Abstract
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.
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References
Adams PR (1990) Mycelial amylase activities of thermophilic species of Rhizomucor, Humicola and Papulaspora. Mycopathologia 112:35–37. https://doi.org/10.1007/BF01795178
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
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
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. https://doi.org/10.1016/0003-2697(76)90527-3
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
Farag MA, Al-Nusarie ST (2014) Production, optimization, characterization and antifungal activity of chitinase produced by Aspergillus terreus. Afr J Biotechnol 13:1567–1578. https://doi.org/10.5897/AJB2014.13628
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. https://doi.org/10.1016/j.ejar.2016.04.004
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. https://doi.org/10.1006/bbrc.1999.1428
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):1458–1465
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 3:126–131
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. https://doi.org/10.4148/1941-4765.1173
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
Khanna P, Sundari SS, Kumar NJ (1995) Production, isolation and partial purification of xylanase from Aspergillus sp. World J Microbiol Biotechnol 11:242–243. https://doi.org/10.1007/BF00704661
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. https://doi.org/10.3389/fmicb.2016.02170
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0
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. https://doi.org/10.1016/j.pep.2008.12.004
Li D-C (2006) Review of fungal chitinases. Mycopathologia 161:345–360. https://doi.org/10.1007/s11046-006-0024-y
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. https://doi.org/10.1155/2014/654913 (Article ID 654913).
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):7102–7108
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428
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. https://doi.org/10.1002/pro.370
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. https://doi.org/10.1016/0304-4165(90)90127-l
Porter CM, Miller BG (2012) Cooperativity in monomeric enzymes with single ligand-binding sites. Bioorg Chem 43:44–50. https://doi.org/10.1016/j.bioorg.2011.11.001
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 56:213–219
Rathore AS, Gupta RD (2015) Chitinases from bacteria to human: properties applications, and perspectives. Enzyme Res 2015:1–8. https://doi.org/10.1155/2015/791907 (Article ID 791907)
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
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. https://doi.org/10.1038/sj/jim/7000107
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):299–308
Rustiguel CB (2014) Comparison of the biochemical properties of the chitinases produced by different isolates of Metarhizium anisopliae. Thesis, University of São Paulo, Brazil
Seidl V (2008) Chitinases of filamentous fungi: a large group of diverse proteins with multiple physiological functions. Fungal Biol Rev 22:36–42. https://doi.org/10.1016/j.fbr.2008.03.002
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 107:990–999
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. https://doi.org/10.2174/1389203715666140512120605
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. https://doi.org/10.1007/s00284-012-0208-2
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. https://doi.org/10.0.6.54/1414-431x20165658
Wiseman A (1975) Industrial practice with enzymes. In: Wiseman A (ed) Handbook of enzyme biotechnology, 1rd edn. Horwood, Chichester, pp 243–272
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. https://doi.org/10.1046/j.1432-1327.2001.02323.x
Acknowledgements
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.
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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.
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Alves, T.B., de Oliveira Ornela, P.H., de Oliveira, A.H.C. et al. Production and characterization of a thermostable antifungal chitinase secreted by the filamentous fungus Aspergillus niveus under submerged fermentation. 3 Biotech 8, 369 (2018). https://doi.org/10.1007/s13205-018-1397-6
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DOI: https://doi.org/10.1007/s13205-018-1397-6