Combining Molecular Imprinting With Peptide Assembly To Construct Artificial Enzymes With Substrate Selectivity

In this thesis,the short peptide(Fmoc-FFH)was used as the basic building block to construct molecularly imprinted peptide-based artificial hydrolase and peroxidase by self-assembly or co-assembly with hemin and combining with molecularly imprinted polymer.The morphologies,structures,catalytic activities and substrate selectivity of molecularly imprinted peptide-based enzyme mimics were characterized and analysed by various methods.The main conclusions are as follows:(1)A peptide-based hydrolase with substrate selectivity was constructed based on molecular imprinting.Driven by non-covalent interaction,Fmoc-FFH was assembled into catalytic nanofibers(SA-H)and exhibited esterase-like hydrolysis activity.Molecular imprinting was used to create the imprinted polymer layer with p-NPA binding sites on the surface of the nanofibers to obtain the molecular imprinting peptide-based hydrolase(AMIP-H),which maintains the nanofiber morphology and?-sheet active secondary structure.AMIP-H follows Michaelis–Menten kinetics,and the catalytic activity of AMIP-H on p-NPA is 2.5 times higher than that of SA-H.AMIP-H is recyclable and exhibits higher catalytic activity in a wider reaction temperature and pH.Furthermore,we confirmed that the substrate selectivity of this artificial enzyme can be customized through changing imprinting template(e.g.p-NPA,p-NPB,p-NPH).(2)A peptide-based peroxidase with substrate selectivity was constructed based on molecular imprinting.Driven by non-covalent interaction,Fmoc-FFH and hemin were co-assembled into nanofibers(SA-H/Hemin),which exhibited peroxidase-like activity in the reaction with ABTS and H₂O₂as substrates.The imprinted polymer layer with ABTS binding sites was formed on the surface of SA-H/Hemin nanofibers by molecular imprinting,then the molecular imprinting peptide-based peroxidase SMIP-H/Hemin was obtained.SMIP-H/Hemin maintains the nanofibers morphology and the?-sheet active secondary structure.The imprinted artificial enzyme has a higher substrate affinity and follows Michaelis–Menten kinetics.Compared to SA-H/Hemin,SMIP-H/Hemin shows a 2.4-flod higher activity in the catalysis of ABTS and retains higher catalytic activity in a wider temperature and pH range.Further,by introducing the cationic monomer in imprinting process,a positively charged SMIP_pos-H/Hemin was obtained.The results showed that the catalytic activity of SMIP_pos-H/Hemin on the negatively charged ABTS was further increased to 6.3times than that of SA-H/Hemin,while the catalytic activity on the positively charged substrate TMB was decreased.Thus,we obtained a peptide-based peroxidase with high catalytic selectivity for ABTS.