Design And Application Of New Mimetic Enzymes/nanozymes Based On Histidine

Nanozyme refers to a class of nanomaterials with enzyme-like catalytic activity.Nanozymes not only have the physical and chemical properties of nanomaterials,but also have enzyme-like biocatalytic activity.Since nanozymes were reported in 2007,there have been more and more researches and nanozymes are also considered to be the third generation enzymes.Compared with biological enzymes,nanozymes have the advantages of easy preparation,convenient storage,and low cost.So they have attracted the attention of domestic and foreign researchers.With the rapid development of nanotechnology,research on nanozymes has also progressed by leaps and bounds.Nanozymes have emerged in the fields of biosensing,immunoassay and biomedicine.They have gradually become candidates for replacing natural enzymes.However,nanozymes also expose many shortcomings.For example,the catalytic activity of nanozymes is generally lower than that of natural enzymes;the types of enzyme activities of nanozymes are few,mainly concentrated in oxidoreductases;enzymes can catalyze a variety of substrates with poor specificity.These are the three most important problems of nanozymes that need to be solved.Histidine(His)is a natural amino acid with an isoelectric point(PI)of 7.59.It presents a zwitterionic ion in a partial physiological environment.It participates in the catalytic activity center of many natural enzymes.Therefore,Histidine plays an important role in the design of new nanozymes.In this study,three His mimetic enzymes/nanozymes were designed using biomimetic design ideas.Depart from His,His single-atom amino acid nanozyme with high catalytic activity,specificity and selectivity,His amyloid peptide nanozymes with new enzymatic activity transferases and His deuterohemin peptide with strong catalytic specificity were constructed respectively.They provide new ideas for solving the problems of low catalytic activity of nanozymes,few types of nanozymes,and poor specificity of catalytic substrates.In the first part,we designed and synthesized His single-atom amino acid nanozyme(Cu-His)at room temperature.Confirmed by spherical aberration electron microscopy,copper had good monodispersity,which provides a new solution to the current problems of single-atom nanozymes requiring high-temperature synthesis and single-atom agglomeration.Cu-His has high peroxidase activity and specific oxidoreductase activity,because it does not have carbonic anhydrase,acetylcholinesterase and other hydrolase activities.In the second part,we designed a His and amyloid peptide N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-phenylalanyl-L-phenylalanine(Fmoc-F-F)co-assembled His amyloid peptide nanozyme(Fmoc-F-F(His)).This was the first reported study on the co-assembly of 20 natural amino acids and Fmoc-F-F.We observed that His can regulate the depolymerization of Fmoc-F-F nanorods into fibrous filaments through projection electron microscopy.Co-assembly also improved the water solubility of Fmoc-F-F.We also found that the co-assembly behavior of His and Fmoc-F-F is dynamic.We used advanced characterization techniques such as cryo-electron microscopy to study the structure of Fmoc-F-F(His)in more detail.At the same time,we found that Fmoc-F-F(His)peptide nanozyme has oxidoreductase,hydrolase,and transferase activities.This was the first reported nanozyme with transferase activity.We provide a new strategy and template for His to endow the activity of peptide nanozyme.Inspired by the above experiments,we also discovered that Amyloid?(A?)in the brains of patients with Alzheimer's disease(AD)can exert enzyme-like activity,which provides new ideas for the pathogenesis of AD.In the third part,we co-immobilized the His deuterohemin peptide(Deuterohemin-?Ala His Thr Val Glu Lys,Dh HP-6)which has the similar catalytic conditions as glucose oxidase(GOx)and synthesized a new glucose sensor GOx&Dh HP-6-Cu₃(PO₄)₂ with strong specificity of catalytic substrate.The linear range of the sensor for detecting glucose was 5-2000?M,the detection limit was as low as 0.5?M,and the detection time was 10 min.Other sugar components and ascorbic acid had little effect on the test.At the same time,we used solid-phase peptide synthesis technology to modify the structure of Dh HP-6,and designed a new his deuterohemin tripeptide(Deuterohemin-?-Ala-His-Glu,Dh-A-H-E)with higher peroxidase catalytic activity.Its catalytic activity was improved in high salt,alcohol,phosphate buffer,and calcium chloride.In summary,the three new mimetic enzymes/nanozymes designed by His provide a good idea for solving the three major problems of nanozymes.Besides,they show extremely broad application prospects.