Construction And Application Of Bio-inspired Oxidase-like Catalytic Systems Based On Biomolecular Self-assembly

Enzymes are highly efficient biocatalysts that have evolved over billions of years,playing essential roles in living organisms by catalyzing various chemical reactions.However,the folded structures of enzymes are sensitive to changes in the environment,making the catalytic performance of enzymes easily affected by external conditions.As a result,mild reaction conditions are necessary for natural enzymes to catalyze reactions efficiently,which often cannot meet the needs of industrial transformation.Moreover,the separation and purification of natural enzymes can be difficult.To expand the application range of enzymes,researchers have developed various enzyme engineering techniques,such as directed evolution.Although effective in regulating enzyme function,directed evolution is time-consuming and laborious.Additionally,this method can only change the natural amino acid species and cannot utilize a wide variety of amino acid variants or derivatives.Base on the intrinsic supramolecular properties of enzymes,molecular self-assembly provides a new strategy for the design and preparation of artificial catalysts with enzyme-like functions.The active site of enzyme is the key area for the catalytic function.It is a challenging research project to recreate the geometric structure and chemical environment of the active site of natural enzymes in artificial systems and construct the enzyme mimic with catalytic activity rivaling natural enzymes.In this thesis,we designed and constructed three oxidase-like catalytic systems through biomolecular self-assembly,drawing inspiration from the active site structure and catalytic principle of natural enzymes.（1）The work in the second chapter was inspired by the active site structure and catalytic principle of natural enzymes,particularly the histidine residue found in the active site of hydrolase and multicopper oxidase enzymes（such as laccase）.Based on this inspiration,three different oxidase-like catalytic systems were designed and constructed through biomolecular self-assembly.The first system was a bifunctional enzyme mimic that can catalyze both hydrolysis and oxidation reactions.This was achieved by self-assembling histidine derivatives with Cu²⁺,which forms a multiple copper active site similar to that found in laccase enzymes.The aggregation of fluorene methoxycarbonyl（Fmoc）was used to enrich the imidazole groups of histidine,leading to high catalytic hydrolysis activity.Guanosine monophosphate was also included to enhance the catalytic oxidation activity.This enzyme mimic exhibited the ability to catalyze the hydrolysis-oxidation（or oxidation-hydrolysis）cascade reaction and demonstrated excellent thermal stability.Therefore,it has potential applications in the decomposition of aromatic ester pollutants.In addition,the catalytic activity of the enzyme mimic could be regulated by methyl-substituting at different nitrogen positions of the imidazole group.When H attached to N_δwas substituted by methyl,the nucleophilic ability of the imidazole group was enhanced,leading to an increase in the coordination-dissociation rate with copper and higher catalytic hydrolysis and oxidation activities.However,substitution of H attached to N_εresulted in a significant decrease in catalytic activity,confirming the heterogeneity of the different N atoms of imidazole in the catalytic process.Overall,this work demonstrated that molecular self-assembly can be used to construct artificial cascade catalytic systems using amphiphilic amino acids,and the catalytic properties can be modulated through amino acid modifications.（2）Based on the inspiration from the enzyme-substrate complex,the third chapter describes the construction of a riboflavin-loaded supramolecular gel-like material via biomolecular self-assembly,which is capable of photo-generating H₂O₂.Guanosine molecules self-assemble to form G-quartets by hydrogen bonding with the assistance of K⁺,and then form fibers throughπ-πstacking.Subsequently,a supramolecular gel is formed with the aid of a crosslinking agent.Riboflavin,which serves as the photocatalytic active center,is loaded into the gel through covalent linking and aromatic stacking.Guanosine serves as both the building block of the gel and the reducing substrate of photocatalytic oxidation.Riboflavin and guanosine are in close proximity to each other through chemical linking and aromatic stacking,forming a functional material similar to an active center-substrate complex that can be activated.Under blue light（460 nm）irradiation,the material can complete the catalytic oxidation cycle without the need for additional components and release H₂O₂.The material exhibits excellent storage and thermal stability.Due to its H₂O₂-producing ability,the material displays good antibacterial properties after irradiation and is biosafe,which makes it a promising candidate for use as a new post-irradiation antibacterial wound dressing.（3）Drawing inspiration from the function of the distal histidine residue in the active site of horseradish peroxidase（HRP）during the catalytic cycle,the work in the fourth chapter presents a design strategy for enhancing the catalytic activity of HRP by co-assembling histidine-rich peptides with HRP to form a supramolecular complex.The histidine-rich peptides can self-assemble intoβ-sheets and interact non-covalently with HRP to alter its conformation.This allows the exogenous histidine provided by the peptides to approach the active site,accelerate the conversion of the resting state and intermediate state of heme,and facilitate the catalytic cycle of the enzyme.This leads to a nearly threefold increase in the maximum catalytic rate of HRP,while also improving the stability of the HRP/peptide hybrid.This chapter proposes a universal principle for regulating heme-peroxidase using histidine-rich peptides.The HRP/peptide hybrid can effectively enhance detection sensitivity in biomolecular detection applications.In this thesis,biomolecular self-assembly inspired by natural enzymes provides a simple and effective way to regulate enzyme function and expand the application range of enzymes.This approach bridges the gap between supramolecular chemistry and enzyme engineering.The bio-inspired design of enzyme mimics is helpful for researchers to understand the catalytic process of enzymes and the evolution of life.