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PreprintCited by 2Biofuel production and bioconversionEnzyme Production and CharacterizationPolysaccharides and Plant Cell Walls

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Biochemical and structural characterisation of a family GH5 cellulase from endosymbiont of shipworm P. megotara

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Junghare, Madan, Manavalan, Tamilvendan, Fredriksen, Lasse, Leiros, Ingar, Altermark, Bjørn, Eijsink, Vincent G.H., Vaaje-Kolstad, Gustav·2023·Research Square
DOI 10.21203/rs.3.rs-2475918/v1

Preprint — published as 10.1186/s13068-023-02307-1

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Abstract

Background Cellulases play a key role in enzymatic conversion of plant cell-wall polysaccharides into simple and economically relevant sugars. The discovery of novel cellulases from exotic biological niches is of interest as they may present properties that are valuable in biorefining of lignocellulose. Results We have characterized a glycoside hydrolase 5 (GH5) domain of a bi-catalytic GH5-GH6 multidomain enzyme from the unusual bacterial endosymbiont Teredinibacter waterbury of the wood-digesting shipworm Psiloteredo megotara. The cellulase enzyme, Tw Cel5, was produced with and without a native C-terminal family 10 carbohydrate-binding module belongs to GH5, subfamily 2. Both variants showed hydrolytic endo-activity on soluble substrates such as, β-glucan, carboxymethylcellulose and konjac glucomannan. However, low activity was observed towards crystalline form of cellulose. Interestingly, when co-incubated with a cellulose active LPMO, a clear synergy was observed that boosted hydrolysis of crystalline cellulose. The crystal structure of the GH5 catalytic domain was solved to 1.0 Å resolution and revealed a substrate binding cleft containing a putative +3 subsite, which is uncommon in this enzyme family. The enzyme Tw Cel5 was active in a wide range of pH and temperatures and showed high tolerance for NaCl. Conclusions This study provides an important advance on discovery new enzymes from shipworm and shed new light on biochemical and structural characterization of cellulolytic cellulase and showed boost in hydrolytic activity of cellulase on crystalline cellulose when co-incubated with cellulose active LPMO. These findings will be relevant for the development of future enzyme cocktail that may be useful for the biotechnological conversion of lignocellulose.

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Research works printed but not linked: 60

  1. Klemm D, Heublein B, Fink HP, Bohn A. Cellulose: fascinating biopolymer and sustainable raw material. Angew Chem Int Ed Engl. 2005;44(22):3358–93; doi: 10.1002/anie.200460587. · DOI · Paper page
  2. Mariano M, El Kissi N, Dufresne A. Cellulose nanocrystals and related nanocomposites: Review of some properties and challenges. Journal of Polymer Science Part B: Polymer Physics. 2014;52(12):791–806; doi: https://doi.org/10.1002/polb.23490. · DOI
  3. Zhang Z, Donaldson AA, Ma X. Advancements and future directions in enzyme technology for biomass conversion. Biotechnol Adv. 2012;30(4):913–9; doi: 10.1016/j.biotechadv.2012.01.020. · DOI
  4. Himmel ME, Ding S-Y, Johnson DK, Adney WS, Nimlos MR, Brady JW, et al. Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production. Science. 2007;315(5813):804–7; doi: doi:10.1126/science.1137016. · DOI
  5. Østby H, Hansen LD, Horn SJ, Eijsink VGH, Várnai A. Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives. Journal of Industrial Microbiology and Biotechnology. 2020;47(9–10):623–57; doi: 10.1007/s10295-020-02301-8. · DOI
  6. Andlar M, Rezić T, Marđetko N, Kracher D, Ludwig R, Šantek B. Lignocellulose degradation: An overview of fungi and fungal enzymes involved in lignocellulose degradation. Eng Life Sci. 2018;18(11):768–78; doi: 10.1002/elsc.201800039. · DOI
  7. Cragg SM, Beckham GT, Bruce NC, Bugg TD, Distel DL, Dupree P, et al. Lignocellulose degradation mechanisms across the Tree of Life. Curr Opin Chem Biol. 2015;29:108–19; doi: 10.1016/j.cbpa.2015.10.018. · DOI
  8. Berlemont R, Martiny AC. Genomic potential for polysaccharide deconstruction in bacteria. Appl Environ Microbiol. 2015;81(4):1513–19; doi: 10.1128/aem.03718-14. · DOI
  9. Bhardwaj N, Kumar B, Agrawal K, Verma P. Current perspective on production and applications of microbial cellulases: a review. Bioresources and Bioprocessing. 2021;8(1):95; doi: 10.1186/s40643-021-00447-6. · DOI
  10. Sabbadin F, Pesante G, Elias L, Besser K, Li Y, Steele-King C, et al. Uncovering the molecular mechanisms of lignocellulose digestion in shipworms. Biotechnology for Biofuels. 2018;11(1):59; doi: 10.1186/s13068-018-1058-3. · DOI
  11. Stravoravdis S, Shipway JR, Goodell B. How Do Shipworms Eat Wood? Screening Shipworm Gill Symbiont Genomes for Lignin-Modifying Enzymes. Frontiers in Microbiology. 2021;12; doi: 10.3389/fmicb.2021.665001. · DOI
  12. Distel DL. The Biology of Marine Wood Boring Bivalves and Their Bacterial Endosymbionts. In: Wood Deterioration and Preservation. American Chemical Society; 2003. p. 253–71. · DOI
  13. Nair NB, Saraswathy M. The Biology of Wood-Boring Teredinid Molluscs. In: Russell FS, Yonge M, editors. Advances in Marine Biology. Academic Press; 1971. p. 335–509.
  14. Distel DL, Roberts SJ. Bacterial Endosymbionts in the Gills of the Deep-Sea Wood-Boring Bivalves Xylophaga atlantica and Xylophaga washingtona. The Biological Bulletin. 1997;192(2):253–61; doi: 10.2307/1542719. · DOI
  15. Lechene CP, Luyten Y, McMahon G, Distel DL. Quantitative Imaging of Nitrogen Fixation by Individual Bacteria Within Animal Cells. Science. 2007;317(5844):1563–6; doi: 10.1126/science.1145557. · DOI
  16. O’Connor RM, Fung JM, Sharp KH, Benner JS, McClung C, Cushing S, et al. Gill bacteria enable a novel digestive strategy in a wood-feeding mollusk. Proceedings of the National Academy of Sciences. 2014;111(47):E5096-E104; doi: doi:10.1073/pnas.1413110111. · DOI
  17. Brito TL, Campos AB, von Meijenfeldt FAB, Daniel JP, Ribeiro GB, Silva GGZ, et al. The gill-associated symbiont microbiome is a main source of woody-plant polysaccharide hydrolase genes and secondary metabolite gene clusters in < em > Neoteredo reynei, a unique shipworm from south Atlantic mangroves. bioRxiv. 2018:357616; doi: 10.1101/357616. · DOI
  18. Pesante G, Sabbadin F, Elias L, Steele-King C, Shipway JR, Dowle AA, et al. Characterisation of the enzyme transport path between shipworms and their bacterial symbionts. BMC Biology. 2021;19(1):233; doi: 10.1186/s12915-021-01162-6. · DOI
  19. 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(D1):D571-d7; doi: 10.1093/nar/gkab1045. · DOI · Paper page
  20. Forsberg Z, Vaaje-Kolstad G, Westereng B, Bunæs AC, Stenstrøm Y, MacKenzie A, et al. Cleavage of cellulose by a CBM33 protein. Protein Sci. 2011;20(9):1479–83; doi: 10.1002/pro.689. · DOI
  21. Howard MB, Ekborg NA, Taylor LE, Hutcheson SW, Weiner RM. Identification and analysis of polyserine linker domains in prokaryotic proteins with emphasis on the marine bacterium Microbulbifer degradans. Protein Sci. 2004;13(5):1422–5; doi: 10.1110/ps.03511604. · DOI
  22. Altamia MA, Shipway JR, Stein D, Betcher MA, Fung JM, Jospin G, et al. Teredinibacter waterburyi sp. nov., a marine, cellulolytic endosymbiotic bacterium isolated from the gills of the wood-boring mollusc Bankia setacea (Bivalvia: Teredinidae) and emended description of the genus Teredinibacter. Int J Syst Evol Microbiol. 2020;70(4):2388–94; doi: 10.1099/ijsem.0.004049. · DOI
  23. Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nat Methods. 2022;19(6):679–82; doi: 10.1038/s41592-022-01488-1. · DOI · Paper page
  24. Song Y-H, Lee K-T, Baek J-Y, Kim M-J, Kwon M-R, Kim Y-J, et al. Isolation and characterization of a novel endo-β-1,4-glucanase from a metagenomic library of the black-goat rumen. Brazilian Journal of Microbiology. 2017;48(4):801–8; doi: https://doi.org/10.1016/j.bjm.2017.03.006. · DOI
  25. Várnai A, Siika-Aho M, Viikari L. Carbohydrate-binding modules (CBMs) revisited: reduced amount of water counterbalances the need for CBMs. Biotechnol Biofuels. 2013;6(1):30; doi: 10.1186/1754-6834-6-30. · DOI
  26. Bernardes A, Pellegrini VOA, Curtolo F, Camilo CM, Mello BL, Johns MA, et al. Carbohydrate binding modules enhance cellulose enzymatic hydrolysis by increasing access of cellulases to the substrate. Carbohydr Polym. 2019;211:57–68; doi: 10.1016/j.carbpol.2019.01.108. · DOI
  27. Chalak A, Villares A, Moreau C, Haon M, Grisel S, d'Orlando A, et al. Influence of the carbohydrate-binding module on the activity of a fungal AA9 lytic polysaccharide monooxygenase on cellulosic substrates. Biotechnol Biofuels. 2019;12:206; doi: 10.1186/s13068-019-1548-y. · DOI
  28. Liberato MV, Silveira RL, Prates ÉT, de Araujo EA, Pellegrini VOA, Camilo CM, et al. Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism. Scientific Reports. 2016;6(1):23473; doi: 10.1038/srep23473. · DOI
  29. Alvarez TM, Paiva JH, Ruiz DM, Cairo JP, Pereira IO, Paixão DA, et al. Structure and function of a novel cellulase 5 from sugarcane soil metagenome. PLoS One. 2013;8(12):e83635; doi: 10.1371/journal.pone.0083635. · DOI
  30. Naas AE, MacKenzie AK, Dalhus B, Eijsink VGH, Pope PB. Structural Features of a Bacteroidetes-Affiliated Cellulase Linked with a Polysaccharide Utilization Locus. Sci Rep. 2015;5:11666; doi: 10.1038/srep11666. · DOI
  31. Lazaridou A, Biliaderis CG. Molecular aspects of cereal β-glucan functionality: Physical properties, technological applications and physiological effects. Journal of Cereal Science. 2007;46(2):101–18; doi: https://doi.org/10.1016/j.jcs.2007.05.003. · DOI
  32. Takigami S. Chapter 18 - Konjac glucomannan. In: Phillips GO, Williams PA, editors. Handbook of Hydrocolloids (Third Edition). Woodhead Publishing; 2021. p. 563–77. · DOI
  33. Witkamp RF. 3.15 - Biologically Active Compounds in Food Products and Their Effects on Obesity and Diabetes. In: Liu H-W, Mander L, editors. Comprehensive Natural Products II. Oxford: Elsevier; 2010. p. 509–45. · DOI
  34. Barras F, Bortoli-German I, Bauzan M, Rouvier J, Gey C, Heyraud A, et al. Stereochemistry of the hydrolysis reaction catalyzed by endoglucanase Z from Erwinia chrysanthemi. FEBS Letters. 1992;300(2):145–8; doi: https://doi.org/10.1016/0014-5793(92)80183-H. · DOI
  35. Bischoff KM, Rooney AP, Li XL, Liu S, Hughes SR. Purification and characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis. Biotechnol Lett. 2006;28(21):1761–5; doi: 10.1007/s10529-006-9153-0. · DOI
  36. Xu P-N, Distel DL. Purification and characterization of an endo-1, 4-β-D glucanase from the cellulolytic system of the wood-boring marine mollusk Lyrodus pedicellatus (Bivalvia: Teredinidae). Marine Biology. 2004;144(5):947–53; doi: 10.1007/s00227-003-1251-0. · DOI
  37. Santos CR, Paiva JH, Sforça ML, Neves JL, Navarro RZ, Cota J, et al. Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168. Biochem J. 2012;441(1):95–104; doi: 10.1042/bj20110869. · DOI
  38. Liu J, Liu WD, Zhao XL, Shen WJ, Cao H, Cui ZL. Cloning and functional characterization of a novel endo-β-1,4-glucanase gene from a soil-derived metagenomic library. Appl Microbiol Biotechnol. 2011;89(4):1083–92; doi: 10.1007/s00253-010-2828-4. · DOI
  39. Annamalai N, Thavasi R, Vijayalakshmi S, Balasubramanian T. A novel thermostable and halostable carboxymethylcellulase from marine bacterium Bacillus licheniformisAU01. World Journal of Microbiology and Biotechnology. 2011;27(9):2111–5; doi: 10.1007/s11274-011-0674-x. · DOI
  40. Hakamada Y, Koike K, Yoshimatsu T, Mori H, Kobayashi T, Ito S. Thermostable alkaline cellulase from an alkaliphilic isolate, Bacillus sp. KSM-S237. Extremophiles. 1997;1(3):151–6; doi: 10.1007/s007920050028. · DOI
  41. Yin YR, Zhang F, Hu QW, Xian WD, Hozzein WN, Zhou EM, et al. Heterologous expression and characterization of a novel halotolerant, thermostable, and alkali-stable GH6 endoglucanase from Thermobifida halotolerans. Biotechnol Lett. 2015;37(4):857–62; doi: 10.1007/s10529-014-1742-8. · DOI
  42. Fowler CA, Sabbadin F, Ciano L, Hemsworth GR, Elias L, Bruce N, et al. Discovery, activity and characterisation of an AA10 lytic polysaccharide oxygenase from the shipworm symbiont Teredinibacter turnerae. Biotechnology for Biofuels. 2019;12(1):232; doi: 10.1186/s13068-019-1573-x. · DOI
  43. Forsberg Z, Mackenzie AK, Sørlie M, Røhr ÅK, Helland R, Arvai AS, et al. Structural and functional characterization of a conserved pair of bacterial cellulose-oxidizing lytic polysaccharide monooxygenases. Proceedings of the National Academy of Sciences. 2014;111(23):8446-51; doi: doi:10.1073/pnas.1402771111. · DOI
  44. Chapon V, Czjzek M, El Hassouni M, Py B, Juy M, Barras F. Type II protein secretion in gram-negative pathogenic bacteria: the study of the structure/secretion relationships of the cellulase cel5 (formerly EGZ) from Erwinia chrysanthemi11Edited by I. B. Holland. Journal of Molecular Biology. 2001;310(5):1055–66; doi: https://doi.org/10.1006/jmbi.2001.4787. · DOI
  45. Holm L, Laakso LM. Dali server update. Nucleic Acids Res. 2016;44(W1):W351-5; doi: 10.1093/nar/gkw357. · DOI
  46. Py B, Bortoli-German I, Haiech J, Chippaux M, Barras F. Cellulase EGZ of Erwinia chrysanthemi: structural organization and importance of His98 and Glu133 residues for catalysis. Protein Eng. 1991;4(3):325–33; doi: 10.1093/protein/4.3.325. · DOI
  47. Shaw A, Bott R, Vonrhein C, Bricogne G, Power S, Day AG. A Novel Combination of Two Classic Catalytic Schemes. Journal of Molecular Biology. 2002;320(2):303–9; doi: https://doi.org/10.1016/S0022-2836(02)00387-X. · DOI
  48. KOSHLAND Jr. DE. STEREOCHEMISTRY AND THE MECHANISM OF ENZYMATIC REACTIONS. Biological Reviews. 1953;28(4):416–36; doi: https://doi.org/10.1111/j.1469-185X.1953.tb01386.x. · DOI
  49. Chang CJ, Lee CC, Chan YT, Trudeau DL, Wu MH, Tsai CH, et al. Exploring the Mechanism Responsible for Cellulase Thermostability by Structure-Guided Recombination. PLoS One. 2016;11(3):e0147485; doi: 10.1371/journal.pone.0147485. · DOI
  50. Garg R, Srivastava R, Brahma V, Verma L, Karthikeyan S, Sahni G. Biochemical and structural characterization of a novel halotolerant cellulase from soil metagenome. Scientific Reports. 2016;6(1):39634; doi: 10.1038/srep39634. · DOI
  51. Zhu Y, Han L, Hefferon KL, Silvaggi NR, Wilson DB, McBride MJ. Periplasmic Cytophaga hutchinsonii Endoglucanases Are Required for Use of Crystalline Cellulose as the Sole Source of Carbon and Energy. Appl Environ Microbiol. 2016;82(15):4835–45; doi: doi:10.1128/AEM.01298-16. · DOI
  52. Wood TM. Preparation of crystalline, amorphous, and dyed cellulase substrates. In: Methods in Enzymology. Academic Press; 1988. p. 19–25.
  53. Zhang H, Yohe T, Huang L, Entwistle S, Wu P, Yang Z, et al. dbCAN2: a meta server for automated carbohydrate-active enzyme annotation. Nucleic Acids Res. 2018;46(W1):W95-W101; doi: 10.1093/nar/gky418. · DOI
  54. Almagro Armenteros JJ, Tsirigos KD, Sønderby CK, Petersen TN, Winther O, Brunak S, et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nature Biotechnology. 2019;37(4):420–3; doi: 10.1038/s41587-019-0036-z. · DOI
  55. Gasteiger E, Hoogland C, Gattiker A, Duvaud Se, Wilkins MR, Appel RD, et al. Protein Identification and Analysis Tools on the ExPASy Server. In: Walker JM, editor. The Proteomics Protocols Handbook. Totowa, NJ: Humana Press; 2005. p. 571–607. · DOI
  56. Miller GL. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry. 1959;31(3):426–8; doi: 10.1021/ac60147a030. · DOI · Paper page
  57. Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 2011;67(Pt 4):235–42; doi: 10.1107/S0907444910045749. · DOI · Paper page
  58. Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 1997;53(Pt 3):240–55; doi: 10.1107/s0907444996012255. · DOI
  59. Cowtan K. The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallographica Section D. 2006;62(9):1002–11; doi: doi:10.1107/S0907444906022116. · DOI
  60. Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004;60(Pt 12 Pt 1):2126–32; doi: 10.1107/s0907444904019158. · DOI · Paper page

Metadata

Authors
Junghare, M., Manavalan, T., Fredriksen, L., Leiros, I., Altermark, B., Eijsink, V. G. H., & Vaaje-Kolstad, G.
Affiliations
  • Norwegian University of Life Sciences (NO)
  • UiT The Arctic University of Norway (NO)
  • Norwegian University of Life Sciences
Corresponding author
Junghare, Madan
ORCID
Venue
Research Square
Publisher
Research Square Platform LLC
Published
2023-01-19
DOI
10.21203/rs.3.rs-2475918/v1
OpenAlex
W4317434589
Cited by
2
References
1
Citations by year
2024: 1 · 2023: 1
Type
posted content
Language
en
Open access
Licence
Locations
  • Research Square (acceptedVersion, cc-by)
  • NORA - Norwegian Open Research Archives (submittedVersion, other-oa)
  • HZB Repository (Helmholtz-Zentrum Berlin für Materialien und Energie GmbH (HZB)) (submittedVersion, other-oa)
Funders
Norges Forskningsråd (269408, 283647, 221568)
Topics
  • Physical Sciences › Engineering › Biomedical Engineering › Biofuel production and bioconversion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Polysaccharides and Plant Cell Walls
Keywords
crystalline cellulose hydrolysis, enzyme crystal structure, substrate-binding cleft

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