介孔二氧化钛固载γ-谷氨酰转肽酶的制备及性质

γ-谷氨酰转肽酶（GGT）在临床诊断和生物催化方面具有重要的应用价值。本文以介孔氧化钛晶须为载体进行GGT的固定化，考察了载体结构特性、吸附时间和给酶量对固定化效果的影响，并对固定化酶的催化特性及其稳定性进行了研究。结果显示，以最可几孔径为30 nm的介孔TiO₂为载体，载体载酶量可达5.07mg·g^-1。在给酶量为18.99 U·g^-1时，经室温吸附2.5 h，固定化酶活性回收率可达73.05%。固定化酶的pH稳定性和热稳定性均显著优于游离酶，在4℃下保温贮藏60 d、转化22个批次后，固定化酶活力仍可保持初始值的71.30%。经测定，游离酶和固定化酶的米氏常数K_m分别为0.79 mmol·L^-1和1.05 mmol·L^-1，酰基化反应活化能分别为13.59 kJ·mol^-1和15.42 kJ·mol^-1；固定化GGT的失活反应活化能E_d为92.80 kJ·mol-1，相比于游离酶（49.61 kJ·mol^-1）有明显的增加。

Immobilization of GGT on meso-Ti02: preparation and properties

γ-Glutamyltranspeptidase (GGT）is an important enzyme with wide applications in biocatalysis and clinical diagnosis.In this work, mesoporous fibrous titania (M-TiO₂）was used for immobilization of GGT from B.subtilis NX-2 and the properties of immobilized GGT were also investigated.When the M-TiO₂ support with average pore diameter of 30 nm was used, the amount of immobilized protein was 5.07 mg·g^-1, and the yield of activity was 73.05% at the ratio of GGT/support was 18.99 U·g^-1 after incubation at room temperature for 2.5 h.The thermal and pH stability of the immobilized GGT was higher than that in its free form.After storage at 4℃ for 60 days and repeated use for 22 batches, the activity of the immobilized GGT remained 71.30% of its initial activity.The kinetic parameters (K_m）for free and immobilized GGT were determined as 0.79 mmol·L^-1 and 1.05 mmol·L^-1, respectively.The activation energy (E_a）values of glutamylation were 15.42 kJ·mol^-1and 13.59 kJ·mol^-1 for immobilized and free GGT.The thermal inactivation energy (E_d）values of GGT for immobilized and free enzyme were also calculated to be 92.80 kJ·mol^-1 and 49.61 kJ·mol^-1, respectively.