Campus Tefé, Federal Institute of Education, Science and Technology of Amazon (IFAM), Tefé, Brazil.
Laboratory of Immunopathology Keizo Asami (LIKA), Federal University of Pernambuco, Recife, Brazil.
Prep Biochem Biotechnol. 2021;51(3):289-299. doi: 10.1080/10826068.2020.1815054. Epub 2020 Sep 9.
β-Galactosidase production, partial purification and characterization by a new fungal were investigated. Partial purification was performed by aqueous two-phase system (ATPS) using polyethylene glycol (PEG) molar mass, PEG concentration, citrate concentration and pH as the independent variables. Purification factor (), partition coefficient () and yield () were the responses. After identification by rDNA sequencing and classification as URM 7803, this isolate achieved a maximum cell concentration and β-galactosidase activity of 0.48 g/L and 462.1 U/mL, respectively. β-Galactosidase partitioned preferentially for bottom salt-rich phase likely due to hydrophobicity and volume exclusion effect caused in the top phase by the high PEG concentration and molar mass. The highest value of (12.94) was obtained using 24% (w/w) PEG 8000 g/mol and 15% (w/w) citrate, while that of (79.76%) using 20% (w/w) PEG 400 g/mol and 25% (w/w) citrate, both at pH 6. The enzyme exhibited optimum temperature in crude and ATPS extracts in the ranges 35-50 °C and 40-55 °C, respectively, and optimum pH in the range 3.0-4.5, with a fall of enzyme activity under alkaline conditions. Some metal ions and detergents inhibited, while others stimulated enzyme activity. Finally, URM 7803 β-galactosidase showed a profile suitable for prebiotics production.
研究了一种新型真菌的β-半乳糖苷酶的生产、部分纯化和特性。通过双水相系统（ATPS），使用聚乙二醇（PEG）分子量、PEG 浓度、柠檬酸浓度和 pH 值作为独立变量进行部分纯化。以纯化因子（）、分配系数（）和产率（）为响应值。经过 rDNA 测序鉴定和分类为 URM 7803 后，该分离株的最大细胞浓度和β-半乳糖苷酶活性分别达到 0.48 g/L 和 462.1 U/mL。β-半乳糖苷酶优先分配到富含底部盐的相中，可能是由于顶部相中高 PEG 浓度和分子量导致疏水性和体积排除效应。使用 24%（w/w）PEG 8000 g/mol 和 15%（w/w）柠檬酸时，获得了最高的 （12.94），而使用 20%（w/w）PEG 400 g/mol 和 25%（w/w）柠檬酸时，获得了最高的 （79.76%），均在 pH 6 时。粗酶和 ATPS 提取物中酶的最适温度范围分别为 35-50°C 和 40-55°C，最适 pH 范围为 3.0-4.5，碱性条件下酶活性下降。一些金属离子和去污剂抑制酶活性，而另一些则刺激酶活性。最后，URM 7803 β-半乳糖苷酶的活性适合生产益生元。
