Skip to main content
SpringerLink
Log in

Fast automated online xylanase activity assay using HPAEC-PAD

  • Paper in Forefront
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

In contrast to biochemical reactions, which are often carried out under automatic control and maintained overnight, the automation of chemical analysis is usually neglected. Samples are either analyzed in a rudimentary fashion using in situ techniques, or aliquots are withdrawn and stored to facilitate more precise offline measurements, which can result in sampling and storage errors. Therefore, in this study, we implemented automated reaction control, sampling, and analysis. As an example, the activities of xylanases on xylotetraose and soluble xylan were examined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The reaction was performed in HPLC vials inside a temperature-controlled Dionex™ AS-AP autosampler. It was started automatically when the autosampler pipetted substrate and enzyme solution into the reaction vial. Afterwards, samples from the reaction vial were injected repeatedly for 60 min onto a CarboPac™ PA100 column for analysis. Due to the rapidity of the reaction, the analytical method and the gradient elution of 200 mM sodium hydroxide solution and 100 mM sodium hydroxide with 500 mM sodium acetate were adapted to allow for an overall separation time of 13 min and a detection limit of 0.35–1.83 mg/L (depending on the xylooligomer). This analytical method was applied to measure the soluble short-chain products (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and longer xylooligomers) that arise during enzymatic hydrolysis. Based on that, the activities of three endoxylanases (EX) were determined as 294 U/mg for EX from Aspergillus niger, 1.69 U/mg for EX from Bacillus stearothermophilus, and 0.36 U/mg for EX from Bacillus subtilis.

Xylanase activity assay automation

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1a–d
Fig. 2
Fig. 3a–d
Fig. 4a–d
Fig. 5a–c
Fig. 6a–b

Similar content being viewed by others

References

  1. Zhao X, Zhang L, Liu D. Biomass recalcitrance. Part I: the chemical compositions and physical structures affecting the enzymatic hydrolysis of lignocellulose. Biofuels Bioprod Biorefin. 2012;6(4):465–82. https://doi.org/10.1002/bbb.1331.

    Article  CAS  Google Scholar 

  2. Viell J, Inouye H, Szekely NK, Frielinghaus H, Marks C, Wang Y, et al. Multi-scale processes of beech wood disintegration and pretreatment with 1-ethyl-3-methylimidazolium acetate/water mixtures. Biotechnol Biofuels. 2016;9:7. https://doi.org/10.1186/s13068-015-0422-9.

  3. Yang B, Dai Z, Ding SY, Wyman CE. Enzymatic hydrolysis of cellulosic biomass. Biofuels. 2011;2:421–49. https://doi.org/10.4155/bfs.11.116.

    Article  CAS  Google Scholar 

  4. Klein-Marcuschamer D, Oleskowicz-Popiel P, Simmons BA, Blanch HW. The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnol Bioeng. 2012;109(4):1083–7. https://doi.org/10.1002/bit.24370.

    Article  CAS  Google Scholar 

  5. Diogo JA, Hoffmam ZB, Zanphorlin LM, Cota J, Machado CB, Wolf LD, et al. Development of a chimeric hemicellulase to enhance the xylose production and thermotolerance. Enzym Microb Technol. 2015;69:31–7. https://doi.org/10.1016/j.enzmictec.2014.11.006.

  6. Nomenclature Committee of the International Union of Biochemistry. Units of enzyme activity. Eur J Biochem. 1979;97:319–20. https://doi.org/10.1111/j.1432-1033.1979.tb13116.x.

  7. Lever M. Colorimetric and fluorometric carbohydrate determination with p-hydroxybenzoic acid hydrazide. Biochem Med. 1973;7:274–81. https://doi.org/10.1016/0006-2944(73)90083-5.

  8. Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem. 1959;31:426–8. https://doi.org/10.1021/ac60147a030.

    Article  CAS  Google Scholar 

  9. König J, Grasser R, Pikor H, Vogel K. Determination of xylanase, beta-glucanase, and cellulase activity. Anal Bioanal Chem. 2002;374(1):80–7. https://doi.org/10.1007/s00216-002-1379-7.

    Article  Google Scholar 

  10. Decker SR, Adney WS, Jennings E, Vinzant TB, Himmel ME. Automated filter paper assay for determination of cellulase activity. Appl Biochem Biotechnol. 2003;105-108:689–703. https://doi.org/10.1385/ABAB.

    Article  CAS  Google Scholar 

  11. Bailey MJ, Biely P, Poutanen K. Interlaboratory testing of methods for assay of xylanase activity. J Biotechnol. 1992;23(3):257–70. https://doi.org/10.1016/0168-1656(92)90074-J.

    Article  CAS  Google Scholar 

  12. Anders N, Schelden M, Simon R, Spiess AC. Automated chromatographic laccase-mediator system activity assay. Anal Bioanal Chem. 2016;409(20):4801–9. https://doi.org/10.1007/s00216-017-0423-6.

    Article  Google Scholar 

  13. Dionex Corporation. Product manual for Dionex CarboPacTM MA1, PA1, PA10 and PA100 columns 031824–08. http://tools.thermofisher.com/content/sfs/manuals/4375-Man-031824-08-CarboPac-Combined-May10.pdf. Accessed 9 May 2017.

  14. He XS, Shyu YT, Nathoo S, Wong SL, Doi RH. Construction and use of a Bacillus subtilis mutant deficient in multiple protease genes for the expression of eukaryotic genes. Ann N Y Acad Sci. 1991;27(646):69–77. https://doi.org/10.1111/j.1749-6632.1991.tb18565.x.

  15. Bähr C, Leuchtle B, Lehmann C, Becker J, Jeude M, Peinemann F, et al. Dialysis shake flask for effective screening in fed-batch mode. Biochem Eng J. 2012;69:182–95. https://doi.org/10.1016/j.bej.2012.08.012.

  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(1–2):248–54. https://doi.org/10.1016/0003-2697(76)90527-3.

    Article  CAS  Google Scholar 

  17. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, et al. Measurement of protein using bicinchoninic acid. Anal Chem. 1985;150:76–85.

  18. Ghose TK, Bisaria VS. Measurement of hemicellulase activities—part 1: xylanases. Pure Appl Chem. 1987;59(12):1739–52. https://doi.org/10.1351/pac198759020257.

  19. Anders N, Humann H, Langhans B, Spiess AC. Simultaneous determination of acid-soluble biomass-derived compounds using high performance anion exchange chromatography coupled with pulsed amperometric detection. Anal Methods. 2015;7:7866–73. https://doi.org/10.1039/c5ay01371b.

    Article  CAS  Google Scholar 

  20. Épshtein N. Structure of chemical compounds, methods of analysis and process control: validation of HPLC techniques. Pharm Chem J. 2004;38:212–28.

    Article  Google Scholar 

  21. Courtin CM, Swennen K, Verjans P, Delcour JA. Heat and pH stability of prebiotic arabinoxylooligosaccharides, xylooligosaccharides and fructooligosaccharides. Food Chem. 2009;112(4):831–7. https://doi.org/10.1016/j.foodchem.2008.06.039.

    Article  CAS  Google Scholar 

  22. Oefner PJ, Lanziner AH, Bonn G, Bobleter O. Quantitative studies on furfural and organic-acid formation during hydrothermal, acidic and alkaline-degradation of deuterium-xylose. Monatsh Chem. 1992;123(6–7):547–56. https://doi.org/10.1007/BF00816848.

    Article  CAS  Google Scholar 

  23. Baumann MJ, Murphy L, Lei N, Krogh KBRM, Borch K, Westh P. Advantages of isothermal titration calorimetry for xylanase kinetics in comparison to chemical-reducing-end assays. Anal Biochem. 2011;410(1):19–26. https://doi.org/10.1016/j.ab.2010.11.001.

    Article  CAS  Google Scholar 

  24. Kumar P, Barrett DM, Delwiche MJ, Stroeve P. Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind Eng Chem. 2009;48(8):3713–29. https://doi.org/10.1021/ie801542g.

    Article  CAS  Google Scholar 

  25. Subramaniyan S, Prema P. Biotechnology of microbial xylanases: enzymology, molecular biology, and application. Crit Rev Biotechnol. 2002;22(1):33–64. https://doi.org/10.1080/07388550290789450.

Download references

Acknowledgments

This work was performed as part of the Cluster of Excellence “Tailor-Made Fuels from Biomass,” which is funded by the Excellence Initiative of the German federal and state governments to promote science and research at German universities. The scientific activities of the Bioeconomy Science Center were financially supported by the Ministry of Innovation, Science, and Research within the framework of the NRW Strategieprojekt BioSC (no. 313/323-400-002 13). Specials thanks go to Priya Philip of the Institute for Biochemical Engineering of RWTH Aachen University for supplying the RAMOS fed-batch technique.

Author information

Authors and Affiliations

  1. Aachener Verfahrenstechnik—Enzyme Process Technology, RWTH Aachen University, Worringer Weg 1, 52074, Aachen, Germany

    Christin Cürten, Nico Anders, Niels Juchem & Antje C. Spiess

  2. Bioeconomy Science Center (BioSC), c/o Forschungszentrum Jülich, D-52425, Jülich, Germany

    Christin Cürten, Nico Anders, Niels Juchem, Nina Ihling, Kristina Volkenborn, Andreas Knapp, Karl-Erich Jaeger, Jochen Büchs & Antje C. Spiess

  3. Aachener Verfahrenstechnik—Biochemical Engineering, RWTH Aachen University, D-52074, Aachen, Germany

    Nina Ihling & Jochen Büchs

  4. Institute of Molecular Enzyme Technology, Heinrich Heine University Düsseldorf, D-40225, Düsseldorf, Germany

    Kristina Volkenborn, Andreas Knapp & Karl-Erich Jaeger

  5. Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, D-52425, Jülich, Germany

    Karl-Erich Jaeger

  6. Institute of Biochemical Engineering, TU Braunschweig, Rebenring 56, D-38106, Braunschweig, Germany

    Antje C. Spiess

Authors
  1. Christin Cürten
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nico Anders
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Niels Juchem
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nina Ihling
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kristina Volkenborn
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Andreas Knapp
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Karl-Erich Jaeger
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jochen Büchs
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Antje C. Spiess
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nico Anders.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

ESM 1

(PDF 382 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cürten, C., Anders, N., Juchem, N. et al. Fast automated online xylanase activity assay using HPAEC-PAD. Anal Bioanal Chem 410, 57–69 (2018). https://doi.org/10.1007/s00216-017-0712-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-017-0712-0

Keywords

Search

Navigation