Extracellular thermostable proteolytic activity of the milk spoilage bacterium Pseudomonas fluorescens PS19 on bovine caseins.
Extracellular thermostable proteolytic activity of the milk spoilage bacterium Pseudomonas fluorescens PS19 on bovine caseins.
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DOI：
10.3168/jds.2016-10894
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发表时间：
2016-06
影响因子：
3.5
通讯作者：
M. Stuknytė;M. Decimo;M. Colzani;T. Silvetti;Milena Brasca;S. Cattaneo;G. Aldini;I. D. Noni
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文献类型：
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作者：
M. Stuknytė;M. Decimo;M. Colzani;T. Silvetti;Milena Brasca;S. Cattaneo;G. Aldini;I. D. Noni
研究了从牛乳中分离的荧光假单胞菌PS19的热稳定蛋白水解活性。热处理的无细胞上清液（HT-CFS）含有约45 kDa的热稳定蛋白酶，如酪蛋白酶谱所示。我们将该酶指定为荧光假单胞菌AprX金属蛋白酶（UniProtKB Acc.No.C9WKP6）。在10 kDa的超滤浓缩后，HT-CFS在酶谱上显示出另外2条热稳定的蛋白水解条带，分子量约为15和25 kDa。前者产生AprX蛋白酶的片段，而25-kDa蛋白酶与假单胞菌属的任何已知蛋白都不同源。随后，我们评估了HT-CFS在7 ° C或22°C体外孵育期间对牛αS-、β-和κ-酪蛋白的蛋白水解活性。通过超高效液相色谱-串联质谱法，我们确定了释放的肽（n=591）。它们中的一些在两种孵育温度下的整个孵育期间抵抗蛋白水解，因此，它们可以被假定为荧光假单胞菌PS 19对牛酪蛋白的蛋白水解作用的指示剂。
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.