title: SHG10 keratinolytic alkaline protease from Bacillus licheniformis SHG10 DSM 28096: Robust stability and unusual non-cumbersome purification.
abstract: Present study underlines an unusual non-cumbersome-powerful strategy for purification of SHG10 keratinolytic alkaline protease from Bacillus licheniformis SHG10 DSM 28096 with robust stability properties. The enzyme was impressively purified to homogeneity with specific activity, purification fold, and yield of 613.82 U mg-1, 58.91 and 99%, respectively, via a sequential two-step purification strategy: precipitation with 65% (NH4)2SO4and flow through fractions of DEAE-cellulose DE 53 column. SDS-PAGE conferred a monomeric enzyme with a molecular mass of 30.4 kDa. The enzyme demonstrated optimal activity at pH (10.0-11.0) and at 65 °C. It exhibited full stability at pH (6.0-11.0) over 38 h at 4 °C and at 65 °C for 15 min. Remarkable enhanced enzyme activity (130.15 and 126.37%) was retained in presence of commercial laundry detergents Oxi and Ariel after 1 h, respectively. Organic solvent stability of the enzyme was verified in butanol, ether, acetonitrile, isopropanol, and chloroform. Imposingly, full storage stability (100%) of the enzyme along 1 year in -20 °C was confirmed. Km-Vmaxwas 0.00174 mM-534.2 mM Sub · min-1· mg protein-1and 1.266 mg-28.89 mg Sub · h-1· mg protein-1on N-Suc-Ala-Ala-Pro-Phe-pNA and keratin azure, respectively. Robust stability properties of SHG10 keratinolytic alkaline protease along with rapid-efficient purification underpin its potential commercialization for industrial exploitation.
authors.affiliation: Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Egypt.
authors.forename: Amira M
authors.initials: AM
authors.lastname: Embaby
authors.affiliation: Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Egypt.
authors.forename: Hesham
authors.initials: H
authors.lastname: Saeed
authors.affiliation: Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Egypt.|Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas, USA.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Hussein
mesh_terms: D000069976:Bacillus licheniformis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D003636:DEAE-Cellulose; D003902:Detergents / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007633:Keratins / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D007633:Keratins; D003636:DEAE-Cellulose; D010450:Endopeptidases; C087822:alkaline protease
keywords: Bacillus licheniformis SHG10 DSM 28096; Non-cumbersome purification; Robust enzyme stability properties; SHG 10 keratinolytic alkaline protease