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 alpha(S)-, beta-, and kappa-casein during in vitro incubation at 7 or 22 degrees 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.

Extracellular thermostable proteolytic activity of Pseudomonas fluorescens PS19 on bovine milk caseins / M. Stuknytė, M. Decimo, M. Colzani, T. Silvetti, M. Brasca, S. Cattaneo, G. Aldini, I. De Noni. - In: JOURNAL OF DAIRY SCIENCE. - ISSN 0022-0302. - 99:6(2016 Jun), pp. 4188-4195. [10.3168/jds.2016-10894]

Extracellular thermostable proteolytic activity of Pseudomonas fluorescens PS19 on bovine milk caseins

M. Stuknytė
Primo
;
M. Colzani;T. Silvetti;S. Cattaneo;G. Aldini
Penultimo
;
I. De Noni
2016

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 alpha(S)-, beta-, and kappa-casein during in vitro incubation at 7 or 22 degrees 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.
AprX protease; Pseudomonas fluorescens; casein; liquid chromatography-mass spectrometry
Settore AGR/15 - Scienze e Tecnologie Alimentari
Settore AGR/16 - Microbiologia Agraria
Settore CHIM/01 - Chimica Analitica
Settore AGR/13 - Chimica Agraria
Settore BIO/11 - Biologia Molecolare
Settore BIO/10 - Biochimica
Settore CHIM/08 - Chimica Farmaceutica
16-mar-2016
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/404957
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