Academic Unit of Garanhuns, Federal Rural University of Pernambuco, Garanhuns, PE, Brazil.
Laboratory of Basic Biology Teaching Azarias Salgado/LABAS, Reference High School Azarias Salgado, Angelim, PE, Brazil.
Prep Biochem Biotechnol. 2020;50(6):619-626. doi: 10.1080/10826068.2020.1721535. Epub 2020 Feb 4.
The protease from Kita UCP1279 extraction by aqueous two-phase PEG-Citrate (ATPS) systems, using a factorial design 2, was investigated. Then, the variables studied were polyethylene glycol (PEG) molar mass (M), concentrations of PEG (C) and citrate (C), and pH. The responses analyzed were the partition coefficient (K), activity yield (Y) and purification factor (PF). The thermodynamic parameters of the ATPS partition were estimated as a function of temperature. ATPS was able to pre-purify the protease (PF = 1.6) and obtained 84% activity yield. The thermodynamic parameters ΔG° (-10.89 kJ mol), ΔH (-5.0 kJ mol) and partition ΔS (19.74 J mol K) showed that the preferential migration of almost all protein contaminants of the crude extract to the salt-rich phase, while the preferred protease was the PEG rich phase. The extracted enzyme presents optimum temperature and pH at range of 40-50 °C and 9.0-11.0, respectively. Moreover, the enzyme was identified as serine protease based on inhibition profile. ATPS showed the satisfactory performance as the first step for Kita UCP1279 protease pre-purification.
采用两水相系统（PEG-柠檬酸）从 Kita UCP1279 中提取蛋白酶，通过析因设计 2 进行研究。然后，研究了聚乙二醇（PEG）的摩尔质量（M）、PEG（C）和柠檬酸（C）浓度以及 pH 等变量。分析的响应是分配系数（K）、活性产率（Y）和纯化因子（PF）。ATPS 相分配的热力学参数随温度而变化。ATPS 能够预纯化蛋白酶（PF=1.6），并获得 84%的活性产率。热力学参数 ΔG°（-10.89 kJ mol）、ΔH（-5.0 kJ mol）和分配 ΔS（19.74 J mol K）表明，几乎所有粗提物中蛋白质污染物优先迁移到富含盐的相，而优选的蛋白酶是富含 PEG 的相。提取的酶在 40-50°C 和 9.0-11.0 的范围内具有最佳温度和 pH 值。此外，根据抑制谱，该酶被鉴定为丝氨酸蛋白酶。ATPS 作为 Kita UCP1279 蛋白酶预纯化的第一步表现出令人满意的性能。
