Animal Bioscience

  • 제37권9호
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  • Pages.1581-1594
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  • 2024
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  • 2765-0189(pISSN)
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  • 2765-0235(eISSN)
아세아태평양축산학회 (Asian Australasian Association of Animal Production Societies)
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Expression and characterization of a novel microbial GH9 glucanase, IDSGLUC9-4, isolated from sheep rumen

  • Yongzhen Zhu (Jilin Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences) ;
  • Shuning Bai (Key Laboratory of Molecular Animal Nutrition, Ministry of Education, Zhejiang University) ;
  • Nuo Li (Key Laboratory of Molecular Animal Nutrition, Ministry of Education, Zhejiang University) ;
  • Jun-Hong Wang (Key Laboratory of Molecular Animal Nutrition, Ministry of Education, Zhejiang University) ;
  • Jia-Kun Wang (Key Laboratory of Molecular Animal Nutrition, Ministry of Education, Zhejiang University) ;
  • Qian Wang (Key Laboratory of Molecular Animal Nutrition, Ministry of Education, Zhejiang University) ;
  • Kaiying Wang (Jilin Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences) ;
  • Tietao Zhang (Jilin Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences)
  • 투고 : 2024.03.06
  • 심사 : 2024.05.20
  • 발행 : 2024.09.01
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초록

Objective: This study aimed to identify and characterize a novel endo-β-glucanase, IDSGLUC9-4, from the rumen metatranscriptome of Hu sheep. Methods: A novel endo-β-glucanase, IDSGLUC9-4, was heterologously expressed in Escherichia coli and biochemically characterized. The optimal temperature and pH of recombinant IDSGLUC9-4 were determined. Subsequently, substrate specificity of the enzyme was assessed using mixed-linked glucans including barley β-glucan and Icelandic moss lichenan. Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), matrix assisted laser desorption ionization time of flight mass spectrometry analyses were conducted to determine the products released from polysaccharides and cello-oligosaccharides substrates. Results: The recombinant IDSGLUC9-4 exhibited temperature and pH optima of 40℃ and pH 6.0, respectively. It exclusively hydrolyzed mixed-linked glucans, with significant activity observed for barley β-glucan (109.59±3.61 µmol/mg min) and Icelandic moss lichenan (35.35±1.55 µmol/mg min). TLC and HPLC analyses revealed that IDSGLUC9-4 primarily released cellobiose, cellotriose, and cellotetraose from polysaccharide substrates. Furthermore, after 48 h of reaction, IDSGLUC9-4 removed most of the glucose, indicating transglycosylation activity alongside its endo-glucanase activity. Conclusion: The recombinant IDSGLUC9-4 was a relatively acid-resistant, mesophilic endo-glucanase (EC 3.2.1.4) that hydrolyzed glucan-like substrates, generating predominantly G3 and G4 oligosaccharides, and which appeared to have glycosylation activity. These findings provided insights into the substrate specificity and product profiles of rumen-derived GH9 glucanases and contributed to the expanding knowledge of cellulolytic enzymes and novel herbivore rumen enzymes in general.

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과제정보

This work was financially supported by the Agricultural Science and Technology Innovation Program of China (Grant CAAS-ASTIP-2021-ISAPS). The authors thank Mr. Weidong Zeng from The Experimental Teaching Center, College of Animal Sciences, Zhejiang University, for facility support.

참고문헌

  1. Beckmann L, Simon O, Vahjen W. Isolation and identification of mixed linked beta -glucan degrading bacteria in the intestine of broiler chickens and partial characterization of respective 1,3-1,4-beta-glucanase activities. J Basic Microbiol 2006;46:175-85. https://doi.org/10.1002/jobm.200510107
  2. Romero JJ, Macias EG, Ma ZX, et al. Improving the performance of dairy cattle with a xylanase-rich exogenous enzyme preparation. J Dairy Sci 2016;99:3486-96. https://doi.org/10.3168/jds.2015-10082
  3. Sztupecki W, Rhazi L, Depeint F, Aussenac T. Functional and nutritional characteristics of natural or modified wheat bran non-starch polysaccharides: a literature review. Foods 2023;12:2693. https://doi.org/10.3390/foods12142693
  4. Drula E, Garron ML, Dogan S, Lombard V, Henrissat B, Terrapon N. The carbohydrate-active enzyme database: functions and literature. Nucleic Acids Res 2022;50:D571-7. https://doi.org/10.1093/nar/gkab1045
  5. Manavalan T, Manavalan A, Heese K. Characterization of lignocellulolytic enzymes from white-rot fungi. Curr Microbiol 2015;70:485-98. https://doi.org/10.1007/s00284-014-0743-0
  6. Masoumi SJ, Mehrabani D, Saberifiroozi M, Fattahi MR, Moradi F, Najafi M. The effect of yogurt fortified with Lactobacillus acidophilus and Bifidobacterium sp. probiotic in patients with lactose intolerance. Food Sci Nutr 2021;9:1704-11. https://doi.org/10.1002/fsn3.2145
  7. Aspeborg H, Coutinho PM, Wang Y, Brumer H 3rd, Henrissat B. Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5). BMC Evol Biol 2012;12:186. https://doi.org/10.1186/1471-2148-12-186
  8. Glasgow EM, Kemna EI, Bingman CA, et al. A structural and kinetic survey of GH5_4 endoglucanases reveals determinants of broad substrate specificity and opportunities for biomass hydrolysis. J Biol Chem 2020;295:17752-69. https://doi.org/10.1074/jbc.RA120.015328
  9. Zhou Y, Wang X, Wei W, et al. A novel efficient β-glucanase from a paddy soil microbial metagenome with versatile activities. Biotechnol Biofuels 2016;9:36. https://doi.org/10.1186/s13068-016-0449-6
  10. Jiang N, Ma XD, Fu LH, Li CX, Feng JX, Duan CJ. Identification of a unique 1,4-β-D-glucan glucohydrolase of glycoside hydrolase family 9 from Cytophaga hutchinsonii. Appl Microbiol Biotechnol 2020;104:7051-66. https://doi.org/10.1007/s00253-020-10731-8
  11. Phakeenuya V, Ratanakhanokchai K, Kosugi A, Tachaapaikoon C. A novel multifunctional GH9 enzyme from Paenibacillus curdlanolyticus B-6 exhibiting endo/exo functions of cellulase, mannanase and xylanase activities. Appl Microbiol Biotechnol 2020;104:2079-96. https://doi.org/10.1007/s00253-020-10388-3
  12. Culp EJ, Goodman AL. Cross-feeding in the gut microbiome: ecology and mechanisms. Cell Host Microbe 2023;31:485-99. https://doi.org/10.1016/j.chom.2023.03.016
  13. He B, Jin S, Cao J, Mi L, Wang J. Metatranscriptomics of the Hu sheep rumen microbiome reveals novel cellulases. Biotechnol Biofuels 2019;12:153. https://doi.org/10.1186/s13068-019-1498-4
  14. Wang Q, Luo Y, He B, Jiang LS, Liu JX, Wang JK. Characterization of a novel xylanase gene from rumen content of Hu sheep. Appl Biochem Biotechnol 2015;177:1424-36. https://doi.org/10.1007/s12010-015-1823-8
  15. Bailey MJ, Biely P, Poutanen K. Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol 1992;23:257-70. https://doi.org/10.1016/0168-1656(92)90074-J
  16. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. https://doi.org/10.1006/abio.1976.9999
  17. Gao DY, Sun XB, Fang Y, et al. Heterologous expression and characterization of two novel glucanases derived from sheep rumen microbiota. World J Microbiol Biotechnol 2022;38:87. https://doi.org/10.1007/s11274-022-03269-6
  18. Xie F, Jin W, Si H, et al. An integrated gene catalog and over 10,000 metagenome-assembled genomes from the gastrointestinal microbiome of ruminants. Microbiome 2021;9:137. https://doi.org/10.1186/s40168-021-01078-x
  19. Tolonen AC, Chilaka AC, Church GM. Targeted gene inactivation in Clostridium phytofermentans shows that cellulose degradation requires the family 9 hydrolase Cphy3367. Mol Microbiol 2009;74:1300-13. https://doi.org/10.1111/j.1365-2958.2009.06890.x
  20. Kurokawa J, Hemjinda E, Arai T, Kimura T, Sakka K, Ohmiya K. Clostridium thermocellum cellulase CelT, a family 9 endoglucanase without an Ig-like domain or family 3c carbohydrate-binding module. Appl Microbiol Biotechnol 2002;59:455-61. https://doi.org/10.1007/s00253-002-1048-y
  21. Kesavulu MM, Tsai JY, Lee HL, Liang PH, Hsiao CD. Structure of the catalytic domain of the Clostridium thermocellum cellulase CelT. Acta Crystallogr D Biol Crystallogr 2012;68:310-20. https://doi.org/10.1107/S0907444912001990
  22. Pereira JH, Sapra R, Volponi JV, Kozina CL, Simmons B, Adams PD. Structure of endoglucanase Cel9A from the thermoacidophilic Alicyclobacillus acidocaldarius. Acta Crystallogr D Biol Crystallogr 2009;65:744-50. https://doi.org/10.1107/S0907444909012773
  23. Petkun S, Rozman Grinberg I, Lamed R, et al. Reassembly and co-crystallization of a family 9 processive endoglucanase from its component parts: structural and functional significance of the intermodular linker. PeerJ 2015;3:e1126. https://doi.org/10.7717/peerj.1126
  24. Kuch NJ, Kutschke ME, Parker A, Bingman CA, Fox BG. Contribution of calcium ligands in substrate binding and product release in the Acetovibrio thermocellus glycoside hydrolase family 9 cellulase CelR. J Biol Chem 2023;299:104655. https://doi.org/10.1016/j.jbc.2023.104655
  25. Dadheech T, Shah R, Pandit R, et al. Cloning, molecular modeling and characterization of acidic cellulase from buffalo rumen and its applicability in saccharification of lignocellulosic biomass. Int J Biol Macromol 2018;113:73-81. https://doi.org/10.1016/j.ijbiomac.2018.02.100
  26. Guerrero EB, de Villegas RMD, Soria MA, Santangelo MP, Campos E, Talia PM. Characterization of two GH5 endoglucanases from termite microbiome using synthetic metagenomics. Appl Microbiol Biotechnol 2020;104:8351-66. https://doi.org/10.1007/s00253-020-10831-5
  27. Cao JW, Deng Q, Gao DY, et al. A novel bifunctional glucanase exhibiting high production of glucose and cellobiose from rumen bacterium. Int J Biol Macromol 2021;173:136-45. https://doi.org/10.1016/j.ijbiomac.2021.01.113
  28. Kalyani DC, Reichenbach T, Aspeborg H, Divne C. A homo-dimeric bacterial exo-β-1,3-glucanase derived from moose rumen microbiome shows a structural framework similar to yeast exo-β-1,3-glucanases. Enzyme Microb Technol 2021;143:109723. https://doi.org/10.1016/j.enzmictec.2020.109723
  29. Li Y, Song W, Han X, et al. Recent progress in key lignocellulosic enzymes: enzyme discovery, molecular modifications, production, and enzymatic biomass saccharification. Bioresour Technol 2022;363:127986. https://doi.org/10.1016/j.biortech.2022.127986
  30. Taguchi H, Hagiwara D, Genma T, et al. Cloning of the Ruminococcus albus cel5D and cel9A genes encoding dockerin module-containing endoglucanases and expression of cel5D in Escherichia coli. Biosci Biotechnol Biochem 2004;68:1557-64. https://doi.org/10.1271/bbb.68.1557
  31. Larsbrink J, Rogers TE, Hemsworth GR, et al. A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes. Nature 2014;506:498-502. https://doi.org/10.1038/nature12907
  32. Gu Y, Zheng F, Wang Y, et al. Characterization of two thermophilic cellulases from Talaromyces leycettanus JCM12802 and their synergistic action on cellulose hydrolysis. PLoS One 2019;14:e0224803. https://doi.org/10.1371/journal.pone.0224803
  33. Sadaqat B, Sha C, Rupani PF, Wang H, Zuo W, Shao W. Man/Cel5B, a bifunctional enzyme having the highest mannanase activity in the hyperthermic environment. Front Bioeng Biotechnol 2021;9:637649. https://doi.org/10.3389/fbioe.2021.637649
  34. Volkov PV, Rubtsova EA, Rozhkova AM, et al. Properties of recombinant endo-β-1,6-glucanase from Trichoderma harzianum and its application in the pustulan hydrolysis. Carbohydr Res 2021;499:108211. https://doi.org/10.1016/j.carres.2020.108211
  35. Li N, Han J, Zhou Y, et al. A rumen-derived bifunctional glucanase/mannanase uncanonically releases oligosaccharides with a high degree of polymerization preferentially from branched substrates. Carbohydr Polym 2024;330:121828. https://doi.org/10.1016/j.carbpol.2024.121828
  36. Li D, Li X, Dang W, et al. Characterization and application of an acidophilic and thermostable β-glucosidase from Thermofilum pendens. J Biosci Bioeng 2013;115:490-6. https://doi.org/10.1016/j.jbiosc.2012.11.009
  37. Curiel JA, de la Bastida AR, Langa S, Peiroten A, Landete JM. Characterization and stabilization of GluLm and its application to deglycosylate dietary flavonoids and lignans. Appl Microbiol Biotechnol 2024;108:80. https://doi.org/10.1007/s00253-023-12956-9
  38. Sawant S, Birhade S, Anil A, Gilbert H, Lali A. Two- way dynamics in β-glucosidase catalysis. J Mol Catal B Enzym 2016;133:161-6. https://doi.org/10.1016/j.molcatb.2016.08.010
  39. Mendez-Liter JA, Nieto-Dominguez M, Fernandez de Toro B, et al. A glucotolerant β-glucosidase from the fungus Talaromyces amestolkiae and its conversion into a glycosynthase for glycosylation of phenolic compounds. Microb Cell Fact 2020;19:127. https://doi.org/10.1186/s12934-020-01386-1