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

在这项研究中，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。2021年和2022年，我们成功制备了介孔二氧化硅，并利用硅烷偶联剂对其表面进行了改性。作为介孔氧化硅的选择，合成了SBA-15，而不是MCM-41。这是由于以下事实：（1）其合成方案在Ogura实验室（进行本研究的小组）中得到良好建立，（2）SBA-15具有与MCM-41类似的孔结构，具有受控的孔径，以及（3）已知SBA-15在其表面上具有高SiOH密度，其允许容易的表面改性以调节其表面与靶酶之间的固定化相互作用。以8 nm，10 nm，为了验证介孔二氧化硅固定化对不稳定酶的稳定作用，首先将一种比原儿茶酸3，4-双加氧酶更常用、应用更广泛的酶脂肪酶固定化到m-SBA-15上。结果表明，通过将脂肪酶固定到m-SBA-15中，成功地观察到脂肪酶稳定性的增加。建立了固定化酶的酶活测定方法和活性测定方法，探索了固定化酶的最佳孔径。

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.