A fungal isolate with capability to grow in keratinous substrate as only source of carbon and nitrogen was identified as Aspergillus niger using the sequencing of the ITS region of the rDNA. This strain produced a slightly acid keratinase and an acid protease during cultivation in feather meal. The peak of keratinolytic activity occurred in 48?h and the maximum proteolytic activity in 96?h. These enzymes were partly characterized as serine protease and aspartic protease, respectively. The effects of feather meal concentration and initial pH on enzyme production were evaluated using a central composite design combined with response surface methodology. The optimal conditions were determined as pH 5.0 for protease and 7.8 for keratinase and 20?g/L of feather meal, showing that both models were predictive. Production of keratinases by A. niger is a less-exploited field that might represent a novel and promising biotechnological application for this microorganism. 1. Introduction Aspergillus niger is one of the most important microorganisms in biotechnology. It has been already used to produce extracellular enzymes such as glucose oxidase, pectinase, α-amylase and glucoamylase, organic acids, and recombinant proteins. In addition, A. niger is used for biotransformations and waste treatment [1–3]. Among the various enzymes produced by the fungus are included proteases. The major extracellular proteolytic activities in A. niger appear to be due to acid proteases [4]. Acid proteases [E.C.3.4.23] are endopeptidases that depend on aspartic acid residues for their catalytic activity and show maximal activity at low pH. These enzymes offer a variety of applications in the food, beverage industry, and medicine [5]. Keratin is a fibrous protein that occurs in vertebrates and exerts protective and structural functions. It is the major component of feathers, wool, scales, hair, stratum corneum, horns, scalps, and nails [6]. Keratin is insoluble and presents high mechanic resistance, as well as recalcitrance to common proteolytic enzymes like pepsin, trypsin, and papain [7]. This resistance is because of the tight folding of protein chain in α-helix (α-keratin) and β-sheets (β-keratin) in a super-coiled polypeptide chain, kept by strong association by disulfide bonds [8, 9]. Keratinases [EC 3.4.21/24/99.11] are specific proteases that display the capability of keratin degradation. These enzymes are gaining importance in the last years, with many applications associated with hydrolysis of keratinous substrates, mainly byproducts of agroindustrial processes [10].
References
[1]
A. H. F. J. Roth and P. Dersch, “A novel expression system for intracellular production and purification of recombinant affinity-tagged proteins in Aspergillus niger,” Applied Microbiology and Biotechnology, vol. 86, no. 2, pp. 659–670, 2010.
[2]
E. Schuster, N. Dunn-Coleman, J. Frisvad, and P. van Dijck, “On the safety of Aspergillus niger—a review,” Applied Microbiology and Biotechnology, vol. 59, no. 4-5, pp. 426–435, 2002.
[3]
P. W. M. van Dijck, G. C. M. Selten, and R. A. Hempenius, “On the safety of a new generation of DSM Aspergillus niger enzyme production strains,” Regulatory Toxicology and Pharmacology, vol. 38, no. 1, pp. 27–35, 2003.
[4]
G. Jarai and F. Buxton, “Nitrogen, carbon, and pH regulation of extracellular acidic proteases of Aspergillus niger,” Current Genetics, vol. 26, no. 3, pp. 238–244, 1994.
[5]
K. S. Vishwanatha, A. G. Appu Rao, and S. A. Singh, “Characterisation of acid protease expressed from Aspergillus oryzae MTCC 5341,” Food Chemistry, vol. 114, no. 2, pp. 402–407, 2009.
[6]
C. Vignardet, Y. C. Guillaume, L. Michel, J. Friedrich, and J. Millet, “Comparison of two hard keratinous substrates submitted to the action of a keratinase using an experimental design,” International Journal of Pharmaceutics, vol. 224, no. 1-2, pp. 115–122, 2001.
[7]
R. C. S. Thys, F. S. Lucas, A. Riffel, P. Heeb, and A. Brandelli, “Characterization of a protease of a feather-degrading Microbacterium species,” Letters in Applied Microbiology, vol. 39, no. 2, pp. 181–186, 2004.
[8]
R. D. B. Fraser and D. A. D. Parry, “Molecular packing in the feather keratin filament,” Journal of Structural Biology, vol. 162, no. 1, pp. 1–13, 2008.
[9]
L. Kreplak, J. Doucet, P. Dumas, and F. Briki, “New aspects of the α-helix to β-sheet transition in stretched hard α-keratin fibers,” Biophysical Journal, vol. 87, no. 1, pp. 640–647, 2004.
[10]
A. Brandelli, “Bacterial keratinases: useful enzymes for bioprocessing agroindustrial wastes and beyond,” Food and Bioprocess Technology, vol. 1, no. 2, pp. 105–116, 2008.
[11]
R. Gupta and P. Ramnani, “Microbial keratinases and their prospective applications: an overview,” Applied Microbiology and Biotechnology, vol. 70, no. 1, pp. 21–33, 2006.
[12]
S. T. Silveira, D. J. Daroit, and A. Brandelli, “Pigment production by Monascus purpureus in grape waste using factorial design,” LWT—Food Science and Technology, vol. 41, no. 1, pp. 170–174, 2008.
[13]
S. Zhu, S. Lee, S. K. Hargrove, and G. Chen, “Prediction of combustion efficiency of chicken litter using an artificial neural network approach,” Fuel, vol. 86, no. 5-6, pp. 877–886, 2007.
[14]
A. K. Casali, L. Goulart, L. K. Rosa e Silva et al., “Molecular typing of clinical and environmental Cryptococcus neoformans isolates in the Brazilian state Rio Grande do Sul,” FEMS Yeast Research, vol. 3, no. 4, pp. 405–415, 2003.
[15]
S. Horisawa, Y. Sakuma, and S. Doi, “Qualitative and quantitative PCR methods using species-specific primer for detection and identification of wood rot fungi,” Journal of Wood Science, vol. 55, no. 2, pp. 133–138, 2009.
[16]
M. A. Larkin, G. Blackshields, N. P. Brown et al., “Clustal W and Clustal X version 2.0,” Bioinformatics, vol. 23, no. 21, pp. 2947–2948, 2007.
[17]
K. Tamura, J. Dudley, M. Nei, and S. Kumar, “MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0,” Molecular Biology and Evolution, vol. 24, no. 8, pp. 1596–1599, 2007.
[18]
L. A. Dedavid e Silva, F. C. Lopes, S. T. Silveira, and A. Brandelli, “Production of cellulolytic enzymes by Aspergillus phoenicis in grape waste using response surface methodology,” Applied Biochemistry and Biotechnology, vol. 152, no. 2, pp. 295–305, 2009.
[19]
AOAC, Official Methods of Analysis, Association of Official Analytical Chemists, Washington, DC, USA, 15th edition, 2005.
[20]
M. P. Sangorrín, E. J. Folco, C. M. Martone, and J. J. Sánchez, “Purification and characterization of a proteinase inhibitor from white croaker skeletal muscle (Micropogon opercularis),” International Journal of Biochemistry and Cell Biology, vol. 33, no. 7, pp. 691–699, 2001.
[21]
A. Riffel, S. Ortolan, and A. Brandelli, “De-hairing activity of extracellular proteases produced by keratinolytic bacteria,” Journal of Chemical Technology and Biotechnology, vol. 78, no. 8, pp. 855–859, 2003.
[22]
A. P. F. Corrêa, D. J. Daroit, and A. Brandelli, “Characterization of a keratinase produced by Bacillus sp. P7 isolated from an Amazonian environment,” International Biodeterioration and Biodegradation, vol. 64, no. 1, pp. 1–6, 2010.
[23]
R. Myers and R. C. Montgomery, Response Surface Methodology: Process and Product Optimizationusing Designed Experiments, Wiley, New York, NY, USA, 2002.
[24]
J. I. Pitt and A. D. Hocking, “Aspergillus and related Teleomorphs,” Fungi and Food Spoilage, Springer Science + Business Media, London, UK, 2009.
[25]
F. Giraud, J. Dupont, M. Haon et al., “Phylogenetic analysis of the Aspergillus niger aggregate in relation to feruloyl esterase activity,” Research in Microbiology, vol. 158, no. 5, pp. 413–419, 2007.
[26]
E. R. Palencia, M. A. Klich, A. E. Glenn, and C. W. Bacon, “Use of a rep-PCR system to predict species in the Aspergillus section Nigri,” Journal of Microbiological Methods, vol. 79, no. 1, pp. 1–7, 2009.
[27]
G. Perrone, A. Susca, F. Epifani, and G. Mulè, “AFLP characterization of Southern Europe population of Aspergillus section Nigri from grapes,” International Journal of Food Microbiology, vol. 111, pp. S22–S27, 2006.
[28]
A. R. El Boushy, A. F. B. van der Poel, and O. E. D. Walraven, “Feather meal—a biological waste: its processing and utilization as a feedstuff for poultry,” Biological Wastes, vol. 32, no. 1, pp. 39–74, 1990.
[29]
A. Grazziotin, F. A. Pimentel, E. V. de Jong, and A. Brandelli, “Nutritional improvement of feather protein by treatment with microbial keratinase,” Animal Feed Science and Technology, vol. 126, no. 1-2, pp. 135–144, 2006.
[30]
P. Anbu, S. C. B. Gopinath, A. Hilda, N. Mathivanan, and G. Annadurai, “Secretion of keratinolytic enzymes and keratinolysis by Scopulariopsis brevicaulis and Trichophyton mentagrophytes: regression analysis,” Canadian Journal of Microbiology, vol. 52, no. 11, pp. 1060–1069, 2006.
[31]
A. Brandelli, D. J. Daroit, and A. Riffel, “Biochemical features of microbial keratinases and their production and applications,” Applied Microbiology and Biotechnology, vol. 85, no. 6, pp. 1735–1750, 2010.
[32]
R. Siala, A. Sellami-Kamoun, M. Hajji, I. Abid, N. Gharsallah, and M. Nasri, “Extracellular acid protease from Aspergillus niger I1: purification and characterization,” African Journal of Biotechnology, vol. 8, no. 18, pp. 4582–4589, 2009.
[33]
F. M. Olajuyigbe, J. O. Ajele, and T. L. Olawoye, “Some physicochemical properties of acid protease produced during growth of Aspergillus niger (NRRL 1785),” Global Journal of Pure and Applied Sciences, vol. 9, pp. 523–528, 2003.
[34]
A. M. Farag and M. A. Hassan, “Purification, characterization and immobilization of a keratinase from Aspergillus oryzae,” Enzyme and Microbial Technology, vol. 34, no. 2, pp. 85–93, 2004.
[35]
P. D. Haaland, Experimental Design in Biotechnology, Marcel Dekker, New York, NY, USA, 1989.
[36]
R. M. D. B. Santos, A. A. P. Firmino, C. M. De Sá, and C. R. Felix, “Keratinolytic activity of Aspergillus fumigatus fresenius,” Current Microbiology, vol. 33, no. 6, pp. 364–370, 1996.
[37]
K. R. Kiran, N. G. Karanth, and S. Divakar, “Preparation of stearoyl lactic acid ester catalyzed by lipases from Rhizomucor miehei and porcine pancreas optimization using response surface methodology,” Applied Microbiology and Biotechnology, vol. 52, no. 4, pp. 579–584, 1999.