Feruloyl Esterase Research Articles

Characterization of heat- and alkali-resistant feruloyl esterase from Humicola insolens and application in the production of high-strength kraft straws

Characterization of heat- and alkali-resistant feruloyl esterase from Humicola insolens and application in the production of high-strength kraft straws

Distinct Adjacent Substrate Binding Pocket Regulates the Activity of a Decameric Feruloyl Esterase from Bacteroides thetaiotaomicron.

Understanding how the human gut microbiota contribute to the metabolism of dietary carbohydrates is of great interest, particularly those with ferulic acid (FA) decorations that have manifold health benefits. Here, we report the crystal structure of a decameric feruloyl esterase (BtFae) from Bacteroides thetaiotaomicron in complex with methyl ferulate (MFA), revealing that MFA is situated in a noncatalytic substrate binding pocket adjacent to the catalytic pocket. Molecular docking and mutagenesis studies further demonstrated that the adjacent pocket affects substrate binding in the active site and negatively regulates the BtFae activity on both synthetic and natural xylan substrates. Additionally, quantum mechanics (QM) calculations were employed to investigate the catalytic process of BtFae from substrate binding to product release, and identified TS_2 in the acylation step is rate-limiting. Collectively, this study unmasks a novel regulatory mechanism of FAE activity, which may contribute to further investigation of FA-conjugated polysaccharides metabolism in the human gut.

Augmentation of Fermentability and Bioavailability Characteristics of Wheat Bran via the Synergistic Interaction between Arabinoxylan-Specific Degrading Enzymes and Lactic Acid Bacteria.

To enhance the use of wheat bran in chicken feed, a solid-state fermentation approach was used with Lactobacillus paracasei LAC28 and Pediococcus acidilactici BCC-1, along with arabinoxylan-specific degrading enzymes (xylanase, arabinofuranosidase, feruloyl esterase, XAF). The effects of the fermentation process were evaluated both in vitro and in vivo. In the in vitro study, XAF supplementation demonstrated superior performance, significantly reducing the pH of the fermented wheat bran (FWB) and increasing lactic, acetic, and butyric acid levels, total phenol content, and free radical scavenging capacity (P < 0.05) compared to the XAF-free group. In the in vivo study, broilers were fed diets containing either unfermented wheat bran (UFWB) or FWB (fermented individually with LAC28 or BCC-1). Broilers fed FWB with BCC-1 exhibited significant improvements in body weight gain, intestinal morphology, and nutrient digestibility (P < 0.05) compared to the control group. Moreover, the FWB established a healthier microbial community in the avian gastrointestinal tract. Overall, this study demonstrated the potential of combining XAF and bacteria to enhance wheat bran fermentation, benefiting broiler intestinal health and growth. This innovative approach holds promise as a cost-efficient and sustainable strategy to improve the nutritional quality of wheat bran for animal feed applications.

Optimization of fermentation conditions for the production of recombinant feruloyl esterase BpFaeT132C-D143C.

Feruloyl esterases (FAEs) are a crucial component of the hemicellulose-degrading enzyme family that facilitates the degradation of lignocellulose while releasing hydroxycinnamic acids such as ferulic acid with high added value. Currently, the low enzyme yield of FAEs is one of the primary factors limiting its application. Therefore, in this paper, we optimized the fermentation conditions for the expression of FAEBpFaeT132C-D143C with excellent thermal stability in Escherichia coli by experimental design. Firstly, we explored the effects of 11 factors such as medium type, isopropyl-β-D-thiogalactopyranoside (IPTG) concentration, and inoculum size on BpFaeT132C-D143C activity separately by the single factor design. Then, the significance of the effects of seven factors, such as post-induction temperature, shaker rotational speed, and inoculum size on BpFaeT132C-D143C activity, was analyzed by Plackett-Burman design. We identified the main factors affecting the fermentation conditions of E. coli expressing BpFaeT132C-D143C as post-induction temperature, pre-induction period, and post-induction period. Finally, we used the steepest ascent path design and response surface method to optimize the levels of these three factors further. Under the optimal conditions, the activity of BpFaeT132C-D143C was 3.58 U/ml, which was a significant 6.6-fold increase compared to the pre-optimization (0.47 U/ml), demonstrating the effectiveness of this optimization process. Moreover, BpFaeT132C-D143C activity was 1.52 U/ml in a 3-l fermenter under the abovementioned optimal conditions. It was determined that the expression of BpFaeT132C-D143C in E. coli was predominantly intracellular in the cytoplasm. This study lays the foundation for further research on BpFaeT132C-D143C in degrading agricultural waste transformation applications.

Charge mediated nano-filtration for recovering sinapic acid from a mustard bran hydrolysate

The recovery of valuable chemicals, such as sinapic acid, from enzymatic hydrolysates is essential to enabling a sustainable biorefinery. In this study, we designed a system to recover sinapic acid produced chemo-enzymatically from waste mustard bran. The process involves sequential separation, beginning with size partitioning using ultrafiltration for enzyme removal and recovery, followed by charge-mediated size partitioning via nanofiltration to isolate sinapic acid from other extracts. Four polyethersulfone (PES) ultrafiltration membranes with molecular weight cut-offs (MWCOs) of 5000 and 10 000 Da and maximum allowable working pressures (MAWPs) of 3 and 10 bar were screened for their effectiveness in removing Bovine Serum Albumin (BSA) as a model compound. Subsequently, these membranes were applied to recover the feruloyl esterase enzyme. The membrane with an MWCO of 5000 Da and an MAWP of 10 bar achieved 97.93 % enzyme recovery with a permeate flow rate of 31.4 L/h/m2 at 6 bar. Next, we evaluated the effects of zeta potential interactions on the preferential rejection of sinapic acid using various nanofiltration membranes with potentially charged surfaces, including polyimide, silicon-based thin film composite, and polypiperazine amide membranes with MWCOs ranging from 150 to 600 Da. The polypiperazine amide membrane demonstrated the highest recovery of sinapic acid, achieving 86 % recovery from a model solution and 64 % recovery from mustard bran hydrolysate. Compositional analysis of the permeate confirmed that the rejection rate (R) is influenced primarily by the pKa rather than molecular size, following the trend: sinapic acid (pKa = 4.58; 224.2 Da; R = 64.0 %), acetic acid (pKa = 4.76; 60.1 Da; R = 23.8 %), xylose (pKa = 12.15; 150.1 Da; R = 13.7 %), glucose (pKa = 12.28; 180.2 Da; R = 8.7 %), and arabinose (pKa = 12.34; 150.1 Da; R = 8.5 %). The zeta potential interactions across nanofiltration membranes enhanced sinapic acid recovery from the mustard bran hydrolysate, hence, charge mediation significantly influenced the membrane separation of these complex mixtures with varying pKa values.

The Effects of Single- or Mixed-Strain Fermentation of Red Bean Sourdough, with or without Wheat Bran, on Bread Making Performance and Its Potential Health Benefits in Mice Model.

The effects of single- (Lactobacillus fermentum) or mixed-strain (Lactobacillus fermentum, Kluyveromyces marxianus) fermentation of red bean with or without wheat bran on sourdough bread quality and nutritional aspects were investigated. The results showed that, compared to unfermented controls, the tannins, phytic acid, and trypsin inhibitor levels were significantly reduced, whereas the phytochemical (TPC, TFC, and gallic acid) and soluble dietary fiber were increased in sourdough. Meanwhile, more outstanding changes were obtained in sourdough following a mixed-strain than single-strain fermentation, which might be associated with its corresponding β-glucosidase, feruloyl esterase, and phytase activities. An increased specific volume, reduced crumb firmness, and greater sensory evaluation of bread was achieved after mixed-strain fermentation. Moreover, diets containing sourdough, especially those prepared with mixed-strain-fermented red bean with wheat bran, significantly decreased serum pro-inflammatory cytokines levels, and improved the lipid profile, HDL/LDL ratio, glucose tolerance, and insulin sensitivity of mice. Moreover, gut microbiota diversity increased towards beneficial genera (e.g., Bifidobacterium), accompanied with a greater increase in short-chain fatty acid production in mice fed on sourdough-based bread diets compared to their controls and white bread. In conclusion, mixed-strain fermentation's synergistic effect on high fiber-legume substrate improved the baking, sensory quality, and prebiotic effect of bread, leading to potential health benefits in mice.

An Exploratory Study of the Enzymatic Hydroxycinnamoylation of Sucrose and Its Derivatives.

Phenylpropanoid sucrose esters are a large and important group of natural substances with significant therapeutic potential. This work describes a pilot study of the enzymatic hydroxycinnamoylation of sucrose and its derivatives which was carried out with the aim of obtaining precursors of natural phenylpropanoid sucrose esters, e.g., vanicoside B. In addition to sucrose, some chemically prepared sucrose acetonides and substituted 3'-O-cinnamates were subjected to enzymatic transesterification with vinyl esters of coumaric, ferulic and 3,4,5-trimethoxycinnamic acid. Commercial enzyme preparations of Lipozyme TL IM lipase and Pentopan 500 BG exhibiting feruloyl esterase activity were tested as biocatalysts in these reactions. The substrate specificity of the used biocatalysts for the donor and acceptor as well as the regioselectivity of the reactions were evaluated and discussed. Surprisingly, Lipozyme TL IM catalyzed the cinnamoylation of sucrose derivatives more to the 1'-OH and 4'-OH positions than to the 6'-OH when the 3'-OH was free and the 6-OH was blocked by isopropylidene. In this case, Pentopan reacted comparably to 1'-OH and 6'-OH positions. If sucrose 3'-O-coumarate was used as an acceptor, in the case of feruloylation with Lipozyme in CH3CN, 6-O-ferulate was the main product (63%). Pentopan feruloylated sucrose 3'-O-coumarate comparably well at the 6-OH and 6'-OH positions (77%). When a proton-donor solvent was used, migration of the 3'-O-cinnamoyl group from fructose to the 2-OH position of glucose was observed. The enzyme hydroxycinnamoylations studied can shorten the targeted syntheses of various phenylpropanoid sucrose esters.

Unlocking the potential of feruloyl esterase from Myceliophthora verrucosa: a key player in efficient conversion of biorefinery-relevant pretreated rice straw.

The online version contains supplementary material available at 10.1007/s13205-024-04013-7.

Intracellular removal of acetyl, feruloyl and p-coumaroyl decorations on arabinoxylo-oligosaccharides imported from lignocellulosic biomass degradation by Ruminiclostridium cellulolyticum

BackgroundXylans are polysaccharides that are naturally abundant in agricultural by-products, such as cereal brans and straws. Microbial degradation of arabinoxylan is facilitated by extracellular esterases that remove acetyl, feruloyl, and p-coumaroyl decorations. The bacterium Ruminiclostridium cellulolyticum possesses the Xua (xylan utilization associated) system, which is responsible for importing and intracellularly degrading arabinoxylodextrins. This system includes an arabinoxylodextrins importer, four intracellular glycosyl hydrolases, and two intracellular esterases, XuaH and XuaJ which are encoded at the end of the gene cluster.ResultsGenetic studies demonstrate that the genes xuaH and xuaJ are part of the xua operon, which covers xuaABCDD’EFGHIJ. This operon forms a functional unit regulated by the two-component system XuaSR. The esterases encoded at the end of the cluster have been further characterized: XuaJ is an acetyl esterase active on model substrates, while XuaH is a xylan feruloyl- and p-coumaryl-esterase. This latter is active on oligosaccharides derived from wheat bran and wheat straw. Modelling studies indicate that XuaH has the potential to interact with arabinoxylobiose acylated with mono- or diferulate. The intracellular esterases XuaH and XuaJ are believed to allow the cell to fully utilize the complex acylated arabinoxylo-dextrins imported into the cytoplasm during growth on wheat bran or straw.ConclusionsThis study reports for the first time that a cytosolic feruloyl esterase is part of an intracellular arabinoxylo-dextrin import and degradation system, completing its cytosolic enzymatic arsenal. This system represents a new pathway for processing highly-decorated arabinoxylo-dextrins, which could provide a competitive advantage to the cell and may have interesting biotechnological applications.

Characterization of a novel multifunctional β-glucosidase/xylanase/feruloyl esterase and its effects on improving the quality of Longjing tea

Herein, a novel multifunctional enzyme β-glucosidase/xylanase/feruloyl esterase (GXF) was constructed by fusion of β-glucosidase and bifunctional xylanase/feruloyl esterase. The activities of β-glucosidase, xylanase, feruloyl esterase and acetyl xylan esterase displayed by GXF were 67.18 %, 49.54 %, 38.92 % and 23.54 %, respectively, higher than that of the corresponding single functional enzymes. Moreover, the GXF performed better in enhancing aroma and quality of Longjing tea than the single functional enzymes and their mixtures. After treatment with GXF, the grassy and floral odors of tea infusion were significantly improved. Moreover, GXF treatment could improve concentrations of flavonoid aglycones of myricetin, kaempferol and quercetin by 68.1-, 81.42- and 77.39-fold, respectively. In addition, GXF could accelerate the release of reducing sugars, ferulic acid and xylo-oligosaccharides by 9.48-, 8.25- and 4.11-fold, respectively. This multifunctional enzyme may have potential applications in other fields such as food production and biomass degradation.

Synergistic enzyme assisted release of ferulic acid from brewer´s spent grain

Brewer’s spent grain (BSG), the most abundant by-product of the beer industry, can be considered a sustainable source of ferulic acid (4-hydroxy-3-methoxycinnamic acid) (FA). FA is esterified to the arabinoxylan in the BSG. FA exhibits a variety of biological activities and is a target for valorization of BSG. We present a novel enzyme-assisted approach that utilizes the synergistic action of a specific bacterial endo-1,4-β-xylanase and a fungal ferulic acid esterase activity to selectively release high yields of FA from BSG. The study compares the efficacy of three different fungal feruloyl esterases (E.C. 3.1.1.73) of carbohydrate esterase family 1 (CE1), HiFae, AoFae1, and AnFae1, acting separately or in combination with two different endo-1,4-β-xylanases (EC 3.2.1.8), a bacterial GH30 enzyme from Dickeya chrysanthemi, and Shearzyme®, a commercial GH10 enzyme from Aspergillus aculeatus, respectively. On an equimolar dosage basis, the AnFae1-GH30 was the most efficient combination in releasing FA from BSG. This combination showed significant synergy in a dose-dependent manner, enabling up to 85% FA recovery from BSG within 30 min. of treatment. The results demonstrate a novel enzymatic approach for valorization of BSG as FA is considered a high-value natural compound for use in e.g. anti-aging skin care products.

Multiple strategies to improve extracellular secretion and activity of feruloyl esterase

Feruloyl esterase has a wide range of applications, but there are still problems with low enzyme yield and activity, and complex purification steps. Our previous research found Lactobacillus amylovorus feruloyl esterase could be secreted extracellular in Escherichia coli. In this study, multiple strategies were implemented to maximize the extracellular production of feruloyl esterase with improved activity in E. coli. Firstly, codon-optimized feruloyl esterase was obtained based on the preference of E. coli, resulting in 41.97 % increase in extracellular secretion. Furthermore, by cascading T7 promoters, replacing the 5′ UTR, randomly mutating the N-terminal sequence, and co-expressing secretory cofactors, the extracellular secretion was increased by 36.46 %, 31.25 %, 20.66 % and 25.75 %, respectively. Moreover, the feruloyl esterase were mutated to improve the substrate affinity and activity. The catalytic efficiency of Fae-Q134T and Fae-Q198A increased by 4.62-fold and 5.42-fold. Combining above strategies, extracellular feruloyl esterase activity was increased from 2013.70 U/L to 10,349.04 U/L. These results indicated that the activity and yield of feruloyl esterase secreted by E. coli were significantly increased, which laid a foundation for its industrial application.

Fermenting Distiller's Grains by the Domesticated Microbial Consortium To Release Ferulic Acid.

The microbial consortium FA12 that can release ferulic acid (FA) by fermenting distiller's grains was screened from Daqu. Taibaiella, Comamonadaceae, and Ochrobacum were highly abundant in FA12 by 16S rRNA gene sequencing. In the process of long-term acclimation with distiller's grains as a medium, the biomass of FA12 remained stable, and the pH value of fermentation liquid was also relatively stable. Meanwhile, the activities of cellulase, xylanase, and feruloyl esterase secreted by FA12 were stable in the ranges of 0.2350-0.4470, 0.1917-0.3078, and 0.1103-0.1595 U/mL, respectively, and the release of FA could reach 133.77 μg/g. It is proven that the microbial consortium has good genetic stability. In addition, the structural changes of lignocellulose in distiller's grains before and after fermentation were analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR), and the changes of distiller's grains weight and lignocellulose content before and after fermentation were also detected. These results all confirmed that FA12 had the function of degrading distiller's grains. In this study, we explored a method to use microbial communities to release FA from distiller's grains and degrade lignocellulose in the waste, which opened up a new way for the application of the high value of lost waste.

Porcine in vitro digestion and solubilization of non-starch polysaccharides in corn and wheat supplemented with xylanase and feruloyl esterase

A study evaluated porcine in vitro digestion and non-starch polysaccharide (NSP) solubilization of corn and wheat without or with xylanase alone or xylanase plus feruloyl esterase (FE). The enzymes supplied 4000 U of xylanase and 35 U of FE per kilogram of sample. Samples were digested with pepsin at a pH of 2.0 and then with pancreatin at a pH of 6.8. The digested samples were filtered to obtain unhydrolyzed residue, which was then washed using alcohol and acetone. The cereal grains (CGs) before in vitro digestion and the washed unhydrolyzed residues were analyzed for dry matter and NSP. Xylanase improved ( P &lt; 0.05) in vitro digestibility of dry matter (IVDDM) for wheat (85% vs. 89%), but not for corn (78% vs. 79%). Addition of FE to the xylanase-supplemented CGs increased ( P &lt; 0.05) IVDDM for both CGs, and reduced ( P &lt; 0.05) the arabinoxylans in the unhydrolyzed residue for corn, but not for wheat. In conclusion, xylanase product used in the current study is more effective in improving the digestibility of nutrients in wheat than in corn. The FE product used in the current study can improve the efficacy of xylanase in improving the nutrient digestibility of wheat and corn.

The secreted feruloyl esterase of Verticillium dahliae modulates host immunity via degradation of GhDFR.

Feruloyl esterase (ferulic acid esterase, FAE) is an essential component of many biological processes in both eukaryotes and prokaryotes. This research aimed to investigate the role of FAE and its regulation mechanism in plant immunity. We identified a secreted feruloyl esterase VdFAE from the hemibiotrophic plant pathogen Verticillium dahliae. VdFAE acted as an important virulence factor during V. dahliae infection, and triggered plant defence responses, including cell death in Nicotiana benthamiana. Deletion of VdFAE led to a decrease in the degradation of ethyl ferulate. VdFAE interacted with Gossypium hirsutum protein dihydroflavanol 4-reductase (GhDFR), a positive regulator in plant innate immunity, and promoted the degradation of GhDFR. Furthermore, silencing of GhDFR led to reduced resistance of cotton plants against V. dahliae. The results suggested a fungal virulence strategy in which a fungal pathogen secretes FAE to interact with host DFR and interfere with plant immunity, thereby promoting infection.

Co-production of ferulic acid and p-coumaric acid from distiller grain by a putative feruloyl esterase discovered in metagenome assembled genomes

The brewing industry generates a large amount of distillation waste, causing environmental pollution and resource wastage. In this study, a putative gene encoding feruloyl esterase detected in 33 bins using metagenomic sequencing, was expressed to exogenously to generate a recombinase with a molecular weight of 40.8 KDa. The feruloyl esterase has been optimized through codon optimization and signal peptide truncation to exhibit optimal activity at pH 6.0 and 50 °C, specifically when using 4-nitrophenyl trans-ferulate as its substrate. By employing this enzyme, p-coumaric acid and ferulic acid were simultaneously produced using brewing waste of distilled spent grain with husk. The yield of the two phenolic acids amounted to 91.2 μg and 89.25 μg from 1 g of distilled spent grain with husk, respectively. Although the yield obtained was lower than that from wheat bran and corn cob, the strategy serves as a model for the high-value utilization of brewing waste and has hold application potential. The integration of enzyme catalysis and metagenomic sequencing revealed a significant importance for clean production from agricultural processing industrial waste.

Efficient secretion of an enzyme cocktail in Escherichia coli for hemicellulose degradation

The use of host to secrete several hemicellulase is a cost-effective way for hemicellulose degradation. In this study, the xylose utilization gene xylAB of Escherichia coli BL21 was knocked out, and the xylanase (N20Xyl), β-xylosidase (Xys), and feruloyl esterase (FaeLam) were co-expressed in this strain. By measuring the content of reducing sugars generated by enzymatic hydrolysis of wheat bran in the fermentation supernatant, the order of the three enzymes was screened to obtain the optimal recombinant strain of E. coli BL21/∆xylAB/pDIII-2. Subsequently, fermentation conditions including culture medium, inducer concentration, induction timing, metal ions, and glycine concentration were optimized. Then, different concentrations of wheat bran and xylan were added to the fermentation medium for degradation. The results showed that the extracellular reducing sugars content reached the highest value of 33.70 ± 0.46 g/L when 50 g/L xylan was added. Besides, the scavenging rates of hydroxyl radical by the fermentation supernatant was 81.0 ± 1.41 %, and the total antioxidant capacity reached 2.289 ± 0.55. Furthermore, it showed the growth promotion effect on different lactic acid bacteria. These results provided a basis for constructing E. coli strain to efficiently degrade hemicellulose, and the strain obtained has great potential application to transform hemicellulose into fermentable carbon source.

Characterization and optimization of 5´ untranslated region containing poly-adenine tracts in Kluyveromyces marxianus using machine-learning model

BackgroundThe 5´ untranslated region (5´ UTR) plays a key role in regulating translation efficiency and mRNA stability, making it a favored target in genetic engineering and synthetic biology. A common feature found in the 5´ UTR is the poly-adenine (poly(A)) tract. However, the effect of 5´ UTR poly(A) on protein production remains controversial. Machine-learning models are powerful tools for explaining the complex contributions of features, but models incorporating features of 5´ UTR poly(A) are currently lacking. Thus, our goal is to construct such a model, using natural 5´ UTRs from Kluyveromyces marxianus, a promising cell factory for producing heterologous proteins.ResultsWe constructed a mini-library consisting of 207 5´ UTRs harboring poly(A) and 34 5´ UTRs without poly(A) from K. marxianus. The effects of each 5´ UTR on the production of a GFP reporter were evaluated individually in vivo, and the resulting protein abundance spanned an approximately 450-fold range throughout. The data were used to train a multi-layer perceptron neural network (MLP-NN) model that incorporated the length and position of poly(A) as features. The model exhibited good performance in predicting protein abundance (average R2 = 0.7290). The model suggests that the length of poly(A) is negatively correlated with protein production, whereas poly(A) located between 10 and 30 nt upstream of the start codon (AUG) exhibits a weak positive effect on protein abundance. Using the model as guidance, the deletion or reduction of poly(A) upstream of 30 nt preceding AUG tended to improve the production of GFP and a feruloyl esterase. Deletions of poly(A) showed inconsistent effects on mRNA levels, suggesting that poly(A) represses protein production either with or without reducing mRNA levels.ConclusionThe effects of poly(A) on protein production depend on its length and position. Integrating poly(A) features into machine-learning models improves simulation accuracy. Deleting or reducing poly(A) upstream of 30 nt preceding AUG tends to enhance protein production. This optimization strategy can be applied to enhance the yield of K. marxianus and other microbial cell factories.

Integration of subcritical water extraction and treatment with xylanases and feruloyl esterases maximises release of feruloylated arabinoxylans from wheat bran

Wheat bran is an abundant and low valued agricultural feedstock rich in valuable biomolecules as arabinoxylans (AX) and ferulic acid with important functional and biological properties. An integrated bioprocess combining subcritical water extraction (SWE) and enzymatic treatments has been developed for maximised recovery of feruloylated arabinoxylans and oligosaccharides from wheat bran. A minimal enzymatic cocktail was developed combining one xylanase from different glycosyl hydrolase families and a feruloyl esterase. The incorporation of xylanolytic enzymes in the integrated SWE bioprocess increased the AX yields up to 75%, higher than traditional alkaline extraction, and SWE or enzymatic treatment alone. The process isolated AX with tailored molecular structures in terms of substitution, molar mass, and ferulic acid, which can be used for structural biomedical applications, food ingredients and prebiotics. This study demonstrates the use of hydrothermal and enzyme technologies for upcycling agricultural side streams into functional bioproducts, contributing to a circular food system.

Boosting sugarcane trash decomposition: synergistic action and proximity effect of xylanase and feruloyl esterase co-displayed on the cell surface of Pichia pastoris (Komagataella phaffi)

Xylanase and feruloyl esterase co-displayed on yeast surface boosts lignocellulosic biomass degradation through synergistic effect caused by their proximity.