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Production and biochemical and molecular characterization of a keratinolytic serine protease from chicken feather-degrading Bacillus licheniformis RPk

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A novel feather-degrading bacterium was isolated from a polluted river and identified as Bacillus licheniformis RPk. The isolate exhibited high proteinase production when grown in chicken-feather media. Complete feather degradation was achieved during cultivation. Maximum protease activity (4150 U/mL with casein as a substrate and 37.35 U/mL with keratin as a substrate) was obtained when the strain was grown in a medium containing 7.5 g/L chicken feathers, 2 g/L yeast extract, 0.5 g/L NaCl, 0.1 g/L MgSO4·7H2O, 0.7 g/L KH2PO4, and 1.4 g/L K2HPO4 for 48 h with agitation of 200 rev/min at 37 °C. The major protease produced by B. licheniformis RPk was purified to homogeneity by a 3-step procedure. The molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE and gel filtration. The optimum pH and temperature for the caseinolytic activity were around 11.0 and 65 °C, respectively. The optimum pH and temperature for the keratinolytic activity were 9.0 and 60 °C, respectively. The activity of the enzyme was totally lost in the presence of phenylmethylsulfonyl fluoride, which suggests that the purified enzyme is a serine protease. The thermostability of the enzyme was considerably enhanced in the presence of Ca2+ at temperatures >50 °C. The kerRP gene, which encodes the keratinolytic protease, was isolated, and its DNA sequence was determined. The deduced amino acid sequence revealed that the keratinase KerRP differs from KerA of B. licheniformis PWD-1, subtilisin Carlsberg, and keratinase of B. licheniformis by 2, 4, and 62 amino acids, respectively.

Une nouvelle souche kératinolytique a été isolée à partir d’une rivière polluée et identifiée comme étant Bacillus licheniformis RPk. Cette souche possède une production importante de protéase sur milieu à base de plumes de poulets. Les plumes sont complètement solubilisées durant 48 h de culture. Une production optimale de kératinase de 4150 U/mL sur caséine et 37,35 U/mL sur la kératine fut obtenue avec la composition du milieu suivant : 7,5 g/L plumes de poulet ; 2 g/L extrait de levure ; 0,5g/L NaCl ; 0,1 g/L MgSO4·7H2O ; 0,7 g/L KH2PO4 ; 1,4 g/L K2HPO4 ; sous agitation à 200 rev/min pour une période d’incubation de 48 h à 37  °C. La kératinase a été purifiée à homogénéité en suivant 3 étapes. La masse moléculaire de la kératinase de RPk est estimée à 32 kDa par SDS-PAGE et gel filtration. L’enzyme purifiée a une activité optimale à 65  °C et à pH 11,0 sur la caséine et à 60  °C et à pH 9,0 sur la kératine. Elle est complètement inhibée par le PMSF, il s’agit donc d’une protéase à serine. La thermostabilité de l’enzyme est considérablement améliorée en présence de calcium à des températures supérieures à 50  °C. Le gène kerRP, qui code pour la kératinase de RPk, a été isolé et sa séquence d’ADN a été déterminée. La séquence en acide amine de la kératinase KerRP montre qu’elle diffère de KerA de B. licheniformis PWD-1, de la subtilisin Carlsberg et de la keratinase de B. licheniformis par 2, 4 et 62 acide aminés, respectivement.

Document Type: Research Article

Publication date: April 1, 2009

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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