Aguilar-Zárate Pedro, Gutiérrez-Sánchez Gerardo, Michel Mariela R, Bergmann Carl W, Buenrostro-Figueroa José J, Ascacio-Valdés Juan A, Contreras-Esquivel Juan C, Aguilar Cristóbal N
Engineering Department, Instituto Tecnológico de Ciudad Valles, Tecnológico Nacional de México, Ciudad Valles, San Luis Potosí C.P. 79010, Mexico.
Complex Carbohydrate Research Center, The University of Georgia, Athens, GA 30602, USA.
Foods. 2023 Feb 20;12(4):903. doi: 10.3390/foods12040903.
The present work describes the purification of an enzyme capable of degrading punicalagin. The enzyme was produced by GH1 by solid-state fermentation, and the enzyme production was induced by using ellagitannins as the sole carbon source. The purification steps included the concentration by lyophilization, desalting, anionic exchange, and gel filtration chromatography. The enzyme kinetic constants were calculated by using punicalagin, methyl gallate, and sugar beet arabinans. The molecular mass of the protein was estimated by SDS-PAGE. The identified bands were excised and digested using trypsin, and the peptides were submitted to HPLC-MS/MS analysis. The docking analysis was conducted, and a 3D model was created. The purification fold increases 75 times compared with the cell-free extract. The obtained values were 0.053 mM, 0.53% and 6.66 mM for punicalagin, sugar beet arabinans and methyl gallate, respectively. The optimal pH and temperature for the reaction were 5 and 40 °C, respectively. The SDS-PAGE and native PAGE analysis revealed the presence of two bands identified as α-l-arabinofuranosidase. Both enzymes were capable of degrading punicalagin and releasing ellagic acid.
本研究描述了一种能够降解石榴皮苷的酶的纯化过程。该酶由GH1通过固态发酵产生，以鞣花单宁作为唯一碳源诱导产酶。纯化步骤包括冻干浓缩、脱盐、阴离子交换和凝胶过滤色谱。使用石榴皮苷、没食子酸甲酯和甜菜阿拉伯聚糖计算酶动力学常数。通过SDS-PAGE估计蛋白质的分子量。将鉴定出的条带切下并用胰蛋白酶消化，肽段进行HPLC-MS/MS分析。进行对接分析并创建三维模型。与无细胞提取物相比，纯化倍数增加了75倍。石榴皮苷、甜菜阿拉伯聚糖和没食子酸甲酯的所得值分别为0.053 mM、0.53%和6.66 mM。反应的最佳pH和温度分别为5和40℃。SDS-PAGE和非变性PAGE分析显示存在两条被鉴定为α-L-阿拉伯呋喃糖苷酶的条带。两种酶都能够降解石榴皮苷并释放鞣花酸。
