title: Purification and characterization of three extracellular proteinases produced by Pseudomonas fluorescens INIA 745, an isolate from ewe's milk.
abstract: Three proteinases were isolated from culture medium of Pseudomonas fluorescens INIA 745 and purified to homogeneity by a combination of Phenyl-Sepharose, DEAE-Sepharose, and Sephadex G-100 chromatography. Optimal temperature for enzymatic activity was 45 degrees C for all three proteinases. The pH optimum of proteinases I and II was found to be 7.0, while that of proteinase III was 8.0. Divalent metal ions like Cu2+, Co2+, Zn2+, Fe2+, and Hg2+ were inhibitory to proteinase activity while Ca2+, Mg2+, and Mn2+ had little or no inhibitory effect. The three enzymes were strongly inhibited by EDTA and 1,10-phenantroline and partially by cysteine. The three enzymes are metalloproteinases since they were inhibited by chelators and reactivated by Co2+, Mn2+, Cu2+, and Zn2+. The Km values of proteinases I, II, and III for casein were calculated to be 3.2, 2.6, and 5.2 mg/ml, respectively. Proteinases II and III rapidly degraded beta-casein, with preference to alphas1-casein, whereas proteinase I hydrolyzed both casein fractions at a slow rate.
authors.affiliation: Departamento de Tecnología de Alimentos, INIA, Madrid, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Fernández
authors.forename: A F
authors.initials: AF
authors.lastname: Mohedano
authors.forename: P
authors.initials: P
authors.lastname: Gaya
authors.forename: M
authors.initials: M
authors.lastname: Medina
authors.forename: M
authors.initials: M
authors.lastname: Nuñez
mesh_terms: D000818:Animals; D002847:Chromatography, Agarose; D010450:Endopeptidases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D005260:Female; D006863:Hydrogen-Ion Concentration; D008892:Milk / Q000382:microbiology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000302:isolation & purification; D012756:Sheep; D013696:Temperature
chemical_list: D011480:Protease Inhibitors; D010450:Endopeptidases