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Li, Zhongyuan, Xianli Xue, Heng Zhao, Peilong Yang, Huiying Luo, Junqi Zhao, Huoqing Huang, and Bin Yao. "A C-Terminal Proline-Rich Sequence Simultaneously Broadens the Optimal Temperature and pH Ranges and Improves the Catalytic Efficiency of Glycosyl Hydrolase Family 10 Ruminal Xylanases." Applied and Environmental Microbiology 80, no. 11 (March 21, 2014): 3426–32. http://dx.doi.org/10.1128/aem.00016-14.
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ABSTRACTEfficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene (xynA) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed inEscherichia coli. The C-terminal oligopeptide had significant effects on the function and structure of XynA. Compared with XynA-Tr, XynA exhibited improved specific activity (12-fold) and catalytic efficiency (14-fold), a higher temperature optimum (50°C versus 45°C), and broader ranges of temperature and pH optima (pH 5.0 to 7.5 and 40 to 60°C versus pH 5.5 to 6.5 and 40 to 50°C). Moreover, XynA released more xylose than XynA-Tr when using beech wood xylan and wheat arabinoxylan as the substrate. The underlying mechanisms responsible for these changes were analyzed by substrate binding assay, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and xylooligosaccharide hydrolysis. XynA had no ability to bind to any of the tested soluble and insoluble polysaccharides. However, it contained more α helices and had a greater affinity and catalytic efficiency toward xylooligosaccharides, which benefited complete substrate degradation. Similar results were obtained when the C-terminal sequence was fused to another GH10 xylanase from sheep rumen. This study reveals an engineering strategy to improve the catalytic performance of enzymes.
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Do, Thi Tuyen, Dinh Thi Quyen, Thi Nuong Nguyen, and Van Thuat Nguyen. "Molecular characterization of a glycosyl hydrolase family 10 xylanase from Aspergillus niger." Protein Expression and Purification 92, no. 2 (December 2013): 196–202. http://dx.doi.org/10.1016/j.pep.2013.09.011.
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Liang, Fangfang, Yi Mo, Suleman Shah, Ying Xie, Arshad Mehmood, Hesheng Jiang, and Yafen Guo. "Characterization of Two Wheat-Derived Glycoside Hydrolase Family-10 Xylanases Resistant to Xylanase Inhibitors." Journal of Food Quality 2022 (April 5, 2022): 1–10. http://dx.doi.org/10.1155/2022/9590243.
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Xylanase inhibitors inhibit the activities of microbial xylanases and seriously compromise the efficacy of microbial xylanases added to modify cereals. Cereal endogenous xylanases are unaffected by these xylanase inhibitors, but little information is available regarding their effects in improving cereal quality, a neglected potential application. As a strategy for circumventing the negative effects of xylanase inhibitors, the objective of this study was to use genetic engineering to obtain sufficient amounts of active endo-1,4-β-D-xylanase from wheat to analyze the characteristics of its structure. The endo-1,4-β-D-xylanase from wheat was heterologously expressed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, MALDI-TOF/TOF (MS) analyses, and enzyme activity determination confirmed 2 active endo-1,4-β-D-xylanases (EXY3 and EXY4) were successfully obtained. The molecular weights (MW) and isoelectric point (pI) of EXY3 were 36.108 kDa and 5.491, while those of the EXY4 protein were 41.933 kDa and 5.726. They both contained the same catalytic domain of GH10 xylanases from G266 to V276 and have the same catalytic site, Glu273. They shared the same putative N-glycosylation sites (N62-T63-S64 and N280–V281–S282) and 3 putative O-glycosylation sites (Ser8, Ser9, and Thr21), but EXY4 had an additional O-glycosylation site (Thr358). EXY3 was smaller than EXY4 by 51 amino acids because of a nonsense mutation and premature termination. They both had the 8-fold beta/alpha-barrel (TIM-barrel) fold. The specific activities of EXY3 and EXY4 were 152.0891 and 67.2928 U/mg, respectively. This work demonstrates a promising way to obtain wheat xylanases by genetic engineering; the properties of the enzymes indicate their potential application in cereal-based industries.
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Kim, Do Young, Jonghoon Kim, Yung Mi Lee, Jong Suk Lee, Dong-Ha Shin, Bon-Hwan Ku, Kwang-Hee Son, and Ho-Yong Park. "Identification and Characterization of a Novel, Cold-Adapted d-Xylobiose- and d-Xylose-Releasing Endo-β-1,4-Xylanase from an Antarctic Soil Bacterium, Duganella sp. PAMC 27433." Biomolecules 11, no. 5 (April 30, 2021): 680. http://dx.doi.org/10.3390/biom11050680.
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Endo-β-1,4-xylanase is a key enzyme in the degradation of β-1,4-d-xylan polysaccharides through hydrolysis. A glycoside hydrolase family 10 (GH10) endo-β-1,4-xylanase (XylR) from Duganella sp. PAMC 27433, an Antarctic soil bacterium, was identified and functionally characterized. The XylR gene (1122-bp) encoded an acidic protein containing a single catalytic GH10 domain that was 86% identical to that of an uncultured bacterium BLR13 endo-β-1,4-xylanase (ACN58881). The recombinant enzyme (rXylR: 42.0 kDa) showed the highest beechwood xylan-degrading activity at pH 5.5 and 40 °C, and displayed 12% of its maximum activity even at 4 °C. rXylR was not only almost completely inhibited by 5 mM N-bromosuccinimide or metal ions (each 1 mM) including Hg2+, Ca2+, or Cu2+ but also significantly suppressed by 1 mM Ni2+, Zn2+, or Fe2+. However, its enzyme activity was upregulated (>1.4-fold) in the presence of 0.5% Triton X-100 or Tween 80. The specific activities of rXylR toward beechwood xylan, birchwood xylan, oat spelts xylan, and p-nitrophenyl-β-d-cellobioside were 274.7, 103.2, 35.6, and 365.1 U/mg, respectively. Enzymatic hydrolysis of birchwood xylan and d-xylooligosaccharides yielded d-xylose and d-xylobiose as the end products. The results of the present study suggest that rXylR is a novel cold-adapted d-xylobiose- and d-xylose-releasing endo-β-1,4-xylanase.
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Tu, Bin, Tao Zhang, Yuping Wang, Li Hu, Jin Li, Ling Zheng, Yi Zhou, et al. "Membrane-associated xylanase-like protein OsXYN1 is required for normal cell wall deposition and plant development in rice." Journal of Experimental Botany 71, no. 16 (April 27, 2020): 4797–811. http://dx.doi.org/10.1093/jxb/eraa200.
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Abstract The rice (Oryza sativa) genome encodes 37 putative β-1,4-xylanase proteins, but none of them has been characterized at the genetic level. In this work, we report the isolation of slim stem (ss) mutants with pleiotropic defects, including dwarfism, leaf tip necrosis, and withered and rolled leaves under strong sunlight. Map-based cloning of the ss1 mutant identified the candidate gene as OsXyn1 (LOC_03g47010), which encodes a xylanase-like protein belonging to the glycoside hydrolase 10 (GH10) family. OsXyn1 was found to be widely expressed, especially in young tissues. Subcellular localization analysis showed that OsXyn1 encodes a membrane-associated protein. Physiological analysis of ss1 and the allelic ss2 mutant revealed that water uptake was partially compromised in these mutants. Consistently, the plant cell wall of the mutants exhibited middle lamella abnormalities or deficiencies. Immunogold assays revealed an unconfined distribution of xylan in the mutant cell walls, which may have contributed to a slower rate of plant cell wall biosynthesis and delayed plant growth. Additionally, water deficiency caused abscisic acid accumulation and triggered drought responses in the mutants. The findings that OsXyn1 is involved in plant cell wall deposition and the regulation of plant growth and development help to shed light on the functions of the rice GH10 family.
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Herold, Silvia, Robert Bischof, Benjamin Metz, Bernhard Seiboth, and Christian P. Kubicek. "Xylanase Gene Transcription in Trichoderma reesei Is Triggered by Different Inducers Representing Different Hemicellulosic Pentose Polymers." Eukaryotic Cell 12, no. 3 (January 4, 2013): 390–98. http://dx.doi.org/10.1128/ec.00182-12.
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ABSTRACTThe ascomyceteTrichoderma reeseiis a paradigm for the regulation and production of plant cell wall-degrading enzymes, including xylanases. Four xylanases, including XYN1 and XYN2 of glycosyl hydrolase family 11 (GH11), the GH10 XYN3, and the GH30 XYN4, were already described. By genome mining, we identified a fifth xylanase, XYN5, belonging to GH11. Transcriptional analysis reveals that the expression of all xylanases butxyn3is induced byd-xylose, dependent on the cellulase and xylanase regulator XYR1 and negatively regulated by the carbon catabolite repressor CRE1. Impairment ofd-xylose catabolism at thed-xylose reductase and xylitol dehydrogenase step strongly enhanced induction byd-xylose. Knockout of thel-xylulose reductase-encoding genelxr3, which connects thed-xylose andl-arabinose catabolic pathways, had no effect on xylanase induction. Besides the induction byd-xylose, theT. reeseixylanases were also induced byl-arabinose, and this induction was also enhanced in knockout mutants inl-arabinose reductase (xyl1),l-arabitol dehydrogenase (lad1), andl-xylulose reductase (lxr3). Induction byl-arabinose was also XYR1 dependent. Analysis of intracellular polyols revealed accumulation of xylitol in all strains only during incubation withd-xylose and accumulation ofl-arabitol only during incubation withl-arabinose. Induction byl-arabinose could be further stimulated by addition ofd-xylose. We conclude that the expression of theT. reeseixylanases can be induced by bothd-xylose andl-arabinose, but independently of each other and by using different inducing metabolites.
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Trong Khoa, Dao, Do Thi Huyen, and Truong Nam Hai. "Probe-mining of endo-1,4-beta-xylanase from goats-rumen bacterial metagenomic DNA data." Vietnam Journal of Biotechnology 19, no. 3 (October 13, 2021): 519–28. http://dx.doi.org/10.15625/1811-4989/16632.
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Endo-1,4-beta-xylanases (xylanases) are classified into 9 glycoside hydrolase families, GH5, 8, 10, 11, 30, 43, 51, 98, and 141 based on the CAZy database. The probe sequences representing the enzymes were constructed from published sequences of actual experimental studies with xylan decomposition activity. From online databases, we found one sequence belonging to the GH5 family, 6 sequences belonging to the GH8 family and 5 sequences belonging to the GH30 family exhibiting xylanase activity. Thus specific probes for xylanase GH8 and GH30 families were designed with the length of 351 and 425 amino acids respectively. The reference values for the probe of the GH8 family were defined as the sequences with maximum score greater than 168, the lowest coverage was 84%, the lowest similarity was 36%; for the probe GH30, the maximum score was greater than 316, the coverage was greater than 98%, the similarity was greater than 41%. Using the built probes, including the probe of the two GH10 and GH11 families, we found 41 xylanase-encoding sequences from the metagenomic DNA data of bacteria in Vietnamese goats’rumen. Of the 41 exploited sequences, 19 were identical to the BGI company's annotation result based on KEGG database, whereas there were 16 sequences that are not annotated by the BGI company. Total 28 of 41 exploited sequences were complete open reading frames, of which the predicted ternary structure was highly similar to the published structures of xylanase.
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Yang, Jiangke, and Zhenggang Han. "Understanding the Positional Binding and Substrate Interaction of a Highly Thermostable GH10 Xylanase from Thermotoga maritima by Molecular Docking." Biomolecules 8, no. 3 (July 30, 2018): 64. http://dx.doi.org/10.3390/biom8030064.
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Glycoside hydrolase family 10 (GH10) xylanases are responsible for enzymatic cleavage of the internal glycosidic linkages of the xylan backbone, to generate xylooligosaccharides (XOS) and xyloses. The topologies of active-site cleft determine the substrate preferences and product profiles of xylanases. In this study, positional bindings and substrate interactions of TmxB, one of the most thermostable xylanases characterized from Thermotoga maritima to date, was investigated by docking simulations. XOS with backbone lengths of two to five (X2–X5) were docked into the active-site cleft of TmxB by AutoDock The modeled complex structures provided a series of snapshots of the interactions between XOS and TmxB. Changes in binding energy with the length of the XOS backbone indicated the existence of four effective subsites in TmxB. The interaction patterns at subsites −2 to +1 in TmxB were conserved among GH10 xylanases whereas those at distal aglycone subsite +2, consisting of the hydrogen bond network, was unique for TmxB. This work helps in obtaining an in-depth understanding of the substrate-binding property of TmxB and provides a basis for rational design of mutants with desired product profiles.
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Teo, Seng Chong, Kok Jun Liew, Mohd Shahir Shamsir, Chun Shiong Chong, Neil C. Bruce, Kok-Gan Chan, and Kian Mau Goh. "Characterizing a Halo-Tolerant GH10 Xylanase from Roseithermus sacchariphilus Strain RA and Its CBM-Truncated Variant." International Journal of Molecular Sciences 20, no. 9 (May 9, 2019): 2284. http://dx.doi.org/10.3390/ijms20092284.
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A halo-thermophilic bacterium, Roseithermus sacchariphilus strain RA (previously known as Rhodothermaceae bacterium RA), was isolated from a hot spring in Langkawi, Malaysia. A complete genome analysis showed that the bacterium harbors 57 glycoside hydrolases (GHs), including a multi-domain xylanase (XynRA2). The full-length XynRA2 of 813 amino acids comprises a family 4_9 carbohydrate-binding module (CBM4_9), a family 10 glycoside hydrolase catalytic domain (GH10), and a C-terminal domain (CTD) for type IX secretion system (T9SS). This study aims to describe the biochemical properties of XynRA2 and the effects of CBM truncation on this xylanase. XynRA2 and its CBM-truncated variant (XynRA2ΔCBM) was expressed, purified, and characterized. The purified XynRA2 and XynRA2ΔCBM had an identical optimum temperature at 70 °C, but different optimum pHs of 8.5 and 6.0 respectively. Furthermore, XynRA2 retained 94% and 71% of activity at 4.0 M and 5.0 M NaCl respectively, whereas XynRA2ΔCBM showed a lower activity (79% and 54%). XynRA2 exhibited a turnover rate (kcat) of 24.8 s−1, but this was reduced by 40% for XynRA2ΔCBM. Both the xylanases hydrolyzed beechwood xylan predominantly into xylobiose, and oat-spelt xylan into a mixture of xylo-oligosaccharides (XOs). Collectively, this work suggested CBM4_9 of XynRA2 has a role in enzyme performance.
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Lu, Lin, Yongwei Liu, and Zengyan Zhang. "Global Characterization of GH10 Family Xylanase Genes in Rhizoctonia cerealis and Functional Analysis of Xylanase RcXYN1 During Fungus Infection in Wheat." International Journal of Molecular Sciences 21, no. 5 (March 6, 2020): 1812. http://dx.doi.org/10.3390/ijms21051812.
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Wheat (Triticum aestivum L.) is an important staple crop. Rhizoctonia cerealis is the causal agent of diseases that are devastating to cereal crops, including wheat. Xylanases play an important role in pathogenic infection, but little is known about xylanases in R. cerealis. Herein, we identified nine xylanase-encoding genes from the R. cerealis genome, named RcXYN1–RcXYN9, examined their expression patterns, and investigated the pathogenicity role of RcXYN1. RcXYN1–RcXYN9 proteins contain two conserved glutamate residues within the active motif in the glycoside hydrolase 10 (GH10) domain. Of them, RcXYN1–RcXYN4 are predicted to be secreted proteins. RcXYN1–RcXYN9 displayed different expression patterns during the infection process of wheat, and RcXYN1, RcXYN2, RcXYN5, and RcXYN9 were expressed highly across all the tested inoculation points. Functional dissection indicated that the RcXYN1 protein was able to induce necrosis/cell-death and H2O2 generation when infiltrated into wheat and Nicotiana benthamiana leaves. Furthermore, application of RcXYN1 protein followed by R. cerealis led to significantly higher levels of the disease in wheat leaves than application of the fungus alone. These results demonstrate that RcXYN1 acts as a pathogenicity factor during R. cerealis infection in wheat. This is the first investigation of xylanase genes in R. cerealis, providing novel insights into the pathogenesis mechanisms of R. cerealis.
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Kiribayeva, Assel, Birzhan Mukanov, Dmitriy Silayev, Zhiger Akishev, Yerlan Ramankulov, and Bekbolat Khassenov. "Cloning, expression, and characterization of a recombinant xylanase from Bacillus sonorensis T6." PLOS ONE 17, no. 3 (March 17, 2022): e0265647. http://dx.doi.org/10.1371/journal.pone.0265647.
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Xylanase is one of industrial enzymes with diverse applications including the paper-bleaching industry and feed additives. Here, a strain having xylanolytic activity and identified as Bacillus sonorensis T6 was isolated from soil. A secretory enzyme was identified by mass-spectrometry as a xylanase of glycosyl hydrolase family 11, with a molecular weight of 23.3 kDa. The xylanase gene of Bacillus sonorensis T6 was cloned and expressed in Escherichia coli (yielding an enzyme designated as rXynT6-E) and in Pichia pastoris (yielding rXynT6-P). The recombinant xylanases were found to have optimal activity at 47–55°C and pH 6.0–7.0. The recombinant xylanase expressed in P. pastoris has 40% higher thermal stability than that expressed in E. coli. The recombinant xylanases retained 100% of activity after 10 h incubation in the pH range 3–11 and 68% of activity after 1 h at pH 2.0. The xylanase activities of rXynT6-E and rXynT6-P under optimal conditions were 1030.2 and 873.8 U/mg, respectively. The good stability in a wide range of pH and moderate temperatures may make the xylanase from Bacillus sonorensis T6 useful for various biotechnological applications, e.g., as an enzyme additive in the feed industry.
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Sizova, M. V., J. A. Izquierdo, N. S. Panikov, and L. R. Lynd. "Cellulose- and Xylan-Degrading Thermophilic Anaerobic Bacteria from Biocompost." Applied and Environmental Microbiology 77, no. 7 (February 11, 2011): 2282–91. http://dx.doi.org/10.1128/aem.01219-10.
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ABSTRACTNine thermophilic cellulolytic clostridial isolates and four other noncellulolytic bacterial isolates were isolated from self-heated biocompost via preliminary enrichment culture on microcrystalline cellulose. All cellulolytic isolates grew vigorously on cellulose, with the formation of either ethanol and acetate or acetate and formate as principal fermentation products as well as lactate and glycerol as minor products. In addition, two out of nine cellulolytic strains were able to utilize xylan and pretreated wood with roughly the same efficiency as for cellulose. The major products of xylan fermentation were acetate and formate, with minor contributions of lactate and ethanol. Phylogenetic analyses of 16S rRNA and glycosyl hydrolase family 48 (GH48) gene sequences revealed that two xylan-utilizing isolates were related to aClostridium clariflavumstrain and represent a distinct novel branch within the GH48 family. Both isolates possessed high cellulase and xylanase activity induced independently by either cellulose or xylan. Enzymatic activity decayed after growth cessation, with more-rapid disappearance of cellulase activity than of xylanase activity. A mixture of xylan and cellulose was utilized simultaneously, with a significant synergistic effect observed as a reduction of lag phase in cellulose degradation.
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Jun, Hyun S., Jong K. Ha, Laercio M. Malburg, Jr., Ann M. Verrinder Gibbins, and Cecil W. Forsberg. "Characteristics of a cluster of xylanase genes inFibrobacter succinogenesS85." Canadian Journal of Microbiology 49, no. 3 (March 1, 2003): 171–80. http://dx.doi.org/10.1139/w03-024.
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Xylanase genes xyn10D, xyn10E, and xyn10B, located sequentially on the Fibrobacter succinogenes S85 chromosome, were separately cloned and their properties characterized. Analysis of the sequences documented that xylanases Xyn10D, Xyn10E, and Xyn10B each consist of an N-terminal catalytic domain (glycosyl hydrolase family 10) and a C-terminal carbohydrate-binding module (CBM, family 6) connected by proline-rich linker sequences. The amino acid sequences exhibited similarities of between 53 and 60%. The xyn10D, xyn10E, and truncated xyn10BΔCBM were expressed in Escherichia coli and purified to homogeneity. The purified Xyn10D, Xyn10E, and Xyn10BΔCBM exhibited the same temperature optimum (40°C) and pH optimum (6.5) and the highest specific activity against arabinoxylan, oat spelt xylan, and birchwood xylan, respectively. Xyn10D exhibited an affinity for cellulose and xylan with 47 and 33% binding, respectively, while the truncated Xyn10DΔCBM did not bind to the substrates. The main hydrolysis products of the three xylanases acting on oat spelt xylan and arabinoxylan were xylose and xylobiose. RT-PCR analysis showed that the three genes were co-transcribed as a single transcript. Western immunoblot analysis revealed that the three xylanases were expressed at a very low level by F. succinogenes grown on either glucose or cellulose as the source of carbohydrate.Key words: Fibrobacter succinogenes S85, xylan, xylanase, clustered genes, RT-PCR.
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Millward-Sadler, S. J., K. Davidson, G. P. Hazlewood, G. W. Black, H. J. Gilbert, and J. H. Clarke. "Novel cellulose-binding domains, NodB homologues and conserved modular architecture in xylanases from the aerobic soil bacteria Pseudomonas fluorescens subsp. cellulosa and Cellvibrio mixtus." Biochemical Journal 312, no. 1 (November 15, 1995): 39–48. http://dx.doi.org/10.1042/bj3120039.
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To test the hypothesis that selective pressure has led to the retention of cellulose-binding domains (CBDs) by hemicellulase enzymes from aerobic bacteria, four new xylanase (xyn) genes from two cellulolytic soil bacteria, Pseudomonas fluorescens subsp. cellulosa and Cellvibrio mixtus, have been isolated and sequenced. Pseudomonas genes xynE and xynF encoded modular xylanases (XYLE and XYLF) with predicted M(r) values of 68,600 and 65000 respectively. XYLE contained a glycosyl hydrolase family 11 catalytic domain at its N-terminus, followed by three other domains; the second of these exhibited sequence identity with NodB from rhizobia. The C-terminal domain (40 residues) exhibited significant sequence identity with a non-catalytic domain of previously unknown function, conserved in all the cellulases and one of the hemicellulases previously characterized from the pseudomonad, and was shown to function as a CBD when fused to the reporter protein glutathione-S-transferase. XYLF contained a C-terminal glycosyl hydrolase family 10 catalytic domain and a novel CBD at its N-terminus. C. mixtus genes xynA and xynB exhibited substantial sequence identity with xynE and xynF respectively, and encoded modular xylanases with the same molecular architecture and, by inference, the same functional properties. In the absence of extensive cross-hybridization between other multiple cel (cellulase) and xyn genes from P. fluorescens subsp. cellulosa and genomic DNA from C. mixtus, similarity between the two pairs of xylanases may indicate a recent transfer of genes between the two bacteria.
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Peterson, Robyn, Jasmine Grinyer, and Helena Nevalainen. "Secretome of the Coprophilous Fungus Doratomyces stemonitis C8, Isolated from Koala Feces." Applied and Environmental Microbiology 77, no. 11 (April 15, 2011): 3793–801. http://dx.doi.org/10.1128/aem.00252-11.
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ABSTRACTCoprophilous fungi inhabit herbivore feces, secreting enzymes to degrade the most recalcitrant parts of plant biomass that have resisted the digestive process. Consequently, the secretomes of coprophilous fungi have high potential to contain novel and efficient plant cell wall degrading enzymes of biotechnological interest. We have used one-dimensional and two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization–time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), and quadrupole time-of-flight liquid chromatography–tandem mass spectrometry (Q-TOF LC-MS/MS) to identify proteins from the secretome of the coprophilous fungusDoratomyces stemonitisC8 (EU551185) isolated from koala feces. As the genome ofD. stemonitishas not been sequenced, cross-species identification,de novosequencing, and zymography formed an integral part of the analysis. A broad range of enzymes involved in the degradation of cellulose, hemicellulose, pectin, lignin, and protein were revealed, dominated by cellobiohydrolase of the glycosyl hydrolase family 7 and endo-1,4-β-xylanase of the glycosyl hydrolase family 10. A high degree of specialization for pectin degradation in theD. stemonitisC8 secretome distinguishes it from the secretomes of some other saprophytic fungi, such as the industrially exploitedT. reesei. In the first proteomic analysis of the secretome of a coprophilous fungus reported to date, the identified enzymes provide valuable insight into how coprophilous fungi subsist on herbivore feces, and these findings hold potential for increasing the efficiency of plant biomass degradation in industrial processes such as biofuel production in the future.
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Verjans, Priscilla, Emmie Dornez, Martien Segers, Steven Van Campenhout, Kristel Bernaerts, Tim Beliën, Jan A. Delcour, and Christophe M. Courtin. "Truncated derivatives of a multidomain thermophilic glycosyl hydrolase family 10 xylanase from Thermotoga maritima reveal structure related activity profiles and substrate hydrolysis patterns." Journal of Biotechnology 145, no. 2 (January 2010): 160–67. http://dx.doi.org/10.1016/j.jbiotec.2009.10.014.
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StJohn, Franz J., John D. Rice, and James F. Preston. "Paenibacillus sp. Strain JDR-2 and XynA1: a Novel System for Methylglucuronoxylan Utilization." Applied and Environmental Microbiology 72, no. 2 (February 2006): 1496–506. http://dx.doi.org/10.1128/aem.72.2.1496-1506.2006.
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ABSTRACT Environmental and economic factors predicate the need for efficient processing of renewable sources of fuels and chemicals. To fulfill this need, microbial biocatalysts must be developed to efficiently process the hemicellulose fraction of lignocellulosic biomass for fermentation of pentoses. The predominance of methylglucuronoxylan (MeGAXn), a β-1,4 xylan in which 10% to 20% of the xylose residues are substituted with α-1,2-4-O-methylglucuronate residues, in hemicellulose fractions of hardwood and crop residues has made this a target for processing and fermentation. A Paenibacillus sp. (strain JDR-2) has been isolated and characterized for its ability to efficiently utilize MeGAXn. A modular xylanase (XynA1) of glycosyl hydrolase family 10 (GH 10) was identified through DNA sequence analysis that consists of a triplicate family 22 carbohydrate binding module followed by a GH 10 catalytic domain followed by a single family 9 carbohydrate binding module and concluding with C-terminal triplicate surface layer homology (SLH) domains. Immunodetection of the catalytic domain of XynA1 (XynA1 CD) indicates that the enzyme is associated with the cell wall fraction, supporting an anchoring role for the SLH modules. With MeGAXn as substrate, XynA1 CD generated xylobiose and aldotetrauronate (MeGAX3) as predominant products. The inability to detect depolymerization products in medium during exponential growth of Paenibacillus sp. strain JDR-2 on MeGAXn, as well as decreased growth rate and yield with XynA1 CD-generated xylooligosaccharides and aldouronates as substrates, indicates that XynA1 catalyzes a depolymerization process coupled to product assimilation. This depolymerization/assimilation system may be utilized for development of biocatalysts to efficiently convert MeGAXn to alternative fuels and biobased products.
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Han, Sung Ok, Hideaki Yukawa, Masayuki Inui, and Roy H. Doi. "Isolation and Expression of the xynB Gene and Its Product, XynB, a Consistent Component of the Clostridium cellulovorans Cellulosome." Journal of Bacteriology 186, no. 24 (December 15, 2004): 8347–55. http://dx.doi.org/10.1128/jb.186.24.8347-8355.2004.
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ABSTRACT The nucleotide sequence of the Clostridium cellulovorans xynB gene, which encodes the XynB xylanase, consists of 1,821 bp and encodes a protein of 607 amino acids with a molecular weight of 65,976. XynB contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 147-amino-acid sequence that is homologous to the family 4-9 (subfamily 9 in family 4) carbohydrate-binding domain. Downstream of this domain is a family 10 catalytic domain of glycosyl hydrolase. The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly. The XynB sequence from mass spectrometry and N-terminal amino acid sequence analyses agreed with that deduced from the nucleotide sequence. XynB was highly active toward xylan, but not active toward carboxymethyl cellulose. The enzyme was optimally active at 40°C and pH 5.0. Northern hybridizations revealed that xynB is transcribed as a monocistronic 1.9-kb mRNA. RNA ligase-mediated rapid amplification of 5′ cDNA ends by PCR (RLM-5′RACE PCR) analysis of C. cellulovorans RNA identified a single transcriptional start site of xynB located 47 bp upstream from the first nucleotide of the translation initiation codon. Alignment of the xynB promoter region provided evidence for highly conserved sequences that exhibited strong similarity to the σA consensus promoter sequences of gram-positive bacteria. Expression of xynB mRNA increased from early to middle exponential phase and decreased during the early stationary phase when the cells were grown on cellobiose. No alternative promoter was observed by RLM-5′RACE PCR and reverse transcriptase PCR analyses during expression. The analysis of the products from xylan hydrolysis by thin-layer chromatography indicated its endoxylanase activity. The results suggest that XynB is a consistent and major cellulosomal enzyme during growth on cellulose or xylan.
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Fredriksen, L., R. Stokke, M. S. Jensen, B. Westereng, J. K. Jameson, I. H. Steen, and V. G. H. Eijsink. "Discovery of a Thermostable GH10 Xylanase with Broad Substrate Specificity from the Arctic Mid-Ocean Ridge Vent System." Applied and Environmental Microbiology 85, no. 6 (January 11, 2019). http://dx.doi.org/10.1128/aem.02970-18.
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ABSTRACT A two-domain GH10 xylanase-encoding gene (amor_gh10a) was discovered from a metagenomic data set, generated after in situ incubation of a lignocellulosic substrate in hot sediments on the sea floor of the Arctic Mid-Ocean Ridge (AMOR). AMOR_GH10A comprises a signal peptide, a carbohydrate-binding module belonging to a previously uncharacterized family, and a catalytic glycosyl hydrolase (GH10) domain. The enzyme shares the highest sequence identity (42%) with a hypothetical protein from a Verrucomicrobia bacterium, and its GH10 domain shares low identity (24 to 28%) with functionally characterized xylanases. Purified AMOR_GH10A showed thermophilic and halophilic properties and was active toward various xylans. Uniquely, the enzyme showed high activity toward amorphous cellulose, glucomannan, and xyloglucan and was more active toward cellopentaose than toward xylopentaose. Binding assays showed that the N-terminal domain of this broad-specificity GH10 binds strongly to amorphous cellulose, as well as to microcrystalline cellulose, birchwood glucuronoxylan, barley β-glucan, and konjac glucomannan, confirming its classification as a novel CBM (CBM85). IMPORTANCE Hot springs at the sea bottom harbor unique biodiversity and are a promising source of enzymes with interesting properties. We describe the functional characterization of a thermophilic and halophilic multidomain xylanase originating from the Arctic Mid-Ocean Ridge vent system, belonging to the well-studied family 10 of glycosyl hydrolases (GH10). This xylanase, AMOR_GH10A, has a surprisingly wide substrate range and is more active toward cellopentaose than toward xylopentaose. This substrate promiscuity is unique for the GH10 family and could prove useful in industrial applications. Emphasizing the versatility of AMOR_GH10A, its N-terminal domain binds to both xylans and glycans, while not showing significant sequence similarities to any known carbohydrate-binding module (CBM) in the CAZy database. Thus, this N-terminal domain lays the foundation for the new CBM85 family.
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Joshi, Johnson Beslin, R. Priyadharshini, and Sivakumar Uthandi. "Glycosyl hydrolase 11 (xynA) gene with xylanase activity from thermophilic bacteria isolated from thermal springs." Microbial Cell Factories 21, no. 1 (April 15, 2022). http://dx.doi.org/10.1186/s12934-022-01788-3.
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Abstract Background Hemicellulose is one of the copious polymer in lignocellulosic biomass (LCB). It is primarily composed of xylan linked by β-1,4 glycosidic bonds. Xylanase preferentially cleaves the β-1,4-glycosidic bonds in the xylan backbone resulting in complete hydrolysis of the biomass. Thermostable variants of glycoside hydrolases act as robust catalysts, not only in degradation but also during processing, to obtain specific carbohydrate-containing chemicals and materials (Ramasamy et al. in Madras Agric J 107(special):1. 10.29321/MAJ.2020.000382, 2020). Results The xylanase production by two thermophilic bacteria isolated from thermal springs was evaluated. In addition, the gene encoding this industrially vital enzyme was isolated and characterized, and its protein structure was analyzed. The thermophilic bacteria producing xylanases were isolated from augmented sawdust and banana fiber biomass from hot springs of Himachal Pradesh and identified as Bacillus subtilis VSDB5 and Bacillus licheniformis KBFB4 using 16S rRNA gene sequencing. The persistent xylanase activity revealed that the enzyme is secreted extracellularly with the maximum activity of 0.76 IU mL−1 and 1.0 IU mL−1 at 6 h and 12 h of growth by KBFB4 and VSDB5, respectively, under submerged fermentation. Both the strains exhibited the maximum activity at pH 6 and a temperature of 50 °C. The xylanases of KBFB4 and VSDB5 were thermostable and retained 40% of their activity at 60 °C after incubation for 30 min. Xylanase of VSDB5 had wide thermotolerance and retained 20% of its activity from 60 to 80 °C, whereas xylanase of KBFB4 showed wide alkali tolerance and retained 80% of its activity until pH 10. The xylanase (xynA)-encoding gene (650 bp) cloned from both the strains using specific primers showed 98 to 99% homology to β-1,4-endoxylanase gene. Further in silico analysis predicted that the xylanase protein, with a molecular weight of 23 kDa, had a high pI (9.44–9.65), which explained the alkaline nature of the enzyme and greater aliphatic index (56.29). This finding suggested that the protein is thermostable. Multiple sequence alignment and homology modeling of the protein sequence revealed that the gene product belonged to the GH11 family, indicating its possible application in bioconversion. Conclusion The strains B. subtilis VSDB5 and B. licheniformis KBFB4 obtained from hot springs of Himachal Pradesh produced potent and alkali-tolerant thermostable xylanases, which belong to the GH11 family. The enzyme can be supplemented in industrial applications for biomass conversion at high temperatures and pH (or in processes involving alkali treatment). Graphical Abstract
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Zhao, Fang, Hai-Yan Cao, Long-Sheng Zhao, Yi Zhang, Chun-Yang Li, Yu-Zhong Zhang, Ping-Yi Li, Peng Wang, and Xiu-Lan Chen. "A Novel Subfamily of Endo-β-1,4-Glucanases in Glycoside Hydrolase Family 10." Applied and Environmental Microbiology 85, no. 18 (June 28, 2019). http://dx.doi.org/10.1128/aem.01029-19.
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ABSTRACTAs classified by the Carbohydrate-Active Enzymes (CAZy) database, enzymes in glycoside hydrolase (GH) family 10 (GH10) are all monospecific or bifunctional xylanases (except a tomatinase), and no endo-β-1,4-glucanase has been reported in the family. Here, we identifiedArcticibacterium luteifluviistationiscarboxymethyl cellulase (AlCMCase) as a GH10 endo-β-1,4-glucanase.AlCMCase originated from an Arctic marine bacterium,Arcticibacterium luteifluviistationisSM1504T. It shows low identity (<35%) with other GH10 xylanases. The gene encodingAlCMCase was overexpressed inEscherichia coli. Biochemical characterization showed that recombinantAlCMCase is a cold-adapted and salt-tolerant enzyme.AlCMCase hydrolyzes cello- and xylo-configured substrates via an endoaction mode. However, in comparison to its significant cellulase activity, the xylanase activity ofAlCMCase is negligible. Correspondingly,AlCMCase has remarkable binding capacity for cello-oligosaccharides but no obvious binding capacity for xylo-oligosaccharides.AlCMCase and its homologs are grouped into a branch separate from other GH10 xylanases in a phylogenetic tree, and two homologs also displayed the same substrate specificity asAlCMCase. These results suggest thatAlCMCase and its homologs form a novel subfamily of GH10 enzymes that have robust endo-β-1,4-glucanase activity. In addition, given the cold-adapted and salt-tolerant characters ofAlCMCase, it may be a candidate biocatalyst under certain industrial conditions, such as low temperature or high salinity.IMPORTANCECellulase and xylanase have been widely used in the textile, pulp and paper, animal feed, and food-processing industries. Exploring novel cellulases and xylanases for biocatalysts continues to be a hot issue. Enzymes derived from the polar seas might have novel hydrolysis patterns, substrate specificities, or extremophilic properties that have great potential for both fundamental research and industrial applications. Here, we identified a novel cold-adapted and salt-tolerant endo-β-1,4-glucanase,AlCMCase, from an Arctic marine bacterium. It may be useful in certain industrial processes, such as under low temperature or high salinity. Moreover,AlCMCase is a bifunctional representative of glycoside hydrolase (GH) family 10 that preferentially hydrolyzes β-1,4-glucans. With its homologs, it represents a new subfamily in this family. Thus, this study sheds new light on the substrate specificity of GH10.
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Wu, Haiyang, Eleni Ioannou, Bernard Henrissat, Cédric Y. Montanier, Sophie Bozonnet, Michael J. O’Donohue, and Claire Dumon. "Investigating the multi-modularity of a GH10 Xylanase found in termite gut metagenome." Applied and Environmental Microbiology, November 13, 2020. http://dx.doi.org/10.1128/aem.01714-20.
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The functional screening of a Pseudacanthotermes militaris termite gut metagenomic library revealed an array of xylan degrading enzymes including Pm25, a multi-modular Glycoside Hydrolase (GH) family 10. Sequence analysis showed details of the unusual domain organization of this enzyme. It consists of one catalytic domain, which is intercalated by two Carbohydrate Binding Modules (CBMs) from family 4. The genes upstream of pm25 are susC-susD-unk suggesting Pm25 is a Xyn10C-like enzyme belonging to a polysaccharide utilization loci. The majority of Xyn10C-like enzymes shared the same interrupted domain architecture, and were vastly distributed in different xylan utilization loci found in gut Bacteroidetes, indicating its importance in glycan foraging for the gut microbiota. In order to understand its unusual multi-modularity and the possible role of the CBMs, a detailed characterization of the full length Pm25 and truncated variants was performed. Results revealed that the GH10 catalytic module is specific towards the hydrolysis of xylan. Ligand binding results indicate that the GH10 module and the CBMs act independently whereas the tandem CBM4s act synergistically with each other and improve enzymatic activity when assayed on insoluble polysaccharides. In addition, we show that the UNK protein upstream of Pm25 is able to bind arabinoxylan. Altogether, these findings contribute to a better understanding of the potential role of Xyn10C-like proteins in xylan utilization systems of gut bacteria. IMPORTANCE: Xylan is the major hemicellulosic polysaccharide in cereals and contributes to the recalcitrance of the plant cell wall toward degradation. Bacteroidetes, one of the main phyla in Rumen and Human gut microbiota, have been shown to encode polysaccharide utilization loci (PUL) dedicated to the degradation of xylan. Here we present the biochemical characterization of a xylanase encoded by a bacteroidetes strain isolated from the termite gut metagenome. This xylanase is a multi-modular enzyme of which sequence is interrupted by the insertion of two CBM from family 4. Our results show that not only this enzyme resemble homologues that were shown to be important for xylan degradation in rumen or human diet but show that the CBM insertion in the middle of the sequence seems to be a common feature in xylan utilisation system. This study shed light on a better understanding toward xylan degradation and plant cell wall deconstruction which can conduct to several applications in food, feed and bioeconomy.
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Liu, Zhaoxing, Tingting Shao, Yan Li, Bin Wu, Honghua Jia, and Ning Hao. "Expression, Characterization and Its Deinking Potential of a Thermostable Xylanase From Planomicrobium glaciei CHR43." Frontiers in Bioengineering and Biotechnology 9 (February 17, 2021). http://dx.doi.org/10.3389/fbioe.2021.618979.
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Genome mining is more and more widely used in identifying new enzymes from database. In the present study, we reported a putative xylanase, Pg-Xyn (WP_053166147.1), which originated from a psychrotolerant strain Planomicrobium glaciei CHR 43, and was identified from Genbank by genome mining. Sequence analysis and homology modeling showed that Pg-Xyn belongs to glycosyl hydrolase family 10. On the basis of heterologous expression in E. coli and biochemical characterization, we found Pg-Xyn was most active at pH 9.0 and 80°C and exhibited good stability from pH 5.0 to 12.0 and below 90°C. Pg-Xyn was slightly activated in the presence of Ca2+ and Mg2+, while it was strongly inhibited by Mn2+. The analysis of hydrolysis products showed that Pg-Xyn was an endo-β-1,4-xylanase. In addition, Pg-Xyn performed good deinking ability in a paper deinking test. In consideration of its unique properties, Pg-Xyn might be a promising candidate for application in the paper and pulp industries.
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Nour, Shaimaa A., Ghada M. El-Sayed, Hanan A. A. Taie, Maha T. H. Emam, Ahmed F. El-Sayed, and Rasha G. Salim. "Safe production of Aspergillus terreus xylanase from Ricinus communis: gene identification, molecular docking, characterization, production of xylooligosaccharides, and its biological activities." Journal of Genetic Engineering and Biotechnology 20, no. 1 (August 12, 2022). http://dx.doi.org/10.1186/s43141-022-00390-9.
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Abstract Background The production of industrial enzymes such as xylanase using sufficient cost-effective substrates from potent microorganisms is considered economically feasible. Studies have reported castor cake (Ricinus communis) as the most potent and inexpensive alternative carbon source for production of xylanase C by using Aspergillus terreus (A. terreus). Results A. terreus strain RGS Eg-NRC, a local isolate from agro-wastes, was first identified by sequencing the internal transcribed spacer region of a nuclear DNA encoding gene cluster deposited in GenBank (accession number MW282328). Before optimization of xylanase production, A. terreus produced 20.23 U/g of xylanase after 7 days using castor cake as a substrate in a solid-state fermentation (SSF) system that was employed to achieve ricin detoxification and stimulate xylanase production. Physicochemical parameters for the production of xylanase were optimized by using a one-variable-at-a-time approach and two statistical methods (two-level Plackett–Burman design and central composite design, CCD). The maximum xylanase yield after optimization was increased by 12.1-fold (245 U/g). A 60–70% saturation of ammonium sulfate resulted in partially purified xylanase with a specific activity of 3.9 IU/mg protein. At 60 °C and pH 6, the partially purified xylanase had the highest activity, and the activation energy (Ea) was 23.919 kJmol. Subsequently, antioxidant capacity and cytotoxicity tests in normal Ehrlich ascites carcinoma human cells demonstrated xylooligosaccharides produced by the xylanase degradation of xylan as a potent antioxidant and moderate antitumor agent. Further investigations with sodium dodecyl sulfate polyacrylamide gel electrophoresis then determined the molecular weight of partially purified xylanase C to be 36 kDa. Based on the conserved regions, observations revealed that xylanase C belonged to the glycosyl hydrolase family 10. Next, the xylanase-encoding gene (xynC), which has an open reading frame of 981 bp and encodes a protein with 326 amino acids, was isolated, sequenced, and submitted to the NCBI GenBank database (accession number LC595779.1). Molecular docking analysis finally revealed that Glu156, Glu262, and Lys75 residues were involved in the substrate-binding and protein-ligand interaction site of modeled xylanase, with a binding affinity of −8.7 kcal. mol−1. Conclusion The high production of safe and efficient xylanase could be achieved using economical materials such as Ricinus communis.