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Abstract: The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 °C was 8.5 and the optimum temperature at pH 8.5 was 55 °C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.

[1] Böckle, B., Galunsky, B., Muller, R., 1995. Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530. Appl. Environ. Microbiol., 61(10):3705-3710.

[2] Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72(1-2):248-254.

[3] Bressollier, P., Letourneau, F., Urdaci, M., Verneuil, B., 1999. Purification and characterization of a keratinolytic serine proteinase from Streptomyces albidoflavus. Appl. Environ. Microbiol., 65(6):2570-2576.

[4] Cai, C.G., Lou, B.G., Zheng, X.D., 2008. Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis. J. Zhejiang Univ. Sci. B, 9(1):60-67.

[5] de Toni, C.H., Richter, M.F., Chagas, J.R., Henriques, J.A.P., Termignoni, C., 2002. Purification and characterization of an alkaline serine endopeptidase from a feather-degrading Xanthomonas maltophilia strain. Can. J. Microbiol., 48(4):342-348.

[6] El-Naghy, M.A., El-Ktatny, M.S., Fadl-Allah, E.M., Nazeer, W.W., 1998. Degradation of chicken feathers by Chrysosporium georgiae. Mycopathologia, 143(2):77-84.

[7] El-Refai, H.A., AbdelNaby, M.A., Gaballa, A., El-Araby, M.H., Abdel Fattah, A.F., 2005. Improvement of the newly isolated Bacillus pumilus FH9 keratinolytic activity. Process Biochem., 40(7):2325-2332.

[8] Friedrich, J., Gradišar, H., Vrecl, M., Pogacnik, A., 2005. In vitro degradation of porcine skin epidermis by a fungal keratinase of Doratomyces microsporus. Enzyme Microb. Technol., 36(4):455-460.

[9] Gessesse, A., Rajni, H.K., Gashe, B.A., 2003. Novel alkaline proteases from alkaliphilic bacteria grown on chicken feather. Enzyme Microb. Technol., 32(5):519-524.

[10] Gradišar, H., Kern, S., Friedrich, J., 2000. Keratinase of Doratomyces microsporus. Appl. Microbiol. Biotechnol., 53(2):196-200.

[11] Gradišar, H., Friedrich, J., Križaj, 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. Microb., 71(7):3420-3426.

[12] Grazziotin, A., Pimentel, F.A., de Jong, E.V., Brandelli, A., 2006. Nutritional improvement of feather protein by treatment with microbial keratinase. Anim. Feed Sci. Technol., 126(1-2):135-144.

[13] Gupta, R., Ramnani, P., 2006. Microbial keratinase and their prospective application: an overview. Appl. Microbiol. Biotechnol., 70(1):21-33.

[14] Gushterova, A., Vasileva-Tonkova, E., Dimova, E., Nedkov, P., Haertle, T., 2005. Keratinase production by newly isolated Antarctic actinomycete strains. World J. Microbiol. Biotechnol., 21(6-7):831-834.

[15] Ignatova, Z., Gousterova, A., Spassov, G., Nedkov, P., 1999. Isolation and partial characterization of extracellular keratinase from a wool degrading Thermophilic actinomycete strain Thermoactinomyces candidus. Can. J. Microbiol., 45(3):217-222.

[16] Kim, J.M., Lim, W.J., Suh, H.J., 2001. Feather-degrading Bacillus species from poultry waste. Process Biochem., 37(3):287-291.

[17] Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T₄. Nature, 227(5259):680-685.

[18] Lal, S., Rajak, R.C., Hasija, S.K., 1999. In vitro degradation of keratin by two species of Bacillus. J. Gen. Appl. Microbiol., 45(6):283-287.

[19] Langeveld, J.P.M., Wang, J.J., van de Wiel, D.F.M., Shih, G.C., Garssen, G.J., Bossers, A., Shih, J.C.H., 2003. Enzymatic degradation of prion protein in brain stem from infected cattle and sheep. J. Infect. Dis., 188(11):1782-1789.

[20] Lin, X., Lee, C.G., Casale, E.S., Shih, J.C.H., 1992. Purification and characterization of a keratinase from a feather-degrading Bacillus licheniformis strain. Appl. Environ. Microbiol., 58(10):3271-3275.

[21] Lucas, F.S., Broennimann, O., Febbraro, I., Heeb, P., 2003. High diversity among feather-degrading bacteria from a dry meadow soil. Microb. Ecol., 45(3):282-290.

[22] Macedo, A.J., da Silva, W.O.B., Gava, R., Driemerier, D., Henriques, J.A.P., Termignoni, C., 2005. Novel keratinase from Bacillus subtilis S14 exhibiting remarkable dehairing capabilites. Appl. Environ. Microbiol., 71(1):594-596.

[23] Mitsuiki, S., Ichikawa, M., Oka, T., Sakai, M., Moriyama, Y., Sameshima, Y., Goto, M., Furukawa, K., 2004. Molecular characterization of a keratinolytic enzyme from an alkaliphilic Nocardiopsis sp. TOA-1. Enzyme Microb. Technol., 34(5):482-489.

[24] Nam, G.W., Lee, D.W., Lee, H.S., Lee, N.J., Kim, B.C., Choe, E.A., Hwang, J.K., Suhartono, M.T., Pyun, Y.R., 2002. Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe. Arch. Microbiol., 178(6):538-547.

[25] Noval, J.J., Nickerson, W.J., 1959. Decomposition of native keratin by Streptomyces fradiae. J. Bacteriol., 77(3):251-259.

[26] Odetallah, N.H., Wang, J.J., Garlich, J.D., Shih, J.C.H., 2003. Keratinase in starter diets improves growth of broiler chicks. Poultry Sci., 82(4):664-670.

[27] Onifade, A.A., A1-Sane, N.A., Al-Musallam, A.A., Al-Zarban, S., 1998. Potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources. Biores. Technol., 66(1):1-11.

[28] Ramnani, P., Gupta, R., 2004. Optimization of medium composition for keratinase production on feather by Bacillus licheniformis RG1 using statistical methods involving response surface methodology. Biotechnol. Appl. Biochem., 40(11):491-496.

[29] Riffel, A., Lucas, F.S., Heeb, P., Brandelli, A., 2003. Characterization of a new keratinolytic bacterium that completely degrades native feather keratin. Arch. Microbiol., 179(4):258-265.

[30] Rozs, M., Manczinger, L., Vagvolgyi, C., Kevei, F., 2001. Secretion of a trypsin-like thiol protease by a new keratinolytic strain of Bacillus licheniformis. FEMS Microb. Lett., 205(2):221-224.

[31] Stahl, W.H., Mcque, B., Siu, R.G.H., 1949. Decomposition of cystine and wool by treatment in the ball mill and autoclave. J. Biol. Chem., 177(11):69-73.

[32] Suh, H.J., Lee, H.K., 2001. Characterization of a keratinolytic serine protease from Bacillus subtilis KS-1. J. Protein Chem., 20(2):165-169.

[33] Takami, H., Nogi, Y., Horikoshi, K., 1999. Reidentification of the keratinase-producing facultativel alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans. Extremophiles, 3(4):293-296.

[34] Thys, R.C.S., Lucas, F.S., Riffel, A., Heeb, P., Brandelli, A., 2004. Characterization of a protease of a feather-degrading Microbacterium species. Lett. Appl. Microbiol., 39(2):181-186.

[35] Wawrzkiewicz, K., Lobarzewski, J., Wolski, T., 1987. Intracellular keratinase of Trichophyton gallinae. J. Med. Veternary Mycol., 25(4):261-268.

[36] Williams, C.M., Richter, C.S., Mackenzie, J.M., Shih, J.C.H., 1990. Isolation, identification, and characterization of a feather-degrading bacterium. Appl. Environl. Microbiol., 56(6):509-1515.

[37] Williams, C.M., Lee, C.G., Garlich, J.D., Shih, J.C.H., 1991. Evaluation of a bacterial feather fermentation product, feather-lysate, as a feed protein. Poultry Sci., 70(1):85-94.