“…Thus, the evaluation of XOS production potential for BvelXyn11 can be included in this study. The molecular weight of rSrocXyn10-Ec and rBvelXyn11-Ec on SDS-PAGE (55 kDa for rSrocXyn10-Ec and 27 kDa for rBvelXyn11-Ec) are consistent with the general characteristics that xylanases belonging to GH10 family show a relative high molecular weight (≥ 30 kDa), and xylanases belonging to the GH11 family share a relative low molecular weight (< 30 kDa) ( Gavaseraei et al, 2021 ; Mendonca et al, 2023 ). For example, xylanase (from B. cereus L −1 ) ( Zhang et al, 2023 ), Xylanase II (from Aspergillus sydowii ) ( Brandt et al, 2020 ) and Xyl11 (from Trichoderma asperellum ND-1) ( Zheng et al, 2022 ) belonging to GH11 showed molecular weights of 23 kDa, 23.52 kDa and 22.47 kDa, respectively; and xylanase from Penicillium menonorum SP10 ( Luong et al, 2023 ), AS1 (from B. altitudinis ) ( Chauhan et al, 2023 ) and SipoEnXyn10A (from S. ipomoeae ) ( Xian et al, 2019 ) belonging to GH10 showed molecular weights of 54 kDa, 43 kDa and 44 kDa, respectively.…”
Endo-xylanase hydrolyzing xylan in cellulosic residues releasing xylobiose as the major product at neutral pH are desirable in the substitute sweeteners industry. In this study, two endo-xylanases were obtained from Streptomyces rochei and Bacillus velezensis. SrocXyn10 showed the highest identity of 77.22%, with a reported endo-xylanase. The optimum reaction temperature and pH of rSrocXyn10-Ec were pH 7.0 and 60°C, with remarkable stability at 45°C or pHs ranging from 4.5 to 11.0. rBvelXyn11-Ec was most active at pH 6.0 and 50°C, and was stable at 35°C or pH 3.5 to 10.5. Both rSrocXyn10-Ec and rBvelXyn11-Ec showed specific enzyme activities on wheat arabinoxylan (685.83 ± 13.82 and 2809.89 ± 21.26 U/mg, respectively), with no enzyme activity on non-xylan substrates. The Vmax of rSrocXyn10-Ec and rBvelXyn11-Ec were 467.86 U mg−1 and 3067.68 U mg−1, respectively. The determined Km values of rSrocXyn10-Ec and rBvelXyn11-Ec were 3.08 g L−1 and 1.45 g L−1, respectively. The predominant product of the hydrolysis of alkaline extracts from bagasse, corncob, and bamboo by rSrocXyn10-Ec and rBvelXyn11-Ec were xylooligosaccharides. Interestingly, the xylobiose content in hydrolysates by rSrocXyn10-Ec was approximately 80%, which is higher than most reported endo-xylanases. rSrocXyn10-Ec and rBvelXyn11-Ec could be excellent candidates to produce xylooligosaccharides at neutral/near-neutral pHs. rSrocXyn10-Ec also has potential value in the production of xylobiose as a substitute sweetener.
“…Thus, the evaluation of XOS production potential for BvelXyn11 can be included in this study. The molecular weight of rSrocXyn10-Ec and rBvelXyn11-Ec on SDS-PAGE (55 kDa for rSrocXyn10-Ec and 27 kDa for rBvelXyn11-Ec) are consistent with the general characteristics that xylanases belonging to GH10 family show a relative high molecular weight (≥ 30 kDa), and xylanases belonging to the GH11 family share a relative low molecular weight (< 30 kDa) ( Gavaseraei et al, 2021 ; Mendonca et al, 2023 ). For example, xylanase (from B. cereus L −1 ) ( Zhang et al, 2023 ), Xylanase II (from Aspergillus sydowii ) ( Brandt et al, 2020 ) and Xyl11 (from Trichoderma asperellum ND-1) ( Zheng et al, 2022 ) belonging to GH11 showed molecular weights of 23 kDa, 23.52 kDa and 22.47 kDa, respectively; and xylanase from Penicillium menonorum SP10 ( Luong et al, 2023 ), AS1 (from B. altitudinis ) ( Chauhan et al, 2023 ) and SipoEnXyn10A (from S. ipomoeae ) ( Xian et al, 2019 ) belonging to GH10 showed molecular weights of 54 kDa, 43 kDa and 44 kDa, respectively.…”
Endo-xylanase hydrolyzing xylan in cellulosic residues releasing xylobiose as the major product at neutral pH are desirable in the substitute sweeteners industry. In this study, two endo-xylanases were obtained from Streptomyces rochei and Bacillus velezensis. SrocXyn10 showed the highest identity of 77.22%, with a reported endo-xylanase. The optimum reaction temperature and pH of rSrocXyn10-Ec were pH 7.0 and 60°C, with remarkable stability at 45°C or pHs ranging from 4.5 to 11.0. rBvelXyn11-Ec was most active at pH 6.0 and 50°C, and was stable at 35°C or pH 3.5 to 10.5. Both rSrocXyn10-Ec and rBvelXyn11-Ec showed specific enzyme activities on wheat arabinoxylan (685.83 ± 13.82 and 2809.89 ± 21.26 U/mg, respectively), with no enzyme activity on non-xylan substrates. The Vmax of rSrocXyn10-Ec and rBvelXyn11-Ec were 467.86 U mg−1 and 3067.68 U mg−1, respectively. The determined Km values of rSrocXyn10-Ec and rBvelXyn11-Ec were 3.08 g L−1 and 1.45 g L−1, respectively. The predominant product of the hydrolysis of alkaline extracts from bagasse, corncob, and bamboo by rSrocXyn10-Ec and rBvelXyn11-Ec were xylooligosaccharides. Interestingly, the xylobiose content in hydrolysates by rSrocXyn10-Ec was approximately 80%, which is higher than most reported endo-xylanases. rSrocXyn10-Ec and rBvelXyn11-Ec could be excellent candidates to produce xylooligosaccharides at neutral/near-neutral pHs. rSrocXyn10-Ec also has potential value in the production of xylobiose as a substitute sweetener.
“…The results show that the MD simulations retain the overall fold and topology of the AlphaFold prediction, and that is maintained at 298 K along the time. The structures reveal the presence of two amino acid residues—Glu133 and Glu238—in close proximity and in opposing directions that could act as catalytic residues, as in other xylanases (Mendonça et al 2023 ). Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Computational methods were applied to predict the Xyl4 structure and the interaction with the substrate, xylohexaose. The xylanases of GH10 family hydrolyze glycosidic bonds by acid-base catalysis through a double displacement mechanism involving two glutamate residues (Mendonça et al 2023 ), positioned at the bottom of the active site cleft. This is consistent with the position observed for Glu133 and Glu238 in the arranged model.…”
Section: Discussionmentioning
confidence: 99%
“…This set of enzymes, collectively named carbohydrate-active enzymes (CAZymes), include hemicellulases (e.g., xylanases, mannanases, and arabinofuranosidases) that degrade the polysaccharides present in hemicellulose. Xylanases, namely endo-1,4-β-xylanases (EC 3.2.1.8), are recognized as important mediators in the hydrolytic catalysis of xylan polymers, more specifically in the cleavage of the internal β-1,4- d -xylosidic bonds (Gupta et al 2022 ; Kukkar et al 2022 ; Mendonça et al 2023 ). This type of enzymes is produced by a wide range of microorganisms, namely bacteria and fungi, and has already been exploited in several biotechnological applications, including in the biofuel, pulp and paper, food, feed animal, and textile industries (Ajeje et al 2021 ).…”
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
“…The effect on growth was possibly connected to CAZymes activities. The GHs families contain hydrolases such as endo-and exo-1,4-beta-glucanase ( Mendonça et al, 2023 ), which play an important role in the ductility of cell wall and affect plant growth and development. We found that although the gene expression of GHs in CAZymes was up-regulated under pH stress, the endo-and exo-1,4-beta-glucanase and β-glucosidase activities were highest at pH 7.…”
IntroductionpH is one of the important factors affecting the growth and performance of microorganisms.MethodsWe studied the pH response and plant growth-promoting (PGP) ability of Rhizopus delemar using cultivation experiments and transcriptomics, and verified the expression profiles using quantitative real-time PCR.ResultspH affected the growth and PGP properties of R. delemar. At pH 7, the growth rate of R. delemar was rapid, whereas pH 4 and 8 inhibited mycelial growth and PGP ability, respectively. In the pot experiment, the plant height was the highest at pH 7, 56 cm, and the lowest at pH 4 and pH 5, 46.6 cm and 47 cm, respectively. Enzyme activities were highest at pH 6 to pH 7. Enzyme activities were highest at pH 6 to pH 7. Among the 1,629 differentially expressed genes (DEGs), 1,033 genes were up-regulated and 596 were down-regulated. A total of 1,623 DEGs were annotated to carbohydrate-active enzyme coding genes.DiscussionThe PGP characteristics, e.g., Phosphorus solubilization ability, of R. delemar were strongest at pH 7. The results provide useful information regarding the molecular mechanism of R. delemar pH response.
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