College of Food Science & Technology, Yunnan Agricultural University, Kunming 650201, Yunnan, China.
College of Food Science & Technology, Yunnan Agricultural University, Kunming 650201, Yunnan, China.
Food Res Int. 2024 May;183:114225. doi: 10.1016/j.foodres.2024.114225. Epub 2024 Mar 12.
The aim of this study was to isolate and identify the main milk-clotting proteases from Prinsepia utilis Royle. Protein isolates obtained using precipitation with 20 %-50 % ammonium sulfate (AS) showed higher milk-clotting activity (MCA) at 154.34 + 0.35 SU. Two milk-clotting proteases, namely P191 and P1831, with molecular weight of 49.665 kDa and 68.737 kDa, respectively, were isolated and identified using liquid chromatography-mass spectrometry (LC-MS/MS). Bioinformatic analysis showed that the two identified milk-clotting proteases were primarily involved in hydrolase activity and catabolic processes. Moreover, secondary structure analysis showed that P191 structurally consisted of 40.85 % of alpha-helices, 15.96 % of beta-strands, and 43.19 % of coiled coil motifs, whereas P1831 consisted of 70 % of alpha-helices, 7.5 % of beta-strands, and 22.5 % of coiled coil motifs. P191 and P1831 were shown to belong to the aspartic protease and metalloproteinase types, and exhibited stability within the pH range of 4-6 and good thermal stability at 30-80 °C. The addition of CaCl (<200 mg/L) increased the MCA of P191 and P1831, while the addition of NaCl (>3 mg/mL) inhibited their MCA. Moreover, P191 and P1831 preferably hydrolyzed kappa-casein, followed by alpha-casein, and to a lesser extent beta-casein. Additionally, cheese processed with the simultaneous use of the two proteases isolated in the present study exhibited good sensory properties, higher protein content, and denser microstructure compared with cheese processed using papaya rennet or calf rennet. These findings unveil the characteristics of two proteases isolated from P. utilis, their milk-clotting properties, and potential application in the cheese-making industry.
本研究旨在从 Prinsepia utilis Royle 中分离和鉴定主要的凝乳蛋白酶。使用 20%-50%硫酸铵（AS）沉淀获得的蛋白质提取物在 154.34+0.35SU 时表现出更高的凝乳酶活性（MCA）。使用液相色谱-质谱联用（LC-MS/MS）分离和鉴定了两种凝乳蛋白酶，分别命名为 P191 和 P1831，分子量分别为 49.665 kDa 和 68.737 kDa。生物信息学分析表明，这两种鉴定出的凝乳蛋白酶主要参与水解酶活性和分解代谢过程。此外，二级结构分析表明，P191 结构上由 40.85%的α-螺旋、15.96%的β-折叠和 43.19%的卷曲螺旋组成，而 P1831 则由 70%的α-螺旋、7.5%的β-折叠和 22.5%的卷曲螺旋组成。P191 和 P1831 属于天冬氨酸蛋白酶和金属蛋白酶类型，在 pH 4-6 范围内表现出稳定性，在 30-80°C 下具有良好的热稳定性。添加 CaCl（<200mg/L）会提高 P191 和 P1831 的 MCA，而添加 NaCl（>3mg/mL）则会抑制其 MCA。此外，P191 和 P1831 优先水解κ-酪蛋白，其次是α-酪蛋白，β-酪蛋白水解程度较低。此外，与使用木瓜凝乳蛋白酶或小牛凝乳蛋白酶加工的奶酪相比，使用本研究中分离的两种蛋白酶同时加工的奶酪具有良好的感官特性、更高的蛋白质含量和更致密的微观结构。这些发现揭示了从 P. utilis 中分离的两种蛋白酶的特性、它们的凝乳特性以及在奶酪制造工业中的潜在应用。
