Abstract

Enzyme immobilization has been reported as a promising approach to improving parameters such as thermal stability, pH and reusability. In this study, a polyacrylamide cryogel functionalized with L-phenylalanine was prepared to be used in the adsorption of β-glucosidase from Thermoascus aurantiacus, aiming at its separation and also its immobilization on the cryogel matrix. The enzyme was produced by solid state fermentation. First, the adsorption was studied as a function of the pH and the resulting yield (Y, %) and purification factor (P_F, dimensionless) were determined (1.57–5.13 and 64.19–91.20, respectively). The P_F and yield from eluate samples obtained at pH 3.0 were the highest (5.13 and 91.20, respectively). Then, β-glucosidase was immobilized on the hydrophobic cryogel and the recovery activities (%) were determined as a function of temperature and in the presence of different saline solutions. The values ranged from 14.45 to 45.97. As expected, salt type and ionic strength affected the activity remained in the immobilized β-glucosidase. The average bioreactor activity was 39.9 U/g of dry cryogel and its operational stability was measured, with no decrease in activity being observed during seven cycles. Kinetic parameters of free and immobilized enzyme were determined according to different models.

摘要

据报道，酶固定化是改善热稳定性、pH 值和可重复使用性等参数的一种很有前途的方法。本研究制备了一种L-苯丙氨酸功能化的聚丙烯酰胺冷冻凝胶，用于吸附橙色嗜热子囊菌中的β-葡萄糖苷酶，旨在将其分离并固定在冷冻凝胶基质上。该酶是通过固态发酵产生的。首先，将吸附作为 pH 的函数进行研究，并确定所得产率（Y，%）和纯化因子（PF ，无量纲）（_{分别为 1.57–5.13 和 64.19–91.20）。}P _F _在 pH 3.0 下获得的洗脱液样品的产量最高（分别为 5.13 和 91.20）。然后，将 β-葡萄糖苷酶固定在疏水性冷冻凝胶上，并在不同盐溶液存在的情况下将回收活性 (%) 确定为温度的函数。值范围从 14.45 到 45.97。正如预期的那样，盐类型和离子强度会影响固定化 β-葡萄糖苷酶中保留的活性。平均生物反应器活性为 39.9 U/g 干冻凝胶，并测量了其操作稳定性，在七个循环期间未观察到活性降低。根据不同的模型确定游离酶和固定化酶的动力学参数。