Institute of Technical Chemistry, Department of Carbohydrate Technology, Technische Universität Braunschweig, Gaußstraße 17, 38106, Braunschweig, Germany.
Appl Biochem Biotechnol. 2020 Jul;191(3):1155-1170. doi: 10.1007/s12010-020-03252-7. Epub 2020 Jan 24.
Two simple and easily reproducible methods for the immobilization of β-galactosidase (β-gal) from Aspergillus oryzae on electrospun gelatin nanofiber mats (GFM) were developed. The process was optimized regarding the electrospinning solvent system and the subsequent cross-linking of GFM in order to increase their stability in water. β-Gal was covalently immobilized on activated gelatin nanofiber mats with hexamethylenediamine (HMDA) as a bifunctional linker and secondly via entrapment into the gelatin nanofibers during the electrospinning process (suspension electrospinning). Optimal immobilization parameters for covalent immobilization were determined to be at pH 7.5, 40 °C, β-gal concentration of 1 mg/mL and immobilization time of 24.5 h. For suspension electrospinning, the optimal immobilization parameters were identified at pH 4.5 and β-gal concentration of 0.027 wt.% in the electrospinning solution. The pH and temperature optima of immobilized β-gal shifted from 30 °C, pH 4.5 (free enzyme) to pH 3.5, 50 °C (covalent immobilization) and pH 3.5, 40 °C (suspension electrospinning). Striking differences in the Michaelis constant (K) of immobilized β-gal compared with free enzyme were observed with a reduction of K up to 50% for immobilized enzyme. The maximum velocity (v) of immobilization by suspension electrospinning was almost 20 times higher than that of covalent immobilization. The maximum GOS yield for free β-gal was found to be 27.7% and 31% for immobilized β-gal.
开发了两种简单且易于重现的方法，可将米曲霉β-半乳糖苷酶（β-gal）固定在静电纺丝明胶纳米纤维垫（GFM）上。为了提高其在水中的稳定性，优化了静电纺丝溶剂体系和 GFM 的后续交联过程。β-gal 与己二胺（HMDA）作为双官能 linker 共价固定在活化的明胶纳米纤维垫上，其次通过在静电纺丝过程中（悬浮静电纺丝）将其包埋在明胶纳米纤维中。确定共价固定的最佳固定化参数为 pH 7.5、40°C、β-gal 浓度为 1mg/mL 和固定化时间为 24.5 h。对于悬浮静电纺丝，确定的最佳固定化参数为 pH 4.5 和 0.027 wt.%的β-gal 在静电纺丝溶液中的浓度。固定化β-gal 的 pH 和温度最适值从游离酶的 30°C、pH 4.5（游离酶）分别转移到 50°C、pH 3.5（共价固定）和 40°C、pH 3.5（悬浮静电纺丝）。与游离酶相比，固定化β-gal 的米氏常数（K）明显降低，固定化酶的 K 降低了高达 50%。悬浮静电纺丝固定化的最大速度（v）几乎比共价固定化高 20 倍。游离β-gal 的最大 GOS 产率为 27.7%，而固定化β-gal 的最大 GOS 产率为 31%。
