Production and biochemical and molecular characterization of a keratinolytic serine protease from chicken feather-degrading Bacillus licheniformis RPk.
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 x 7H2O, 0.7 g/L KH2PO4, and 1.4 g/L K2HPO4 for 48 h with agitation of 200 rev/min at 37 degrees 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 degrees C, respectively. The optimum pH and temperature for the keratinolytic activity were 9.0 and 60 degrees 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 degrees 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.
Fakhfakh
Nahed
N
Laboratoire de Genie Enzymatique et de Microbiologie, Ecole Nationale d'Ingenieurs de Sfax, B.P. << W >> 3038 Sfax, Tunisia. Nahedfakh_zouari@yahoo.fr
Kanoun
Safia
S
Manni
Laila
L
Nasri
Moncef
M
D000595:Amino Acid Sequence; D000818:Animals; D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D002645:Chickens; D004795:Enzyme Stability; D005241:Feathers / Q000378:metabolism* / Q000382:microbiology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D045483:Rivers / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014416:Tunisia; D014876:Water Pollution
D010447:Peptide Hydrolases; C035855:keratinase; D012697:Serine Endopeptidases