Development of Highly Selective and Efficient Enzymatic Degradation Catalysis by Immobilizing Protocatechuate 3,4-Dioxygenase in Mesoporous Silica

在本研究中，一种酶将被固定在化学修饰的介孔二氧化硅内，以提高其稳定性和反应性。本研究关注的酶是原儿茶酚酸3,4-双加氧酶，它可以选择性地切割儿茶酚芳香环的强C-C键。在2021年和2022年，实现了介孔二氧化硅的制备和硅烷偶联剂对其表面的改性。选用SBA-15代替MCM-41合成介孔二氧化硅。这是由于：(1)其合成方案在Ogura实验室（本研究开展的小组）中得到了完善，(2)SBA-15具有与MCM-41相似的孔隙结构，孔径可控，(3)已知SBA-15表面具有高SiOH密度，这使得易于表面修饰以调整其表面与目标酶之间的固定化相互作用。将制备的SBA-15平均孔径分别为8 nm、10 nm和12 nm，通过硅烷偶联进行表面改性，得到改性后的SBA-15、m-SBA-15。为了证明不稳定酶通过固定在介孔二氧化硅中的稳定作用，首先将一种比原儿茶酸3,4-双加氧酶（脂肪酶）更常见和广泛使用的酶固定在m-SBA-15中。结果，通过固定在m-SBA-15中成功观察到脂肪酶稳定性的增加。建立了测定其酶活性和活性脂肪酶定量的方法，并对固定化的最佳孔径进行了探索。

In this research, an enzyme will be immobilized inside the chemically modified mesoporous silica to enhance both its stability and reactivity. The enzyme of interest in this work is protocatechuate 3,4-dioxygenase, which can selectively cleave a strong C-C bond of catechol aromatic rings.In year 2021 and 2022, preparation of mesoporous silica and modification of its surface using silane coupling agent were achieved. As a choice of mesoporous silica, SBA-15 was synthesized, instead of MCM-41. This was due to the fact that (1) the protocol of its synthesis was well-established in the Ogura lab, the group where this research was carried out, (2) SBA-15 has a similar pore structure as MCM-41 with controlled pore size, and (3) SBA-15 is known to possess high SiOH density on its surface, which allows the easy surface modification to tune the immobilization interactions between its surface and target enzymes.The SBA-15 prepared in three different average pore sizes of 8 nm, 10 nm, and 12 nm were subjected to surface modification by silane coupling to obtain the modified SBA-15, m-SBA-15.To prove the stabilizing effect of the unstable enzyme via immobilization into mesoporous silica, a more commonly and widely used enzyme compared to protocatechuate 3,4-dioxygenase, lipase, was immobilized first into m-SBA-15. As a result, the increase in stability of lipase was successfully observed through immobilization into m-SBA-15. Moreover, methods to measure its enzymatic activity and quantification of active lipase were established, and optimized pore size for immobilization was explored.