Thermostable, Thiol Activated Keratinase from Pseudomonas aeruginosa KS-1 for Prospective Applications in Prion Decontamination
Fifty proteolytic bacterial cultures were screened for their potential to degrade chicken feather. Eight strains which degraded chicken feather within 24 h were further evaluated for degradation of surrogate yeast prion protein, Sup35 NM. Prion degradation was studied by congo red binding assay and a strain of Pseudomonas aeruginosa KS-1 was selected for further evaluation as it exhibited maximal shift in absorbance of congo red from 0.268 to 0.926. Keratinase from this strain was purified using Q-sepharose chromatography with 60% recovery and a purification fold of 2.24. It was identified as a 33 kDa monomeric protein having a pH and temperature optima of 7 and 50°C, respectively. It was stable over a broad range of pH ranging from pH 2-11 and was highly thermostable with a t1/2 of >2 h at 80°C. It was a serine, thiol activated protease with two fold increase in activity in presence of β-mercaptoethanol. It also hydrolyzed a range of complex protein substrates including casein, feather, fibrin. Amidolytic activity on synthetic substrates revealed that it efficiently cleaved phenylalanine and arginine. It hydrolyzed insulin B-chain at cystein, glycine, histidine, phenylalanine and leucine as revealed by ESI-MS analysis.
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