Production and characterization of thermostable metallo-keratinase from newly isolated Bacillus subtilis NRC 3

• Published in: International journal of biological macromolecules Vol. 55; pp. 169 - 175
• Main Authors: Tork, Sanaa E., Shahein, Yasser E., El-Hakim, Amr E., Abdel-Aty, Azza M., Aly, Magda M.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2013
• Subjects: 16S rDNA; Bacillus subtilis; Bacillus subtilis - classification; Bacillus subtilis - genetics; Bacillus subtilis - isolation & purification; Bacillus subtilis - metabolism; barium; calcium; cation exchange chromatography; cations; chickens; Enzyme Activation; enzyme activity; Enzyme Stability; enzymes; feather meal; feathers; gel chromatography; Hydrogen-Ion Concentration; Hydrolysis; keratin; Kinetics; magnesium; Metallo-keratinase; PCR-based RFLP; Peptide Hydrolases - biosynthesis; Peptide Hydrolases - chemistry; Peptide Hydrolases - isolation & purification; Peptide Hydrolases - metabolism; Phylogeny; potassium; RNA, Ribosomal, 16S; SDS-PAGE; sodium; Temperature; thermal stability; Thermostability; Thermostability; SDS-PAGE; PCR-based RFLP; 16S rDNA; Metallo-keratinase; Bacillus subtilis
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2013.01.002

Novel keratinolytic enzyme (32kDa) secreted by a newly isolated Bacillus strain (Bacillus subtilis NRC3) cultivated in medium containing chicken feather meal was purified and partially characterized in a set of biochemical assays. The purification was carried out by applying a protocol of two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-75 columns. The purified enzyme showed a specific activity of 5233units/mg protein against 169units/mg protein for crude extract with 31 fold purification. The enzymatic activity of the purified keratinolytic enzyme stimulated by Na+, K+, Mg2+, Ba2+, Ca2+, and inhibited by entire tested cations and metalloproteinase inhibitors, indicating that it belongs to metallo-keratinase enzymes. The optimum pH and temperature for the purified enzyme were (7.5, 8.0) and (50, 40°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. These results suggest that this keratinase may be a useful alternative and ecofriendly route for handling the abundant amount of waste feathers.