Insight into the eminent biotechnological applications of xylanolytic enzymes for sustainable bioprocessing

Abstract

Xylan is one of the most abundant polysaccharides in nature and presents a structural complexity characterized by a heterogeneous polymer composition. Comprising various sugar subunits and associated acids linked through a diverse array of bonds, xylan poses challenges for complete degradation. This review article provides a comprehensive overview of xylan's structure, the role of xylanolytic enzymes in its degradation, and the industrial applications of xylanases in sectors, such as paper and pulp, food, textiles, and pharmaceuticals. Furthermore, it also discusses the use of advanced biotechnology tools, such as nano-biotechnology and genetic engineering, particularly through CRISPR/CAS technology, for enhancing the thermostability of xylanases. This article also provides insights into emerging trends in xylanase research, including bioprospecting novel thermostable xylanases from metagenomes, protein engineering, synthetic biology, and the integration of biorefinery. Finally, it highlights the importance of regulatory frameworks and standardization initiatives for ensuring the quality and the sustainability of xylanase-based technologies. Overall, this review offers valuable insights into the multifaceted role of xylanases in biotechnology and industrial bioprocessing while outlining future directions for research and innovation in this field.

Introduction

Enzymes are vital biological catalysts that facilitate efficient chemical reactions in various processes (Abd-Elhalem et al. 2015; Abd-Elhalim et al. 2023; Ahmed et al. 2023; Mwaheb et al. 2024). However, their sensitivity to harsh operating conditions limits their broad industrial application. Lignocellulosic biomass comprises approximately 20–30% hemicellulose and is a promising renewable resource for producing valuable chemical feedstocks (Zhang et al. 2023). Among hemicelluloses, xylan is the second most abundant polysaccharide on earth, playing a crucial role in the plant cell wall’s structural integrity and flexibility. The structure of xylan is highly diverse across plant species. In hardwoods, xylan exists predominantly as O-acetyl-4-O-methylglucuronoxylan, while in softwoods, it is primarily arabino-4-O-methylglucuronoxylan (Bhardwaj et al. 2019). In grasses and cereals, xylan is present as arabinoxylans containing significant arabinose side chains. This structural variability is not only species-dependent but also impacts how xylan interacts with other cell wall components, contributing to its role in biomass recalcitrance and the challenges of enzymatic hydrolysis (Walia et al. 2017; Curry et al. 2023). Understanding the diversity of xylan structures is essential for developing effective enzymatic processes for biomass conversion (Zhang et al. 2023).

Xylanases (EC 3.2.1.8) are the enzymes responsible for the hydrolysis of β-1,4-glycosidic bonds of xylopyranosyl units from xylan (Liu et al. 2023a, b). Pentosanases and endo-1,4-β-xylanase represent alternative terms for xylanases. Additionally, common synonyms for xylanase include β-xylanase, β-1,4-xylanase, endo-xylanase, endo-1,4-β-d-xylanase, and β-1,4-d-xylan-xylanohydrolase (Kumar et al. 2018a, b). Xylanases, produced by various microorganisms, such as bacteria, filamentous fungi, yeasts, and actinomycetes, are industrially significant enzymes (Akpinar and Karaoglu 2024). These enzymes are classified into several glycoside hydrolase (GH) families, including GH families 5, 7, 8, 10, 11, 26, 30, and 43 (Motta et al. 2013; Chadha et al. 2019). Xylanases exhibit a wide range of applications in various industrial processes. Cellulase-free xylanase preparations are extensively used in the paper and pulp industries for pulping and bleaching processes, where they aid in the breakdown of hemicellulose components, thereby improving the quality of paper products. In the biofuel industry, xylanases play a crucial role in the saccharification step of biomass conversion, facilitating the release of fermentable sugars from hemicellulose for subsequent ethanol production (Basu et al. 2017). Furthermore, these enzymes find applications in organic waste treatment processes where they contribute to the degradation of hemicellulosic waste materials. In food industry, particularly in baking, xylanases are employed to enhance the stability and the flexibility of dough by acting on soluble and insoluble pentosans, thereby improving the overall quality of baked products (Ahmed et al. 2014).

This article aims to explore the potential of thermostable bacterial xylanases in various industrial sectors. Industries are shifting toward eco-friendly technologies, and xylanases are poised to replace old, traditional chemical processes across sectors like pulp and paper, textiles, pharmaceuticals, and food. It highlights their unique properties, including stability and activity at elevated temperatures, making them suitable for industrial processes. Additionally, it addresses current challenges and prospects, highlighting their importance in advancing industrial processes toward greater sustainability and efficiency.

Structure of xylan

Xylan, a complex hetero-polymeric structure, comprises a backbone of d-xylose linked with β-1,4-glycosidic bonds, often accompanied by traces of l-arabinose. Its composition varies depending on the biomass source, with forms, such as O-acetyl-4-O-methylglucuronoxylan in hardwoods, arabino-4-O-methylglucuronoxylan in softwoods, and arabinoxylans in grasses and annual plants. Additionally, xylan residues may bear various side-chain groups, including acetyl, feruloyl, glucopyranosyl, 4-O-methyl-d-glucuronopyranosyl, p-coumaroyl, or α-l-arabinofuranosyl (Curry et al. 2023). The degradation of the complex xylan structure necessitates the concerted action of xylanolytic enzymes, targeting specific bonds for thorough hydrolysis. These enzymes include endo-1,4-β-d-xylanases, which cleave the xylan backbone randomly, and β-d-xylosidases, responsible for breaking down xylose polymers into monomeric forms. Additionally, α-glucuronidase and acetylxylan esterase act to remove acetyl and phenolic side branches, while α-l-arabinofuranosidases catalyze the removal of side groups. Furthermore, p-coumaric esterase and ferulic acid esterase cleave ester bonds present on xylan (Gómez et al. 2016; Bhardwaj et al. 2019).

The presence of specific side-chain groups, such as acetyl and feruloyl, attached to the xylan backbone plays a crucial role in determining its functional properties and applications. These side-chain groups influence xylan’s solubility, enzymatic degradability, and material properties, making it valuable in industries ranging from biofuels to biomaterials. Zhang et al. (2011) have explained how the presence of acetyl groups can impact the enzymatic degradation of xylan. The acetyl side chain in xylan hinders the accessibility of xylanases to the xylan backbone. Therefore, acetyl xylan esterases are used in the process of degradation to remove the acetyl group and improve the accessibility of xylanases to the xylan backbone. Studies on aspen wood and wheat straw have shown that eliminating acetyl groups can increase xylan solubilization and enhance cellulose accessibility (Zhang et al. 2011; Selig et al. 2009). Furthermore, in the process of biofuel production, a process of deacetylation takes place to enhance the efficiency of bioethanol production (Dodd and Cann 2009). Comprehending the structure of xylan is pivotal for engineering microorganisms capable of enhanced xylanase production with desired properties. Moreover, this understanding informs the optimization of strategies for xylanase production and purification, facilitating its application across various industrial processes. The structure of xylan, highlighting the bonds that are targeted by different xylanolytic enzymes for complete hydrolysis, is shown in Fig. 1

Fig. 1.

This diagram illustrates the step-by-step mechanism of the enzymatic hydrolysis of xylan by seven different enzymes: endo-xylanase, β-xylosidase, acetyl xylan esterase, α-glucuronidase, α-arabinofuranosidase, and feruloyl and p-coumaroyl esterases, which results in the formation of simple sugar molecules (Bhardwaj et al. 2019)

Xylanolytic enzymes in xylan degradation

Numerous xylanolytic enzymes are needed for the degradation of the intricate structure of xylan as it is a complicated process. The list of enzymes involved in the degradation of xylan molecules is represented in Table 1. Endo-1,4-β-d-xylanases play a key role by randomly cleaving bonds along the xylan backbone, dismantling the larger polymer (Kaur et al. 2023). β-d-Xylosidases then work to further reduce the xylose polymer into its basic monosaccharide form through stepwise removal. Acetyl and phenolic side chains are removed by two important enzymes, α-glucuronidase and acetyl xylan esterase. These enzymes act to detach branches from the core xylan structure. Likewise, α-l-arabinofuranosidases catalyze the separation of arabinose side groups through the hydrolysis of linkages. Lastly, xylan holds ester linkages that must be targeted for specific ester-cleaving enzymes. p-Coumaric esterase and ferulic acid esterase ease this process by cleaving the ester bonds between hydroxycinnamic acid residues and xylan backbone (Chakdar et al. 2016; Walia et al. 2017). The complex plant cell wall part xylan is deconstructed into simpler sugar monomers through the coordinated and sequential activities of these various enzymes. Each enzyme plays a crucial role in dismantling distinct aspects of the xylan structure through selective catalytic mechanisms.

Table 1.

Shows the key enzymes responsible for the breakdown of xylan with their EC number and mode of action

Enzyme	EC Classification	Action
Endo-1,4-β-xylanase	EC 3.2.1.8	Cleaves internal bonds linking xylose units within the xylan backbone
β-Xylosidase	EC 3.2.1.37	Removes consecutive xylose residues from the non-reducing ends of xylooligosaccharides
α-Glucuronidase	EC 3.2.1.139	Hydrolyzes ester linkages between glucuronic acid and the xylan core
Acetylxylan esterase	EC 3.1.1.72	Breaks acetyl groups from substituted xylose units along the xylan chain
α-l-Arabinofuranosidase	EC 3.2.1.55	Detaches l-arabinofuranose side chains attached to the xylan backbone
p-Coumarate esterase	EC 3.1.1. B10	Cleaves ester bonds connecting p-coumaric acid to hydroxycinnamic acids within xylan
Ferulate esterase	EC 3.1.1.73	Breaks ester bonds linking ferulate groups to other hydroxycinnamic acids on xylan

Various thermophilic bacteria, especially from Geobacillus sp. and Caldicellulosiruptor sp. strains, have been analyzed under different temperatures, pH, and substrate conditions, which support their described mechanisms of action. For instance, studies on xylanases from Geobacillus species, like Geobacillus sp. WSUCEF1 and Geobacillus thermodenitrificans, show impressive thermal stability, with these enzymes working optimally at temperatures as high as 60–90 °C (Bhalla et al. 2015; Huang et al. 2017). Notably, the half-life of these enzymes varies. For example, at 60 °C, they can last for around 12 days, while at 70 °C, the duration shortens to about 13 days. Such durability makes these enzymes ideal for high-temperature industrial processes. Additionally, when these are tested against beechwood xylan, these enzymes outperformed popular commercial xylanases like Cellic HTEC 2 and Accellerase XY, proving their superior efficiency in breaking down substrates.

Similarly, xylanases from Caldicellulosiruptor species, including C. bescii and C. owensensis, have also shown significant thermostability. These enzymes remain active at temperatures up to 90 °C and function across a wide pH spectrum (An et al. 2015; Liu et al. 2017). They are versatile and can degrade not only xylan but also complex polysaccharides like barley β-glucan and even crystalline cellulose (Haq and Akram, 2019). This range of activities underscores their potential in bioconversion applications. Another interesting example comes from Thermopolyspora flexuosa. Its GH10 xylanase performs well at temperatures between 65 and 80 °C, maintaining stability even in the presence of ionic liquids, which are often used during biomass processing (Anbarasan et al. 2017). This stability, even in challenging conditions, highlights the enzyme’s value for industrial use. The thermostability and the broad substrate specificity of these enzymes make them highly promising for industrial applications like biomass conversion. Their ability to perform under various conditions validates their potential, and continued research could unlock even more uses for these robust biocatalysts.

Sources of thermostable xylanases

Xylanase is found richly throughout nature, showcasing its captivating presence across a broad spectrum of living organisms from bacteria present in marine and terrestrial environments to those inhabiting the intricate ecosystems of the rumen. Its ubiquity underscores the fascinating diversity of this enzymatic marvel (Chakdar et al. 2016). Bacillus, Staphylococcus, Cellulomonas, Micrococcus, Paenibacillus, Arthrobacter, Pseudoxanthomonas, Microbacterium, and Rhodothermus are major species of bacteria that produce xylanase enzymes (Chapla et al. 2012; Dutta et al. 2020). Among all the species of bacteria, Bacillus spp. are the most prominent, including B. pumilus, B. circulans, B. stearothermophilus, B. halodurans, B. subtilis, and B. amyloliquefaciens (Banka et al. 2014; Gupta et al. 2015).

Clostridium thermocellum, Stenotrophomonas maltophila, Streptomyces spp., Rhodothermus marinus, Thermotoga sp., and Bacillus spp. are thermostable bacterial species reported to produce xylanases enzyme at 60–70 °C in neutral or alkaline conditions (Thomas et al. 2014). Some common habitats of thermostable xylanase-producing bacteria are marine self-solfataric fields, thermal springs, terrestrial and hot pools (Soni et al. 2020). Most of the thermostable xylanases belong to (glycoside hydrolases) GH family 10 and GH11, while GH5, 7, 8, 10, and 43 are a few other classes that also produce thermostable xylanases (Kumar et al. 2016; Ghosh et al. 2020). The names of different microorganisms that produce thermostable xylanases of GH10 and 11 are listed in Table 2.

Table 2.

Different types of thermostable xylanase-producing microbes of GH families 10 and 11

Organism	GH family	References
Bacillus stearothermophilus	GH10	Chakdar et al. (2016)
Caldicellulosiruptor sp.	GH10	An et al. (2015)
Caldicoprobacter algeriensis strain TH7C1	GH11	Amel et al. (2016)
Chaetomium thermophilum	GH11	Liu et al. (2022)
Clostridium thermocellum	GH10 and 11	Hamann and Noronha (2022)
Malbranchea cinnamomea strain S168	GH11	Fan et al. (2014)
Paecilomyces variotii	GH10 and 11	Abdella et al. (2021)
Thermoascus aurantiacus	GH10	De Oliveira Nascimento et al. (2022)

Enhanced thermostability of xylanase is most likely because of slight modifications in its protein structure, i.e., an increase in the number of hydrogen bonds and salt bridges, an increase in the number of charged surface residues, an improvement in internal packing, and an increase in the number of charged surface residues. Disulfide bridges introduced, notably at the N- or C-termini or in the helix regions of the xylanase, improve their stability at high temperatures (Zhang et al. 2016).

Various microorganisms have been found to produce stable xylanases with optimal pH and temperature ranges suitable for industrial applications. Caldicoprobacter algeriensis produces an enzyme called xynBCA with an optimum pH of 6.5 and temperature of 80 °C, exhibiting a specific activity of 117 U/mg. The xylanase from C. algeriensis was shown to retain 20% of its activity after 20 min of incubation at 80 °C (Mhiri et al. 2020). Furthermore, Streptomyces griseorubens LH-3 also produces an endo-xylanase had an optimal pH and temperature of 5.0 and 60 °C, respectively, with a specific activity of 767.2 U/mg (Wu et al. 2018). Thermotoga species, such as T. maritima and T. neapolitana, have been found to produce xylanases and hemicellulases functioning around pH 5.0–6.0 and temperatures from 90 to 100 °C (Benedetti et al. 2019; Yang et al. 2020).

Classification of xylanase

Based on molecular mass and isoelectric point, crystal structure, and catalytic/kinetic properties, xylanase may be generically categorized into three categories (Li and Kokare 2017). Prior classification systems for xylanases focused on molecular weight and isoelectric point, allocating two categories: high molecular weight with low isoelectric (acidic) point (HMWLI; above 30 k Da), and low molecular weight with high isoelectric (basic) point (LMWHI; below 30 k Da). However, exceptions emerged where not all xylanases cleanly fit into weights above or below 30 (Bhardwaj et al. 2019). As such, a more robust approach considers primary and tertiary structures in tandem with catalytic properties, kinetic traits, substrate specificity, and product profiles. This facilitates comparisons to mechanistic characteristics (De Souza Vandenberghe et al. 2020). Features like genomic sequences, three-dimensional crystal structures, and functional roles are cataloged by glycoside hydrolase (GH) families within the Carbohydrate-Active Enzymes database (CAZy). This repository synthesizes such intricate details on individual xylanases. Its integrated classification scheme accounts for inconsistencies in earlier frameworks dependent solely on molecular mass and isoelectric point. By incorporating diverse structural and enzymatic data, a more comprehensive system has superseded older paradigms (Bhardwaj et al. 2019).

The Carbohydrate-Active Enzymes database (CAZy) is a carefully curated knowledge resource focused on enzymes integral to the breakdown, alteration, and formation of glycosidic bonds within carbohydrates and glycoconjugates. It synthesizes publicly accessible information sources like the National Center for Biotechnology Information (NCBI) to provide genomic sequences, functional annotations, structural classifications, three-dimensional protein structures, and biochemical details on these carbohydrate-active enzymes (Lombard et al. 2014). CAZy centralizes data on family classifications and phylogenetic relationships, as well as catalytic mechanisms and molecular interactions gleaned from published scientific literature. By integrating diverse experimental data, it aims to further understand enzymatic functionality at the sequence and structural level for these important carbohydrate-modifying proteins. The classification of xylanase enzymes based on the Glycoside Hydrolase Family is summarized in Table 3.

Table 3.

Properties and structures of GH family xylanases

Family	Domain/subunit structure	Catalytic mechanism/properties	References
GH8	Multi-subunit	Induced fit mechanism. Conformational changes during catalysis. Investigation of pXyl from Pseudoalteromonas haloplanktis TAH3a revealed − 3 to + 3 subsites through xylopentaose/xylotriose complexes	Mendonca et al. (2023)
GH10	Dimeric	Coupling the impact of temperature-induced structural changes, xylanase (TpXyl10B) displayed a temperature-dependent action mode	An et al. (2015); Mendonca et al. (2023)
GH11	Monomeric	Bacillus circulans BCX contains nucleophile Glu78 and acid/base catalyst Glu172. N35D mutant stabilizes the transition state through the Asp35-Glu172 H-bond, increasing efficiency. "Reverse protonation" mechanism	Paës et al. (2012); Han et al. (2013)
GH19	Modular	Globitermes brachycerastes Xyl-ORF19 contains GH10 and Big_2 domains. Big_2 domain impacts kinetics/properties without reducing catalytic activity	Han et al. (2013)

Enhancing the thermostability of xylanases by advanced biotechnology tools

Enzyme immobilization

Immobilization of enzymes is studied for significant improvements in the life cycle of an enzyme as it can be reused after the end of the reaction, which helps in achieving cost-effectiveness in industrial applications (Moehlenbrock and Minteer 2016). Immobilization also strengthens enzyme structure against various stressors like high temperature, solvents, and extreme pH (Kumar et al. 2018a, b). When enzymes are immobilized, their conformation becomes more rigid due to bonding with the support material. This hinders thermal distortions to the enzyme architecture (Aissaoui et al. 2013). Recent advances in molecular biology enable the introduction of additional binding domains onto enzymes to promote stronger immobilization. Entrapment within polymers, encapsulation in inorganic matrices, covalent attachment to carrier materials, and self-immobilization through fusion tags are various immobilization methods (T.sriwong and Matsuda 2022).

In optimizing xylanases reuse and recovery cycles for cost-effective industrial applications, various key factors should be considered. We can ensure efficient recovery of xylanases by optimizing the enzyme’s stability under different operational conditions (Dhaver et al. 2022). For example, a study by Dhaver et al. (2022) has shown that using Fe²⁺, Mg²⁺, and Zn²⁺ as cofactors can improve xylanase stability significantly. Keeping enzymes active under these conditions ensures better recovery of enzymes and facilitates multiple reuse cycles without a substantial loss of activity (Dhaver et al. 2022). Furthermore, immobilization of enzymes onto solid supports such as magnetic nanoparticles (MNPs) facilitates easier recovery of enzymes through magnetic separation (Bilal et al. 2018). This significantly reduces the operational cost as the enzyme can be recovered and reused without loss of activity over multiple cycles (Dhaver et al. 2022).

Numerous studies have immobilized xylanases to boost thermal resilience and activity (Cakmak & Ertunga 2016). Researchers prepared a hybrid of levan–xylanase (LXy) nanoparticles, immobilizing them within alginate beads. This system maintained 80% activity over a wide pH range from 3–10 (Basu et al. 2017) and temperatures from 20–90 °C (Jampala et al. 2017). Similarly, xylanase from B. licheniformis was entrapped in calcium-alginate beads using glutaraldehyde for crosslinking. The immobilized form exhibited optimal pH from 8 to 9 and temperatures 50–60 °C (Kumar et al. 2018a, b). Another group of researchers isolated the xynTF16 xylanase gene from a thermophile called Geobacillus sp. The gene was recombinantly expressed in E. coli, and the purified enzyme was immobilized on chitosan. Remarkably, this immobilized recombinant xylanase improved its optimal temperature from 55 to 65 °C. It could also be reused for six cycles, retaining 80% initial activity (Cakmak and Ertunga 2016).

Nanotechnology for enzyme improvement

Nanotechnology has greatly contributed to advances in the biotechnology field, particularly regarding enzyme engineering applications. One approach involves immobilizing enzymes onto nanomaterials to improve characteristics, such as catalytic activity, thermostability, and recyclability (Rai et al. 2019; Gkantzou et al. 2021). Nanoparticles exhibit unique properties for immobilization due to their large surface area and functional groups available for enzyme attachment. Several recent studies have successfully utilized nanotechnology to enhance xylanase thermostability (Jampala et al. 2017; Kumar et al. 2017). In one experiment, a levan–xylanase nano-hybrid was immobilized within alginate beads, showing increased activity and heat tolerance. Separately, xylanase isolated from Aspergillus niger was immobilized on iron oxide-coated chitosan magnetic nanoparticles, retaining 87.5% activity even at high temperatures (Liu et al. 2014). Similarly, graphene oxide nanosheets decorated with superparamagnetic iron oxide (SPGO) particles were used to covalently immobilize xylanase, demonstrating improved thermal, pH, and storage stability (Royvaran et al. 2016).

Beyond xylanase applications, nanotechnology has benefited from other economically important enzymes. One group engineered a thermostable chitin-binding domain onto beta-glucosidase from Thermotoga maritima (Tm-β-Glu), immobilizing the enzyme fusion onto magnetic nanoparticles (MNPs). Compared to free enzymes, this novel system exhibited greater galacto-oligosaccharide synthesis capacity and binding efficiency (Ajeje et al. 2021). Magnetic separation also allowed repetitive reuse without loss of activity (Alnadari et al. 2020). In another study, thermostable beta-glucosidase from T. petrophila was immobilized on a macro-porous resin modified with polyethylenimine and glutaraldehyde. Results showed drastically enhanced thermo- and pH-stability, as well as glucose tolerance. Activity was 21% higher than free enzymes at 85 °C (Shi et al. 2018). Additionally, immobilizing beta-galactosidase on amino- or cyanuric chloride-modified silica nanoparticles significantly boosted thermal resilience, retaining 72% activity versus 35% for free enzyme after incubation at 60 °C for 12 h (Banjanac et al. 2016). The application of nanobiotechnology functionalizes enzyme immobilization matrices, augmenting heat resistance and recyclability. This proves invaluable for industrial applications demanding robust, cost-effective biocatalysts suited to harsh processing conditions. Ongoing research continues optimizing such strategies to fully unlock nanotechnological potential for industrial enzymology.

Regardless of these advancements, various challenges hinder the large-scale industrial application of nanobiotechnology for enzyme immobilization. When immobilizing enzymes as nanoparticles, especially with porous materials, we face the challenge of diffusional limitation, and in such cases, immobilization through encapsulation and entrapment is not feasible (Zanuso et al. 2021; De Araújo et al. 2023; Bai et al. 2025). Complex substances such as lignocellulosic biomass encounter limited access to the active site of an enzyme, which limits their hydrolysis efficiency (De Araújo et al. 2023). Another challenge faced is mass transfer limitation in real-world reactors (Patti et al. 2024). Immobilized enzymes often encounter reduced interactions with substrates due to limited diffusion, particularly in industrial processes involving large heterogeneous substrates, such as in bioethanol production (De Araújo et al. 2023). Furthermore, the cost of nanomaterials and the scalability of the immobilization present another significant challenge when applying nanotechnology for enzyme immobilization in real-world industrial settings (Clauser et al. 2022; De Araújo et al. 2023). The cost of nanomaterials, such as magnetic nanoparticles, is high, which reduces the cost-effectiveness of industrial processes using nanotechnology. These challenges underscore the need for continued research to optimize nanomaterials for industrial settings. Overcoming these challenges will allow industries to avail the benefits of nanotechnology in enzyme immobilization.

Genetic engineering

Genetic engineering techniques have effectively improved enzymatic thermostability through targeted protein modifications. Directed evolution mimics natural selection in the lab to rapidly evolve desirable traits in enzymes. This approach was used to enhance the thermostability of endo-β-1,4-glucanase III from Trichoderma reesei QM94141, retaining activity up to 50 °C for 30 min over a wide pH range (4.4–8.0) (Miao et al. 2022). Similarly, a GH11 family xylanase variant showed 820-fold greater thermostability at 70 °C for 410 min compared to the wild-type (Xing et al. 2021). Directed evolution also increased the specific activity of a Bacillus stearothermophilus xylanase A (BaxA) by 3.5-fold (Xu et al. 2016). Semi-rational design utilizes bioinformatics to identify conserved residues or catalytic sites for guided mutagenesis (Wang et al. 2018, 2020). One study generated a chitinase PpChil cloned from Paenibacillus pasadenensis CS06611 mutant with introduced disulfide bonds and proline substitution, exhibiting significantly higher thermostability and activity (Ajeje et al. 2021). Separately, a single point mutation in a Penicillium canescens xylanase improved its thermostability 2.5-fold at 50–60 °C (Denisenko et al. 2017). Structure-guided rational design entails generating targeted mutations based on crystal structures to minimize structural disruption (De Simone et al. 2015). Combining structural data with SCHEMA recombination generated chimeric proteins for a novel hyper-thermophilic cellulase (Heinzelman et al. 2013). Disulfide bonds also critically impact thermostability—their strategic placement in xylanases boosted melting temperatures.

Modern genome editing tools enable precise direct modifications (Kumar et al. 2018a, b). Zinc finger nucleases (ZFNs), TALENs, and CRISPR-Cas9 introduce double-strand DNA breaks, allowing knock-ins or knockouts (Yadav et al. 2016). CRISPR is highly specific and versatile, directing Cas9 to targeted sequences (Kumar et al. 2018a, b). It has been applied to generate microbial cell factories through multiplex gene editing. CRISPR has significantly contributed to the design of robust thermostable xylanases. In CRISPR-Cas9, specific amino acids are replaced with amino acids that can improve the enzyme’s thermostability and activity (Garg et al. 2023). This targeted approach allows scientists to change the genetic code of microbes, which results in improved xylanase production and activity in industrial settings. For example, the manufacturing of bioethanol requires the use of high temperatures. In such cases, genetically modified thermostable enzymes performed better as compared to normal enzymes (Garg et al. 2023). In addition to this, CRISPR/Cas9 has been used to improve the resistance of biofuel-producing organisms to various fermentation inhibitors, which is essential in enhancing overall production yields (Garg et al. 2023). The thermostability of S. cerevisiae is enhanced by editing it with CRISPR-Cas9. It can withstand higher temperatures and produce more ethanol after modifying amino acids in NADH dehydrogenase and pyruvate kinase through CRISPR-Cas9 editing (Sánchez-Muñoz et al. 2022; Garg et al. 2023). This demonstrates how genetic engineering not only boosts the enzyme's thermostability but also improves tolerance to environmental stress, making these enzymes highly suitable for industrial applications.

Another study has explained that five mutations in the N-terminus of mesophilic xylanases induced thermostability in xylanase enzymes and enabled them to function effectively at higher temperatures in industries (Zhang et al. 2010). Recent research has demonstrated that disrupting the xylanase inhibitor protein (XIP) gene in common wheat using CRISPR/Cas9 technology can significantly enhance dough quality (Sun et al. 2022a). This genetic modification leads to increased SDS-sedimentation values (SV) and improved stability time (ST), both of which are critical indicators of dough strength and elasticity (Sun et al. 2022a, b). This study suggests that targeting the TaXip gene could be a promising strategy for developing high-quality wheat varieties with superior baking properties (Sun et al. 2022b). Continued optimization exploiting enzyme engineering holds promise to design highly robust biocatalysts suited for industries requiring harsh conditions like biofuel and animal feed processing.

Bioprospecting novel thermostable xylanases from metagenomes

Metagenomic approaches have significantly contributed to the discovery of novel thermostable xylanases with diverse catalytic properties (Zhong et al. 2021). It allows the study of microbial communities directly from environmental samples without the need for cultivation (Ariaeenejad et al. 2018). Therefore, this is very useful for microbes that live in extreme conditions and are often difficult to culture in labs. Function-based metagenomic screening effectively identifies xylanase-encoding genes from uncultured microbiomes inhabiting extreme environments, such as deserts, hydrothermal vents, hot springs, and hydrocarbon reservoirs (Hebal et al. 2021). The ability of microorganisms to thrive under such stressful conditions equips their enzymes with heightened thermal resilience. Several thermophilic xylanases have been uncovered this way. One example is the discovery of PersiXyn1, a novel xylanase identified through metagenomic sequencing of camel rumen microbiota (Ariaeenejad et al. 2018). With the help of metagenomics, researchers can sequence and analyze microbial DNA from the rumen of the camel, and this leads to the discovery of a novel PersiXyn1 coding gene (Ariaeenejad et al. 2018). This was later expressed in E. coli, which proved the stability of this enzyme across a wide range of temperatures and pH levels, making it a good candidate for industrial applications (Ariaeenejad et al. 2018). Xylanase was also discovered in a chicken cecum metagenome, isolating xylanase highly suited to that habitat's high salt concentrations (Al-Darkazali et al. 2017). Considering poultry feed consists mainly of non-starch polysaccharides like xylans and arabinoxylans, microbial communities within the chicken intestine would abundantly express enzymes degrading such substrates (Ajeje et al. 2021).

Another investigation applied function-based screening to a compost soil metagenomic library, characterizing a novel GH11 endo-xylanase gene encoding an alkali-stable, hyperthermophilic enzyme (Ajeje et al. 2021). The recombinant protein demonstrated maximal activity at an unusually high pH of 9.0 and temperature of 80 °C (Verma et al. 2013). Similarly, sequence-based searches amplified a xylanase gene from metagenomic DNA derived from cow dung microbes. The expressed enzyme showed optimal thermostability around pH 7 and 75 °C, well-suited to the thermal tolerance of ruminal microflora (Sun et al. 2015). Further investigations are exploring whether such metagenome-sourced xylanases harbinger novel catalytic mechanisms evolved under other demanding environmental stresses. Metagenomics not only helps in the discovery of enzymes but also facilitates in modifying these enzymes to improve their performance in harsh industrial conditions. Future metagenomic strategies are poised to revolutionize the discovery of xylanases with novel catalytic mechanisms, especially from organisms living under extreme conditions. Through this, we can bypass the limitations of traditional culturing methods and can access the whole genetic material of microbes, including unique and unculturable organisms from extreme temperature, salinity, and pH (Shikha et al. 2020). With advancements in high-throughput sequencing, future metagenomic strategies will allow for the rapid screening of environmental samples, leading to the discovery of new xylanases that exhibit unique catalytic activities (Saini et al. 2022). Furthermore, metagenomic approaches can uncover microbial species that are present in low abundance but may harbor highly efficient enzymes for breaking down lignocellulosic biomass (De Fátima Alves et al. 2018).

Innovating bioprospecting strategies and frequently optimizing genomic access will unlock microbial adaptations from diverse uncultured reservoirs. This strengthens the biotechnological toolkit with thermostable biocatalysts like xylanases applicable across industries requiring high-temperature processing. Continued metagenomics-guided discovery could engineer even more robust enzymes extending functionality under extreme industrial conditions.

Xylanase production and optimization

Various fermentation processes are used to produce xylanase enzymes using different organisms under optimized conditions. Mainly SmF (submerged fermentation) and SSF (solid-state fermentation) are used for xylanase production (Motta et al. 2013; Walia et al. 2017; Dhaver et al. 2022). Generally, culture and media in which microorganisms are grown play a significant role in producing xylanase. The growth environment utilized for fermentative xylanase production must be tailored deliberately. The cultivation medium must optimally support high levels of the target xylanase while also maintaining cost-effectiveness and preventing unnecessary waste (Dhaver et al. 2022). Incubation time, temperature, carbon and nitrogen sources, agitation, pH, and concentration are some important physical and chemical parameters for the optimum growth of xylanase (Kereh et al. 2018). Readily available and low-cost agro-industrial residues, like wheat bran, corncob, corn stover, and wheat straw, have demonstrated potential as suitable substrates capable of yielding increased amounts of enzyme through fermentation (Naik et al. 2023). Careful design of the microbial conditions is pivotal to fully achieve the dual aims of maximized enzymatic output alongside sound economic and environmental stewardship throughout the process (Dhaver et al. 2022).

Furthermore, the fermentation chosen is often based on the microbe involved in the process of production. Bacteria grow through a bacterial growth cycle and need abundant water to carry out their metabolic functions. As a result, submerged fermentation (SmF) is typically more suitable for bacteria since this process utilizes a liquid growth medium and keeps the microbes fully submerged. In contrast, fungi like molds and yeasts propagate using a mycelial network structure that does not demand as much moisture to thrive. Mycelium can penetrate and break down solid fermentation substrates when moisture is present (Walia et al. 2017). Therefore, fungi are commonly suited to solid-state fermentation (SSF) which utilizes a moist solid material as the fermentation platform and does not require complete submersion of the microbe (Wang et al. 2023). In general, the water needs and the physical growth habits of the microbial variant dictate whether SmF or SSF will be the preferred fermentation setup. According to statistics almost 90% of the total xylanase is produced through SmF. (Bhardwaj et al. 2019).

Solid-state fermentation (SSF)

Solid-state fermentation differs from other types as it does not contain freely flowing or excess water within the system. Rather than being suspended in an aqueous solution, the microbes carry out fermentation using components present in a moist solid substrate or culture medium (Lizardi-Jiménez and Martínez 2017). In SSF systems, the solid material acts both as a nutrient source and support structure for microbial growth (Kumar et al. 2013). The carbon, nitrogen, minerals, and other growth factors necessary for the microbes are derived directly from the solid particles. The advantages of solid-state fermentation over submerged fermentation for growing microbes are demonstrated in Fig. 2. Solid-state fermentation for microbial growth requires less water and can easily grow on affordable agro-waste; these solid substrates provide important physical properties that allow microbial attachment and anchorage (Kumar et al. 2013; Behera and Ray 2016).

Fig. 2.

This figure highlights the benefits of solid-state fermentation over submerged fermentation to produce the xylanase enzyme. Using the SSF downstream method is easier, and in this method, we can use agro-waste as substrate

Numerous physical, chemical, and biochemical factors are considered before conducting any Solid-State Fermentation. Several key factors influence the microbial fermentation process in solid-state systems. First, the chosen microorganism and substrate must be appropriately selected based on their compatibility (Kumar et al. 2013; Behera and Ray 2016). Pre-treatment of the substrate can impact its digestibility. Moisture content and water activity of the solid substrate are also important since microbes require sufficient hydration. Environmental parameters like relative humidity and temperature must be controlled. An even temperature helps prevent issues from metabolic heat buildup. Inoculation quantity and cultivation period will affect microbial establishment and fermentation efficiency. Maintaining uniformity of conditions across the fermenting material is important as well. Lastly, gas atmosphere composition, such as oxygen uptake and carbon dioxide production rates, influences either aerobic or anaerobic nature of growth (Costa et al. 2018). Close monitoring and optimization of these interrelated process variables lead to successful microbial fermentations using solid-state systems (Kumar et al. 2013).

Submerged fermentation (SmF)

Submerged fermentation (SmF) involves the cultivation of microorganisms fully immersed in an aqueous growth medium. During SmF, components of the growth medium remain evenly solubilized, allowing the microbe to derive nutrients directly. This submersion permits precise regulation and monitoring of critical process parameters, such as pH, temperature, and dissolved oxygen concentration. The capacity for stringent control enables predictive and reproducible scale-up of fermentation operations. Moreover, research has indicated the carbon and nitrogen sources optimally supporting the metabolism of individual microbial strains are often quite specific (Kumar et al. 2013; Bhardwaj et al. 2019). Through the selection of tailored nutrient formulations, SmF maximizes productivity (Bhardwaj et al. 2019). The defined, homogenous conditions produced in SmF also facilitate consistent manufacturing on a large scale. In a nutshell, the distinctive regulated environment afforded by submerged fermentation provides an advantageous platform for the standardized generation of enzymes through controlled microbial biosynthesis. Further optimization of strain-specific growth media promises to augment the applicability and productivity of this industrial biotechnology technique.

Media composition and optimizing strategies

The composition of the growth medium or substrate can be optimized to provide an ideal mix of nutrients for increased metabolism and production. Physical parameters like temperature, moisture level, aeration, etc. may also be regulated to establish conditions conducive to robust microbial activity (Singh et al. 2017). The microbial strain itself could potentially be enhanced through various biotechnological applications. Traditional strain improvement methods like mutagenesis may allow isolates with better-producing capabilities to be obtained. Metabolic engineering and genetic modification techniques provide avenues to upregulate pathways for heightened biosynthesis of the desired product. Advanced molecular tools now facilitate such targeted manipulations at the genetic level. Adopting a multi-pronged strategy combining media optimization, process refinement, and strain amplification through modern biotechnology has promised to boost fermentation productivity (Sharma 2017).

To optimize the economic feasibility of xylanase production through solid-state fermentation and submerged fermentation, we can use different approaches, such as one-factor-at-a-time (OFAT) method and statistical methods (Bhardwaj et al. 2019; Dhaver et al. 2022). OFAT is a classical method in which one variable, such as substrate concentration, pH, or temperature, is adjusted while all other factors are kept constant (Bhardwaj et al. 2019). This allows researchers to find the optimal conditions step by step for xylanase production (Long et al. 2017). Fusarium sp. BVKT R2 is a strain that is evaluated at different temperatures, incubation times, pH, substrate concentrations, and agitation in submerged fermentation by OFAT (Ramanjaneyulu et al. 2017). Although OFAT is the most used method in initial optimization studies, it has several drawbacks. One of the major drawbacks is that it does not account for the interactions between different factors (Zhang and Wu 2021) and requires many experiments, making it time-consuming and labor-intensive. Furthermore, this method can lead to unreliable conclusions as it does not provide a comprehensive view of how multiple factors may influence the overall enzyme production (Dhaver et al. 2022). Another method used to overcome the limitations of the OFAT method is a statistical approach such as Response Surface Methodology (RSM) (Bhardwaj et al. 2019; Dhaver et al. 2022). We can identify key variables by simultaneously screening multiple factors, such as temperature, incubation time, and nutrient concentration in PBD (Ekpenyong et al. 2017). This makes the process more efficient by reducing the number of experiments (Ekpenyong et al. 2017), but it does not explain how different variables interact with each other (Dhaver et al. 2022). Once important factors are identified, RSM is applied to further optimize the condition (Dhaver et al. 2022). RSM allows for the exploration of the interactions between variables and creates mathematical models to predict how changes in multiple factors will affect enzyme yield (Wu and Ahn 2018). This approach is more efficient than OFAT and results in improved enzyme production, which in turn helps reduce costs. By optimizing conditions using RSM, industries can maximize xylanase yields while minimizing operational costs, making the production process more economically feasible (Dhaver et al. 2022).

Xylanases applications

Bacterial xylanases exhibit several characteristics that render them well-suited for industrial applications (Paul and Thatoi 2022). Additionally, bacterial xylanases commonly lack cellulolytic side activity, conferring substrate-specificity (Chakdar et al. 2016). These advantageous properties have facilitated the widespread commercial proliferation of bacterial xylanases. Their production methodology and use have been protected intellectually via numerous patents globally. Continued metabolic engineering and high-throughput screening efforts further optimized such enzymes for commercial functionality. As such, bacterial xylanases represent promising biocatalysts for myriad applications benefiting from thermostability and alkaline resilience (Chakdar et al. 2016). Their proliferation exemplifies how industrially amenable qualities and protective patenting incentivize commercialization. Various industrial applications of xylanase enzymes are represented in Fig. 3.

Fig. 3.

Major industrial applications of xylanases enzyme (pharmaceutical industry, paper and pulp industry, textile industry, and food industry)

Paper and pulp industry

Bio-bleaching

Traditionally, in paper pulp industries, pulp is cooked at 170 °C with alkali and then chemically bleached by chlorine agents to remove lignin (Kumar et al. 2016; Nayeem et al. 2023). An alternative, eco-friendly approach is switching to bio-bleaching by microbial xylanases (Kumar 2020). Ligno-hemicellulolytic xylanases work on the cross-linking layer of xylan in pulp fiber, distorting the intact layer and facilitating the extraction of lignin from the structure (Thomas et al. 2015; Kumar et al. 2016). Various studies have shown that the use of xylanases in the process of bio-bleaching has dramatically reduced the amount of chlorine needed, from 10 to 50% (Kumar et al. 2016). Several microbes have been researched for their ability to produce xylanases beneficial for the bio-bleaching of pulp and reducing reliance on chemicals. The process of bioleaching in the paper and pulp industries is demonstrated in Fig. 4. Studies have shown that Talaromyces thermophilus can decrease the kappa number (lignin content measure) of industrial kraft pulp by 27.4% (Maalej-Achouriet al. 2012). B. halodurans FNP 135 was found to reduce chlorine consumption by 20% while increasing the brightness of eucalyptus pulp by 4.3% and decreasing the kappa number by 35% (Sharma et al. 2015). B. halodurans C-125 treatment led to 10% less chlorine usage and 5.2% higher brightness of wheat straw pulp (Lin et al. 2013). T. lanuginosus CBS 288.54 and B. stearothermophilus SDX increased the brightness of wheat straw pulp by 1.8–7.8% and 1.76%, respectively (Kumar et al. 2016; Bhardwaj et al. 2019). Studies also demonstrated the effectiveness of xylanases from Bacillus spp., B. pumilus, and Paenibacillus campinasensis G1 for bio-bleaching different wood and non-wood pulps (Zheng et al. 2012; Irfan et al. 2016).

Fig. 4.

This diagram represents all the steps involved in the process of bio-bleaching in the paper and pulp industry by incorporating xylanase enzymes. The kraft pulp is added to the mixer tank along with xylanases enzyme, and later it is moved to the reaction tank for the process of bio-bleaching (Kumar et al. 2018a, b)

These findings provide evidence that strategically selected microbial strains can supplement existing bleaching processes and reduce reliance on chlorine-based chemicals. Furthermore, variability in microbial strains also influences the performance of xylanases in the bio-bleaching process by producing enzymes with specific and unique traits. Xylanases from different microorganisms show varying levels of stability at different temperatures and pH conditions (Bhardwaj et al. 2019). For example, B. licheniformis, which is a Gram-positive, spore-forming bacterium, is an excellent candidate for the bio-bleaching process in industries because it can work at 60 °C, 6–10 pH; ideal conditions for kraft pulp bio-bleaching (Malhotra and Chapadgaonkar 2023; Raj et al. 2018). On the other hand, the xylanase produced by Sporotrichum thermophilum, which is active at 50–70 °C, is particularly useful for bio-bleaching of bagasse pulp (Joshi & Khare 2011), demonstrating that temperature tolerance of enzymes can vary based on their microbial source. The use of xylanases for bio-bleaching provides advantages beyond just reducing costs. It also leads to improvements in paper quality. One factor contributing to higher kappa numbers and pulp yellowing is 4-O-methylglucuronic acid substitutions on xylan polymers (Alokika and Singh 2019). During alkaline cooking, this is transformed into hexenuronic acid (Wang et al. 2024). Many global pulp producers are slowly replacing chemical bleaching with a more sustainable enzymatic approach. Countries like Japan, South America, North America, and parts of Europe have incorporated xylanase treatment. Canada, a leading pulp exporter, bleaches over 10% of its pulp using xylanases. Also, pretreating pulps with xylanases before chemical bleaching enhances the properties of generated cellulosic nanofibers (CNF). Studies find enzymatic pretreatment of unbleached bagasse and eucalyptus improves CNF crystallinity (Nie et al. 2018; Tao et al. 2019). Overall, bio-bleaching demonstrates benefits beyond cost savings alone by also enhancing paper attributes and enabling downstream applications like CNF production. Its gradual adoption globally underscores these multifaceted advantages.

Deinking of paper

In the recycling industry of paper, chemicals, such as chlorine, chlorine-based derivatives, sodium hydroxide, sodium carbonate, sodium silicate, hydrogen peroxide, hypochlorite, and chelating agents, are used for the dislodgement of ink from wastepaper. The release of these toxic effluents required labor-intensive treatment to mitigate potential damage (Maity et al. 2012). Recently, an enzyme-based alternative approach has been reported by Chandra and Singh (2012) and Dhiman et al. (2014) in which xylanase and laccase enzymes have been proposed for removing printing residue from effluents in these industries. A study conducted by Gupta et al. (2015) sought to assess the potential of combined enzymatic treatments to deink aged newsprint materials. When xylanase and laccase acted on old newsprint, measurable improvements were observed, such as a substantial increase in brightness levels (11.8%), whiteness ratings (39%), and physical characteristics including breaking length (34.8%), burst factor (2.77%), and tear factor (2.4%) (Dhiman et al. 2014; Chakdar et al. 2016). The synergistic interplay between xylanase and laccase during joint processing appeared to positively impact these material properties (Gupta et al. 2015). In contrast, conventional chemical-based deinking methods for such recycled paper stocks have been shown to degrade aspects of freeness and tensile strength. The collaborative activity of enzymes during remediation exhibited enhanced efficacy compared to solely chemical-centric approaches, offering brighter and higher quality recycled paper fibers with maintained or improved mechanical attributes.

Another study has found that using commercial cellulase and xylanase preparations derived from B. halodurans TSEV1, dosages of 1.2 units per milligram achieved the most effective results (Kumar and Satyanarayana 2014). Separate work investigated a cellulase-xylanase complex produced by E. coli SD5. Applying this enzymatic mixture led to reductions in hexenuronic acid and kappa number parameters, along with a 10% increase in brightness levels and gains in tear strength characteristics for recycled paper stocks. The capacity of strategically applied cellulolytic and xylanolytic activities, both alone and in coordinated combination, to enhance the deinking performance and fiber qualities of wastepaper substrates warrants ongoing examination within the recycling sector (Kumar et al. 2018a, b). Continued refinement of enzyme selection and application protocols may further optimize process efficiencies.

Food industry

Bakery products

Xylanases find wide applications in baking industry due to their ability to improve dough properties and bread quality attributes (Tekkol et al. 2017). Hemicelluloses like arabinoxylans present in wheat flour pose challenges for producing high-quality dough and bread (Kaur et al. 2019). Xylanases act on these polymers, solubilizing water-unextractable arabinoxylan and transforming it into a soluble form (Chakdar et al. 2016; Kaur et al. 2019; Sun et al. 2022a, b). This allows for more uniform water distribution and enhances gluten network formation in the dough (Dahiya and Singh 2019). Studies have demonstrated xylanases, such as GH11 enzymes from B. subtilis, can increase dough viscosity and volume while decreasing gluten aggregation and dough firmness (Chakdar et al. 2016). When added to bread recipes, xylanases yield improvements, such as higher rising, loaf volume, and specific volume (Driss et al. 2013; De Queiroz Brito Cunha et al. 2018). Additionally, traits like texture, taste, flavor, and overall acceptability are enhanced (Ghoshal et al. 2013). Researchers have shown xylanases sourced from Penicillium occitanis and microbial consortia yield softer bread with better shelf life and reduced staling (Driss et al. 2013; Ghoshal et al. 2013). Newer enzymes like those from B. licheniformis and recombinant xylanases (r-XynBS27) expressed in Pichia pastoris also enhance dough properties. It helps in achieving the required shape, crumb structure, loaf volume, and texture (Bajpai 2014; De Queiroz Brito Cunha et al. 2018). Combining xylanases with amylases can further boost the effects on bread attributes (Kim and Yoo 2020; Liu et al. 2023a, b). Xylanases also enrich bread with prebiotic arabinoxylan oligosaccharides during production (Rudjito et al. 2023). In brewing, xylanases help clarify beer viscosity and appearance by hydrolyzing barley cell walls (Yu et al. 2021). Across food applications, xylanases prove invaluable biocatalysts for optimizing processes and final product quality through their targeted impact on complex plant polymers.

Food processing and juice clarification

Xylanases play an important role in fruit juice extraction and clarification. Raw juice has various polysaccharides like cellulose, hemicellulose, pectin, and lignin that increase viscosity and haziness. Xylanases help remove these compounds, lowering viscosity and preventing cluster formation to yield clearer, brighter juices through enhanced centrifugation and filtration (Danalache et al. 2018). Xylanases from Streptomyces sp. clarified orange, mousambi, and pineapple juices by 20.9%, 23.6%, and 27.9% respectively (Rosmine et al. 2017). Immobilized Bacillus pumilus xylanase clarified orange and grape juices by 29% and 26% (Kumar et al. 2014). Shahrestani et al. (2016) reported clarification of various fruit juices using xylanase immobilized on magnetic nanoparticles. Streptomyces sp. AOA40 xylanase increased the clarity of apple, orange, and grape juices by 17.8%, 18.4%, and 17.9% (Adıgüzel and Tunçer 2016). P. acidilactici GC25 xylanase treated various juices, lowering turbidity and increasing sugar levels (Adıgüzel et al. 2019).

Textile industry

Xylanases have many applications in textile processing, it is used for the key processes of de-sizing and scouring (Malhotra and Chapadgaonkar 2018). Adhesive materials from plant fibers, such as hemp, flax, ramie, and jute, are removed in the de-sizing process while in scouring inhibitory materials are dislodged from de-sized fibers (Subash and Perumalsamy 2021; Elfaleh et al. 2023). Conventionally, high heat and oxidizing agents are used in alkali which compromises fiber strength over time (Bhardwaj et al. 2019). Research was conducted on Bacillus pumilus xylanase to jute, boosting whiteness, and brightness while lowering yellowness (Garg et al. 2013). Testing revealed higher whiteness, brightness, and less yellowness when combining xylanase and pectinase than standard alkaline scouring on cotton (Singh et al. 2018). Another study found a Thermomyces longibrachiatum xylanase enhanced de-sizing and scouring without additives (Aty et al. 2018). Stability at elevated temperatures allows certain xylanases to function beyond textiles, for example in stone washing, biopolishing, or detergents (Escuder-Rodríguez et al. 2018). In summary, the research highlighted xylanases as eco-friendly textile processing agents with industrial potential.

Pharmaceutical industry

Xylanases play a key role in the generation of XOS through biomass conversion. Xylo-oligosaccharides (XOS) are a mixture of oligosaccharides composed of D-xylose units joined by β-1,4-glycosidic linkages (Chakdar et al. 2016). Based on the number of xylose monomers, they are classified as xylotriose, xylotetrose, xylobiose, and so on (Chakdar et al. 2016). XOS plays an important role as a prebiotic because it is not broken down or absorbed within the gastrointestinal tract (Bhardwaj et al. 2019). As a result, XOS selectively promotes the growth of significant microorganisms in the gastrointestinal system, aiding in the regulation of human digestive health (Marim & Gabardo 2021). The advantages of XOS in pharmaceutical industries are listed in Table 4. Specifically, XOS favors the proliferation of bacteria such as Bifidobacterium sp. that are beneficial to the digestive system (Li et al. 2015). XOS can be generated from xylan-containing lignocellulosic biomass through chemical means, enzymatic hydrolysis, or a combined approach (De Freitas et al. 2019; Santibáñez et al. 2021). The enzyme complex used for XOS production must exhibit low exoxylanase and β-xylosidase activity to hinder the formation of excessive xylose, as high levels can impede XOS synthesis (Verma et al. 2013; Jain et al. 2015).

Table 4.

Advantages of xylo-oligosaccharides (XOS) usage in different sectors

Advantage	Details	References
Prebiotic properties	Selectively stimulates beneficial gut bacteria, regulates digestive health	Marim and Gabardo (2021)
Functional food applications	Reduces cholesterol, hinders starch retrogradation, improves calcium bioavailability, enhances food nutrition/sensory qualities	Motta et al. (2013)
Pharmaceutical applications	Immunomodulatory, anti-cancer, antimicrobial, antioxidant, anti-allergic, anti-inflammatory effects	Gupta et al. (2018)
Phytopharmaceutical and feed uses	Shows growth in regulatory activity in aquaculture and poultry	Bhardwaj et al. (2019)

Future of xylanases for industrial applications

Looking ahead, the future of xylanases appears promising, with several avenues for advancement and innovation on the horizon. One area of focus is the enhancement of xylanase specificity and efficiency through advanced protein engineering techniques. By tailoring xylanases for specific industrial applications, researchers aim to improve performance while reducing costs. Additionally, novel production systems are being explored, including synthetic biology approaches and microbial consortia, which offer scalability and sustainability benefits. These innovations could revolutionize the way xylanases are produced, making them more accessible and cost-effective. Moreover, we can utilize recombinant DNA technology to extract DNA from microbial strains that are present in extreme environmental conditions. These sources have the potential to produce extremely stable xylanases enzymes. Recently, Ali et al (2025) demonstrated a thermostable xylanase from Geobacillus stearothermophilus strain NASA267 and investigated the production of recombinant thermostable xylanase for the saccharification of agro-waste plant biomass into fermentable sugars. Using recombinant technology, we can produce numerous thermostable xylanases enzymes for different industrial processes. Bioinformatic tools can also be used for increasing the accessibility of metabolomics, genomics, and proteomics data. Furthermore, the concept of biorefineries also gains momentum; xylanases are expected to play a pivotal role in the efficient utilization of lignocellulosic biomass. Integration of xylanase-based processes into biorefinery platforms could unlock new opportunities to produce biofuels, biochemicals, and biopolymers from renewable sources. Furthermore, the development of tailored enzyme cocktails comprising xylanases and other lignocellulolytic enzymes holds promise for synergistic biomass conversion. By optimizing enzyme mixtures, researchers aim to maximize the yield of fermentable sugars from diverse feedstocks.

Beyond traditional industries, xylanases are poised to find applications in emerging fields, such as nanotechnology, biomedicine, and environmental remediation. Leveraging the catalytic properties of xylanases in these novel contexts could drive sustainable innovation and technological advancement. Bioprospecting efforts and metagenomic analyses offer opportunities for the discovery of novel xylanase-producing microorganisms and enzymes. By exploring diverse environmental niches, researchers can uncover valuable enzymatic resources with unique properties and functionalities. There is a growing need for regulatory frameworks and standardization initiatives to ensure the quality, safety, and environmental sustainability of xylanase-derived products and processes. Establishing guidelines and certifications will facilitate the widespread adoption of xylanase-based technologies. In summary, the future of xylanases is characterized by innovation, collaboration, and sustainability, with the potential to drive significant advancements in biotechnology and industrial processes.

Conclusion

In conclusion, the comprehensive understanding of xylan structure and the intricate mechanisms of xylanolytic enzymes illuminate the path toward unlocking its vast potential across various industrial sectors. Through meticulous research and technological advancements, xylanases continue to emerge as indispensable biocatalysts, offering sustainable solutions for biomass conversion, food processing, textile industry, and pharmaceutical applications. The synergistic efforts in enzyme engineering, optimization strategies, and biotechnological innovations pave the way for enhanced xylanase production with tailored properties, further propelling its efficacy and applicability in diverse industrial processes. As we delve deeper into the realm of xylanase research, the horizon brims with promises of greener, more efficient, and economically viable solutions, poised to shape the landscape of industrial biotechnology soon.

Funding

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All data generated or analyzed during this study are included in this article [and there is no supplementary information file].

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

Not applicable. This research did not involve human participants and/or animals.

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Consent to publication

We assure the integrity and quality of our research work. It is also stated that there is no plagiarism in this work and all points taken from other authors are well cited in the text. Our study did not require ethical board approval because it did not contain human or animal trials. This study is completely independent and impartial.