Research On Acylpeptide Hydrolase's Modification,Real-time Regulation And Treatment Of Alzheimer's Disease

With the development of science and technology recently,researches of microorganisms from the extreme environments have received extensive attention.Among the rapidly developing fields of enzymology,enzymes derived from thermophiles not only have good stability at high temperature,but also have amazing potential catalytic capabilities.These characteristics benefit for exploring the new biocatalysts and new options.The acylpeptide hydrolase derived from the thermophilic Archaea has high research value and exhibits excellent non-specific ability to catalyze the synthesis of C-C bonds in synthetic applications.A key role of acylpeptide hydrolase is cleaning the toxic proteins in cells.This work mainly used a series of the acylpeptide hydrolases cloned and expressed by our laboratory for three aspects:(1)Our researches investigated the effects of mutations on structure and function and screened better mutants,whose structure of the enzyme enhances the yield and catalytic efficiency of the non-specific catalytic in Aldol addition reaction.(2)Our researches designed the thermophilic acylpeptide hydrolase with nanomaterial-immobilization and constructed a real-time catalytic system to investigate the Aldol addition reaction.(3)To explore the application of thermophilic acylpeptide hydrolase in Alzheimer's disease,we proposed a therapeutic strategy combined with targeted single-chain antibody,and constructed a composite of thermophilic acylpeptide hydrolase and nanomaterials to apply to treatment of Alzheimer's disease.First,we selected acylpeptide hydrolases(mesophilic acylpeptide hydrolase BSU32230,thermophilic acylpeptide hydrolase ST0779,hyperthermophilic acylpeptide hydrolase APE1547)with different temperature sources in our laboratory as the research objects.Comparing the results of the alignment and structural simulation,it is speculated that the bridge structure of acylpeptide hydrolase plays an important role in the structure and function.However,there are few reports on the bridge structure.The bridge region is a hinge structure connecting two domains,and consists of two structural elements,including a linker region directly connecting the two domains and an N-terminal region extending from the N-terminus to the C-terminal domain.The mutation of region was engineered by inserting and deleting amino acids in it.The results showed that the linker region directly affects the stability and catalytic ability of the enzyme.It showed that the bridge structure has a great influence on the catalytic performance and plays a vital role in the structural stability.Therefore,through the rational mutation design of the bridge structure,the mutant ST-4A with good stability and better catalytic ability was successfully screened,and the yield of the catalytic Aldol reaction was improved.It is expected to be further applied in the field of synthesis.On the other hand,the researches selected thermophilic acylpeptide hydrolase APE1547 and ST0779 as research objects to construct a low-energy catalytic system with real-time regulation,high efficiency,high specificity and high reaction controllability.By using nanomaterials to achieve local rapid temperature rise for activating the enzyme to convert to the active catalytic state and heat the reaction system,the self-assembly composite of thermophilic acylpeptide hydrolase and gold nanomaterial is used to convert the near-infrared laser into the photothermal effect of internal energy.And it was applied to catalysis Aldol reaction.The system constructed the property of real-time regulation using infrared irradiation as a switch of the reaction.It quickly controlled the opening and termination of the reaction.Therefore,it achieved the controllability of the reaction.The system effectively improved the non-specific catalysis rate of the Aldol reaction,and the catalytic rate is increased by 8 folds.The constructed complex has high reusability and stability;it successfully achieved a real-time regulation catalytic system.In addition,due to the high research value of acylpeptide hydrolase,it plays a key role in the clearance of cytotoxic proteins.It is known to report human acylpeptide hydrolase as a potential A? degrading enzyme,which effectively eliminates toxic A? in vivo.It has potential application in Alzheimer's disease.Due to the low stability of human acylpeptide hydrolase after purification,subsequent application research and the development of drugs is difficult.Therefore,based on the potential research value of acylpeptide hydrolase,in-depth research and new drug development will be carried out by the exogenous acylpeptide hydrolase with the same functional properties.Therefore,in this study,we attempted to use the thermophilic acylpeptide hydrolase ST0779 from our laboratory to detect its effective degradation of A? peptide and reduce A?-mediated toxicity.Furthermore,a combination of thermophilic acylpeptide hydrolase ST0779 and single-chain antibody 12B4 was proposed,which may degrade A? monomer and toxic oligomers and inhibit A?-mediated related toxicity.It may achieve treatment of Alzheimer's disease.Finally,the targeted nano-material complex was constructed,which was supplemented by the photothermal effect of near-infrared to enhance the therapeutic effect.It degraded A? in the Alzheimer's nematode model and inhibited A?-mediated toxicity,delaying the nematode rate.In this work,an in-depth study of acylpeptide hydrolase not only demonstrates the relationship between the structure of the bridge region and the catalytic mechanism through mutation,but also optimizes the stability and catalysis of the acylpeptide hydrolase.Mutation provided a new direction to the organic synthesis reactions;on the other hand,construction of nanomaterial immobilization with near-infrared irradiation achieved the real-time regulation of the catalytic system,and finally applied to Aldol reaction.The value of acylpeptide hydrolase is potential in synthetic applications.Finally,the application of thermophilic acylpeptide hydrolase in the degradation of A? was explored.The combination therapeutic strategy of thermophilic acylpeptide hydrolase ST0779 and single-chain antibody 12B4 was proposed.It was better to degrade A? and inhibits A?-mediated toxicity and for treatment of Alzheimer's disease.Through the research,the nano-material complex was finally constructed and supplemented by the photothermal effect of near-infrared to enhance the therapeutic effect.And nematode model assay was used to degrade A?,inhibit A?-mediated toxicity and delay the paralysis rate.It provides the new strategy for drug development in Alzheimer's disease.Moreover,it reveals the application value of acylpeptide hydrolase in Alzheimer's disease.It shows the great potential application for new enzyme from thermophilic Archaea.