Skip to main content

Advertisement

SpringerLink
Log in

EcXyl43 β-xylosidase: molecular modeling, activity on natural and artificial substrates, and synergism with endoxylanases for lignocellulose deconstruction

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Biomass hydrolysis constitutes a bottleneck for the biotransformation of lignocellulosic residues into bioethanol and high-value products. The efficient deconstruction of polysaccharides to fermentable sugars requires multiple enzymes acting concertedly. GH43 β-xylosidases are among the most interesting enzymes involved in hemicellulose deconstruction into xylose. In this work, the structural and functional properties of β-xylosidase EcXyl43 from Enterobacter sp. were thoroughly characterized. Molecular modeling suggested a 3D structure formed by a conserved N-terminal catalytic domain linked to an ancillary C-terminal domain. Both domains resulted essential for enzymatic activity, and the role of critical residues, from the catalytic and the ancillary modules, was confirmed by mutagenesis. EcXyl43 presented β-xylosidase activity towards natural and artificial substrates while arabinofuranosidase activity was only detected on nitrophenyl α-L-arabinofuranoside (pNPA). It hydrolyzed xylobiose and purified xylooligosaccharides (XOS), up to degree of polymerization 6, with higher activity towards longer XOS. Low levels of activity on commercial xylan were also observed, mainly on the soluble fraction. The addition of EcXyl43 to GH10 and GH11 endoxylanases increased the release of xylose from xylan and pre-treated wheat straw. Additionally, EcXyl43 exhibited high efficiency and thermal stability under its optimal conditions (40 °C, pH 6.5), with a half-life of 58 h. Therefore, this enzyme could be a suitable additive for hemicellulases in long-term hydrolysis reactions. Because of its moderate inhibition by monomeric sugars but its high inhibition by ethanol, EcXyl43 could be particularly more useful in separate hydrolysis and fermentation (SHF) than in simultaneous saccharification and co-fermentation (SSCF) or consolidated bioprocessing (CBP).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Brunzelle JS, Jordan DB, McCaslin DR, Olczak A, Wawrzak Z (2008) Structure of the two-subsite β-D-xylosidase from Selenomonas ruminantium in complex with 1,3 bis[tris(hydroxymethyl)methylamino]propane. Arch Biochem Bioph 474:157–166

    Article  CAS  Google Scholar 

  • Brüx C, Ben-David A, Shallom-Shezifi D, Leon M, Niefind K, Shoham G, Shoham Y, Schomburg D (2006) The structure of an inverting GH43 β-xylosidase from Geobacillus stearothermophilus with its substrate reveals the role of the three catalytic residues. J Mol Biol 359(1):97–109

    Article  PubMed  CAS  Google Scholar 

  • Campos E, Negro Alvarez M, di Lorenzo G, Gonzalez S, Rorig M, Talia P, Grasso D, Sáez F, Manzanares Secades P, Ballesteros Perdices M, Cataldi A (2014) Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria. Microbiol Res 169(2–3):213–220

    Article  PubMed  CAS  Google Scholar 

  • Diogo JA, Hoffmam ZB, Zanphorlin LM, Cota J, Machado CB, Wolf LD, Squina F, Dámasio A, Murakami M, Ruller R (2014) Development of a chimeric hemicellulase to enhance the xylose production and thermotolerance. Enzym Microb Technol 69:31–37

    Article  CAS  Google Scholar 

  • Dodd D, Cann IK (2009) Enzymatic deconstruction of xylan for biofuel production. GCB Bioenergy 1(1):2–17

    Article  PubMed  CAS  Google Scholar 

  • Falck P, Linares-Pastén JA, Adlercreutz P, Karlsson EN (2016) Characterization of a family 43 β-xylosidase from the xylooligosaccharide utilizing putative probiotic Weissella sp. strain 92. Glycobiology 26(2):193–202

    Article  PubMed  CAS  Google Scholar 

  • Fan Z, Yuan L, Jordan DB, Wagschal K, Heng C, Braker JD (2010) Engineering lower inhibitor affinities in β-D-xylosidase. Appl Microbiol Biotechnol 86(4):1099–1113

  • Ghio S, Ontañon OM, Piccinni FE, Marrero R, Talia P, Grasso DH, Campos E (2018) Paenibacillus sp. A59 GH10 and GH11 extracellular Endoxylanases: application in biomass bioconversion. BioEnergy Res 11:174–190

    Article  CAS  Google Scholar 

  • Gírio FM, Fonseca C, Carvalheiro F, Duarte LC, Marques S, Bogel-Łukasik R (2010) Hemicelluloses for fuel ethanol: a review. Bioresour Technol 101(13):4775–4800

    Article  PubMed  CAS  Google Scholar 

  • Jordan DB, Li XL (2007) Variation in relative substrate specificity of bifunctional β-d-xylosidase/α-l-arabinofuranosidase by single-site mutations: roles of substrate distortion and recognition. Biochim Biophys Acta 1774(9):1192–1198

    Article  PubMed  CAS  Google Scholar 

  • Jordan DB, Wagschal K, Grigorescu AA, Braker JD (2013) Highly active β-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates. Appl Microbiol Biotechnol 97(10):4415–4428

    Article  PubMed  CAS  Google Scholar 

  • Jørgensen H, Pinelo M (2017) Enzyme recycling in lignocellulosic biorefineries. Biofuels Bioprod Biorefin 11(1):150–167

    Article  CAS  Google Scholar 

  • Lagaert S, Pollet A, Delcour JA, Lavigne R, Courtin CM, Volckaert G (2011) Characterization of two β-xylosidases from Bifidobacterium adolescentis and their contribution to the hydrolysis of prebiotic xylooligosaccharides. Appl Microbiol Biotechnol 92(6):1179–1185

    Article  PubMed  CAS  Google Scholar 

  • Lagaert S, Pollet A, Courtin CM, Volckaert G (2014) β-Xylosidases and α-L-arabinofuranosidases: accessory enzymes for arabinoxylan degradation. Biotechnol Adv 32(2):316–332

    Article  PubMed  CAS  Google Scholar 

  • Lee CC, Braker JD, Grigorescu AA, Wagschal K, Jordan DB (2013) Divalent metal activation of a GH43 β-xylosidase. Enzym Microb Technol 52(2):84–90

    Article  CAS  Google Scholar 

  • Miller GL, Blum R, Glennon WE, Burton AL (1960) Measurement of carboxymethylcellulase activity. Anal Biochem 1(2):127–132

    Article  CAS  Google Scholar 

  • Morais S, Salama-Alber O, Barak Y, Hadar Y, Wilson DB, Lamed R, Bayer EA (2012) Functional association of catalytic and ancillary modules dictates enzymatic activity in glycoside hydrolase family 43 β-xylosidase. J Biol Chem 287(12):9213–9221

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Nurizzo D, Nagy T, Gilbert HJ, Davies GJ (2002) The structural basis for catalysis and specificity of the Pseudomonas cellulosa alpha-glucuronidase GlcA67A. Structure 10(4):547–556

    Article  PubMed  CAS  Google Scholar 

  • Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF chimera-a visualization system for exploratory research and analysis. J Comput Chem 25(13):1605–1612

    Article  PubMed  CAS  Google Scholar 

  • Proctor MR, Taylor EJ Nurizzo D, Turkenburg JP, Lloyd RM, Vardakou M, Davies GJ, Gilbert HJ (2005) Tailored catalysts for plant cell-wall degradation: redesigning the exo/endo preference of Cellvibrio japonicus arabinanase 43A. Proc Natl Acad Sci U S A 102(8):2697–2702

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5(4):725–738

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Saha BC (2003) Purification and properties of an extracellular β-xylosidase from a newly isolated Fusarium proliferatum. Bioresour Technol 90(1):33–38

    Article  PubMed  CAS  Google Scholar 

  • Selig MJ, Knoshaug EP, Adney WS, Himmel ME, Decker SR (2008) Synergistic enhancement of cellobiohydrolase performance on pretreated corn Stover by addition of xylanase and esterase activities. Bioresour Technol 99(11):4997–5005

    Article  PubMed  CAS  Google Scholar 

  • Shallom D, Leon M, Bravman T, Ben-David A, Zaide G, Belakhov V, Shoham G, Schomburg D, Baasov T, Shoham Y (2005) Biochemical characterization and identification of the catalytic residues of a family 43 β-D-xylosidase from Geobacillus stearothermophilus T-6. Biochemistry 44(1):387–397

    Article  PubMed  CAS  Google Scholar 

  • Singh SK, Heng C, Braker JD, Chan VJ, Lee CC, Jordan DB, Yuan L, Wagschal K (2014) Directed evolution of GH43 β-xylosidase XylBH43 thermal stability and L186 saturation mutagenesis. J Ind Microbiol Biotechnol 41(3):489–498

    Article  PubMed  CAS  Google Scholar 

  • Smaali I, Rémond C, O’Donohue MJ (2006) Expression in Escherichia coli and characterization of β-xylosidases GH39 and GH-43 from Bacillus halodurans C-125. Appl Microbiol Biotechnol 73(3):582–590

    Article  PubMed  CAS  Google Scholar 

  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12):2725–2729

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Umemoto Y, Onishi R, Araki T (2008) Cloning of a novel gene encoding β-1,3-xylosidase from a marine bacterium, Vibrio sp. strain XY-214, and characterization of the gene product. Appl Environ Microbiol 74:305–308

    Article  PubMed  CAS  Google Scholar 

  • Viborg AH, Sørensen KI, Gilad O, Steen-Jensen DB, Dilokpimol A, Jacobsen S, Svensson B (2013) Biochemical and kinetic characterisation of a novel xylooligosaccharide-upregulated GH43 β-D-xylosidase/α-L-arabinofuranosidase (BXA43) from the probiotic Bifidobacterium animalis subsp. lactis BB-12. AMB Express 3(1):56

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Wagschal K, Heng C, Lee CC, Wong DW (2009) Biochemical characterization of a novel dual-function arabinofuranosidase/xylosidase isolated from a compost starter mixture. Appl Microbiol Biotechnol 81(5):855–863

    Article  PubMed  CAS  Google Scholar 

  • Wagschal K, Jordan B, Braker JD (2012) Catalytic properties of β-D-xylosidase XylBH43 from Bacillus halodurans C-125 and mutant XylBH43-W147G. Process Biochem 47(3):366–372

    Article  CAS  Google Scholar 

  • Yang X, Shi P, Huang H, Luo H, Wang Y, Zhang W, Yao B (2014) Two xylose-tolerant GH43 bifunctional β-xylosidase/α-arabinosidases and one GH11 xylanase from Humicola insolens and their synergy in the degradation of xylan. Food Chem 148:381–387

    Article  PubMed  CAS  Google Scholar 

  • 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(1):7–8

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Yeoman CJ, Han Y, Dodd D, Schroeder CM, Mackie RI, Cann IK (2010) Thermostable enzymes as biocatalysts in the biofuel industry. Adv Appl Microbiol 70:1–55

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgments

Authors thank MINCYT and CONICET for financial support. OO and FP hold post-doctoral and doctoral fellowships from the Argentinean National Council of Research (CONICET). SG holds an INTA doctoral fellowship. EC, PT, and MC acknowledge CONICET as Research Career members and RM belongs to National Institute of Agriculture Technology (INTA). Pre-treated wheat straw was gently supplied by Dr. Mercedes Ballesteros from CIEMAT, Spain. We thank Igor Polikarpov from USP, Brazil, for critical reading of the manuscript.

Funding

This study was funded by projects MINCYT PICT2014-0791 and CONICET PIP2015-11220150100121CO.

Author information

Authors and Affiliations

  1. Instituto de Biotecnología, CICVyA, Instituto Nacional de Tecnología Agropecuaria (INTA), Dr. N. Repetto y Los Reseros s/n, Hurlingham (1686), Buenos Aires, Argentina

    Ornella M. Ontañon, Rubén Marrero Díaz de Villegas, Florencia E. Piccinni, Paola M. Talia & Eleonora Campos

  2. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina

    Ornella M. Ontañon, Florencia E. Piccinni, Paola M. Talia, María L. Cerutti & Eleonora Campos

  3. Instituto de Suelos, CIRN, Instituto Nacional de Tecnología Agropecuaria (INTA), Buenos Aires, Argentina

    Silvina Ghio

  4. Fundación Instituto Leloir, IIBBA-CONICET, Avenida Patricias Argentinas 435, C1405BWE, Buenos Aires, Argentina

    María L. Cerutti

Authors
  1. Ornella M. Ontañon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Silvina Ghio
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rubén Marrero Díaz de Villegas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Florencia E. Piccinni
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Paola M. Talia
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. María L. Cerutti
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Eleonora Campos
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Eleonora Campos.

Ethics declarations

Conflict of interest

Ornella Ontañon declares that she has no conflict of interest. Silvina Ghio declares that she has no conflict of interest. Rubén Marrero Díaz de Villegas declares that he has no conflict of interest. Florencia E Piccinni declares that she has no conflict of interest. Paola M Talia declares that she has no conflict of interest. María L Cerutti declares that she has no conflict of interest. Eleonora Campos declares that she has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors

Electronic supplementary material

ESM 1

(PDF 448 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ontañon, O.M., Ghio, S., Marrero Díaz de Villegas, R. et al. EcXyl43 β-xylosidase: molecular modeling, activity on natural and artificial substrates, and synergism with endoxylanases for lignocellulose deconstruction. Appl Microbiol Biotechnol 102, 6959–6971 (2018). https://doi.org/10.1007/s00253-018-9138-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-018-9138-7

Keywords

Search

Navigation