title: Biochemical and molecular characterization of new keratinoytic protease from Actinomadura viridilutea DZ50.
abstract: A new extracellular thermostable keratinolytic protease, designated KERDZ, was purified and characterized from a thermophilic actinomycetes Actinomadura viridilutea DZ50 isolated from Algerian fishing port. The isolate exhibited high keratinase production when grown in chicken-feather meal media (18,000U/ml) after 96-h of incubation at 45°C. The enzyme was purified by ammonium sulfate precipitation (35-55%)-dialysis and heat treatment (30min at 75°C) followed by UNO S-1 FPLC cation exchange chromatography and size exclusion HPLC column. The biochemical characterizations carried on include physico-chemical determination and spectroscopic analysis. The MALDI-TOF/MS analysis revealed that the purified enzyme was a monomer with a molecular mass of 19536.10-Da. The sequence of the 25 N-terminal residues of KERDZ showed high homology with those of actinomycetes keratinases. Optimal activity was achieved at pH 11 and 80°C. KERDZ was completely inhibited by PMSF and DFP suggested its belonging to the serine keratinase family. KERDZ displayed higher levels of hydrolysis and catalytic efficiency than bacterial keratinases (KERAK-29, Actinase E, and KERAB) and subtilisins (subtilisin Carlsberg and subtilisin Novo). The kerDZ gene encoding KERDZ was isolated and its DNA sequence was determined. These properties make KERDZ a potential, promising and eco-friendly alternative to the conventional chemicals used for industrial applications.
authors.affiliation: 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.
authors.forename: Mouna
authors.initials: M
authors.lastname: Ben Elhoul
authors.affiliation: 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.
authors.forename: Nadia
authors.initials: N
authors.lastname: Zaraî Jaouadi
authors.affiliation: 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.
authors.forename: Hatem
authors.initials: H
authors.lastname: Rekik
authors.affiliation: 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.
authors.forename: Maroua
authors.initials: M
authors.lastname: Omrane Benmrad
authors.affiliation: 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.
authors.forename: Sondes
authors.initials: S
authors.lastname: Mechri
authors.affiliation: 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.
authors.forename: Emna
authors.initials: E
authors.lastname: Moujehed
authors.affiliation: National Centre for Research and Development of Fisheries and Aquaculture (CNRDPA) 11, Bd Amirouche PO Box 67, Bou Ismaïl, 42415, Tipaza, Algeria.
authors.forename: Sidali
authors.initials: S
authors.lastname: Kourdali
authors.affiliation: Laboratory of Natural Products Chemistry and Biomolecules (LNPCB), University of Blida 1, Road of Soumaâ, PO Box 270, 09000 Blida, Algeria.
authors.forename: Mohamed
authors.initials: M
authors.lastname: El Hattab
authors.affiliation: National Centre for Research and Development of Fisheries and Aquaculture (CNRDPA) 11, Bd Amirouche PO Box 67, Bou Ismaïl, 42415, Tipaza, Algeria; Laboratory of Natural Products Chemistry and Biomolecules (LNPCB), University of Blida 1, Road of Soumaâ, PO Box 270, 09000 Blida, Algeria.
authors.forename: Abdelmalek
authors.initials: A
authors.lastname: Badis
authors.affiliation: 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.
authors.forename: Samir
authors.initials: S
authors.lastname: Bejar
authors.affiliation: 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. Electronic address: bassem.jaouadi@cbs.rnrt.tn.
authors.forename: Bassem
authors.initials: B
authors.lastname: Jaouadi
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010802:Phylogeny; D059748:Proteolysis / Q000187:drug effects; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D013379:Substrate Specificity / Q000187:drug effects; D020860:Subtilisin / Q000378:metabolism; D013696:Temperature
chemical_list: D003902:Detergents; D004791:Enzyme Inhibitors; D007477:Ions; D008670:Metals; D012336:RNA, Ribosomal, 16S; D010447:Peptide Hydrolases; C035855:keratinase; D020860:Subtilisin
keywords: Actinomycetes; Homology modeling; Keratinase