Substrate specificity characterization of a thermostable keratinase from Pseudomonas aeruginosa KS-1.
A feather-degrading strain of Pseudomonas aeruginosa KS-1 was used in the present study. Its crude cell-free fermentation broth completely degraded chicken feather within 12 h, in the absence of disulphide reductase activity. Keratinase from its extracellular broth was purified and characterized, assuming that it would be a potential beta-keratin-degrading enzyme with prospective applications in degradation of beta-plaques of prions. The keratinase was purified by using Q-Sepharose anion exchange chromatography and its molecular weight, as determined by SDS-PAGE analysis, was 45 kDa. It was an alkaline, serine protease with pH and temperature optima of 9 and 60 degrees C, respectively. The enzyme was highly thermostable with a t (1/2) > 2 h at 80 degrees C and had a very high K to C (keratinolytic to caseinolytic) ratio of 2.5. Besides feather keratin, it also hydrolyzed a variety of other complex substrates including fibrin, gelatin and meat protein. Its activity on synthetic substrates revealed that it efficiently cleaves them in the order phenylalanine > lysine > alanine > leucine p-nitroanilides. It also cleaved insulin B chain between Val(12)-Glu(13), Ala(14)-Leu(15), Gly(20)-Glu(21) and Arg(22)-Gly(23) residues.
Sharma
Richa
R
Department of Microbiology, University of Delhi, South Campus, New Delhi 110021, India.
Gupta
Rani
R
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002645:Chickens; D002852:Chromatography, Ion Exchange; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005241:Feathers / Q000378:metabolism / Q000382:microbiology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010447:Peptide Hydrolases; C035855:keratinase