Iannucci Nancy B, Navarro del Cañizo Agustín A, Cascone Osvaldo
Cátedra de Microbiología Industrial y Biotecnología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina.
Appl Biochem Biotechnol. 2003 Mar;104(3):173-84. doi: 10.1385/abab:104:3:173.
Twenty triazinic dyes were assayed as ligands for the chromatographic affinity purification of a neutral protease from Flavourzyme, a commercial preparation. Screening at pH 4.0 allowed the selection of eight dyes on the basis of their high protease adsorption. When the pH was set to 5.0 in order to increase selectivity, only Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A maintained protease adsorption at high values. Neither maximum capacities nor dissociation constants calculated from isotherms measured at 8 and 25 degrees C showed great differences. By contrast, a strong temperature effect was evidenced in the elution step: elution at 8 degrees C allowed 70, 81, and 98% recovery of adsorbed protease with Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A, respectively, whereas only 20% recovery was attained at 25 degrees C. Based on the results obtained, a purification process for the neutral protease contained in Flavourzyme with Cibacron Blue F3G-A as the affinity ligand was developed, yielding 96% of electrophoretically pure enzyme in a single step, the specific activity rising from 850 to 3650 U/mg.
对20种三嗪类染料作为配体用于从商业制剂风味酶中色谱亲和纯化中性蛋白酶进行了测定。在pH 4.0下进行筛选，基于其对蛋白酶的高吸附能力选出了8种染料。当将pH调至5.0以提高选择性时，只有黄HE - 4R、红HE - 3B和汽巴克隆蓝F3G - A保持对蛋白酶的高吸附。在8℃和25℃下根据等温线计算得到的最大容量和解离常数均未显示出很大差异。相比之下，洗脱步骤中温度效应明显：在8℃下洗脱时，黄HE - 4R、红HE - 3B和汽巴克隆蓝F3G - A对吸附蛋白酶的回收率分别为70%、81%和98%，而在25℃下回收率仅为20%。基于所得结果，开发了以汽巴克隆蓝F3G - A为亲和配体从风味酶中纯化中性蛋白酶的工艺，一步即可得到96%的电泳纯酶，比活性从850 U/mg提高到3650 U/mg。
