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Research Journal of Microbiology

Year: 2010  |  Volume: 5  |  Issue: 10  |  Page No.: 954 - 965

Thermostable, Thiol Activated Keratinase from Pseudomonas aeruginosa KS-1 for Prospective Applications in Prion Decontamination

R. Sharma and R. Gupta

Abstract

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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