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Isolation and characterization of a protease from Pseudomonas fluorescens RO98
Author(s) -
Koka R.,
Weimer B.C.
Publication year - 2000
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1046/j.1365-2672.2000.01108.x
Subject(s) - pseudomonas fluorescens , protease , casein , chromatography , size exclusion chromatography , chemistry , enzyme , hydrolysis , enzyme assay , biochemistry , biology , bacteria , genetics
Pseudomonas fluorescens RO98, a raw milk isolate, was inoculated into McKellar's minimal salts medium and incubated at 25 °C for 48 h to allow production of protease. A zinc‐metalloacid protease was purified from the cell‐free concentrate by anion exchange and gel filtration chromatography. The purified protease was active between 15 and 55 °C, and pH 4·5 and 9·0, and was stable to pasteurization. The enzyme had pH and temperature optima for activity of 5·0 and 35 °C, respectively. It was heat stable with a D 55 of 41 min and a D 62·5 of 18 h. Molecular weight of the enzyme was estimated to be 52 kDa by SDS PAGE and size exclusion chromatography. Values for k M of 144·28, 18·73, 110·20 and 35·23 µmol were obtained for whole, α‐, β‐ and κ‐casein, with a V max of 8·26, 0·09, 0·42 and 0·70 µmol mg −1 min −1 , respectively. The enzyme hydrolysed κ‐casein preferentially when incubated with artificial casein micelles.