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

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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.