Purification of keratinase from poultry farm isolate-Scopulariopsis brevicaulis and statistical optimization of enzyme activity |
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| Institution: | 1. Centre for Advanced studies in Botany, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu, India;2. Department of Mathematics, SBK College, Aruppukottai, Tamil Nadu, India;3. Department of Chemical Engineering, Anna University, Chennai 600025, Tamil Nadu, India;1. Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, Sfax 3018, Tunisia;2. Laboratory of Natural Products Chemistry and Biomolecules (LNPCB), University of Blida, 1, Road of Soumaâ, P.O. Box 270, 09000 Blida, Algeria;3. National Centre for Research and Development of Fisheries and Aquaculture (CNRDPA), 11, Bd Amirouche PO Box 67, Bou Ismaïl, 42415 Tipaza, Algeria;4. National Leather and Shoe Center (CNCC), 17 Road of Leather, Z.I. Sidi Rezig, 2033 Mégrine, Ben Arous, Tunisia;1. Laboratory of Cellular and Molecular Biology (LCMB), Microbiology Team, Faculty of Biological Sciences, University of Sciences and Technology of Houari Boumediene (USTHB), PO Box 32, El Alia, Bab Ezzouar, 16111 Algiers, Algeria;2. Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, PO Box 1177, Sfax 3018, Tunisia;3. Aix Marseille Université, IRD, Université de Toulon, CNRS, Mediterranean Institute of Oceanography (MIO), UM 110, 163 Avenue of Luminy, 13288 Marseille Cedex 9, France;1. PG & Research Department of Biotechnology, Mahendra Arts and Science College, Kalippatti, Namakkal 637501, Tamil Nadu, India;2. Centre for Biotechnology, Muthayammal College of Arts and Science, Rasipuram, Namakkal 637 408, Tamil Nadu, India;3. Division of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan 570752, South Korea;1. Department of Biotechnology, KLE Technological University, Hubballi, Karnataka, 580031, India;2. Department of Biochemistry, Karnatak University, Dharwad, Karnataka, 580003, India;3. Key Laboratory of Urban Pollutant Conversion, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, China;1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Pharmaceutical Sciences, Jiangnan University, Wuxi 214122, PR China;2. National Engineering Laboratory for Cereal Fermentation Technology, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China;3. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark;4. Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, Wuxi 214122, PR China |
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| Abstract: | The fungus Scopulariopsis brevicaulis was isolated from poultry farm soil at Namakkal, India. The extracellular keratinase from this fungus was purified to homogeneity by ammonium sulphate precipitation and procedure involving DEAE-Cellulose and Sephadex G-100 chromatographic techniques. The purified enzyme was formed from a monomeric protein with molecular masses of 39 and 36 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 8.0 and the optimum temperature at pH 8.0 was 40 °C. The activity of purified keratinase with respect to pH, temperature and salt concentration was optimized by Box–Behnken design experiment. It was shown that a second-order polynominal regression model could properly interpret the experimental data with an R2-value of 0.9957 and an F-value of 178.32, based on the maximum enzyme activity examined. Calculated optimum conditions were predicted to confer a 100% yield of keratinase activity with 5 mM CaCl2, pH 8.0 and at a temperature of 40 °C. The enzyme was strongly inhibited by PMSF, which suggests a serine residue at or near an active site. The purified keratinase was examined with its potential for dehairing the skin. |
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