Extracellular expression of keratinase Ker P from Pseudomonas aeruginosa in E. coli.
Keratinase from Pseudomonas aeruginosa KS-1 was expressed constitutively as an extracellular protein in Escherichia coli with high specific activity of 3.7 kU/mg. It was purified fourfold as a 33 kDa monomeric protein by Q-Sepharose ion exchange chromatography with a recovery of 95%. It is a serine protease with optimal activity at pH 9 and 50°C. It was stable from pH 4 to 12 for 1 h with a t(1/2) of 12 min at 70°C. It hydrolyzed haemoglobin > fibrin > feather keratin > azo-casein > casein > meat protein > gelatin. Among synthetic substrates, it efficiently hydrolyzed N-Suc-ala-ala-pro-phe-pNA, N-Suc-ala-ala-ala-pNA, N-Suc-ala-ala-pro-leu-pNA and also plasmin substrate, D: -Val-Leu-Lys-pNA.
Sharma
Richa
R
Department of Microbiology, University of Delhi, South Campus, New Delhi, 110021, India.
Gupta
Rani
R
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012685:Sepharose; D010447:Peptide Hydrolases; C035855:keratinase