Isolation of Chrysosporium indicum from poultry soil for keratinase enzyme, its purification and partial characterization
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Abstract
Keratinases are produced by microorganisms as fungi, actinomycetes and bacteria and have the capacity to degrade tough insoluble keratin proteins, including feathers. Feathers are waste produced from the poultry industry worldwide and accumulated as solid waste. Therefore, keratinolytic fungal strains Chrysosporium indicum were isolated by hair baiting method from poultry farm soil of Punjab, India. Isolated C. indicum were screened for proteolytic activity on skimmed milk agar. Field Emission Scanning Electron Microscopy (FeSEM) analysis confirmed morphological characters as C. indicum. Fourier transform infrared spectroscopy analysis was studied for the structural and mechanism analysis of feather degraded during keratinase production. Keratinase enzyme was purified 48.03% recovery by ammonium sulphate precipitation, dialysis for desalting and chromatography. Diethylaminoethyl sepharose (DEAE sepharose) and Sephadex-G75 column were used to perform chromatography and partial characterization of the keratinase for temperature, pH, and substrate. The maximum keratinase activity was observed at 500C, at pH 10. The maximum enzyme activity of 289.1 U/ml was observed with keratin powder as substrate and minimum enzyme activity 67.1 U/ml with keratin azure. This is the first report on the purification and characterization of keratinase by C. indicum using DEAE sepharose as affinity chromatography for the purification of the keratinase enzyme.
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Keywords
Chrysosporium indicum, Fourier transform infrared spectroscopy, Keratinase, Purification, Scanning electron microscopy
References
Allpress, J.D., Mountain, G. & Gowland P.C. (2002). Production, purification and characterization of an extracellular keratinase from Lysobacter NCIMB 9497. Letters in Applied Microbiology, 34, 337-342. doi: 10.1046/j.1472-765x.2002.01093.x.
Anitha, T.S. & Palanivelu, P. (2012). Production and characterization of keratinolytic proteases from the fungus Aspergillus parasiticus. International Journal of Research in Biological Sciences, 2(2), 87-93.
Benedek, T. (1962). Fragmenta mycologia I. Some historical remarks on the development of “hair baiting” of Toma-Karling-Vanbreuseghem (The To-Ka-Va hair baiting method). Mycopathol Mycol Appl. 16:104-106.
Cai, C., Ji-shuang, C., Jiong-jiong, Q., Yun, Y. & Xiao-dong, Z. (2008). Purification and characterization of keratinase from a new Bacillus subtilis strain. Journal of Zhejiang University, 9 (9):713-720. doi: 10.1631/jzus.B0820128.
De Toni, C.H., Richter, M.F., Chagas, J.R., Henriques, J.A. & Termignoni, C. (2002). Purification and characterization of an alkaline serine endopeptidase from a feather-degrading Xanthomonas maltophilia strain. Can J Microbiol., 48:342-348.
Eslahi, N., Dadashian, F. & Nejad, N.H. (2013). An investigation on keratin extraction from wool and feather waste by enzymatic hydrolysis. Prep Biochem. Biotechnol., 43:624-648. doi: 10.1080/10826068.2013.763826.
Esawy, M.A. (2007). Isolation and partial characterization of extracellular keratinase from a novel mesophilic Streptomyces albus AZA. Research Journal of Agriculture and Biological Sciences, 3(6):808-817. DOI: 10.108 0/10826068.2013.763826
Friedrich, A.B. & Antranikian, G. (1996). Keratin degradation by Fervidobacterium pennavorans, a novel thermophilic anaerobic species of the order Thermotogales. Appl Environ Microbiol., 62:2875-2882. doi: 10.1128/aem.62.8.2875-2882.1996.
Gradisar, H., Friedrich, J., Krizaj, I. & Jerala, R. (2005). Similarities and specificities of fungal keratinolytic proteases: comparison of keratinase of Paecilomyces marquandii and Doratomyces microsporus to some known proteases. Appl Environ Microbiol., 71(7): 3420–3426. doi: 10.1128/AEM.71.7.3420-3426.2005.
Gupta, R. & Ramnani, P. (2006). Microbial keratinases and their prospective applications: an overview. Appl. Microbial Biotechnology, 70(1):21-33. https://doi.org/10.1007/s00253-005-0239-8.
Hamiche, S., Mechri, S., Khelouia, L., Annane, R., El Hattab, M., Badis, A. & Jaouadi, B. (2019). Purification and biochemical characterization of two keratinases from Bacillus amyloliquefaciens S13 isolated from marine brown alga Zonaria tournefortii with potential keratin-biodegradation and hide-unhairing activities. Int. J. Biol. Macromol., 122(1):758-769. doi: 10.1016/j.ijbiomac.2018.10.174.
Kainoor, P.S. & Naik, G.R. (2010). Production and characterization: of feather degrading keratinase from Bacillus sp. JB 99. Indian journal of Biotechnology, 9:384-390.
Kanchana, R. (2012). Farm waste recycling through microbial keratinases. Journal of Applied Sciences in Environmental Sanitation, 7 (2): 103–108.
Kim, J.D. (2007). Purification and Characterization of a Keratinase from a Feather-Degrading Fungus, Aspergillus flavus Strain K-03. Microbiology, 35(4):219-225. doi: 10.4489/MYCO.2007.35.4.219. doi: 10.4489/MYCO.200 7.35.4.219
Kumar, J. & Kushwaha, R.K.S. (2014). Screening of fungi efficient in feather degradation and keratinase production. Arch. Appl. Sci. Res., 6(1): 73-78.
Kumar, J. & Kushwaha, R.K.S. (2012). Optimization of media composition for keratinase production on feather by Acremonium strictum RKS1. Advances in Applied Science Research, 3(5):3233-3243.
Kumar, J., Kumar, P. & Kushwaha, R.K.S. (2020). Recycling of chicken feather protein into compost by Chrysosporium indicum JK14 and their effect on the growth promotion of Zea mays. Plant Cell Biotechnology and Molecular Biology, 21(37&38): 75-80.
Kumar, J. & Yadav, R. (2020). Keratinolysis of human hair and chicken feather by nondermatophytic keratinophilic fungi isolated from soil. Journal of Applied and Natural Sciences, 12(4):568-574. https://doi.org/10.31018/jans.v12i4.2398.
Kumar J., Kumar P. & Kushwaha RKS (2021). Significance of keratinophiles in biofertilizer development from keratinous waste: upcoming perspective. In: Biofertilizers Vol. 1, 1st Edition (eds. Rakshit A, Meena V, Singh HB, Singh AK, Pahihar M). (Elesvier). pp. 95-101. https://doi.org/10.1016/B978-0-12-821667-5.00006-3.
Kumari, M. & Kumar J. (2020). Chicken feather waste degradation by Alternaria tenuissima and its application on plant growth. Journal of Applied and Natural Sciences, 12(3): 411-414. https://doi.org/10.31018/jans.v12i3.2345
Lee KH., Park K.K., Park S.H. & Lee J.B. (1987). Isolation purification and characterization of keratinolytic proteinase from Microsporum canis. 28(2): 131-137.
Lin, X., Inglis, G.D., Yanke, L.J. & Cheng, K.J. (1999). Selection and characterization of feather-degrading bacteria from canola meal compost. J Indust Micro & Biotech., 23:149.
Langeveld, J.P.M. & Wang, J., Wiel, DFMV., Shih, G.C., et al. (2003). Enzymatic degradation of prion protein in brain stem from infected cattle and sheep. The Journal of infectious diseases, 188:1782-1789. doi: 10.1086/379664.
Ma, B., Qiao, X., Hou, X. & Yang, Y. (2016). Pure keratin membrane and fibers from chicken feather. Int. J. Biol. Macromolecules, 89:614-621. doi: 10.1016/j.ijbioma c.2016.04.039.
Mehta, R.S., Jholapara, R.J. & Sawant, C.S. (2014). Isolation of a novel feather degrading bacterium and optimization of its cultural conditions for enzyme production. International Journal of Pharmacy and Pharmaceutical Sciences, 6(1):194-201.
Mohanty, A.K., Misra, M. & Drzal, L.T. (2005). Natural Fibers, Biopolymers and Bio Composites, Ist Edition, CRC press, pages 896.
More, S.S., Sridhar, D.L., Prakash, S.N., Vishwakarma, J. & Umashankar S. (2013). Purification and properties of novel fungal alkaline keratinase from Cunnighamella echinulata. Turkish J. of Biochemistry, 38(1): 68-74.
Nnolim, N.E. & Nwodo, U.U. (2020). Bacillus sp. CSK2 produced thermostable alkaline keratinase using agro-wastes: keratinolytic enzyme characterization. BMC Biotechnol., 20:65 doi.org/10.1186/s12896-020-00659-2
Onifade, A.A., Al-Sane, N.A., Al-Musallam, A.A. & Al-Zarban, S. (1998). A Review: Potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources. Bioresour Techno., 66: 1-11.
Pavia, D.L., Lampman, G.M., Kriz, G.S. & Vyvyan, J.A. (2008). Introduction to spectroscopy, fifth edition, Cengage learning, USA, pages 786.
Poopathi, S., Thirugnanasambantham, K., Mani, C., Lakshmi, P.V. & Ragul, K. (2014). Purification and characterization of keratinase from feather degrading bacterium useful for mosquito control-a new report. Trop. Biomed., 31(1):97-109. PMID: 24862049.
Ramnani, P. & Gupta, R. (2004). Optimization of medium composition for keratinase production on feather by Bacillus licheniformis RGI using statistical methods involving reponse surface methodology. Biotechnol. Appl. Biochem., 40:191-196. doi: 10.1042/BA20030228.
Ramnani, P., Singh, R. & Gupta, R. (2005). Keratinolytic potential of Bacillus licheniformis RG1: Structural and biochemical mechanism of feather degradation. Can J Microbiol., 51(3):191-196. doi: 10.1139/w04-123.
Raju, K.C., Neogi, U., Saumya, R. & Goud, N.R. (2007). Studies on extracellular enzyme keratinase from Dermatophyte Microsporum gypseum. International Journal of Biological Chemisry. 1(3): 174-178.
Riffel, A. & Brandelli, A. (2006). Keratinolytic bacteria isolated from feather waste. Brazilian Journal of Microbiology, 37, 395-399. https://doi.org/10.1590/S1517-8382200 6000 300036
Saibabu, V., Niyonzima F.N. & More S.S. (2013). Isolation, partial purification and characterization of keratinase from Bacillus megaterium. International Research Journal of Biological Sciences, 2(2):13-20.
Schrooyen, P.M.M., Dijkstra. P.J., Oberthur, R.C., Bantjes, A. & Feijen, J. (2001). Partially carboxymethylated feather keratins and thermal and mechanical properties of films. Journal of Agricultural and Food Chemistry, 49:221–230. doi: 10.1021/jf9913155.
Singh, C.J. (1997). Characterization of an extracellular keratinase of Trichophyton simii and its role in keratin degradation. Mycopathologia, 135: 13-16.
Sivakumar, T., Shankar, T. & Ramasubramanian, V. (2012). Purification properties of Bacillus thuringiensis TS2 keratinase enzyme. American-Eurasian J. Agric. & Environ. Science, 12(12):1553-1557.
Suntornsuk, W., Tongjun, J., Onnim, P., Ayama, H. & Ratanakanokchai, K. (2005). Purification and characterization of keratinase from a thermotolerant feather degrading bacterium. World Journal of Microbiology & Biotechnology, 21:1111-1117.
Vasconcelos, A., Fredid, G. & Cavaco-Paulo, A. (2008). Biodegradable materials based on silk fibroin and keratin. Biomacromolecules, 1299-1305. doi: 10.1021/bm7012789.
Vigneshwaran, C., Shanmugam, S. & Sathish, K.T. (2010). Screening and characterization of keratinase from Bacillus licheniformis isolated from Namakkal poultry farm. Researcher, 2(4):89-96.
Veerapura, N.Y.M., Veerapura, N.M. & Mahesh, M. (2019). Production and purification of keratinase enzyme from Serratia sp. Isolated from poultry waste. Journal of Applied Science, 19:789-796.DOI: 10.3923/jas.2019.7 89.796.
Ya-peng, C., Xi, F.H., Yang, J., Lu, J.H. & Qian, S.J. (2007). Screening for a new Streptomyces strain capable of efficient keratin degradation. J Environ Sci (China), 19(9):1125-8. doi: 10.1016/s1001-0742(07)60183-1
Anitha, T.S. & Palanivelu, P. (2012). Production and characterization of keratinolytic proteases from the fungus Aspergillus parasiticus. International Journal of Research in Biological Sciences, 2(2), 87-93.
Benedek, T. (1962). Fragmenta mycologia I. Some historical remarks on the development of “hair baiting” of Toma-Karling-Vanbreuseghem (The To-Ka-Va hair baiting method). Mycopathol Mycol Appl. 16:104-106.
Cai, C., Ji-shuang, C., Jiong-jiong, Q., Yun, Y. & Xiao-dong, Z. (2008). Purification and characterization of keratinase from a new Bacillus subtilis strain. Journal of Zhejiang University, 9 (9):713-720. doi: 10.1631/jzus.B0820128.
De Toni, C.H., Richter, M.F., Chagas, J.R., Henriques, J.A. & Termignoni, C. (2002). Purification and characterization of an alkaline serine endopeptidase from a feather-degrading Xanthomonas maltophilia strain. Can J Microbiol., 48:342-348.
Eslahi, N., Dadashian, F. & Nejad, N.H. (2013). An investigation on keratin extraction from wool and feather waste by enzymatic hydrolysis. Prep Biochem. Biotechnol., 43:624-648. doi: 10.1080/10826068.2013.763826.
Esawy, M.A. (2007). Isolation and partial characterization of extracellular keratinase from a novel mesophilic Streptomyces albus AZA. Research Journal of Agriculture and Biological Sciences, 3(6):808-817. DOI: 10.108 0/10826068.2013.763826
Friedrich, A.B. & Antranikian, G. (1996). Keratin degradation by Fervidobacterium pennavorans, a novel thermophilic anaerobic species of the order Thermotogales. Appl Environ Microbiol., 62:2875-2882. doi: 10.1128/aem.62.8.2875-2882.1996.
Gradisar, H., Friedrich, J., Krizaj, I. & Jerala, R. (2005). Similarities and specificities of fungal keratinolytic proteases: comparison of keratinase of Paecilomyces marquandii and Doratomyces microsporus to some known proteases. Appl Environ Microbiol., 71(7): 3420–3426. doi: 10.1128/AEM.71.7.3420-3426.2005.
Gupta, R. & Ramnani, P. (2006). Microbial keratinases and their prospective applications: an overview. Appl. Microbial Biotechnology, 70(1):21-33. https://doi.org/10.1007/s00253-005-0239-8.
Hamiche, S., Mechri, S., Khelouia, L., Annane, R., El Hattab, M., Badis, A. & Jaouadi, B. (2019). Purification and biochemical characterization of two keratinases from Bacillus amyloliquefaciens S13 isolated from marine brown alga Zonaria tournefortii with potential keratin-biodegradation and hide-unhairing activities. Int. J. Biol. Macromol., 122(1):758-769. doi: 10.1016/j.ijbiomac.2018.10.174.
Kainoor, P.S. & Naik, G.R. (2010). Production and characterization: of feather degrading keratinase from Bacillus sp. JB 99. Indian journal of Biotechnology, 9:384-390.
Kanchana, R. (2012). Farm waste recycling through microbial keratinases. Journal of Applied Sciences in Environmental Sanitation, 7 (2): 103–108.
Kim, J.D. (2007). Purification and Characterization of a Keratinase from a Feather-Degrading Fungus, Aspergillus flavus Strain K-03. Microbiology, 35(4):219-225. doi: 10.4489/MYCO.2007.35.4.219. doi: 10.4489/MYCO.200 7.35.4.219
Kumar, J. & Kushwaha, R.K.S. (2014). Screening of fungi efficient in feather degradation and keratinase production. Arch. Appl. Sci. Res., 6(1): 73-78.
Kumar, J. & Kushwaha, R.K.S. (2012). Optimization of media composition for keratinase production on feather by Acremonium strictum RKS1. Advances in Applied Science Research, 3(5):3233-3243.
Kumar, J., Kumar, P. & Kushwaha, R.K.S. (2020). Recycling of chicken feather protein into compost by Chrysosporium indicum JK14 and their effect on the growth promotion of Zea mays. Plant Cell Biotechnology and Molecular Biology, 21(37&38): 75-80.
Kumar, J. & Yadav, R. (2020). Keratinolysis of human hair and chicken feather by nondermatophytic keratinophilic fungi isolated from soil. Journal of Applied and Natural Sciences, 12(4):568-574. https://doi.org/10.31018/jans.v12i4.2398.
Kumar J., Kumar P. & Kushwaha RKS (2021). Significance of keratinophiles in biofertilizer development from keratinous waste: upcoming perspective. In: Biofertilizers Vol. 1, 1st Edition (eds. Rakshit A, Meena V, Singh HB, Singh AK, Pahihar M). (Elesvier). pp. 95-101. https://doi.org/10.1016/B978-0-12-821667-5.00006-3.
Kumari, M. & Kumar J. (2020). Chicken feather waste degradation by Alternaria tenuissima and its application on plant growth. Journal of Applied and Natural Sciences, 12(3): 411-414. https://doi.org/10.31018/jans.v12i3.2345
Lee KH., Park K.K., Park S.H. & Lee J.B. (1987). Isolation purification and characterization of keratinolytic proteinase from Microsporum canis. 28(2): 131-137.
Lin, X., Inglis, G.D., Yanke, L.J. & Cheng, K.J. (1999). Selection and characterization of feather-degrading bacteria from canola meal compost. J Indust Micro & Biotech., 23:149.
Langeveld, J.P.M. & Wang, J., Wiel, DFMV., Shih, G.C., et al. (2003). Enzymatic degradation of prion protein in brain stem from infected cattle and sheep. The Journal of infectious diseases, 188:1782-1789. doi: 10.1086/379664.
Ma, B., Qiao, X., Hou, X. & Yang, Y. (2016). Pure keratin membrane and fibers from chicken feather. Int. J. Biol. Macromolecules, 89:614-621. doi: 10.1016/j.ijbioma c.2016.04.039.
Mehta, R.S., Jholapara, R.J. & Sawant, C.S. (2014). Isolation of a novel feather degrading bacterium and optimization of its cultural conditions for enzyme production. International Journal of Pharmacy and Pharmaceutical Sciences, 6(1):194-201.
Mohanty, A.K., Misra, M. & Drzal, L.T. (2005). Natural Fibers, Biopolymers and Bio Composites, Ist Edition, CRC press, pages 896.
More, S.S., Sridhar, D.L., Prakash, S.N., Vishwakarma, J. & Umashankar S. (2013). Purification and properties of novel fungal alkaline keratinase from Cunnighamella echinulata. Turkish J. of Biochemistry, 38(1): 68-74.
Nnolim, N.E. & Nwodo, U.U. (2020). Bacillus sp. CSK2 produced thermostable alkaline keratinase using agro-wastes: keratinolytic enzyme characterization. BMC Biotechnol., 20:65 doi.org/10.1186/s12896-020-00659-2
Onifade, A.A., Al-Sane, N.A., Al-Musallam, A.A. & Al-Zarban, S. (1998). A Review: Potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources. Bioresour Techno., 66: 1-11.
Pavia, D.L., Lampman, G.M., Kriz, G.S. & Vyvyan, J.A. (2008). Introduction to spectroscopy, fifth edition, Cengage learning, USA, pages 786.
Poopathi, S., Thirugnanasambantham, K., Mani, C., Lakshmi, P.V. & Ragul, K. (2014). Purification and characterization of keratinase from feather degrading bacterium useful for mosquito control-a new report. Trop. Biomed., 31(1):97-109. PMID: 24862049.
Ramnani, P. & Gupta, R. (2004). Optimization of medium composition for keratinase production on feather by Bacillus licheniformis RGI using statistical methods involving reponse surface methodology. Biotechnol. Appl. Biochem., 40:191-196. doi: 10.1042/BA20030228.
Ramnani, P., Singh, R. & Gupta, R. (2005). Keratinolytic potential of Bacillus licheniformis RG1: Structural and biochemical mechanism of feather degradation. Can J Microbiol., 51(3):191-196. doi: 10.1139/w04-123.
Raju, K.C., Neogi, U., Saumya, R. & Goud, N.R. (2007). Studies on extracellular enzyme keratinase from Dermatophyte Microsporum gypseum. International Journal of Biological Chemisry. 1(3): 174-178.
Riffel, A. & Brandelli, A. (2006). Keratinolytic bacteria isolated from feather waste. Brazilian Journal of Microbiology, 37, 395-399. https://doi.org/10.1590/S1517-8382200 6000 300036
Saibabu, V., Niyonzima F.N. & More S.S. (2013). Isolation, partial purification and characterization of keratinase from Bacillus megaterium. International Research Journal of Biological Sciences, 2(2):13-20.
Schrooyen, P.M.M., Dijkstra. P.J., Oberthur, R.C., Bantjes, A. & Feijen, J. (2001). Partially carboxymethylated feather keratins and thermal and mechanical properties of films. Journal of Agricultural and Food Chemistry, 49:221–230. doi: 10.1021/jf9913155.
Singh, C.J. (1997). Characterization of an extracellular keratinase of Trichophyton simii and its role in keratin degradation. Mycopathologia, 135: 13-16.
Sivakumar, T., Shankar, T. & Ramasubramanian, V. (2012). Purification properties of Bacillus thuringiensis TS2 keratinase enzyme. American-Eurasian J. Agric. & Environ. Science, 12(12):1553-1557.
Suntornsuk, W., Tongjun, J., Onnim, P., Ayama, H. & Ratanakanokchai, K. (2005). Purification and characterization of keratinase from a thermotolerant feather degrading bacterium. World Journal of Microbiology & Biotechnology, 21:1111-1117.
Vasconcelos, A., Fredid, G. & Cavaco-Paulo, A. (2008). Biodegradable materials based on silk fibroin and keratin. Biomacromolecules, 1299-1305. doi: 10.1021/bm7012789.
Vigneshwaran, C., Shanmugam, S. & Sathish, K.T. (2010). Screening and characterization of keratinase from Bacillus licheniformis isolated from Namakkal poultry farm. Researcher, 2(4):89-96.
Veerapura, N.Y.M., Veerapura, N.M. & Mahesh, M. (2019). Production and purification of keratinase enzyme from Serratia sp. Isolated from poultry waste. Journal of Applied Science, 19:789-796.DOI: 10.3923/jas.2019.7 89.796.
Ya-peng, C., Xi, F.H., Yang, J., Lu, J.H. & Qian, S.J. (2007). Screening for a new Streptomyces strain capable of efficient keratin degradation. J Environ Sci (China), 19(9):1125-8. doi: 10.1016/s1001-0742(07)60183-1
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Isolation of Chrysosporium indicum from poultry soil for keratinase enzyme, its purification and partial characterization . (2021). Journal of Applied and Natural Science, 13(2), 744-751. https://doi.org/10.31018/jans.v13i2.2609
