Biomimetic Construction And Performance Evaluation Of Supramolecular Enzymes Based On The Chaperonin GroEL And Zinc Tetraphenylporphyrin

Enzymes are a class of substances with high catalytic ability,which are widely used in the field of biomedicine,food hygiene,and chemical engineering.However,there are still many limitations in the application of natural enzymes,such as high production cost,poor stability,and storage difficulties and so on.Therefore,many scholars are committed to developing mimic enzymes with excellent performance.With the introduction of new theories such as supramolecular chemistry and host-guest chemistry,the development of mimetic enzyme systems has been accelerated,and the new research field of supramolecular enzyme mimic enzyme has been widely concerned.Porphyrin mimetic enzymes are a kind of traditional mimetic enzymes,which are widely used in photodynamic therapy,biomimetic catalysis and other fields.Among them,metalloporphyrins are endowed by redox due to their stable large conjugated structure and variable metal valence.Metalloporphyrins can simulate the catalytic ability of biological enzymes;at the same time,due to the strong absorption coefficient of metalloporphyrin in the visible region and excellent photoelectron transfer ability,it is used as an excellent photosensitizer in photodynamic therapy.However,there are still two problems in the application of metalloporphyrin.One is that metalloporphyrins cannot dissolve in aqueous phase due to their strong hydrophobicity,and the other is that they are easily photodegraded under light,which limits their wide application.Therefore,it is very important to solve the two problems of limiting the wide application of metalloporphyrin.In this paper,we used supramolecular chemistry theory to construct GroEL-Zn TPP supramolecular enzyme by hydrophobic interaction,using chaperone GroEL as protein framework and Zinc Tetraphenylporphyrin(Zn TPP)as the catalytic guest.By introducing water-soluble protein GroEL,the water solubility of Zn TPP was enhanced and the light stability of Zn TPP was improved,which solved the problem of restricting the application of metalloporphyrin,At the same time,chaperone GroEL not only provided a stable microenvironment for Zn TPP,but also ensured its efficient redox ability by its axial coordination with Zn²⁺,thus enabling GroEL-Zn TPP supramolecular enzymes to possess zinc-containing metalloproteinase catalytic activity.At the same time,the supramolecular enzymes constructed by different protein backbones may have different physicochemical properties.In order to explore the difference in the performance of supramolecular enzymes constructed by different protein backbones,we constructed SR1-Zn TPP supramolecular enzymes by single Ring of GroEL(SR1),and evaluated the performance differences between GroEL-Zn TPP supramolecular enzymes and SR1-Zn TPP supramolecular enzymes.The main research contents of this topic are as follows:1)GroEL(SR1)-Zn TPP supramolecular enzymes were constructed by surfactant dialysis.The properties of GroEL(SR1)-Zn TPP supramolecular enzymes were characterized by ultraviolet spectrophotometer,fluorescence spectrometer and nanoparticle size analyzer.The experimental results showed that the prepared artificial enzyme showed new physical properties,such as red shift in the UV-visible absorption spectrum,red shift in the fluorescence emission spectrum and new emission peaks,and the particle size of the constructed supramolecular enzyme showed the particle size of the protein skeleton.2)The photochemical properties of artificial enzymes were investigated,including the photostability,electron transfer ability,and singlet oxygen production of Zn TPP encapsulated in GroEL(SR1).The results showed that the light stability of Zn TPP encapsulated in the protein skeleton was significantly enhanced,and the photoprotection efficiency was 80-90%.The addition of protein skeleton made the Zn TPP with strong hydrophobicity directly exist in the aqueous environment,which greatly enhanced water solubility of Zn TPP.At the same time,it had good electron transfer ability,high singlet oxygen yield,good biocompatibility,biodegradability and other advantages,which meet the requirements of excellent photosensitizer,and is expected to be used in photodynamic therapy.At the same time,the performance of artificial enzymes in different protein skeleton construction was different.SR1-Zn TPP artificial enzymes showed better photochemistry than GroEL-Zn TPP artificial enzymes.3)The ability of supramolecular enzymes to mineralize CO₂ as artificial carbonic anhydrase enzymes was explored.Carbonic anhydrase is a kind of widely distributed zinc-containing metalloenzyme,which can efficiently and reversibly catalyze the formation of bicarbonate and hydrogen ions by CO₂.The catalytic active center structure is a deformed four-sided structure composed of three lysine residues and Zn²⁺coordination.This structure is similar to the structure of Zn TPP,so we expect that the constructed GroEL(SR1)-Zn TPP supramolecular enzyme has the catalytic ability of carbonic anhydrase.The experiment mainly includes the determination of enzyme activity,the evaluation of CO₂ capture ability and the experiment of calcium carbonate precipitation.The results showed that the constructed artificial enzymes possessed partial carbonic anhydrase activity and could complete the process of catalyzing CO₂,especially at 60?,the catalytic activity is better than that of the natural enzyme.In the experiment of enzymatic precipitation of calcium carbonate,artificial enzymes also showed good catalytic ability.In addition,the artificial enzymes constructed by different protein skeletons also exhibit different catalytic abilities.Among them,SR1-Zn TPP artificial enzymes exhibit better catalytic abilities than GroEL-Zn TPP artificial enzymes.The experimental results show that the artificial enzymes constructed by us are expected to be applied in the capture and storage of CO₂.