8 xylanase. At the same time, it has strict substrate specificity, is only active against xylan, and its hydrolysis products include xylobiose, xylotrinose, xytetranose, xylenanose, and a small amount of xylose. DgeoXyn is most active at 70℃ and pH 6.0. It is very stable at 10, 20, and 30℃, retaining more than 80% of its maximum enzyme activity. The enzyme activity of DgeoXyn increased by 10% after the addition of Mn2+ and decreased by 80% after the addition of Cu2+. The Km and Vmax of dgeox were 42mg/ml and 20,000 U/mg, respectively, at a temperature of 70℃ and pH of 6.0 using 10mg/ml beechwood xylan as the substrate. This research on DgeoXyn will provide a theoretical basis for the development and application of low-temperature xylanase.

Screening, Production and Characterization of Potential Lignocellulolytic Actinomycetes from Agricultural Field

Actinomycetes are a suitable microbial group for the synthesis of lignocellulose-degrading enzymes. Enzymes that may degrade organic material, including cellulose, hemicellulose, and lignin, are released by actinomycetes. The aim of this research was to isolate actinomycetes from Rajkot, Gujarat, India’s soil and evaluate the activity of their cellulase and Xylanase enzymes. Starch Casein Agar (SCA) was used to identify a total of 30 isolates of actinomycetes. A qualitative plate assay (CMC-Na, Congo red) revealed that the highest zone of catalysis for MMD1 was 36 mm. Five strains were discovered to be effective for quantitative quantification of endoglucanase utilising filter paper and CMC as substrates: MMD1, MMD2, MMD3, MMD4, and MMD8. Following MMD 1 (endoglucanase 5.4 IU; FPase 4.4 IU), MMD 2 (endoglucanase 4.5 IU; FPase 3.4 IU) has demonstrated considerable endoglucanase and FPase activity. Beechwood xylan was used to treat sugarcane bagasse in order to test Xylanase, and 45% of the xylan (hemicellulose) fraction was obtained. MMD1 and MMD2 measured the xylanase enzyme activity (4.8IU and 4.2IU) in quantitative and qualitative assays (34 mm and 22 mm for BWX and 32 mm and 14 mm for agro-waste xylan). The strain MMD1 was identified as Streptomyces chartreusis through morphological, biochemical, and finally molecular characterization by 16S rRNA sequencing. It was then submitted to NCBI GenBank with the accession number MT254830.

Antioxidant capacity of xylooligosaccharides generated from beechwood xylan by recombinant family GH10 Aspergillus niger xylanase A and insights into the enzyme's competitive inhibition by riceXIP

In this study, the family GH10 xylanase AnXylA10 derived from Aspergillus niger JL15 strain was expressed in Pichia pastoris X33. The recombinant xylanase, reAnXylA10 exhibited optimal activity at 40 ℃ and pH 5.0. The hydrolysates generated from beechwood xylan using reAnXylA10 primarily consisted of xylobiose (X2) to xylohexaose (X6) and demonstrated remarkable antioxidant capacity. Furthermore, the rice xylanase inhibitory protein (riceXIP) was observed to competitively inhibit reAnXylA10, exhibiting an inhibition constant (Ki) of 140.6 nM. Molecular dynamics (MD) simulations of AnXylA10-riceXIP complex revealed that the α-7 helix (Q225-S238) of riceXIP intruded into the catalytic pocket of AnXylA10, thereby obstructing substrate access to the active site. Specifically, residue K226 of riceXIP formed robust interactions with E136 and E242, the two catalytic sites of AnXylA10, predominantly through high-occupied hydrogen bonds. Based on QTAIM, electron densities for the atom pairs K226riceXIP@HZ1-E136AnXylA10@OE2 and K226riceXIP@HZ3-E242AnXylA10@OE1 were determined to be 0.04628 and 0.02914 a.u., respectively. Binding free energy of AnXylA10-riceXIP complex was −59.0±7.6 kcal/mol, significantly driven by electrostatic and van der Waals forces. Gaining insights into the interaction between xylanase and its inhibitors, and mining the inhibition mechanism in depth, will facilitate the design of innovative GH10 family xylanases that are both highly efficient and resistant to inhibitors.

A fast, reliable, low‐cost, and efficient xylan extraction for xylooligosaccharides production

AbstractA fast, reliable, low‐cost, and efficient method for the recovery of xylan from agro‐residue materials is needed to improve the use of xylan in industrial processes. Xylan was extracted from two agrowastes (corncobs and wheat bran) using a cost‐effective alkali extraction method. The characteristic band patterns and bending vibrations associated with hemicellulose and beta glycoside linkage could be seen in the corncob‐extracted xylan (CCEx) and wheatbran‐extracted xylan (WBEx). Scanning electron microscopy (SEM) analysis showed that the extracted xylans consisted of aggregated and nonaggregated particles with irregular morphology and rough surfaces. Particle‐size analysis displayed average sizes of 205.6, 210.8, and 794.2 nm for commercial beechwood xylan (Bx), CCEx, and WBEx, respectively. Alkali extraction was performed with different concentrations of NaOH, from 5% to 20%, with a solid–liquid ratio of 1:20. It was found that 82.90 and 76.77% of xylan were extracted from corncob and wheat bran, respectively, which represents a higher yield obtained in a shorter duration in comparison with studies in the literature. The enzymatic hydrolysis of wheat bran and corncob that used 250 IU/mL of Enterobacter hormaechie KS1 xylanase yielded 334.38 and 162.35 mg/g of xylooligosaccharides. Their properties were studied and were found to be very similar to those of commercial xylan. They could reduce costs if applied in industry.

Enzymatic characterization and thermostability improvement of an acidophilic endoxylanase PphXyn11 from Paenibacillus physcomitrellae XB

GH11 enzyme is known to be specific and efficient for the hydrolysis of xylan. It has been isolated from many microorganisms, and its enzymatic characteristics and thermostability vary between species. In this study, a GH11 enzyme PphXyn11 from a novel xylan-degrading strain of Paenibacillus physcomitrellae XB was characterized, and five mutants were constructed to try to improve the enzyme's thermostability. The results showed that PphXyn11 was an acidophilic endo-β-1,4-xylanase with the optimal reaction pH of 3.0–4.0, and it could deconstruct different kinds of xylan substrates efficiently, such as beechwood xylan, wheat arabinoxylan and xylo-oligosaccharides, to produce xylobiose and xylotriose as the main products at the optimal reaction temperature of 40 °C. Improvement of the thermal stability of PphXyn11 using site-directed mutagenesis revealed that three mutants, W33C/N47C, S127C/N174C and S49E, designed by adding the disulfide bonds at the N-terminal, C-terminal and increasing the charged residues on the surface of PphXyn11 respectively, could increase the enzymatic activity and thermal stablility significantly and make the optimal reaction temperature reach 50 °C. Molecular dynamics simulations as well as computed the numbers of salt bridges and hydrogen bonds indicated that the protein structures of these three mutants were more stable than the wild type, which provided theoretical support for their improved thermal stability. Certainly, further research is necessary to improve the enzymatic characteristics of PphXyn11 to achieve the bioconversion of hemicellulosic biomass on an applicable scale.

PH-Induced structural changes in xylanase GH11 from Thermoanaerobacterium saccharolyticum

Background Glycosyl hydrolase 11 (GH11) xylanase is utilized in various in industrial applications such as baking, fruit juice production, pulp processing, and animal feed. Thermophilic GH11 from Thermoanaerobacterium saccharolyticum (TsaGH11) exhibits maximum activity at acid pH with high catalytic efficiency toward beechwood xylan. TsaGH11 activity is pH dependent, exhibiting relative low hydrolase activity at basic pH. However, the effect of a basic pH environment on the structure of TsaGH11 correlated with enzyme activity remains unknown. To understand pH-dependent activity changes, the crystal structure of TsaGH11 at basic pH was determined and compared with that of TsaGH11 at acid pH. Methods TsaGH11 was crystallized at basic pH of 8.5, and the crystal structure was determined at 1.95 Å resolution. The structure, flexibility, and water molecules of TsaGH11 at pH 8.5 and pH 4.3 were compared. Results The open and closed conformations of TsaGH11 at pH 8.5 are reported. Subtle movements of the side chains of amino acids involved in the substrate-binding cleft and catalytic residues were observed. The overall temperature factor of TsaGH11 at pH 8.5 was higher than that at pH 4.6. The position of water molecules near the catalytic residues in TsaGH11 exhibited variations in different pH environments. Conclusions The structural comparison of TsaGH11 at basic and acidic pH offers valuable insights into the pH-dependent functionality of TsaGH11, enhancing our understanding of these structural alterations.

Valorization of agro-wastes by Bacillus altitudinis XYL17 through simultaneous production of xylanase, xylooligosaccharides, and antioxidant compounds

Bacterial strains were isolated from undisturbed regions of Sikkim Himalaya for the production of highly active xylanase and xylooligosaccharides (XOS). Seven bacterial isolates showed xylanase production while fermenting beechwood xylan (BX) and the agrowastes corncob (CC), rice straw (RS), and sugarcane bagasse (SB). Bacillus altitudinis XYL17, isolated from soil at an elevation of 4083 m, produced the highest amount of xylanase. Maximum xylanase production was observed during BX fermentation after 24 h with the an observed activity of 6576.96 ± 82.75 U/mg. CC was observed to be the best agrowaste substrate for xylanase production with an observed activity of 4150.25 ± 94.64 U/mg. Xylopentaose was the most prominent XOS, with a highest observed content of 11.206 ± 0.331 mg/mL in RS hydrolysate. Significant increase of total phenolic content was observed for hydrolysates of B. altitudinis XYL17 fermented agro-wastes as compared to the control, while there was no significant difference (p < 0.05) in phenolic content between the control and fermented BX. The biotechnological potential of the XOS-rich hydrolysate was demonstrated by the observed radical scavenging, HOCl reducing, and myeloperoxidase (MPO) inhibitory activity. MPO inhibitory IC50 of 6.88 mg/mL and 9.4 mg/mL was observed for hydrolysates of SB and CC, respectively. We conclude that B. altitudinis XYL17 can be used for the valorization of agricultural wastes for the simultaneous production of highly efficient xylanase along with XOS and other antioxidant compounds.

Unlocking the potential of xylooligosaccharides: Nanofiltration for fractionation of hardwood hydrolysates

The biotechnological and functional food applications of xylooligosaccharides (XOS) are determined by their degree of polymerisation (DP) and their substituents' characteristics. Separation of XOS presents a formidable challenge for their large-scale production due to their complexity and similarity. This study employs nanofiltration membranes to fractionate beechwood xylan hydrolysates containing XOS. Tubular ceramic membranes with molecular weight cut-offs of 750, 450, and 200 Da effectively separated XOS into three distinct DP ranges, namely X2-X5, X2-X4, and X2-X3. The impacts of operating pressure and pH were evaluated to find the conditions leading to high permeate flux while maintaining high selectivity. Operating pressure of 10 bar with pH 7.0 for the 750 and 450 Da membranes and pH 4.2 for the 200 Da membrane yielded purities of 85%, 71%, and 74% for X2-X5, X2-X4, and X2-X3, respectively.

Optimization of xylanase production by Bacillus sp. MCC2212 under solid-state fermentation using response surface methodology

Xylanases have become increasingly significant due to their versatile applications in the food, paper, and pharmaceutical industries. Nevertheless, the current production of these enzymes relies on costly substrates, with estimates indicating that over 30% of the production expenses are attributed to these substrates. The objective of this study is to optimize the physico-chemical parameters for obtaining the maximum production of xylanase enzyme from Bacillus sp. MCC2212. The production conditions were statistically optimized using Plackett-Burman design (PBD) and Central Composite design (CCD). The significant variables identified through PB design including temperature, substrate to moisture ratio, MgSO4, peptone, tween 20, inoculum size, inoculum age, incubation time, and extracted xylan, were further optimized using the CCD approach. This optimization process revealed the most influential factors affecting xylanase production, with optimal conditions observed at a temperature, 35 °C; a substrate to moisture ratio, 1:1.75 g:mL; MgSO4, 1.1%; peptone, 1.5%; tween 20, 1.25%; inoculum size, 10 % (v/w); inoculum age, 20 h; an incubation time, 50 h; and crude xylan extracted from wheat bran at a concentration of 6% v/w. The R2 value of the model was found to be 0.9901, indicating its effectiveness. The optimized CCD model displayed a 1.84-fold greater xylanase production than the PB design approach. These findings suggest that wheat bran, a readily available agro-waste, could be a feasible alternative to the conventional substrate, beechwood xylan, for the production of xylanase enzyme with the possibility of achieving higher production levels optimized by using statistical design approach.

A novel endo-1,4-β-xylanase from Alicyclobacillus mali FL18: Biochemical characterization and its synergistic action with β-xylosidase in hemicellulose deconstruction

A novel endo-1,4-β-xylanase-encoding gene was identified in Alicyclobacillus mali FL18 and the recombinant protein, named AmXyn, was purified and biochemically characterized. The monomeric enzyme worked optimally at pH 6.6 and 80 °C on beechwood xylan with a specific activity of 440.00 ± 0.02 U/mg and a good catalytic efficiency (kcat/KM = 91.89 s−1mLmg−1). In addition, the enzyme did not display any activity on cellulose, suggesting a possible application in paper biobleaching processes. To develop an enzymatic mixture for xylan degradation, the association between AmXyn and the previously characterized β-xylosidase AmβXyl, deriving from the same microorganism, was assessed. The two enzymes had similar temperature and pH optima and showed the highest degree of synergy when AmXyn and AmβXyl were added sequentially to beechwood xylan, making this mixture cost-competitive and suitable for industrial use. Therefore, this enzymatic cocktail was also employed for the hydrolysis of wheat bran residue. TLC and HPAEC-PAD analyses revealed a high conversion rate to xylose (91.56 %), placing AmXyn and AmβXyl among the most promising biocatalysts for the saccharification of agricultural waste.

High-Molecular-Weight Xylanase from B. pumilus US570 Strain: Purification, Characterization and Application in Banana and Orange Peels Hydrolysis and Breadmaking.

New xylanase (XylUS570) was purified from the Bacillus pumilus US570 strain. It has a molecular mass of about 232kDa. This is the first report on the highest molecular weight monomeric xylanase produced by bacteria. The optimum pH and temperature recorded for enzyme activity were 7 and 55°C, respectively with a half-life time of 10min at 60°C. At 37°C, the enzyme retains more than 50% of its activity at a pH ranging from 6 to 9.5 for 24h. The XylUS570 exhibited a high activity on xylan, but no activity was detected for cellulosic substrates. The Vmax and Km values exhibited by the purified enzyme on beechwood xylan were 37.05 U mL-1 and 4.189mg mL-1, respectively. The XylUS570 was used in banana and orange peels hydrolysis and showed potential efficiency to liberate reducing sugars. It could be a good candidate for bio-ethanol production from fruit waste. The purified enzyme was used also as an additive in breadmaking. A decrease in water absorption, an increase in dough rising and improvements in volume and specific volume of the bread were recorded.

Reassigning the role of a mesophilic xylan hydrolysing family GH43 β-xylosidase from Bacteroides ovatus, BoExXyl43A as exo-β-1,4-xylosidase

The recombinant 40 kDa BoExXyl43A glycoside hydrolase family 43 (GH43) from bacterium Bacteroides ovatus exhibited highest specific activity (U/mg) against corn cob xylan (136.8), followed by Beechwood xylan (81.1), Carbosynth xylan (69.3), 4-O-D-methylglucuronoxylan (61.4) and Birchwood xylan (59.9). BoExXyl43A demonstrated optimal performance at 37 °C and pH 7.6 with Vmax and Km of 141.8 U/mg and 4.0 mg/mL as well as 64.1 U/mg and 6.0 mg/mL against corn cob and Birchwood xylan, respectively. The activity of BoExXyl43A increased by 48 % by addition of 10 mM Ca2+ ions, while 1 mM EDTA or 1 mM EGTA decreased its activity by 100 % or 42.5 %, respectively, highlighting its calcium-ion dependence. Thin-layer chromatography (TLC) analysis of BoExXyl43A hydrolysates of Birchwood and Beechwood xylan as well as that of various xylooligosaccharides (DP2-DP9) from corn cob xylan showed the release of D-xylose, identifying it as an exo-β-1,4-xylosidase/exo-β-1,4-xylanase (EC 3.2.1.-/3.2.1.37). Moreover, the time-dependent TLC analysis of xylobiose hydrolysis showed release of D-xylose units, confirming its β-xylosidase activity. BoExXyl43A also exhibited exo-1,4-β-xylosidase activity on Larchwood and Carbosynth xylans. Notably, it released D-xylose from α-L-Araf2-xylotriose demonstrating its activity against decorated xylooligosaccharides. BoExXyl43A's exo-1,4-β-xylosidase and residual β-xylosidase activity on xylan and xylobiose, respectively, could potentially enhance xylan saccharification efficiency in bioethanol-based refineries. The molecular modeling showed that BoExXyl43A has 5-bladed β-propeller structure with a very shallow active-site having −1, +1 and + 2 subsites, which could accommodate three D-xylose units of longer xylan like xylododecaose thus supporting its exoxylosidase activity.

Transformation of corncob into high-value xylooligosaccharides using glycoside hydrolase families 10 and 11 xylanases from Trichoderma asperellum ND-1

Effective production of xylooligosaccharides (XOS) with lower proportion of xylose entails unique and robust xylanases. In this study, two novel xylanases from Trichoderma asperellum ND-1 belonging to glycoside hydrolase families 10 (XynTR10) and 11 (XynTR11) were over-expressed in Komagataella phaffii X-33 and characterized to be robust enzymes with high halotolerance and ethanol tolerant. Both enzymes displayed strict substrate specificity towards beechwood xylan and wheat arabinoxylan. (Glu153/Glu258) and (Glu161/Glu252) were key catalytic sites for XynTR10 and XynTR11. Notably, XynTR11 could rapidly degrade xylan/XOS into xylobiose without xylose via transglycosylation. Direct degradation of corncob using XynTR10 and XynTR111 displayed that while XynTR10 yielded 77% xylobiose and 25% xylose, XynTR11 yielded much less xylose (11%) and comparable amounts of xylobiose (63%). XynTR10 or XynTR111 has great potential as a catalyst for bioconversion of xylan-containing agricultural waste into high-value products (biofuel or XOS), which is of significant benefit for the economy and environment.

Biochemical unravelling of the endoxylanase activity in a bifunctional GH39 enzyme cloned and expressed from thermophilic Geobacillus sp. WSUCF1

The glycoside hydrolase family 39 (GH39) proteins are renowned for their extremophilic and multifunctional enzymatic properties, yet the molecular mechanisms underpinning these unique characteristics continue to be an active subject of research. In this study, we introduce WsuXyn, a GH39 protein with a molecular weight of 58 kDa, originating from the thermophilic Geobacillus sp. WSUCF1. Previously reported for its exceptional thermostable β-xylosidase activity, WsuXyn has recently demonstrated a significant endoxylanase activity (3752 U·mg−1) against beechwood xylan, indicating towards its bifunctional nature. Physicochemical characterization revealed that WsuXyn exhibits optimal endoxylanase activity at 70 °C and pH 7.0. Thermal stability assessments revealed that the enzyme is resilient to elevated temperatures, with a half-life of 168 h. Key kinetic parameters highlight the exceptional catalytic efficiency and strong affinity of the protein for xylan substrate. Moreover, WsuXyn-mediated hydrolysis of beechwood xylan has achieved 77 % xylan conversion, with xylose as the primary product. Structural analysis, amalgamated with docking simulations, has revealed strong binding forces between xylotetraose and the protein, with key amino acid residues, including Glu278, Tyr230, Glu160, Gly202, Cys201, Glu324, and Tyr283, playing pivotal roles in these interactions. Therefore, WsuXyn holds a strong promise for biodegradation and value-added product generation through lignocellulosic biomass conversion.