Journal of Polymers and the Environment

, Volume 27, Issue 12, pp 2895–2905 | Cite as

An Eco-friendly Method of Purification for Xylanase from Aspergillus niger by Polyelectrolyte Precipitation

  • María Victoria Podestá
  • Esteban Amador Morilla
  • María Belén Allasia
  • Nadia Woitovich Valetti
  • Gisela Tubio
  • María Julia BoggioneEmail author
Original paper


The separation of xylanase (Xyl) from a fungal extract obtained by submerged fermentation (SmF) was carried out using precipitation with natural polyelectrolytes ɩ-carrageenan (Carr) and chitosan (CHS). Xyl activity determined from the fungal extract at different times was modeled by mixed linear model for longitudinal data considering the factors as fixed effects and the experimental units as random effects. A spline regression was used to model the Xyl activity with knots at days 2 and 3 and interaction between three factors (p < 0.0001 in each section). High purification factors were reached with both polymers. Mass spectrometry analysis and zymogram analysis revealed that the Xyl that precipitated with both polymers had a molecular weight of 24 kDa and an isoelectric point (pI) of 5.45. The purification factor (PF) of Xyl in the precipitate of the fungal extract from SmF with 0.05% w/v CHS (pH 8.00) was around 5.6 for a mass ratio of 1.8 × 10−5 mg CHS/mg protein and with 0.5% w/v Carr (pH 7.00) it was 9.0 for a mass ratio of 0.03 mg Carr/mg protein. These results make this precipitation method suitable to be included in a downstream process, providing such advantages as simplicity, scalability, ability to concentrate the enzyme in a single step process as well as the use non-toxic reagents. Taking into account that the process of purification of macromolecules is a decisive step with a profound impact on their final market cost, the developed methodology has a high potential to be used in the purification of Xyl.


Xylanase Polyelectrolyte Carrageenan Chitosan 



This work was supported by the Grants from Agencia Nacional de Promoción Científica y Tecnológica (PICT 2016–1170 and 1712/17), from CONICET (PIP 505/15) and from the Secretary of Science and Technology of National University of Rosario (1BIO589 and BIO520). Authors would like to thank to Antonela Taddia for her colaboration and the Staff from the English Department (Faculty of Biochemical and Pharmaceutical Sciences, National University of Rosario), for correcting the language of this manuscript.


  1. 1.
    Selinheimo E, Kruus K, Buchert J, Hopia A, Autio K (2006) Effects of laccase, xylanase and their combination on the rheological properties of wheat doughs. J Cereal Sci 43:152–159Google Scholar
  2. 2.
    Song W, Zhang K, Chen Z, Hong G, Lin J, Hao C, Zhang S (2018) Effect of xylanase-laccase synergistic pretreatment on physical-mechanical properties of environment-friendly self-bonded bamboo particleboards. J Polym Environ 26:4019–4033Google Scholar
  3. 3.
    Chen L, Wang Z, Zhang B, Ge M, Ng H, Niu Y, Liu L (2019) Production, structure and morphology of exopolysaccharides yielded by submerged fermentation of Antrodia cinnamomea. Carbohyd Polym 205:271–278Google Scholar
  4. 4.
    Zhang J, Pan J, Guan G, Li Y, Xue W, Tang G, Wang A, Wang H (2008) Expression and high-yield production of extremely thermostable bacterial xylanaseB in Aspergillus niger. Enzyme Microb Technol 43:513–516Google Scholar
  5. 5.
    Heinen P, Bauermeister A, Ribeiro L, Messias J, Almeida P, Moraes L, Vargas-Rechia C, de Oliveira A, Ward R, Kadowaki M (2018) GH11 xylanase from Aspergillus tamarii Kita: purification by one-step chromatography and xylooligosaccharides hydrolysis monitored in real-time by mass spectrometry. Int J Biol Macromol 108:291–299PubMedGoogle Scholar
  6. 6.
    Mussatto SI, Dragone G, Roberto IC (2006) Brewers' spent grain: generation, characteristics and potential applications. J Cereal Sci 43:1–14Google Scholar
  7. 7.
    Raj A, Kumar S, Singh SK, Prakash J (2018) Production and purification of xylanase from alkaliphilic Bacillus licheniformis and its pretreatment of eucalyptus kraft pulp. Biocatal Agric Biotechnol 15:199–209Google Scholar
  8. 8.
    Kumar PS, Yaashikaa P, Saravanan A (2018) Isolation, characterization and purification of xylanase producing bacteria from sea sediment. Biocatal Agric Biotechnol 13:299–303Google Scholar
  9. 9.
    Mohamed MA, Ghanem MM, Abd-Elaziz AM, Shams-Eldin IM (2018) Purification and characterization of xylanase isoenzymes from red palm weevil Rhynchophorus ferrugineus. Biocatal Agric Biotechnol 14:321–327Google Scholar
  10. 10.
    Ding C, Li M, Hu Y (2018) High-activity production of xylanase by Pichia stipitis: Purification, characterization, kinetic evaluation and xylooligosaccharides production. Int J Biol Macromol 117:72–77PubMedGoogle Scholar
  11. 11.
    Valetti NW, Lombardi J, Boeris V, Picó G (2012) Precipitation of chymotrypsin from fresh bovine pancreas using ι-carrageenan. Process Biochem 47:2570–2574Google Scholar
  12. 12.
    Valetti NW, Picó G (2013) A friendly method for Raphanus sativus L (wild radish) peroxidase purification by polyelectrolyte precipitation. Sep Purif Technol 119:1–6Google Scholar
  13. 13.
    Boggione MJ, Becher R, Farruggia B (2016) Single method of purification for endoglucanase from Aspergillus niger by polyelectrolyte precipitation. Biocatal Agric Biotechnol 7:118–126Google Scholar
  14. 14.
    Begum ERA, Rajaiah S, Bhavani K, Devi M, Karthika K, Priya CG (2017) Evaluation of extracted chitosan from Portunus pelagicus for the preparation of chitosan alginate blend scaffolds. J Polym Environ 25:578–585Google Scholar
  15. 15.
    Dayarian S, Zamani A, Moheb A, Masoomi M (2014) Physico-mechanical properties of films of chitosan, carboxymethyl chitosan, and their blends. J Polym Environ 22:409–416Google Scholar
  16. 16.
    Sila A, Mlaik N, Sayari N, Balti R, Bougatef A (2014) Chitin and chitosan extracted from shrimp waste using fish proteases aided process: efficiency of chitosan in the treatment of unhairing effluents. J Polym Environ 22:78–87Google Scholar
  17. 17.
    Muraleedharan K, Alikutty P, Mujeeb VA, Sarada K (2015) Kinetic studies on the thermal dehydration and degradation of chitosan and citralidene chitosan. J Polym Environ 23:1–10Google Scholar
  18. 18.
    Fabian C, Huynh L, Ju Y (2010) Precipitation of rice bran protein using carrageenan and alginate. LWT-Food Sci Technol 43:375–379Google Scholar
  19. 19.
    Roy I, Gupta MN (2003) κ-Carrageenan as a new smart macroaffinity ligand for the purification of pullulanase. J Chromatogr A 998:103–108PubMedGoogle Scholar
  20. 20.
    Van-Soest PJ (1983) Nutritional ecology of the ruminants. Corvallis, OregonGoogle Scholar
  21. 21.
    Pinheiro J, Bates D, DebRoy S, Sarkar D, Team RC (2007) Linear and nonlinear mixed effects models. R Package Version 3:1–89Google Scholar
  22. 22.
    Team RC (2017) R: a language and environment for statistical computing. R Foundation for Statistical Computing; 2017. Vienna, Austria. https://www.R-project. org.Google Scholar
  23. 23.
    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428Google Scholar
  24. 24.
    Brown RE, Jarvis KL, Hyland KJ (1989) Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal Biochem 180:136–139PubMedGoogle Scholar
  25. 25.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680Google Scholar
  26. 26.
    Chávez R, Schachter K, Navarro C, Peirano A, Aguirre C, Bull P, Eyzaguirre J (2002) Differences in expression of two endoxylanase genes (xynA and xynB) from Penicillium purpurogenum. Gene 293:161–168PubMedGoogle Scholar
  27. 27.
    Santos M, Jiménez J, Bartolomé B, Gómez-Cordovés C, Del Nozal M (2003) Variability of brewer’s spent grain within a brewery. Food Chem 80:17–21Google Scholar
  28. 28.
    Mussatto SI, Fernandes M, Dragone G, Mancilha IM, Roberto IC (2007) Brewer’s spent grain as raw material for lactic acid production by Lactobacillus delbrueckii. Biotech Lett 29:1973–1976Google Scholar
  29. 29.
    Valetti NW, Boeris V, Picó G (2013) Characterization of chymotrypsin–ι-carrageenan complex in aqueous solution: a solubility and thermodynamical stability study. Int J Biol Macromol 52:45–51PubMedGoogle Scholar
  30. 30.
    Bedade D, Berezina O, Singhal R, Deska J, Shamekh S (2017) Extracellular xylanase production from a new xylanase producer Tuber maculatum mycelium under submerged fermentation and its characterization. Biocatal Agric Biotechnol 11:288–293Google Scholar
  31. 31.
    Terrone CC, de Freitas C, Terrasan CRF, de Almeida AF, Carmona EC (2018) Agroindustrial biomass for xylanase production by Penicillium chrysogenum: purification, biochemical properties and hydrolysis of hemicelluloses. Electron J Biotechnol 33:39–45Google Scholar
  32. 32.
    Lu X, Sun J, Nimtz M, Wissing J, Zeng A-P, Rinas U (2010) The intra-and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate. Microb Cell Fact 9:23PubMedPubMedCentralGoogle Scholar
  33. 33.
    Shi C, He J, Yu J, Yu B, Mao X, Zheng P, Huang Z, Chen D (2016) Physicochemical properties analysis and secretome of Aspergillus niger in fermented rapeseed meal. PLoS ONE 11:e0153230PubMedPubMedCentralGoogle Scholar
  34. 34.
    Cooper C, Dubin P, Kayitmazer A, Turksen S (2005) Polyelectrolyte–protein complexes. Curr Opin Colloid Interface Sci 10:52–78Google Scholar
  35. 35.
    Deng P, Li D, Cao Y, Lu W, Wang C (2006) Cloning of a gene encoding an acidophilic endo-β-1, 4-xylanase obtained from Aspergillus niger CGMCC1067 and constitutive expression in Pichia pastoris. Enzyme Microbial Technol 39:1096–1102Google Scholar
  36. 36.
    Chang PG, Gupta R, Timilsena YP, Adhikari B (2016) Optimisation of the complex coacervation between canola protein isolate and chitosan. J Food Eng 191:58–66Google Scholar
  37. 37.
    Schmitt C, Turgeon SL (2011) Protein/polysaccharide complexes and coacervates in food systems. Adv Coll Interface Sci 167:63–70Google Scholar
  38. 38.
    Ahmed KF, Aschi A, Nicolai T (2018) Formation and characterization of chitosan-protein particles with fractal whey protein aggregates. Colloids Surf B 169:257–264Google Scholar
  39. 39.
    Li G, Huang J, Chen T, Wang X, Zhang H, Chen Q (2017) Insight into the interaction between chitosan and bovine serum albumin. Carbohyd Polym 176:75–82Google Scholar
  40. 40.
    Gupta M, Guoqiang D, Kaul R, Mattiasson B (1994) Purification of xylanase from Trichoderma viride by precipitation with an anionic polymer Eudragit S 100. Biotechnol Tech 8:117–122Google Scholar
  41. 41.
    Gawande P, Kamat M (1999) Purification of Aspergillus sp xylanase by precipitation with an anionic polymer Eudragit S100. Process Biochem 34:577–580Google Scholar
  42. 42.
    Lali A, Aruna N, John R, Thakrar D (2000) Reversible precipitation of proteins on carboxymethyl cellulose. Process Biochem 35:777–785Google Scholar
  43. 43.
    Khanahmadi M, Arezi I, Amiri M-s, Miranzadeh M (2018) Bioprocessing of agro-industrial residues for optimization of xylanase production by solid-state fermentation in flask and tray bioreactor. Biocatal Agric Biotechnol 13:272–282Google Scholar
  44. 44.
    Karunakaran S, Saravanan A, Dhanasekaran S, Senbagam D, Senthil B (2014) Xylanase production from Aspergillus niger. Int J Chem Technol Res 6:4207–4211Google Scholar
  45. 45.
    Do TT, Quyen DT, Nguyen TN (2013) Molecular characterization of a glycosyl hydrolase family 10 xylanase from Aspergillus niger. Protein Expr Purif 92:196–202PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.IPROByQ-CONICET, Faculty of Biochemical and Pharmaceutical SciencesNational University of RosarioRosarioArgentina
  2. 2.Area of Statistics and Data Processing, Faculty of Biochemical and Pharmaceutical SciencesNational University of RosarioRosarioArgentina

Personalised recommendations