Isolation and characterization of a protease from Pseudomonas fluorescens RO98.
Pseudomonas fluorescens RO98, a raw milk isolate, was inoculated into McKellar's minimal salts medium and incubated at 25 degrees 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 degrees 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 degrees C, respectively. It was heat stable with a D55 of 41 min and a D62.5 of 18 h. Molecular weight of the enzyme was estimated to be 52 kDa by SDS PAGE and size exclusion chromatography. Values for kM of 144.28, 18.73, 110.20 and 35.23 micromol were obtained for whole, alpha-, beta- and kappa-casein, with a Vmax of 8.26, 0.09, 0.42 and 0.70 micromol mg-1 min-1, respectively. The enzyme hydrolysed kappa-casein preferentially when incubated with artificial casein micelles.
Koka
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Center for Microbe Detection & Physiology, Department of Nutrition and Food Sciences, Utah State University, Logan 84322, USA.
Weimer
B C
BC
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D003470:Culture Media; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008892:Milk / Q000382:microbiology; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000254:growth & development; D013696:Temperature
D002364:Caseins; D003470:Culture Media; D011480:Protease Inhibitors; D010450:Endopeptidases