Department of Life Sciences, University of Limerick, Limerick, Ireland.
J Agric Food Chem. 2013 Apr 3;61(13):3193-204. doi: 10.1021/jf305274e. Epub 2013 Mar 21.
Purified bovine β-casein was digested with glutamyl endopeptidase (GE) at 37 and 50 °C for 4 h. The peptides generated were determined using nano-LC-ESI-qTOF-MS/MS. GE was highly specific and hydrolyzed peptide bonds in β-casein predominantly on the carboxy terminal of Glu and Asp. Pro residues were not preferred, while Met was poorly preferred at the P1' position. Glu-Met hydrolysis was less preferred in comparison to Asp-Met hydrolysis. Five synthetic peptides corresponding to specific sequences in β-casein were incubated with GE at 37 °C to further characterize the substrate specificity. MS analysis of the digestion products indicated that GE hydrolyzed Glu-Ser in Glu-Glu-Ser. Furthermore, hydrolysis of Glu-Met and Glu-Pro was observed. The presence of multiple-phosphorylated Ser residues upstream from the scissile bond did not appear to affect hydrolysis of Glu-Ser. The results herein are relevant to our understanding of the substrate specificity of GE and the peptides that may be expected during the hydrolysis of β-casein.
将纯化的牛β-酪蛋白用谷氨酰内肽酶（GE）在 37 和 50°C 下消化 4 小时。使用纳升液相色谱-电喷雾-四极杆飞行时间串联质谱（nano-LC-ESI-qTOF-MS/MS）测定生成的肽。GE 具有高度的特异性，主要在 Glu 和 Asp 的羧基末端水解β-酪蛋白中的肽键。Pro 残基不是首选，而 Met 在 P1'位置的优先性较差。与 Asp-Met 水解相比，Glu-Met 水解的优先性较低。将五个对应于β-酪蛋白特定序列的合成肽与 GE 在 37°C 下孵育，以进一步表征其底物特异性。消化产物的 MS 分析表明，GE 在 Glu-Glu-Ser 中水解 Glu-Ser。此外，还观察到 Glu-Met 和 Glu-Pro 的水解。在切割位点上游存在多个磷酸化的 Ser 残基似乎不会影响 Glu-Ser 的水解。本文的结果与我们对 GE 的底物特异性以及在β-酪蛋白水解过程中可能产生的肽的理解有关。
