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Biochemical characterization and low-resolution SAXS shape of a novel GH11 exo-1,4-β-xylanase identified in a microbial consortium

  • Danilo Elton Evangelista
  • Vanessa de Oliveira Arnoldi Pellegrini
  • Melissa Espirito Santo
  • Simon McQueen-Mason
  • Neil C. Bruce
  • Igor PolikarpovEmail author
Biotechnologically relevant enzymes and proteins
  • 60 Downloads

Abstract

Biotechnologies that aim to produce renewable fuels, chemicals, and bioproducts from residual ligno(hemi)cellulosic biomass mostly rely on enzymatic depolymerization of plant cell walls (PCW). This process requires an arsenal of diverse enzymes, including xylanases, which synergistically act on the hemicellulose, reducing the long and complex xylan chains to oligomers and simple sugars. Thus, xylanases play a crucial role in PCW depolymerization. Until recently, the largest xylanase family, glycoside hydrolase family 11 (GH11) has been exclusively represented by endo-catalytic β-1,4- and β-1,3-xylanases. Analysis of a metatranscriptome library from a microbial lignocellulose community resulted in the identification of an unusual exo-acting GH11 β-1,4-xylanase (MetXyn11). Detailed characterization has been performed on recombinant MetXyn11 including determination of its low-resolution small-angle X-ray scattering (SAXS) molecular envelope in solution. Our results reveal that MetXyn11 is a monomeric globular enzyme that liberates xylobiose from heteroxylans as the only product. MetXyn11 has an optimal activity in a pH range from 6 to 9 and an optimal temperature of 50 °C. The enzyme maintained above 65% of its original activity in the pH range 5 to 6 after being incubated for 72 h at 50 °C. Addition of the enzyme to a commercial enzymatic cocktail (CelicCtec3) promoted a significant increase of enzymatic hydrolysis yields of hydrothermally pretreated sugarcane bagasse (16% after 24 h of hydrolysis).

Keywords

GH11 exo-β-1,4-xylanase Metatranscriptome Biochemical characterization Synergism Small-Angle X-ray scattering 

Notes

Acknowledgments

The authors acknowledge Dr. Marco A. S. Kadowaki for his help with HPAEC analysis and Dr. Evandro Ares de Araújo for assistance with SAXS data collection and processing.

Authors’ contributions

I.P and D.E.E designed the experiments and wrote the manuscript. D.E.E and V.O.A.P performed MetXyn11 biochemical and biophysical characterization. D.E.E performed the SAXS experiments. M.E.S provided pretreated bagasse samples. I.P., S.M.M., N.C.B., D.E.E., and V.O.A.P. contributed to discussion of the results and editing of the manuscript. All the authors approved the final version.

Funding

This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) via grant nos. 10/52362-5, 11/20505-4, 11/21608-1, and 15/13684-0; the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) via grant nos. 405191/2015-4, 303988/2016-9, 440977/2016-9, and 151963/2018-5; and the BBSRC of the UK Research and Innovation (grant number: BB/I018492/1).

Compliance with ethical standards and ethical approval

This article does not contain any studies with human or animal participants.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2019_10033_MOESM1_ESM.pdf (186 kb)
ESM 1 (PDF 186 kb)

References

  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410.  https://doi.org/10.1016/S0022-2836(05)80360-2 CrossRefGoogle Scholar
  2. Beaugrand J, Paës G, Reis D, Takahashi M, Debeire P, O'Donoghue M, Chabbert B (2005) Probing the cell wall heterogeneity of micro-dissected wheat caryopsis using both active and inactive forms of a GH11 xylanase. Planta 222:246–257.  https://doi.org/10.1007/s00425-005-1538-0 CrossRefGoogle Scholar
  3. Biely P, Singh S, Puchart V (2016) Towards enzymatic breakdown of complex plant xylan structures: State of the art. Biotechnol Adv 34:1260–1274.  https://doi.org/10.1016/j.biotechadv.2016.09.001 CrossRefGoogle Scholar
  4. Boisset C, Pétrequin C, Chanzy H, Henrissat B, Schülein M (2001) Optimized mixtures of recombinant Humicola insolens cellulases for the biodegradation of crystalline cellulose. Biotechnol Bioeng 72:339–345.  https://doi.org/10.1002/1097-0290(20010205)72:3<339::AID-BIT11>3.0.CO;2-%23 CrossRefGoogle Scholar
  5. Busse-Wicher M, Gomes TCF, Tryfona T, Nikolovski N, Stott K, Grantham NJ, Tryfona Bolam DN, Skaf MS and Dupree P (2014) The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana. The Plant J 79: 492–506.  https://doi.org/10.1111/tpj.12575
  6. Camilo C, Polikarpov I (2014) High-throughput cloning, expression and purification of glycoside hydrolases using ligation-independent cloning (LIC). Protein Expr Purif 99:35–42.  https://doi.org/10.1016/j.pep.2014.03.008 CrossRefGoogle Scholar
  7. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37:D233–D238.  https://doi.org/10.1093/nar/gkn663 CrossRefGoogle Scholar
  8. Castillo JM, Romero E, Nogales R (2013) Dynamics of microbial communities related to biochemical parameters during vermicomposting and maturation of agroindustrial lignocellulose wastes. Bioresour Technol 146:345–354.  https://doi.org/10.1016/j.biortech.2013.07.093 CrossRefGoogle Scholar
  9. Curtis TP, Head IM, Graham DW (2003) Theoretical ecology for engineering biology. Environ Sci Technol 37:64A–70A.  https://doi.org/10.1021/es0323493 CrossRefGoogle Scholar
  10. DeLano WL (2002) The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto http://www.pymol.org Google Scholar
  11. Duan CJ, Feng JX (2010) Mining metagenomes for novel cellulase genes. Biotechnol Lett 32:1765–1775.  https://doi.org/10.1007/s10529-010-0356-z CrossRefGoogle Scholar
  12. Ericsson UB, Hallberg BM, Detitta GT, Dekker N, Nordlund P (2006) Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal Biochem 357:289–298.  https://doi.org/10.1016/j.ab.2006.07.027 CrossRefGoogle Scholar
  13. Evangelista DE, de Paula FF, Rodrigues A, Henrique-Silva F (2015) Pectinases from Sphenophorus levis Vaurie, 1978 (Coleoptera: Curculionidae): putative accessory digestive enzymes. J Insect Sci 15:1536–2442.  https://doi.org/10.1093/jisesa/ieu168 CrossRefGoogle Scholar
  14. Evangelista DE, Kadowaki MAS, Mello BL, Polikarpov I (2018) Biochemical and biophysical characterization of novel GH10 xylanase prospected from a sugar cane bagasse compost-derived microbial consortia. Int J Biol Macromol 109:560–568.  https://doi.org/10.1016/j.ijbiomac.2017.12.099 CrossRefGoogle Scholar
  15. Franke D, Svergun D (2009) DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J Appl Crystallogr 42:342–346.  https://doi.org/10.1107/S0021889809000338 CrossRefGoogle Scholar
  16. Ghio S, Ontañon O, Piccinni FE, Díaz de Villegas RM, Talia P, Grasso DH, Campos E (2018) Paenibacillus sp. A59 GH10 and GH11 extracellular endoxylanases: application in biomass bioconversion. BioEnergy Res 11:174–190.  https://doi.org/10.1007/s12155-017-9887-7 CrossRefGoogle Scholar
  17. Guinier A and Fournet G (1955) Small-angle scattering of X-rays. John Wiley and Sons. pp. 267Google Scholar
  18. Hu J, Davies J, Mok YK, Gene B, Lee QF, Arato C, Saddler JN (2016) Enzymatic hydrolysis of industrial derived xylo-oligomers to monomeric sugars for potential chemical/biofuel production. ACS Sustain Chem Eng 4:7130–7136.  https://doi.org/10.1021/acssuschemeng.6b02008 CrossRefGoogle Scholar
  19. Isikgor F, Becer C (2015) Lignocellulosic biomass: a sustainable platform for the production of bio-based chemicals and polymers. Polym Chem 6:4497–4559.  https://doi.org/10.1039/C5PY00263J CrossRefGoogle Scholar
  20. Johansson K, El-Ahmad M, Friemann R, Jörnvall H, Markovic O, Eklund H (2002) Crystal structure of plant pectin methylesterase. FEBS Lett 514:243–249.  https://doi.org/10.1016/S0014-5793(02)02372-4 CrossRefGoogle Scholar
  21. Johnson E (2016) Integrated enzyme production lowers the cost of cellulosic ethanol. Biofuels Bioprod Biorefin 10:164–174.  https://doi.org/10.1002/bbb.1634 CrossRefGoogle Scholar
  22. Kabel MA, den Borne H, Vincken JP, Voragen AGJ, Schols HA (2007) Structural differences of xylans affect their interaction with cellulose. Carbohydr Polym 69:94–105.  https://doi.org/10.1016/j.carbpol.2006.09.006 CrossRefGoogle Scholar
  23. Kalim B, Böhringer N, Ali N, Schäberle TF (2015) Xylanases–from microbial origin to industrial application. Br Biotechnol J 7:1–20.  https://doi.org/10.9734/BBJ/2015/15982 CrossRefGoogle Scholar
  24. Keegstra K (2010) Plant cell walls. Plant Physiol 154:483–486.  https://doi.org/10.1104/pp.110.161240 CrossRefGoogle Scholar
  25. Konarev P, Svergun D (2015) A posteriori determination of the useful data range for small-angle scattering experiments on dilute monodisperse systems. IUCrJ 2:352–360.  https://doi.org/10.1107/S2052252515005163 CrossRefGoogle Scholar
  26. Kont R, Kurašin M, Teugjas H, Väljamäe P (2013) Strong cellulase inhibitors from the hydrothermal pretreatment of wheat straw. Biotechnol Biofuels 6:135.  https://doi.org/10.1186/1754-6834-6-135 CrossRefGoogle Scholar
  27. Kozak M (2006) Solution scattering studies of conformation stability of xylanase XYNII from Trichoderma longibrachiatum. Biopolymers 83:95–102.  https://doi.org/10.1002/bip.20531 CrossRefGoogle Scholar
  28. Kozin M, Svergun D (2001) Automated matching of high- and low-resolution structural models. J Appl Crystallogr 34:33–41.  https://doi.org/10.1107/S0021889800014126 CrossRefGoogle Scholar
  29. Kumar G, Pushpa A, Prabha H (2012) A review on xylooligosaccharides. Int Res J Pharm 3:71–74Google Scholar
  30. Kumar R, Bhagia S, Smith MD, Petridis L, Ong RG, Cai CM, Mittal A, Himmel MH, Balan V, Dale BE, Ragauskas AJ, Smith JC, Wyman CE (2018) Cellulose–hemicellulose interactions at elevated temperatures increase cellulose recalcitrance to biological conversion. Green Chem 20:921–934.  https://doi.org/10.1039/c7gc03518g CrossRefGoogle Scholar
  31. Lombard V, Golaconda RH, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490–D495.  https://doi.org/10.1093/nar/gkt1178 CrossRefGoogle Scholar
  32. Mello BL, Alessi AM, McQueen-Mason S, Bruce NC, Polikarpov I (2016) Nutrient availability shapes the microbial community structure in sugarcane bagasse compost-derived consortia. Sci Rep 6:38781.  https://doi.org/10.1038/srep38781 CrossRefGoogle Scholar
  33. Mello BL, Alessi AM, Riaño-Pachón DM, deAzevedo ER, Guimarães FEG, Espirito-Santo MC, McQueen-Mason S, Bruce NC, Polikarpov I (2017) Targeted metatranscriptomics of compost-derived consortia reveals a GH11 exerting an unusual exo-1,4-β-xylanase activity. Biotechnol Biofuels 10:254.  https://doi.org/10.1186/s13068-017-0944-4 CrossRefGoogle Scholar
  34. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  35. Notredame C, Higgins DG, Heringa J (2000) T-coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302:205–217.  https://doi.org/10.1006/jmbi.2000.4042 CrossRefGoogle Scholar
  36. de Oliveira LC, da Silva VM, Colussi F, Cabral AD, de Oliveira NM, Squina FM, Garcia W (2015) Conformational changes in a hyperthermostable glycoside hydrolase: enzymatic activity is a consequence of the loop dynamics and protonation balance. PLoS One 10:e0118225.  https://doi.org/10.1371/journal.pone.0118225 CrossRefGoogle Scholar
  37. Paës G, Berrin JG, Beaugrand J (2012) GH11 xylanases: structure/function/properties relationships and applications. Biotechnol Adv 30:564–592.  https://doi.org/10.1016/j.biotechadv.2011.10.003 CrossRefGoogle Scholar
  38. Pauchet Y, Wilkinson P, Chauhan R, French-Constant RH (2010) Diversity of beetle genes encoding novel plant cell wall degrading enzymes. PLoS One 5:e15635.  https://doi.org/10.1371/journal.pone.0015635 CrossRefGoogle Scholar
  39. Pellegrini VOA, Bernardes A, Rezende CA, Polikarpov I (2018) Cellulose fiber size defines efficiency of enzymatic hydrolysis and impacts degree of synergy between endo- and exoglucanases. Cellulose 25:1865–1881.  https://doi.org/10.1007/s10570-018-1700-z CrossRefGoogle Scholar
  40. Perry J, Tainer J (2013) Developing advanced X-ray scattering methods combined with crystallography and computation. Methods 59:363–371.  https://doi.org/10.1016/j.ymeth.2013.01.005 CrossRefGoogle Scholar
  41. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786.  https://doi.org/10.1038/nmeth.1701 CrossRefGoogle Scholar
  42. Polizeli ML, Rizzatti AC, Monti R, Terenzi HF, Jorge JA, Amorim DS (2005) Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 67:577–591.  https://doi.org/10.1007/s00253-005-1904-7 CrossRefGoogle Scholar
  43. Pollet A, Delcour JA, Courtin CM (2010) Structural determinants of the substrate specificities of xylanases from different glycoside hydrolase families. Crit Rev Biotechnol 30:176–191.  https://doi.org/10.3109/07388551003645599 CrossRefGoogle Scholar
  44. Rambo RP, Tainer JA (2011) Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers 95:559–571.  https://doi.org/10.1002/bip.21638 CrossRefGoogle Scholar
  45. Rittmann BE, Hausner M, Löffler F, Love NG, Muyzer G, Okabe S, Oerther DB, Peccia J, Raskin L, Wagner M (2006) A vista for microbial ecology and environmental biotechnology. Environ Sci Technol 40:1096–1103.  https://doi.org/10.1021/es062631k CrossRefGoogle Scholar
  46. Santo ME, Rezende CA, Bernardinelli OD, Pereira N, Curvelo AAS, Deazevedo ER, Guimarães FEG, Polikarpov I (2018) Structural and compositional changes in sugarcane bagasse subjected to hydrothermal and organosolv pretreatments and their impacts on enzymatic hydrolysis. Ind Crop Prod 113:64–74.  https://doi.org/10.1016/j.indcrop.2018.01.014 CrossRefGoogle Scholar
  47. Schomburg I, Jeske L, Ulbrich M, Placzek S, Chang A, Schomburg D (2017) The BRENDA enzyme information system-from a database to an expert system. J Biotechnol 261:194–206.  https://doi.org/10.1016/j.jbiotec.2017.04.020 CrossRefGoogle Scholar
  48. Silva COG, Vaz RP, Filho EXF (2018) Bringing plant cell wall degrading enzymes into the lignocellulosic biorefinery concept. Biofuels Bioprod Biorefin 12:277–289.  https://doi.org/10.1002/bbb.1832 CrossRefGoogle Scholar
  49. Sims RE, Mabee W, Saddler JN, Taylor M (2010) An overview of second generation biofuel technologies. Bioresour Technol 101:1570–1580.  https://doi.org/10.1016/j.biortech.2009.11.046 CrossRefGoogle Scholar
  50. Slabinski L, Jaroszewski L, Rychlewski L, Wilson IA, Lesley SA, Godzik A (2007) XtalPred: a web server for prediction of protein crystallizability. Bioinform 23:3403–3405.  https://doi.org/10.1093/bioinformatics/btm477 CrossRefGoogle Scholar
  51. Sun FF, Hong J, Hu J, Saddler JN, Fang X, Zhang Z, Shen S (2015) Accessory enzymes influence cellulase hydrolysis of the model substrate and the realistic lignocellulosic biomass. Enzym Microb Technol 79:42–48.  https://doi.org/10.1016/j.enzmictec.2015.06.020 CrossRefGoogle Scholar
  52. Suzuki M, Kato A, Nagata N, Komeda Y (2002) A xylanase, AtXyn1, is predominantly expressed in vascular bundles, and four putative xylanase genes were identified in the Arabidopsis thaliana genome. Plant Cell Physiol 43:759–767.  https://doi.org/10.1093/pcp/pcf088 CrossRefGoogle Scholar
  53. Svergun DI (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Crystallogr 25:495–503.  https://doi.org/10.1107/S0021889892001663 CrossRefGoogle Scholar
  54. Svergun DI, Barberato C, Koch M (1995) CRYSOL - A program to evaluate x-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Crystallogr 28:768–773.  https://doi.org/10.1107/S0021889895007047 CrossRefGoogle Scholar
  55. Väljamäe P, Sild V, Nutt A, Pettersson G, Johansson G (1999) Acid hydrolysis of bacterial cellulose reveals different modes of synergistic action between cellobiohydrolase I and endoglucanase I. Eur J Biochem 266:327–334.  https://doi.org/10.1046/j.1432-1327.1999.00853.x CrossRefGoogle Scholar
  56. Vázquez MJ, Alonso JL, Domínguez H, Parajó JC (2000) Xylooligosaccharides: manufacture and applications. Trends Food Sci Technol 11:387–393.  https://doi.org/10.1016/S0924-2244(01)00031-0 CrossRefGoogle Scholar
  57. Volkov V, Svergun D (2003) Uniqueness of ab initio shape determination in small-angle scattering. J Appl Crystallogr 36:860–864.  https://doi.org/10.1107/S0021889803000268 CrossRefGoogle Scholar
  58. Walia A, Guleria S, Mehta P, Chauhan A, Parkash J (2017) Microbial xylanases and their industrial application in pulp and paper biobleaching: a review. 3 Biotech 7:11–22.  https://doi.org/10.1007/s13205-016-0584-6 CrossRefGoogle Scholar
  59. Watanabe H, Tokuda G (2001) Animal cellulases. Cell Mol Life Sci 58:1167–1178.  https://doi.org/10.1007/PL00000931 CrossRefGoogle Scholar
  60. Wilkins M, Gasteiger E, Bairoch AM, Sanchez JE, Williams K, Appel RD, Hochstrasser D (1999) Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 112:531–552Google Scholar
  61. Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y (2015) The I-TASSER Suite: protein structure and function prediction. Nat Methods 12:7–8.  https://doi.org/10.1038/nmeth.3213 CrossRefGoogle Scholar
  62. Zhang J, Tuomainen P, Siika-aho M, Viikari L (2011) Comparison of the synergistic action of two thermostable xylanases from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis. Bioresour Technol 102:9090–9095.  https://doi.org/10.1016/j.biortech.2011.06.085 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Instituto de Física de São CarlosUniversidade de São PauloSão CarlosBrazil
  2. 2.Department of BiologyUniversity of YorkYorkUK

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