3 Biotech

, 8:369 | Cite as

Production and characterization of a thermostable antifungal chitinase secreted by the filamentous fungus Aspergillus niveus under submerged fermentation

  • Thaís Barboni Alves
  • Pedro Henrique de Oliveira Ornela
  • Arthur Henrique Cavalcanti de Oliveira
  • João Atílio Jorge
  • Luis Henrique Souza GuimarãesEmail author
Original Article


The filamentous fungus Aspergillus niveus produced extracellular antifungal chitinase when cultured under submerged fermentation (SbmF) using crab shells as the carbon source. Maximal chitinase production was achieved at 192 h of cultivation using minimal medium containing 1% chitin. The enzyme was purified 1.97-fold with 40% recovery by ammonium sulfate precipitation and Sephadex G-100 gel filtration. The molecular mass was estimated to be 44 kDa by both 12% SDS-PAGE and Sepharose CL-6B gel filtration. Maximal A. niveus chitinase activity was obtained at 65 °C and pH 5.0. The enzyme was fully stable at 60 °C for up to 120 min and the enzymatic activity was increased by Mn2+. In the presence of reducing and denaturing compounds, the enzyme activity was not drastically affected. The chitinase was able to hydrolyze colloidal chitin, azure chitin, and 4-nitrophenyl N-acetyl-β-D glucosaminide; for the latter, the K0.5 and maximal velocity (Vmax) were 3.51 mM and 9.68 U/mg of protein, respectively. The A. niveus chitinase presented antifungal activity against Aspergillus niger (MIC = 84 µg/mL), A. fumigatus (MIC = 21 µg/mL), A. flavus (MIC = 24 µg/mL), A. phoenicis (MIC = 24 µg/mL), and Paecilomyces variotii (MIC = 21 µg/mL). The fungus A. niveus was able to produce a thermostable and denaturation-resistant chitinase able to inhibit fungal development, signaling its biotechnological potential.


Aspergillus niveus Antifungal activity Chitin Chitinase Fungal hydrolases 



We thank Mauricio de Oliveira for technical assistance. The authors also kindly acknowledge the financial support from FAPESP (Process no. 2011/50880-1) and the research scholarships from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This manuscript is part of the doctoral thesis by T.B.A.

Author contributions

LHSG and TBA designed the study; TBA performed the experiments on chitinase production, purification, and characterization; PHOO performed the experiments on antifungal activity; LHSG, TBA, AHCO, and JAJ analyzed the results; LHSG and TBA wrote the manuscript.

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.


  1. Adams PR (1990) Mycelial amylase activities of thermophilic species of Rhizomucor, Humicola and Papulaspora. Mycopathologia 112:35–37. CrossRefGoogle Scholar
  2. Berini F, Presti I, Beltrametti F, Pedroli M, Varum KM, Pellegioni L, Sjöling S, Marinelli F (2017) Production and characterization of a novel antifungal chitinase identified by functional screening of a suppressive-soil metagenome. Microb Cell Fact 16:16. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Binod P, Pusztaheyi T, Nagy N, Sandhya C, Szakács G, Pócsi I, Pandey A (2005) Production and purification of extracellular chitinases from Penicillium aculeatum NRRL 2129 under solid-state fermentation. Enzyme Microbial Technol 36:880–887. CrossRefGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilization the principle of protein-dye binding. Anal Biochem 72:248–254. CrossRefGoogle Scholar
  5. Duong-Ly KC, Gabelli SB (2014) Salting out of proteins using ammonium sulfate precipitation. Methods Enzymol 541:85–94. CrossRefPubMedGoogle Scholar
  6. Farag MA, Al-Nusarie ST (2014) Production, optimization, characterization and antifungal activity of chitinase produced by Aspergillus terreus. Afr J Biotechnol 13:1567–1578. CrossRefGoogle Scholar
  7. Farag AM, Abd-Elnabey HM, Ibrahim HAH, El-Shenawy M (2016) Purification, characterization and antimicrobial activity of chitinase from marine derived Aspergillus terreus. Egypt J Aquat Res 42:185–192. CrossRefGoogle Scholar
  8. Gun Lee D, Shin SY, Maeng CY, Jin ZZ, Kim KL, Hahm K-S (1999) Isolation and characterization of a novel antifungal peptide from Aspergillus niger. Biochem Biophys Res Commun 263:646–651. CrossRefPubMedGoogle Scholar
  9. Guo R-F, Shi B-S, Li D-C, Ma W, Wei Q (2008) Purification and characterization of a novel thermostable chitinase from Thermomyces lanuginosus SY2 and cloning of its encoding gene. Agric Sci China 7(12):1458–1465CrossRefGoogle Scholar
  10. Hameed AAA, Ayesh AM, Mohamed MAR, Mawla HFA (2012) Fungi and some mycotoxins producing species in the air of soybean and cotton mills: a case study. Atmos Pollut Res 3:126–131CrossRefGoogle Scholar
  11. Hill TW, Kafer E (2001) Improved protocols for Aspergillus minimal medium: trace element and minimal medium salt stock solutions. Fungal Genet Newsl 48:20–21. CrossRefGoogle Scholar
  12. Khan MA, Hamid R, Ahmad M, Abdin MZ, Javed S (2010) Optimization of culture media for enhanced chitinase production from a novel strain of Stenotrophomonas maltophilia using response surface methodology. J Microbiol Biotechnol 20(11):1597–1602. CrossRefPubMedGoogle Scholar
  13. Khanna P, Sundari SS, Kumar NJ (1995) Production, isolation and partial purification of xylanase from Aspergillus sp. World J Microbiol Biotechnol 11:242–243. CrossRefPubMedGoogle Scholar
  14. Kumar P, Mahato DK, Kamle M, Mohanta TK, Kang SG (2017) Aflatoxins: a global concern for food safety, human health and their management. Front Microbiol 7:2170. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. CrossRefPubMedGoogle Scholar
  16. Lee YG, Chung KC, Wi SG, Lee JC, Bae HJ (2009) Purification and properties of a chitinase from Penicillium sp. LYG 0704. Protein Expr Purif 65:244–250. CrossRefPubMedGoogle Scholar
  17. Li D-C (2006) Review of fungal chitinases. Mycopathologia 161:345–360. CrossRefGoogle Scholar
  18. Lodhi G, Kim YS, Hwang JW, Kim SK, Jeon YD, Je JY, Ahn BB, Moon SH, Jeon BT, Park PJ (2014) Chitooligosaccharide and its derivatives: preparation and biological applications. Biomed Res Int 2014:1–13. (Article ID 654913).CrossRefGoogle Scholar
  19. Mania D, Hilpert K, Ruden S, Fischer R, Takeshita N (2010) Screening for antifungal peptides and their modes of action in Aspergillus nidulans. Appl Environ Microbiol 76(21):7102–7108CrossRefPubMedPubMedCentralGoogle Scholar
  20. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428CrossRefGoogle Scholar
  21. Pace CN, Huyghues-Despointes BMP, Fu H, Takano K, Scholtz JM, Grimsley GR (2010) Urea denatured state ensembles contain extensive secondary structure that is increased in hydrophobic proteins. Protein Sci 19:929–943. CrossRefGoogle Scholar
  22. Peralta RM, Terenzi HF, Jorge JA (1990) β-d-glycosidase activities of Humicola grisea var. thermoidea: biochemical and kinetic characterization of a multifunctional enzyme. Biochim Biophys Acta 1033:243–249. CrossRefPubMedGoogle Scholar
  23. Porter CM, Miller BG (2012) Cooperativity in monomeric enzymes with single ligand-binding sites. Bioorg Chem 43:44–50. CrossRefPubMedGoogle Scholar
  24. Gómez Ramírez M, Rojas Avelizapaa LI, Rojas Avelizapab NG, Cruz Camarillo R (2004) Colloidal chitin stained with Remazol Brilliant Blue RR, a useful substrate to select chitinolytic microorganisms and to evaluate chitinases. J Microbiol Methods 56:213–219CrossRefGoogle Scholar
  25. Rathore AS, Gupta RD (2015) Chitinases from bacteria to human: properties applications, and perspectives. Enzyme Res 2015:1–8. (Article ID 791907) CrossRefGoogle Scholar
  26. Rattanakit N, Yano S, Plikomol A, Wakayama M, Tachiki T (2007) Purification of Aspergillus sp. S1-13 chitinases and their role in saccharification of chitin in mash of solid-state culture with shellfish waste. J Biosci Bioeng 103:535–541. CrossRefPubMedGoogle Scholar
  27. Rizatti AC, Jorge JA, Terenzi HF, Rechia CG, Polizeli ML (2001) Purification and properties of a thermostable extracellular β-d-xylosidase produced by a thermotolerant Aspergillus phoenicis. J Ind Microbiol Biotechnol 3:156–160. CrossRefGoogle Scholar
  28. Rojas-Avelizapa LI, Cruz-Camarillo R, Guerrero MI, Rodríguez-Vázquez R, Ibarra JE (1999) Selection and characterization of a proteo-chitinolytic strain of Bacillus thuringiensis, able to grow in shrimp waste media. World J Microbiol Biotechnol 15(2):299–308CrossRefGoogle Scholar
  29. Rustiguel CB (2014) Comparison of the biochemical properties of the chitinases produced by different isolates of Metarhizium anisopliae. Thesis, University of São Paulo, BrazilGoogle Scholar
  30. Seidl V (2008) Chitinases of filamentous fungi: a large group of diverse proteins with multiple physiological functions. Fungal Biol Rev 22:36–42. CrossRefGoogle Scholar
  31. Shehataa AN, Abd El Aty AA, Darwishc DA, Wahabb WAA, Mostafa FA (2018) Purification, physicochemical and thermodynamic studies of antifungal chitinase with production of bioactive chitosan-oligosaccharide from newly isolated Aspergillus griseoaurantiacus KX010988. Int J Biol Macromol 107:990–999CrossRefGoogle Scholar
  32. Singh B, Singh S, Asif AR, Oellerich M, Sharma GL (2014) Allergic aspergillosis and the antigens of Aspergillus fumigatus. Curr Protein Pept Sci 15(5):403–423. CrossRefPubMedGoogle Scholar
  33. Swiontek Brzezinska M, Jankiweicz U (2012) Production of chitinase by Aspergillus niger LOCK 62 and Its potential role in the biological control. Curr Microbiol 65:666–672. CrossRefGoogle Scholar
  34. Thimoteo SS, Glogauer A, Faoro H, Souza EM, Huergo LF, Moerschbacher BM, Pedrosa FO (2017) A broad pH range and processive chitinase from metagenome library. Braz J Med Biol Res 50(1):e5658. Scholar
  35. Wiseman A (1975) Industrial practice with enzymes. In: Wiseman A (ed) Handbook of enzyme biotechnology, 1rd edn. Horwood, Chichester, pp 243–272Google Scholar
  36. Xia G, Jin CZ, Hou JY, Ang SZ, Hang S, Jin C (2001) A novel chitinase having a unique model of action from Aspergillus fumigatus YJ-407. Eur J Biochem 208:4079–4085. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Thaís Barboni Alves
    • 1
  • Pedro Henrique de Oliveira Ornela
    • 1
  • Arthur Henrique Cavalcanti de Oliveira
    • 1
  • João Atílio Jorge
    • 2
  • Luis Henrique Souza Guimarães
    • 1
    • 2
    Email author
  1. 1.Instituto de QuímicaUniversidade Estadual PaulistaAraraquaraBrazil
  2. 2.Faculdade de Filosofia Ciências e Letras de Ribeirão PretoUniversidade de São PauloRibeirão PretoBrazil

Personalised recommendations