Production and characterization of a thermostable antifungal chitinase secreted by the filamentous fungus Aspergillus niveus under submerged fermentation
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.
KeywordsAspergillus 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.
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.
- 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. https://doi.org/10.1186/s12934-017-0634-8 CrossRefPubMedPubMedCentralGoogle Scholar
- 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. https://doi.org/10.1016/j.enzmictec.2004.12.031 CrossRefGoogle Scholar
- Duong-Ly KC, Gabelli SB (2014) Salting out of proteins using ammonium sulfate precipitation. Methods Enzymol 541:85–94. https://doi.org/10.1016/B978-0-12-420119-4.00007-0 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.4014/jmb.0909.09040 CrossRefPubMedGoogle Scholar
- 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. https://doi.org/10.1263/jbb.103.535 CrossRefPubMedGoogle Scholar
- 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
- 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
- Wiseman A (1975) Industrial practice with enzymes. In: Wiseman A (ed) Handbook of enzyme biotechnology, 1rd edn. Horwood, Chichester, pp 243–272Google Scholar