| Enzymes,as proteins or RNAs produced by living cells,have a high degree of specificity and catalytic efficiency for their substrates.Owing to the character of high efficiency,specificity and mild reaction conditions,enzymatic catalysis has been widely used in industrial production,clinical treatment and diagnosis of diseases.Although enzymes have satisfactory catalytic efficiency,there are inherent barriers in their practical application.For example,as biological macromolecules,enzymes have a short blood half-life and poor ability to actively cross cell membranes in clinical therapy.What’s more,the poor stability of enzymes leads to difficulties in reuse in industrial production.Therefore,we are striving to explore effective methods to improve the stability of enzymes.Enzymatic assembly technology,which has the advantages of maintaining the catalytic activity of enzymes,improving the stability of enzymes and facilitating the regulation of enzymatic activity,has been widely used among other methods such as enzymatic structure modification,protein engineering and enzymatic micro-environment regulation.Metal-organic frameworks(MOFs)are framework structures composed of metal nodes and organic ligands linked through coordination bonds.Due to their high porosity,large specific surface areas,convenient surface modification or adjustment of pores’size,MOFs have been used as ideal materials for enzyme assembly in recent years.The manner of enzyme assembly by MOFs includes covalent linkage,surface attachment,Pore encapsulation,co-precipitation and biomimetic mineralization.Among them,biomimetic mineralization has been widely used because of the advantages such as mild synthesis condition,excellent protective effect on enzymes and capable of immobilizing enzymes with high molecular weight.Biomimetic mineralization,which imitates the formation process of inorganic materials under the modulation of organic substances in the process of natural biological mineralization,can facilitate the assembly of proteins,nucleic acids or lipids into MOFs.Based on the above considerations,two types of MOF-enzyme assemblies were contructed,namely lipase@Bio-MOF and SOD@ZIF-8,which were used in the preparation of biodiesel and treatment of Parkinson’s disease,respectively.The specific results are as follows:1.Immobilized lipase in bio-based metal-organic frameworks:A sustainable biocatalyst for biodiesel synthesisBiodiesel refers to fatty acid methyl esters or ethyl esters formed by transesterification of bio-oils with methanol or ethanol.Its fuel performance is similar to that of diesel and has obvious advantages such as versatile,eco-friendly,energy-saving and so on.At present,biodiesel preparation by lipase is an environmental method.In this chapter,we constructed biology-based MOF-lipase composite by biomimetic mineralization technology,termed lipase@Bio-MOF.Lipase participated in the crystallization and assembly process of Bio-MOF and assisted Bio-MOF to form the spherical morphology.The lipase content in lipase@Bio-MOF was about 15%and the secondary structure of lipase would not be influenced obviously after mineralization.More importantly,Bio-MOF could protect lipase and improve the enzymatic stability in high temperature,acid or alkali and metal ion environments after assembly.Finally,using lipase@Bio-MOF as the catalyst,biodiesel was successfully synthesized after the transesterification reaction of methanol and sunflower oil with ideal conversion rates under different oil-alcohol ratios.What’s more,after reusing lipase@Bio-MOF for multiple times,the composite could still maintain the complete structural morphology and excellent catalytic ability.In short,lipase@Bio-MOF provides an environmental means for the preparation of biodiesel and offers an excellent platform for the application of MOF-enzyme composites in the fields of biocatalysis.2.Construction of SOD@ZIF-8 assembly and the research on its antioxidant activityReactive oxygen species(ROS)are types of single-electron reduction products of oxygen produced by living cells in the body,which can be generated by normal metabolism of living cells,chemical factors or radiation.Oxidative damage caused by excessive ROS in the body has a close relationship with a variety of diseases including tumors,aging,inflammation and neurological diseases.As a vital enzyme in the antioxidant system,the effective delivery of SOD in vivo is of great significance for the treatment of ROS-related diseases.In this research,nano-scale assembly—SOD@ZIF-8 was successfully constructed by biomimetic mineralization technology.The formation of this assembly was“complementary and co-existing”,which means that the crystallization and morphology formation of ZIF-8 was promoted by SOD and the resistance ability of SOD against high temperature or trypsin was improved after assembly with ZIF-8.Different from the other enzymatic immobilization methods of MOFs such as surface attachment or pore adsorption,SOD molecules were uniformly distributed inside the ZIF-8 and can maintain the stability of the enzymatic secondary structure as well as high catalytic activity after biomimetic mineralization.The activity of SOD@ZIF-8 in nematodes was systematically studied using N2 wild-type C.elegans as the model organism.It was found that SOD@ZIF-8 could effectively clean up ROS in organisms,thereby improving the health status and stress resistance capacity of nematodes.Further research fould that after treatment with SOD@ZIF-8,the life of nematodes could be extended by optimizing the expression of aging-related genes.The above results illustrated the successful construction of the SOD@ZIF-8 assembly and its excellent antioxidant activity in vitro and in nematodes’bodies,thus opening up a new path for the construction of antioxidant and anti-aging nanocomposites based on ROS scavenging.3.The research of SOD@ZIF-8 in the treatment of Parkinson’s diseaseParkinson’s disease(PD),as a common neurodegenerative disease,its pathogenesis is not very clear,but oxidative stress in the brain plays an important role in the onset and deterioration of PD.Therefore,anti-oxidation therapy is expected to be an effective method for the prevention and treatment of PD.In the research,we successfully constructed SOD@ZIF-8 assembly with ideal antioxidant activity both in vitro and in vivo through biomimetic mineralization,which was further used for the treatment of PD.At the cellular level,PD cell models were built after stimulating SHSY-5Y cells with MPP~+.SOD@ZIF-8 had a significantly stronger ability of entering into cells than free SOD,thus having a better effect on the elimination of intracellular ROS and alleviating cell death caused by excessive ROS after MPP~+stimulation.Meanwhile,SOD@ZIF-8 protected cells by reducing the apoptosis rate.What’s more,the nanocomposites could exert antioxidant effect in cells for a long time.At the animal level,SOD@ZIF-8 had longer half-life than free SOD,which could be evidenced by slower attenuation speed of enzymatic activity after SOD@ZIF-8 injection.MPTP was used to stimulate C57BL/6 male mice for the inducing of PD animal models.The behavioral symptoms of PD could be alleviated effectively after SOD@ZIF-8’s treatment.What’s more,the expression of TH was elevated,as well as the DA contents in striatum.Meanwhile,the levels of ROS and MDA in the brain were significantly reduced after administration,suggesting that the protective effect on nerve cells and the relief of PD symptoms by SOD@ZIF-8 were due to the remission of oxidative stress in the brain.In normal mice,treatment of SOD@ZIF-8 after tail vein injection would not cause toxicity in liver and kidney,neither lesions in major organs,indicated that SOD@ZIF-8 is biocompatible.The above results illustrated that SOD@ZIF-8 could eliminate ROS in cells and organisms effectively and achieve therapeutic effects on PD by alleviating oxidative damage,thus providing insight for the application of MOFs-enzymes composites in ROS related biological diseases.In conclusion,we have constructed MOF-enzyme assemblies with nanometer and micron dimensions by biomimetic mineralization technology,which were then used for biodiesel synthesis and PD treatment,respectively.The above studies have successfully overcome the main difficulties in the application of enzyme molecules,and paved the way for promoting the widely application of assembled enzyme molecules based on MOFs in industrial production and biomedical fields. |
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