title: Extracellular thermostable proteolytic activity of the milk spoilage bacterium Pseudomonas fluorescens PS19 on bovine caseins.
abstract: We studied the thermostable proteolytic activity of Pseudomonas fluorescens PS19 isolated from raw bovine milk. The heat-treated cell-free supernatant (HT-CFS) contained a thermostable protease of approximately 45 kDa, as revealed by casein zymography. We assigned this enzyme to P. fluorescens AprX metalloprotease (UniProtKB Acc. No. C9WKP6). After concentration by ultrafiltration at 10 kDa, the HT-CFS showed 2 other thermostable proteolytic bands on zymogram, with molecular masses of approximately 15 and 25 kDa. The former resulted a fragment of the AprX protease, whereas the 25-kDa protease was not homologous to any known protein of Pseudomonas spp. Subsequently, we assessed the proteolytic activity of the HT-CFS on bovine αS-, β-, and κ-casein during in vitro incubation at 7 or 22°C. By means of ultra-performance liquid chromatography-tandem mass spectrometry we identified the released peptides (n=591). Some of them resisted proteolysis during the whole incubation period at both incubation temperatures and, therefore, they could be assumed as indicators of the proteolytic action of P. fluorescens PS19 on bovine caseins.
authors.affiliation: Dipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente (DeFENS), Università degli Studi di Milano, via G. Celoria 2, 20133 Milan, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Stuknytė
authors.affiliation: Consiglio Nazionale delle Ricerche (CNR), Istituto di Scienze delle Produzioni Alimentari (ISPA), via G. Celoria 2, 20133 Milan, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Decimo
authors.affiliation: Dipartimento di Scienze Farmaceutiche (DiSFarm), Università degli Studi di Milano, via L. Mangiagalli 25, 20133 Milan, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Colzani
authors.affiliation: Consiglio Nazionale delle Ricerche (CNR), Istituto di Scienze delle Produzioni Alimentari (ISPA), via G. Celoria 2, 20133 Milan, Italy.
authors.forename: T
authors.initials: T
authors.lastname: Silvetti
authors.affiliation: Consiglio Nazionale delle Ricerche (CNR), Istituto di Scienze delle Produzioni Alimentari (ISPA), via G. Celoria 2, 20133 Milan, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Brasca
authors.affiliation: Dipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente (DeFENS), Università degli Studi di Milano, via G. Celoria 2, 20133 Milan, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Cattaneo
authors.affiliation: Dipartimento di Scienze Farmaceutiche (DiSFarm), Università degli Studi di Milano, via L. Mangiagalli 25, 20133 Milan, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Aldini
authors.affiliation: Dipartimento di Scienze per gli Alimenti, la Nutrizione e l'Ambiente (DeFENS), Università degli Studi di Milano, via G. Celoria 2, 20133 Milan, Italy. Electronic address: ivano.denoni@unimi.it.
authors.forename: I
authors.initials: I
authors.lastname: De Noni
mesh_terms: D000818:Animals; D002364:Caseins / Q000378:metabolism*; D002417:Cattle; D010450:Endopeptidases / Q000378:metabolism; D008892:Milk / Q000382:microbiology*; D011549:Pseudomonas / Q000378:metabolism; D011551:Pseudomonas fluorescens / Q000302:isolation & purification
chemical_list: D002364:Caseins; D010450:Endopeptidases
keywords: AprX protease; Pseudomonas fluorescens; casein; liquid chromatography-mass spectrometry