基于DNA链置换反应的新型固定化酶反应器制备及应用

建立了一种新型的酶固定化方法,采用DNA链置换反应成功地在单链DNA标记的磁性纳米粒子上实现了酶的链置换无损更替。该技术可实现目标酶的再利用,节约了生产成本。制备的固定化胰蛋白酶微反应器具有较好的重复利用性和高酶切效率,重复使用10次后仍可保持原酶活性的86%;利用链置换反应制备的MNPs@DNATrypsin酶切马心肌红蛋白5 min后,即可获得95%±0%(n=3)的氨基酸序列覆盖率,远超过相同条件下自由酶酶切12 h的结果。实验表明,发展的固定化酶技术具有高磁响应性,便于从反应体系中回收固定化酶和重复使用,同时此技术可显著提高酶活性,因此可用于固定各种重要的酶,同时可将其广泛应用于各种酶促反应中。

Preparation and application of a novel immobilization enzyme reactor based on DNA strand displacement reactions

A novel enzyme immobilization method was developed. The enzyme was immobilized using single-stranded DNA modified magnetic nanoparticles as carriers through DNA strand displacement reactions. Alkaline phosphatase (ALP) was immobilized with partly complementary capture DNA-functionalized nanoparticles (MNPs@capDNA) by highly specific Watson-Crick hybridization to obtain the MNPs@DNA-ALP. For trypsin immobilization, the MNPs@DNA-Trypsin was obtained by the incubation of MNPs@DNA-ALP with trypsin-target DNA conjugates to trigger a toehold-mediated DNA strand displacement reactions. The enzymes can be replaced without damage by the immobilized enzyme procedure. Therefore, the strategy gave the possibility to recycle the enzymes and save the production cost. The immobilized trypsin exhibited excellent reusability and improved digestion performance. The enzymatic activity remained more than 86% after 10 cycles. And the sequence coverage of myoglobin (Myo) was 95%±0% (n=3), which was higher than that obtained by the free enzyme for 12 h. The strategy provides a promising alternative platform for the enzyme immobilization and can be used in a large variety of enzymatic reactions.