title: Purification and partial characterization of serine-metallokeratinase from a newly isolated Bacillus pumilus NRC21.
abstract: A serine metallokeratinase enzyme (30 kDa) produced by a newly isolated Bacillus strain (Bacillus pumilus NRC21) cultivated under optimized conditions in medium containing chicken feather meal was purified and characterized in a set of biochemical assays. The purification was carried out using two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-100 columns. The purified enzyme showed a specific activity of 2000 units/mg protein against 170 units/mg protein for crude extract with 12 fold purification. The enzymatic activity of the keratinase stimulated by (Na(+), K(+), Mg(2+)), Hg(+2) had no effect, and inhibited by entire tested cations, serine and metalloproteinase inhibitors, therefore it can be considered as a serine metalloenzyme. The optimum pH and temperature for the purified enzyme were (7.5, 8.5) and (50, 45 °C) when using keratin azure and azocasein as substrates, respectively. The purified enzyme was highly stable at broad pH and temperature ranged (5-10) and (20-60 °C), respectively and its thermoactivity and thermostability were enhanced in the presence of 5 mM Mg(+2). These results suggest that the purified keratinase may be used in several industrial applications.
authors.affiliation: Biological Sciences Department, King Abdulaziz University, Saudi Arabia; Microbial Genetics Department, National Research Centre, Dokki, Egypt.
authors.forename: Sanaa E
authors.initials: SE
authors.lastname: Tork
authors.affiliation: Molecular Biology Department, National Research Centre, Dokki, Egypt; Biology Department, Hail University, Saudi Arabia. Electronic address: yassershahein_nrc@yahoo.com.
authors.forename: Yasser E
authors.initials: YE
authors.lastname: Shahein
authors.affiliation: Molecular Biology Department, National Research Centre, Dokki, Egypt.
authors.forename: Amr E
authors.initials: AE
authors.lastname: El-Hakim
authors.affiliation: Molecular Biology Department, National Research Centre, Dokki, Egypt; Clinical Laboratory Department, Al-Gad International Colleges for Medical Science, Saudi Arabia.
authors.forename: Azza M
authors.initials: AM
authors.lastname: Abdel-Aty
authors.affiliation: Biological Sciences Department, King Abdulaziz University, Saudi Arabia; Botany Department, Faculty of Science, Kafr El-Sheikh University, Egypt.
authors.forename: Magda M
authors.initials: MM
authors.lastname: Aly
mesh_terms: D000069979:Bacillus pumilus / Q000145:classification / Q000201:enzymology*; D005591:Chemical Fractionation / Q000379:methods*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D012995:Solubility
chemical_list: D004791:Enzyme Inhibitors; D008670:Metals; D011506:Proteins; D012694:Serine; D010447:Peptide Hydrolases; C035855:keratinase
keywords: 16S rRNA; Bacillus; Keratinase; SDS-PAGE