Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity
Organic-inorganic hybrid silica monolith based immobilized trypsin reactor with high enzymatic activity
复制标题
基于有机-无机杂化二氧化硅整体的高酶活性固定化胰蛋白酶反应器
DOI：
10.1021/ac702343a
复制
发表时间：
2008-04-15
影响因子：
7.4
通讯作者：
Zhang, Yukui
中科院分区：
文献类型：
--
作者：
Ma, Junfeng;Liang, Zhen;Zhang, Yukui
以有机-无机杂化硅胶为载体，研制了一种新型固定化胰蛋白酶反应器。在聚合混合物中存在十六烷基三甲基溴化铵（CTAB）的情况下，在100 μ m i.d.毛细管通过溶胶-凝胶法与正硅酸乙酯（TEOS）和3-氨丙基三乙氧基硅烷（APTES）作为前体。随后，用戊二醛活化整体柱，并共价固定胰蛋白酶。通过对十肽C-myc（EQKLISEEDL）反应的监测，计算了固定化胰蛋白酶的酶活，结果表明，固定化胰蛋白酶的酶解速度比游离胰蛋白酶快6600倍。通过消化肌红蛋白进一步证明了这种微反应器的性能，通过微流反相液相色谱-串联质谱法（μ RPLC-MS/MS）分析消化产物。在严格的阈值下，柱上酶切的序列覆盖率为92%，与不溶酶切相同，而肌红蛋白在前一种情况下的停留时间仅为30 s，约为后者（12 h）的1/1440。此外，这种固定化胰蛋白酶反应器还成功地应用于模型蛋白和大肠杆菌蛋白质的混合物的消化。杆菌
A novel kind of immobilized trypsin reactor based on organic-inorganic hybrid silica monoliths has been developed. With the presence of cetyltrimethyl ammonium bromide (CTAB) in the polymerization mixture, the hybrid silica monolithic support was prepared in a 100 mu m i.d. capillary by the sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltrietboxysilane (APTES) as precursors. Subsequently, the monolith was activated by glutaraldehyde, and trypsin was covalently immobilized. By monitoring the reaction of a decapeptide, C-myc (EQKLISEEDL), the enzymatic activity of the immobilized trypsin was calculated, and the results showed that the digestion speed was about 6600 times faster than that performed in free solution. The performance of such a microreactor was further demonstrated by digesting myoglobin, with the digested products analyzed by microflow reversed-phase liquid chromatography coupled with tandem mass spectrometry (mu RPLC-MS/MS). With a stringent threshold for the unambiguous identification of the digests, the yielding sequence coverage for on-column digestion was 92%, the same as that obtained by insolution: digestion, whereas the residence time of myoglobin in the former case was only 30 s, about 1/1440 Of that performed in the latter case (12 h). Moreover, such an immobilized trypsin reactor was also successfully applied to the digestion of a mixture of model proteins and proteins extracted from E. coli.