The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
Institute of Applied Biotechnology, Taizhou Vocational & Technical College, Taizhou, Zhejiang 318000, China.
Int J Biol Macromol. 2020 Jun 15;153:1220-1230. doi: 10.1016/j.ijbiomac.2019.10.252. Epub 2019 Nov 19.
In this study, three active alkaline proteases (AprEs) (BaApr1, BaApr2, and BaApr9) from Bacillus altitudinis W3 were obtained through bioinformatics analysis and verification. Multiple sequence alignment showed low identity of 64.60% and suggested that the three AprEs belonged to the S8A subfamily of serine proteases. They showed maximal activity with pH of 9.5 at 55 °C, 8.5 at 50 °C, and 10.5 at 45 °C, respectively. They were stable at alkaline condition and below 50 °C. In the presence of Ca, the optimal temperatures and thermostability of them were significantly improved. They were activated by Ca and Mg but inhibited by ethylenediaminetetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Surfactants had little effect on them, but most organic solvents had some inhibitory effect except for n-hexane. They were effective in hydrolyzing natural proteins such as casein and NON-fat powdered milk. BaApr1 exhibited the highest catalytic efficiency towards casein and showed an excellent effect on the desensitization of milk proteins. The present study reveals some useful characteristics of the three AprEs, and indicates that AprEs have potential application values in the desensitization process of milk proteins.
在这项研究中，通过生物信息学分析和验证，从嗜盐芽孢杆菌 W3 中获得了三种活性碱性蛋白酶（AprE）（BaApr1、BaApr2 和 BaApr9）。多重序列比对显示它们的同一性低至 64.60%，表明这三种 AprE 属于丝氨酸蛋白酶 S8A 亚家族。它们的最适 pH 值分别为 9.5（55°C）、8.5（50°C）和 10.5（45°C）。它们在碱性条件下和低于 50°C 时稳定。在 Ca 的存在下，它们的最适温度和热稳定性显著提高。它们被 Ca 和 Mg 激活，但被乙二胺四乙酸（EDTA）和苯甲基磺酰氟（PMSF）抑制。表面活性剂对它们影响不大，但除了正己烷外，大多数有机溶剂都有一定的抑制作用。它们对天然蛋白质如酪蛋白和非脂奶粉有很好的水解作用。BaApr1 对酪蛋白表现出最高的催化效率，并对牛奶蛋白脱敏具有极好的效果。本研究揭示了这三种 AprE 的一些有用特性，表明 AprE 在牛奶蛋白脱敏过程中具有潜在的应用价值。
