Xylose Release Research Articles

Xylitol production from passion fruit peel hydrolysate: Optimization of hydrolysis and fermentation processes

The passion fruit peel (PFP) has a high cellulose and hemicellulose content, which can be used to produce fermentable sugars. In this context, this study aims to optimize the release of xylose and the production of xylitol from PFP. The optimized conditions were 0.71 M dilute sulfuric acid and a 21.84-minute treatment, yielding 19.03 g/L of xylose (PFP-1). Different PFP hydrolysates were evaluated to improve xylitol production by the yeast Kluyveromyces marxianus ATCC 36907: PFP-2 (PFP1 treated with Ca(OH)2), PFP-3 (PFP-1 treated with Ca(OH)2 and activated carbon), PFP-4 (PFP-3 with biological elimination of glucose with S. cerevisiae, and concentrated at different xylose concentrations). The applied methods resulted in higher xylitol production (14.97 g/L), when PFP hydrolysate was detoxified with Ca(OH)2, treated with activated charcoal for 1 h, biotreated for glucose removal, and concentrated to 40 g/L of xylose.

Arabinoxylo-oligosaccharides production from unexploited agro-industrial sesame (Sesamum indicum L.) hulls waste

This work demonstrates that sesame (Sesamum indicum L.) hull, an unexploited food industrial waste, can be used as an efficient source for the extraction of hemicellulose and/or pectin polysaccharides to further obtain functional oligosaccharides. Different polysaccharides extraction methods were surveyed including alkaline and several enzymatic treatments. Based on the enzymatic release of xylose, arabinose, glucose, and galacturonic acid from sesame hull by using different enzymes, Celluclast®1.5 L, Pectinex®Ultra SP-L, and a combination of them were selected for the enzymatic extraction of polysaccharides at 50 °C, pH 5 up to 24 h. Once the polysaccharides were extracted, Ultraflo®L was selected to produce arabinoxylo-oligosaccharides (AXOS) at 40 °C up to 24 h. Apart from oligosaccharides production from extracted polysaccharides, alternative approaches for obtaining oligosaccharides were also explored. These were based on the analysis of the supernatants resulting from the polysaccharide extraction, alongside a sequential hydrolysis performed with Celluclast®1.5 L and Ultraflo®L of the starting raw sesame hull. The different fractions obtained were comprehensively characterized by determining low molecular weight carbohydrates and monomeric compositions, average Mw and dispersity, and oligosaccharide structure by MALDI-TOF-MS. The results indicated that sesame hull can be a useful source for polysaccharides extraction (pectin and hemicellulose) and derived oligosaccharides, especially AXOS.

Modifying lignin composition and xylan O-acetylation induces changes in cell wall composition, extractability, and digestibility

BackgroundLignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing.ResultsArabidopsis plant co-harbouring mutation in FERULATE 5-HYDROXYLASE (F5H) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 cross was named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular regulatory processes.ConclusionsSimultaneous modification in xylan and lignin enhances cellulose content with improved saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose and xylobiose release with or without pretreatment after xylanase digestion in both parent and HrGHypAc. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection.

Genomic mining of Geobacillus stearothermophilus GF16 for xylose production from hemicellulose-rich biomasses using secreted enzymes

The valorization of lignocellulosic biomass, derived from various bio-waste materials, has received considerable attention as a sustainable approach to improve production chains while reducing environmental impact. Microbial enzymes have emerged as key players in the degradation of polysaccharides, offering versatile applications in biotechnology and industry. Among these enzymes, glycoside hydrolases (GHs) play a central role. Xylanases, in particular, are used in a wide range of applications and are essential for the production of xylose, which can be fermented into bioethanol or find use in many other industries. Currently, fungal secretomes dominate as the main reservoir of lignocellulolytic enzymes, but thermophilic microorganisms offer notable advantages in terms of enzyme stability and production efficiency. Here we present the genomic characterization of Geobacillus stearothermophilus GF16 to identify genes encoding putative enzymes involved in lignocellulose degradation. Thermostable GHs secreted by G. stearothermophilus GF16 were investigated and found to be active on different natural polysaccharides and synthetic substrates, revealing an array of inducible GH activities. In particular, the concentrated secretome possesses significant thermostable xylanase and β-xylosidase activities (5 ×103 U/L and 1.7 ×105 U/L, respectively), highlighting its potential for application in biomass valorization. We assessed the hemicellulose hydrolysis capabilities of various agri-food wastes using the concentrated secretome of the strain cultivated on xylan. An impressive 300-fold increase in xylose release compared to a commercially available cocktail was obtained with the secretome, underscoring the remarkable efficacy of this approach.

Optimization of xylitol production through Candida tropicalis in xylose hydrolysate from rice husk

AbstractOwing to the extensive cultivation and production of rice, which consequently generates a high amount of rice husk, rice husk is an easily available lignocellulosic waste; however, the applications it currently has do not entirely exploit its potential. This study aims the production of xylitol as an opportunity to value this residue through fermentation of rice husk hydrolysate. For it, the best particle size of rice husk among 0.25–0.6 mm, 0.6–1.2 mm, and unmilled rice husk (URH) for dilute acid hydrolysis was determined by using a randomized complete block design (RCBD) to select the particle size that produced the highest amount of xylose release; subsequently, adaptation of Candida tropicalis was carried out as an alternative to common detoxification methods, by increasing the concentration of hydrolysate in the medium in 20% every 2 days, and then, xylitol fermentation from the best particle size hydrolysate with C. tropicalis was optimized performing a central composite design (CCD) 22. The best particle size for dilute acid hydrolysis was URH and produced a xylose concentration of 12.85 g/L, and the optimal conditions for xylitol fermentation were 4.41 g/L of inoculum and 68.28 g/L of xylose and accomplished a concentration of 36.74 g/L of xylitol, a yield of 0.58 g/g xylose, and a volumetric productivity of 0.34 g/L/h, providing evidence that rice husk has the potential to be valorized as a source of fermentable sugars for the production of xylitol as a high-value metabolite and proving adaptation method as a viable alternative for the strain’s growth as well as xylitol production.

Fungal loosenin-like proteins boost the cellulolytic enzyme conversion of pretreated wood fiber and cellulosic pulps

Microbial expansin-related proteins, including loosenins, can disrupt cellulose networks and increase enzyme accessibility to cellulosic substrates. Herein, four loosenins from Phanerochaete carnosa (PcaLOOLs), and a PcaLOOL fused to a family 63 carbohydrate-binding module, were compared for ability to boost the cellulolytic deconstruction of steam pretreated softwood (SSW) and kraft pulps from softwood (ND-BSKP) and hardwood (ND-BHKP). Amending the Cellic® CTec-2 cellulase cocktail with PcaLOOLs increased reducing products from SSW by up to 40 %, corresponding to 28 % higher glucose yield. Amending Cellic® CTec-2 with PcaLOOLs also increased the release of glucose from ND-BSKP and ND-BHKP by 82 % and 28 %, respectively. Xylose release from ND-BSKP and ND-BHKP increased by 47 % and 57 %, respectively, highlighting the potential of PcaLOOLs to enhance hemicellulose recovery. Scanning electron microscopy and fiber image analysis revealed fibrillation and curlation of ND-BSKP after PcaLOOL treatment, consistent with increasing enzyme accessibility to targeted substrates.

The role of CE16 exo-deacetylases in hemicellulolytic enzyme mixtures revealed by the biochemical and structural study of the novel TtCE16B esterase

Acetyl esterases belonging to the carbohydrate esterase family 16 (CE16) is a growing group of enzymes, with exceptional diversity regarding substrate specificity and regioselectivity. However, further insight into the CE16 specificity is required for their efficient biotechnological exploitation. In this work, exo-deacetylase TtCE16B from Thermothelomyces thermophila was heterologously expressed and biochemically characterized. The esterase targets positions O-3 and O-4 of singly and doubly acetylated non-reducing-end xylopyranosyl residues, provided the presence of a free vicinal hydroxyl group at position O-4 and O-3, respectively. Crystal structure of TtCE16B, the first representative among the CE16 enzymes, in apo- and product-bound form, allowed the identification of residues forming the catalytic triad and oxyanion hole, as well as the structural elements related to the enzyme preference for oligomers. The role of TtCE16B in hemicellulose degradation was investigated on acetylated xylan from birchwood and pre-treated beechwood biomass. TtCE16B exhibited complementary activity to commercially available OCE6 acetylxylan esterase. Moreover, it showed synergistic effects with SrXyl43 β-xylosidase. Overall, supplementation of xylan-targeting enzymatic mixtures with both TtCE16B and OCE6 esterases led to a 3-fold or 4-fold increase in xylose release, when using TmXyn10 and TtXyn30A xylanases respectively.

Comparative analysis of Chrysoporthe cubensis exoproteomes and their specificity for saccharification of sugarcane bagasse

The phytopathogenic fungus Chrysoporthe cubensis is a relevant source of lignocellulolytic enzymes. This work aimed to compare the profile of lignocellulose-degrading proteins secreted by C. cubensis grown under semi-solid state fermentation using wheat bran (WB) and sugarcane bagasse (SB). The exoproteomes of the fungus grown in wheat bran (WBE) and sugarcane bagasse (SBE) were qualitative and quantitatively analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Data are available via ProteomeXchange with identifier PXD046075. Label-free proteomic analysis of WBE and SBE showed that the fungus produced a spectrum of carbohydrate-active enzymes (CAZymes) with exclusive characteristics from each extract. While SBE resulted in an enzymatic profile directed towards the depolymerization of cellulose, the enzymes in WBE were more adaptable to the degradation of biomass rich in hemicellulose and other non-lignocellulosic polymers. Saccharification of alkaline pre-treated sugarcane bagasse with SBE promoted glucose release higher than commercial cocktails (8.11 g L−1), while WBE promoted the higher release of xylose (5.71 g L−1). Our results allowed an in-depth knowledge of the complex set of enzymes secreted by C. cubensis responsible for its high lignocellulolytic activity and still provided the identification of promising target proteins for biotechnological applications in the context of biorefinery.

Intensification of corn fiber saccharification using a tailor made enzymatic cocktail

The transition from an economic model based on resource extraction to a more sustainable and circular economy requires the development of innovative methods to unlock the potential of raw materials such as lignocellulosic biomasses. Corn fiber differs from more traditional lignocellulosic biomasses due to its high starch content, which provides additional carbohydrates for fermentation-based biomanufacturing processes. Due to its unique chemical composition, this study focused on the development of a tailor made enzymatic cocktail for corn fiber saccharification into monosaccharides. Three commercially available hydrolytic enzymes (Cellic® CTec2, Pentopan® Mono BG, and Termamyl® 300 L) were combined to hydrolyze the polysaccharide structure of the three main carbohydrate fractions of corn fiber (cellulose, hemicellulose and starch, respectively). Prior to saccharification, corn fiber was submitted to a mild hydrothermal pretreatment (30 min at 100 °C). Then, two experimental designs were used to render an enzymatic cocktail capable of providing efficient release of monosaccharides. Using 60 FPU/g DM of Cellic® CTec2 and 4.62 U/g DM of Termamyl® 300 L, without addition of Pentopan® Mono BG, resulted in the highest efficiencies for glucose and xylose release (66% and 30%, respectively). While higher enzyme dosages could enhance the saccharification efficiency, adding more enzymes would have a more pronounced effect on the overall process costs rather than in increasing the efficiency for monosaccharides release. The results revealed that the recalcitrance of corn fiber poses a problem for its full enzymatic degradation. This fact combined with the unique chemical composition of this material, justify the need for developing a tailor made enzymatic cocktail for its degradation. However, attention should also be given to the pretreatment step to reduce even more the recalcitrance of corn fiber and improve the performance of the tailored cocktail, as a consequence.

Life cycle assessment of fermentative xylitol production from wheat bran: A comparative evaluation of sulphuric acid and chemical-free wet air oxidation-based pretreatment

Xylitol, a sugar alcohol is a low-calorie sweetener has wide applications in food and pharmaceutical industry and hence considered as a high value premium product. The conventional methods for fermentative xylitol production uses chemicals (acid/alkali) which generates inhibitors affecting fermentation and downstream purification of xylitol. In the present study, wet air oxidation was evaluated as a chemical-free pretreatment option of wheat bran for xylose release which is subsequently fermented to xylitol. Life cycle assessment was used as a tool to compare wet air oxidation with conventional methods like dilute acid pretreatment for valorization of wheat bran, a model agro-industrial remnant. A comparative evaluation of wet air oxidation and dilute acid pretreatment was investigated for fermentative production of xylitol (1 g/L as functional unit) with the aim of identifying the process involved with the lowest environmental impact, while also demonstrating the higher xylose recovery of 13.52 g/L coupled with FO based concentration unit. The inputs and outputs of the process involving energy use, chemicals employed, and emissions produced were evaluated with respect to food and environment perspective. Environmental impacts of wet air oxidation and dilute acid pretreatment with fermentative xylitol production of 6.134 and 1.42 g/L, respectively showed emission results of 1.476 and 5.288 kg CO2 equivalent, respectively. Though, primary energy demand of wet air oxidation was almost found to be equivalent to dilute acid, due to energy penalty for reactor mixing was higher in wet air oxidation. However, energy demand of wet air oxidation-based pretreatment was found to be energy efficient by 30% as compared to dilute acid. Coupling biogas reduces energy demand by 3.5%. The reduction in impacts relevant to food safety namely, marine aquatic and freshwater ecotoxicity potential were found by 74.29% and 84.97% substantially lower in wet air oxidation as compared to dilute acid based xylitol production. The effect of sulphate, as input form of sulphuric acid in dilute acid showed an impact of 3.83 folds higher on human toxicity potential as compared to wet air oxidation pretreatment process.

Enhanced energy savings in enzymatic refining of hardwood and softwood pulp

BackgroundPulp refining is an energy consuming, but integral part of paper production with the aim to increase tensile strength and smoothness of paper sheets. Commercial enzyme formulations are used to lower the energy requirements by pre-treatment of pulp before refining. However, a high number of different commercial enzyme products are available on the market containing enzymes of varying origin and composition, which complicates the prediction of their behavior, especially using different pulp types.ResultsEndoglucanase-rich enzyme formulations were characterized regarding enzyme activity at different temperatures, resulting in a significant decrease of activity above 70 °C. Some enzyme preparations additionally contained arabinosidase, xylanase and β-glucosidase activity consequently resulting in a release of xylose and glucose from pulp as determined by high-performance liquid chromatography. Interestingly, one enzyme formulation even showed lytic polysaccharide monooxygenase (LPMO) activity of 3.05 nkat mg−1. A correlation between enzyme activity using the endoglucanase specific derivatized cellopentaose (CellG5) substrate and enzyme performance in laboratory PFI (Papirindustriens forskningsinstitut) refining trials was observed on softwood pulp resulting in a maximum increase in the degree of refining values from 27.7°SR to 32.7°SR. When added to a purified endoglucanase enzyme (31.6°SR), synergistic effects were found for cellobiohydrolase II (34.7°SR) or β-glucosidase enzymes (35.7°SR) in laboratory refining. Comparison with previously obtained laboratory refining results on hardwood pulp allowed differences in enzyme performance based on varying pulp types to be elucidated.ConclusionsInterestingly, the individual enzymes indeed showed different refining effects on softwood and hardwood pulp. This difference could be predicted after development of an adapted enzyme activity assay by combination of the derivatized cellopentaose CellG5 substrate with either softwood or hardwood sulfate pulp.

Investigating the role of AA9 LPMOs in enzymatic hydrolysis of differentially steam-pretreated spruce

BackgroundTo realize the full potential of softwood-based forest biorefineries, the bottlenecks of enzymatic saccharification of softwood need to be better understood. Here, we investigated the potential of lytic polysaccharide monooxygenases (LPMO9s) in softwood saccharification. Norway spruce was steam-pretreated at three different severities, leading to varying hemicellulose retention, lignin condensation, and cellulose ultrastructure. Hydrolyzability of the three substrates was assessed after pretreatment and after an additional knife-milling step, comparing the efficiency of cellulolytic Celluclast + Novozym 188 and LPMO-containing Cellic CTec2 cocktails. The role of Thermoascus aurantiacus TaLPMO9 in saccharification was assessed through time-course analysis of sugar release and accumulation of oxidized sugars, as well as wide-angle X-ray scattering analysis of cellulose ultrastructural changes.ResultsGlucose yield was 6% (w/w) with the mildest pretreatment (steam pretreatment at 210 °C without catalyst) and 66% (w/w) with the harshest (steam pretreatment at 210 °C with 3%(w/w) SO2) when using Celluclast + Novozym 188. Surprisingly, the yield was lower with all substrates when Cellic CTec2 was used. Therefore, the conditions for optimal LPMO activity were tested and it was found that enough O2 was present over the headspace and that the reducing power of the lignin of all three substrates was sufficient for the LPMOs in Cellic CTec2 to be active. Supplementation of Celluclast + Novozym 188 with TaLPMO9 increased the conversion of glucan by 1.6-fold and xylan by 1.5-fold, which was evident primarily in the later stages of saccharification (24–72 h). Improved glucan conversion could be explained by drastically reduced cellulose crystallinity of spruce substrates upon TaLPMO9 supplementation.ConclusionOur study demonstrated that LPMO addition to hydrolytic enzymes improves the release of glucose and xylose from steam-pretreated softwood substrates. Furthermore, softwood lignin provides enough reducing power for LPMOs, irrespective of pretreatment severity. These results provided new insights into the potential role of LPMOs in saccharification of industrially relevant softwood substrates.

Biorefining of corn stover for efficient production of bioethanol, biodiesel, biomethane, and value-added byproducts

The present study investigated an integrated biorefinery that employed corn stover as the feedstock for sustainable bioethanol, biodiesel, biogas, chitosan, glycerol, and animal feed production. Corn stover was initially subjected to dilute acid pretreatment (1.8 % v/v H2SO4, 121 °C, and 22 min) followed by enzymatic hydrolysis with a commercial cellulase (37 °C, 72 h) to promote the release of glucose (∼93 wt%) and xylose (∼89 wt%). Mucor indicus fungus was then employed to convert the released sugars into bioethanol, glycerol, and fungal biomass with yields of 0.38 g g−1, 36 mg g−1, and 0.51 g g−1, respectively. The biomass of M. indicus was processed to extract chitosan (6 mg g−1 fungal biomass) and lipids (297 mg g−1 fungal biomass). The lipid was subsequently converted to biodiesel via transesterification in the presence of HCl/ MeOH with the yield of 0.54 g g−1 fungal lipid. The defatted biomass residue was then converted to biogas with 81 % theoretical yield through anaerobic digestion. To ensure process circularity, the nutritional values of pretreated and hydrolyzed corn stover were also investigated with their suitability as livestock. It was determined that 158.1 thousand tons of dry corn stover, which was annually collectible in Iran, could be used for the production of 137.6 kg chitosan, 10.4 ton animal feed, 870.0 kg glycerol, 40.7 million litters ethanol, 2.8 million m3 biodiesel, and 449.2 million m3 biomethane. The utilization of the produced ethanol, biodiesel, and biomethane in transporting sector was shown to have the potential of facilitating 4.3 million tons of equivalent carbon dioxide and a 197.8 million dollars reduction of associated social costs.

Bioconversion of Dilute Acid Pretreated Corn Stover to L-Lactic Acid Using Co-Culture of Furfural Tolerant Enterococcus mundtii WX1 and Lactobacillus rhamnosus SCJ9

This study demonstrated the bioconversion of lignocellulosic by-product corn stover (CS) to the value-added fermentative product L-lactic acid using the furfural tolerant Enterococcus mundtii WX1 and Lactobacillus rhamnosus SCJ9. The efficacy of dilute acid pretreatment by sulfuric and formic acids varying from 1% to 4% (v/v) concentration was compared. CS pretreated with 1% (v/v) sulfuric acid was selected for L-LA fermentation regarding the highest efficacy of fermentable sugar release when combined with the enzymatic hydrolysis process. Optimal conditions achieved a highest sugar release of 24.5 g/L glucose and 11.2 g/L of xylose from 100 g/L pretreated CS with 1% (v/v) sulfuric acid at 121 °C for 30 min, followed by enzymatic hydrolysis with Cellic CTec2 30 FPU/g pretreated CS at 50 °C for 48 h. The maximum L-LA titer, yield, and average productivity reached 31.4 g/L, 0.90 g/g, and 1.73 g/L/h, respectively. Moreover, addition of a hemicellulose-degrading enzyme complex combined with Cellic CTec2 led to an increase in xylose release, which resulted in a higher L-LA titer of 36.7 g/L at 48 h fermentation. Moreover, the purification of LA from culture broth by a process of electrodialysis with 331 g/L of LA and purity of 99.7% (w/w), was successful, with an optically pure L-LA of 99.9%. This study not only presents a feasible process for L-LA production from lignocellulose hydrolysate derived from abundant corn stover; this study also showed an alternative approach for solving the problem of haze air pollution caused by inappropriate management of corn production residuals.

β-Xylosidase SRBX1 Activity from Sporisorium reilianum and Its Synergism with Xylanase SRXL1 in Xylose Release from Corn Hemicellulose.

Sposisorium reilianum is the causal agent of corn ear smut disease. Eleven genes have been identified in its genome that code for enzymes that could constitute its hemicellulosic system, three of which have been associated with two Endo-β-1,4-xylanases and one with α-L-arabinofuranosidase activity. In this study, the native protein extracellular with β-xylosidase activity, called SRBX1, produced by this basidiomycete was analyzed by performing production kinetics and its subsequent purification by gel filtration. The enzyme was characterized biochemically and sequenced. Finally, its synergism with Xylanase SRXL1 was determined. Its activity was higher in a medium with corn hemicellulose and glucose as carbon sources. The purified protein was a monomer associated with the sr16700 gene, with a molecular weight of 117 kDa and optimal activity at 60 °C in a pH range of 4-7, which had the ability to hydrolyze the ρ-nitrophenyl β-D-xylanopyranoside and ρ-Nitrophenyl α-L-arabinofuranoside substrates. Its activity was strongly inhibited by silver ions and presented Km and Vmax values of 2.5 mM and 0.2 μmol/min/mg, respectively, using ρ-nitrophenyl β-D-xylanopyranoside as a substrate. The enzyme degrades corn hemicellulose and birch xylan in combination and in sequential synergism with the xylanase SRXL1.

An integrated process for co-producing fermentable sugars and xylonate from sugarcane bagasse based on xylonic acid assisted pretreatment

In this study, a renewable organic acid (xylonic acid), which can be prepared by the biooxidation of xylose, is used for pretreating sugarcane bagasse. The effects of reaction temperature and time on the release of fermentable xylose and glucose were investigated. On the basis of guaranteeing the good enzymatic hydrolysis efficiency and minimizing the effects of inhibitors, the pretreatment with 1 % xylnoic acid at 190 °C for 30 min was selected after optimization. In this case, 70 % xylose was released, while enzymatic hydrolysis yield was also up to 86.5 %. Meanwhile, the pretreated hydrolysate liquor was proved that it could be used for producing xylonate by biooxidation of Gluconobacter oxydans. Finally, the sequential process of the xylonic acid pretreatment and saccharification also clear the path for recycling the lignin as value-added bioproducts. Overall, this study presents a green-like strategy for fully exploiting sugarcane bagasse.

Expression and Characterization of a Small, Xylan/Cellulose-degrading GH43 Protein Derived from Biofertilizer Metagenome

Background: A putative glycosyl hydrolase gene biof1_09 was identified from a metagenomic fosmid library of local biofertilizers in previous report [1]. The gene is renamed as gh43kk in this study. Methods: The gene gh43kk, encoding a putative β-D-xylosidase was amplified by polymerase chain reaction (PCR) and successfully cloned and expressed in Escherichia coli. The expressed recombinant protein was purified by metal affinity chromatography. Its properties were initially verified by enzyme assay and thin layer chromatography (TLC). Results: The purified recombinant protein showed the highest catalytic activities at acidic pH 4 and 50°C toward beechwood xylan, followed by carboxymethylcellulose (CMC). TLC analysis indicated a release of xylose and glucose when xylan and CMC were treated with Gh43kk protein, respectively, whereas glucose and cellobiose were detected when avicel, cellulose and filter paper were used as substrates, suggesting its dual function as xylanase with cellulase activity. The enzyme indicated great stability in a temperature between 10 to 50 °C and a wide range of pH from 4 to 8. Enzyme activity of Gh43kk was enhanced in the presence of magnesium and manganese ions, while calcium ions, Ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) inhibited the enzyme activity. Conclusion: These results suggest that Gh43kk could be a potential candidate for application in various bioconversion processes.

The effects of exogenous fibrolytic enzymes on the in vitro release of xylooligosaccharides and monosaccharides varies across six varieties of wheat

Wheat is a widely used cereal grain for pig and poultry feeds globally. Despite this, there are noticeable differences in its nutritive value, potentially due to varying characteristics like inherent non-starch polysaccharide (NSP) content and composition. Fibrolytic enzymes help degrade NSP and thereby improve feed efficiency in livestock. However, it has been suggested that these enzymes have different effects dependent upon the characteristics of the wheat variety used in a feed. This study investigated the efficacy of different enzyme-wheat variety combinations, by quantifying the release of NSP-derived xylooligosaccharides (XOS) and monosaccharides from six varieties of wheat (Maris Huntsman, Highbury, Paragon, Sinuelo, Chinese Spring and Pavon 76) over a 24 h in vitro incubation with commercially available fibrolytic enzymes (Econase XT, Econase MP1000 or Barley P700). Complete non-starch acid hydrolysis showed there were differences between varieties in their total monosaccharide contents (P < 0.001). There were significant wheat variety x enzyme x incubation time interactions for the release of xylobiose, galactose and glucose (all P < 0.001) and significant enzyme x wheat variety interactions for the release of xylotriose (P = 0.022), xylose (P < 0.001) and arabinose (P = 0.028). Clear differences in release of XOS were observed between the different combinations of enzyme and wheat variety. Econase XT increased xylotriose release from Highbury wheat, and xylobiose release from Sinuelo, with both wheat varieties showing comparable release of xylose. These findings suggest that the fibrolytic enzymes tested have some specificity for the wheat varieties. Hence it might be possible to optimise the combinations of wheat variety and enzyme used in animal feeds, to help maximise the feed efficiency of livestock.

Production of xylose through enzymatic hydrolysis of glucuronoarabinoxylan from brewers’ spent grain

Xylose is an abundant bioresource for obtaining diverse chemicals and added-value products. The production of xylose from green alternatives like enzymatic hydrolysis is an important step in a biorefinery context. This research evaluated the synergism among four classes of hydrolytic purified enzymes—endo-1,4-β-xylanase, α-l-arabinofuranosidase, β-xylosidase, and α-d-glucuronidase—over hydrolysis of glucuronoarabinoxylan (GAX) obtained from brewers’ spent grain (BSG) after alkaline extraction and ethanol precipitation. First, monosaccharides, uronic acids and glycosidic-linkages of alkaline extracted GAX fraction from BSG were characterized, after that different strategies based on the addition of one or two families of enzymes—endo-1,4-β-xylanase (GH10 and GH11) and α-l-arabinofuranosidase (GH43 and GH51)—cooperating with one β-xylosidase (GH43) and one α-d-glucuronidase (GH67) into enzymatic hydrolysis were assessed to obtain the best yield of xylose. The xylose release was monitored over time in the first 90 min and after a prolonged reaction up to 48 h of reaction. The highest yield of xylose was 63.6% (48 h, 40 ℃, pH 5.5), using a mixture of all enzymes devoid of α-l-arabinofuranosidase (GH43) family. These results highlight the importance of GH51 arabinofuranosidase debranching enzyme to allow a higher cleavage of the xylan backbone of GAX from BSG and their synergy with 2 endo-1,4-β-xylanase (GH10 and GH11), one β-xylosidase (GH43) and the inclusion of one α-d-glucuronidase (GH67) in the reaction system. Therefore, this study provides an environmentally friendly process to produce xylose from BSG through utilization of enzymes as catalysts.Graphical

Kinetics of the Release of Sugars from the Enzymatic and Physico-Chemical Pre-treated Sugarcane Bagasse and Residual Forest Biomass

Several pre-treatments are used to release sugars from lignocellulosic materials that are used to produce second-generation ethanol (2G). This study aimed to evaluate the kinetic release of glucose and xylose through the enzymatic and physical treatments of sugarcane bagasse and residual forest biomass, focusing on the ratio between hexose and pentose. Enzymatic hydrolysis after hydrothermal pre-treatment under different conditions, at 170, 170 and 190 °C, 170 and 190 °C with sulfuric acid, and 170 and 190 °C with the Organosolv solvent, all of them for 10 min, were performed with sugarcane bagasse and residual forest biomass, and the kinetic parameters of sugar release were evaluated. The results indicated that compared to hydrothermal and combined hydrothermal and dilute acid hydrolysis, organosolvation process led to higher release of glucose in hydrolysates from both biomasses, with a maximum yield of 14.12 and 33.33 g L–1, respectively. On the other hand, the highest glucose/xylose ratio (about 19), which will facilitate its subsequent use for fermentation, was obtained from sugarcane bagasse after hydrothermal treatment at 170 and 190 °C. This ratio was higher for all treatments when compared to untreated biomass, which indicated that temperature and acid affected xylose instead of glucose.