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Xylanases are key biocatalysts in the degradation of the β‐1,4‐glycosidic linkages in the xylan backbone of hemicellulose. These enzymes are potentially applied in a wide range of bioprocessing industries under harsh conditions. Metagenomics has emerged as powerful tools for the bioprospection and discovery of interesting bioactive molecules from extreme ecosystems with unique features, such as high temperatures. In this study, an innovative combination of function-driven screening of a compost metagenomic library and automatic extraction of halo areas with in-house MATLAB functions resulted in the identification of a promising clone with xylanase activity (LP4). The LP4 clone proved to be an effective xylanase producer under submerged fermentation conditions. Sequence and phylogenetic analyses revealed that the xylanase, Xyl4, corresponded to an endo-1,4-β-xylanase belonging to glycosyl hydrolase family 10 (GH10). When xyl4 was expressed in Escherichia coli BL21(DE3), the enzyme activity increased about 2-fold compared to the LP4 clone. To get insight on the interaction of the enzyme with the substrate and establish possible strategies to improve its activity, the structure of Xyl4 was predicted, refined, and docked with xylohexaose. Our data unveiled, for the first time, the relevance of the amino acids Glu133 and Glu238 for catalysis, and a close inspection of the catalytic site suggested that the replacement of Phe316 by a bulkier Trp may improve Xyl4 activity. Our current findings contribute to enhancing the catalytic performance of Xyl4 towards industrial applications. Key points • A GH10 endo-1,4-β-xylanase (Xyl4) was isolated from a compost metagenomic library • MATLAB’s in-house functions were developed to identify the xylanase-producing clones • Computational analysis showed that Glu133 and Glu238 are crucial residues for catalysis Graphical abstract

Xylanases have received great attention in the development of environment-friendly technologies in the paper and pulp industry. Their use could greatly improve the overall lignocellulosic materials for the generation of liquid fuels and chemicals. Fungi are widely used as xylanase producers and are generally considered as more potent producers of xylanases than bacteria and yeasts. Large-scale production of xylanases is facilitated with the advent of genetic engineering. Recent breakthroughs in genomics have helped to overcome the problems such as limited enzyme availability, substrate scope, and operational stability. Genes encoding xylanases have been cloned in homologous and heterologous hosts with the objectives of overproducing the enzyme and altering its properties to suit commercial applications. Owing to the industrial importance of xylanases, a significant number of studies are reported on cloning and expression of the enzymes during the last few years. We, therefore, have reviewed recent knowledge regarding cloning of fungal xylanase genes into various hosts for heterologous production. This will bring an insight into the current status of cloning and expression of the fungal xylanases for industrial applications.

Xylanases are hydrolytic enzymes which randomly cleave the β 1,4 backbone of the complex plant cell wall polysaccharide xylan. Diverse forms of these enzymes exist, displaying varying folds, mechanisms of action, substrate specificities, hydrolytic activities (yields, rates and products) and physicochemical characteristics. Research has mainly focused on only two of the xylanase containing glycoside hydrolase families, namely families 10 and 11, yet enzymes with xylanase activity belonging to families 5, 7, 8 and 43 have also been identified and studied, albeit to a lesser extent. Driven by industrial demands for enzymes that can operate under process conditions, a number of extremophilic xylanases have been isolated, in particular those from thermophiles, alkaliphiles and acidiphiles, while little attention has been paid to cold-adapted xylanases. Here, the diverse physicochemical and functional characteristics, as well as the folds and mechanisms of action of all six xylanase containing families will be discussed. The adaptation strategies of the extremophilic xylanases isolated to date and the potential industrial applications of these enzymes will also be presented.

Xylanase is an industrial enzyme with diverse applications, including nutritional supplements, agro-waste valorization, and paper pulp bleaching. This study aims to investigate the production of recombinant thermostable xylanase for converting plant biomass into fermentable sugars, a key step in various industrial processes. Geobacillus stearothermophilus strain NASA267, a Gram-positive, thermophilic bacterium, was identified as the top xylanase producer from samples collected in Egypt and Saudi Arabia. The xylanase gene xyl267 was successfully cloned from the NASA267 strain and heterologously expressed in E. coli under the control of a Lambda promoter. Optimal expression conditions were determined, with the highest enzyme activity (40 U/ml) achieved after 4 h of induction at 42 ℃. SDS-PAGE analysis revealed that the molecular weight of the recombinant xylanase was approximately 40 kDa, consistent with the calculated molecular weight (38.6 kDa) based on its amino acid sequence (331 aa). Enzyme sequence and structural analysis revealed that xyl267 shows typical TIM barrel fold where Glu134 and Glu241 constitute the enzyme active site. The xyl267 demonstrated optimal activity at 65 ℃ and maintained full stability up to 60 ℃, while it displayed a half-life of 8 min at 80 ℃. It remained stable at - 20 ℃ for up to 50 days and was most active at pH 8. Although the enzyme was active in presence of various salts, solvents, and cations, the exposure to Cu ⁺, Zn ⁺, Mn ⁺, and methanol reduced the enzyme activity by 47%, 37%, 31%, and 8%, respectively. The enzyme was effective in saccharifying agro-waste, particularly pretreated banana peel, which produced the highest sugar content. These findings highlight xyl267s potential for biomass conversion and industrial applications in high-temperature and alkaline environment. The xyl267 from a NASA strain was cloned and successfully overexpressed in E. coli, producing a ~ 40 kDa recombinant enzyme. It showed optimal activity at 65 ℃, and was most active at pH 8. While it retained activity in various salts and solvents, it was inhibited by some heavy metals. Xyl267 effectively released fermentable sugars from pretreated banana peel, making it a promising candidate for industrial applications in high-temperature, alkaline environments and agro-waste saccharification.

The xylanolytic enzymes Clocl_1795 and Clocl_2746 from glycoside hydrolase (GH) family 30 are highly abundant in the hemicellulolytic system of Acetivibrio clariflavus ( Hungateiclostridium , Clostridium clariflavum ). Clocl_1795 has been shown to be a xylobiohydrolase Ac Xbh30A releasing xylobiose from the non-reducing end of xylan and xylooligosaccharides. In this work, biochemical characterization of Clocl_2746 is presented. The protein, designated Ac Xyn30B, shows low sequence similarity to other GH30 members and phylogenetic analysis revealed that Ac Xyn30B and related proteins form a separate clade that is proposed to be a new subfamily GH30_12. Ac Xyn30B exhibits similar specific activity on glucuronoxylan, arabinoxylan, and aryl glycosides of linear xylooligosaccharides suggesting that it is a non-specific xylanase. From polymeric substrates, it releases the fragments of degrees of polymerization (DP) 2-6. Hydrolysis of different xylooligosaccharides indicates that Ac Xyn30B requires at least four occupied catalytic subsites for effective cleavage. The ability of the enzyme to hydrolyze a wide range of substrates is interesting for biotechnological applications. In addition to subfamilies GH30_7, GH30_8, and GH30_10, the newly proposed subfamily GH30_12 further widens the spectrum of GH30 subfamilies containing xylanolytic enzymes. Key points Bacterial GH30 endoxylanase from A. clariflavus (AcXyn30B) has been characterized AcXyn30B is non-specific xylanase hydrolyzing various xylans and xylooligosaccharides Phylogenetic analysis placed AcXyn30B in a new GH30_12 subfamily

Xylanolytic enzyme systems in ascomycetous yeasts remain underexplored, despite the presence of yeasts in various xylan-rich ecological niches. In this study, we investigated the secreted xylanolytic machineries of three Blastobotrys species— B. mokoenaii , B. illinoisensis , and B. malaysiensis —by integrating genome annotation, bioinformatics, and secretome analyses of cultures grown on beechwood glucuronoxylan. Our findings demonstrate that these yeasts effectively hydrolyze xylan through the secretion of xylanases from the glycoside hydrolase (GH) family 11, which play a central role in cleaving the xylan backbone. Additionally, the yeasts produce a diverse array of other CAZymes, including members of GH families 3, 5, and 67, with putative roles in xylan degradation. We also report on the heterologous expression and functional characterization of the GH30_7 xylanase Bm Xyn30A from B. mokoenaii , which exhibits both glucuronoxylanase and xylobiohydrolase activities. We demonstrate additive effects between GH family 30 Bm Xyn30A and GH family 11 Bm Xyn11A during the hydrolysis of beechwood glucuronoxylan, where the enzymes exhibit complementary roles that enhance the deconstruction of this complex hemicellulose substrate. These findings broaden our understanding of the xylanolytic systems in yeasts and underscore the potential of Blastobotrys species as cell factories and natural xylanase producers. The enzymes they produce hold promise for biorefining applications, enabling efficient utilization of renewable xylan-rich plant biomass resources. Key points • Extracellular GH11 xylanases dominate glucuronoxylan degradation in Blastobotrys yeasts. • Yeast GH30_7 enzyme shows multifaceted activity, supporting complex xylan breakdown. • Blastobotrys yeasts show promise as cell factories for industrial biotechnology applications.

Endo-1,4-β-xylanase is a key xylanolytic enzyme, and our study aimed to enhance the catalytic properties of Alteromones Macleadii xylanase (Xyn ZT-2) through sequence-guided design approach. Analysis of the amino acid sequence revealed highly conserved residues near the active site, with few differences. Introducing various mutations allowed us to modify the enzyme's catalytic performance. Particularly, the A152G mutation led to a 9.8-fold increase in activity and a 23.2-fold increase in catalytic efficiency. Moreover, A152G exhibited an optimal temperature of 65 °C, 20 °C higher than that of Xyn ZT-2, while the T287S mutant showed a 4.9-fold increase in half-life. These results underscore the role of amino acid evolution in shaping xylanase catalysis. Through targeted sequence analysis and a focused mutation library, we effectively improved catalytic performance, providing a straightforward approach for enhancing enzyme efficiency.

BackgroundXylanase with high specific activity plays a crucial role in hemicellulose biodegradation and has important industrial application. The amino acids located in the active site determine the enzyme biological characterization. In this study, structure bioinformatics analysis and alanine screening experiments were performed to explore the roles of amino acids at each subsite of the GH11 xylanase active site.ResultsThere are highly conserved amino acids at − 2 to + 1 subsites, and the network of the interactions is concentrated near the catalytic sites (E86, E178). However, the amino acids at relatively distal subsites, especially at the + 2 and + 3 subsites, are few but diverse. Alanine substitution of amino acids in the active site architecture exerted different impacts on catalytic efficiency. Interestingly, mutants Y180A at the + 2 subsite and Y96A at the + 3 subsite had reduced enzymatic activities by almost 95%, which indicate that these two aromatic residues are necessary for the catalysis of substrates in addition to the highly conserved residues at the − 2 and + 1 subsites. Moreover, in these two subsites, aromatic amino acids with different side-chain properties also affected enzyme activity. The mutants Y180W and Y96W showed 6.2% and 12.8% increase in specific activities by comparison with wild-type enzyme at 50 °C, respectively.ConclusionWe elucidated the interaction between amino acids and substrates in the active site, which will aid understanding of the protein-ligand interaction in enzyme engineering.Key points• Xylanase of GH11 family is a good industrial candidate.• The roles of residues at each subsite of GH11 xylanase active site are explored.• The two aromatic residues at the + 2 and + 3 subsites are necessary for the catalysis.• Y180W and Y96W increased the enzymatic activity by 6.2% and 12.8% at low temperature.

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

Xylanases (EC 3.2.1.8) are hydrolytic enzymes, which randomly cleave the β-1,4-linked xylose residues from xylan. The synthetic gene xynBS27 from Streptomyces sp. S27 was successfully cloned and expressed in Pichia pastoris. The full-length gene consists of 729 bp and encodes 243 amino acids including 51 residues of a putative signal peptide. This enzyme was purified in two steps and was shown to have a molecular weight of 20 kDa. The purified r-XynBS27 was active against beechwood xylan and oat spelt xylan as expected for GH 11 family. The optimum pH and temperature values for the enzyme were 6.0 and 75 °C, respectively. The Km and Vmax were 12.38 mg/mL and 13.68 μmol min/mg, respectively. The r-XynBS27 showed high xylose tolerance and was inhibited by some metal ions and by SDS. r-XynBS27 was employed as an additive in the bread making process. A decrease in firmness, stiffness and consistency, and improvements in specific volume and reducing sugar content were recorded.