Green Synthesis And Application Of Nanocrystalline Enzyme Preparations Based On Metal-organic Frameworks (MOFs)

Enzymes exhibit high efficiency in degrading of herbicide contamination.However,their instability and difficulty in resue limit enzymes in agricultural.Enzyme immobilization can improve stability and catalytic efficiency in the catalytic process,increase the reusebility and reduce the cost of enzyme.In recent years,a new type of microporous nanomaterial has become an ideal candidates for immobilized enzyme,which has been widely applied in the fields,such as gas storage,biomedicine,sensing,and catalysis.Herein,an aryloxyphenoxypropionate herbicide-hydrolyzing enzyme,QpeH,was purified via one-step affinity chromatography and embedded into two types of zeolitic imidazolate frameworks（ZIFs）through biomimetic mineralization.The structural characteristics of enzyme preparation displayed that rhombic dodecahedral morphology（QpeH@ZIF-8）when the concentration of Zn²⁺was 0.4 mol·L^-1,as the concentration increased 0.8 mol·L^-1 was cruciate flowerlike morphology（QpeH@ZIF-10）.Fourier Transform Infrared Spectroscopy（FT-IR）indicated that esterase QpeH was successfully embedded in ZIFs.Meanwhile,X-ray diffraction（XRD）results showed that the esterase QpeH added to ZIFs has no effect on the formation of crystal structure.Thermogravimetric Analysis（TGA）result demonstrated the enzyme loading capacity was higher in ZIF-10 than that in ZIF-8,which was 91.1%and 89.7%,respectively.The catalytic activities of free QpeH and enzyme preparation QpeH@ZIF were studied by using quizalofo-p-ethyl（Qp E）as AOPP herbicide analytical model.Compared with free QpeH,the enzyme activities of QpeH@ZIF-8 and QpeH@ZIF-10 were 86%and 93%,respectively.Therefore,the mechanism of the activity loss of embedded QpeH in ZIF structure was further studied,The results confirmed that the K_m of free QpeH（37.6μm）was lower than QpeH@ZIF-8（66.2μm）and QpeH@ZIF-10（52.1μm）,which was indicated that the structure of ZIFs reduced the affinity between substrate;However,V_m is similar,it is proposed that the decrease in activity of both QpeH@ZIF composites is possibly caused by the hindered mass transfer in the ZIF framework.Meanwhile,the catalytic activities of QpeH@ZIF-8 and QpeH@ZIF-10 on the herbicide Qp E was compared.The results were confirmed that the same concentration of herbicide,compared with the free enzyme,the two enzyme preparations showed higher catalytic activity under the action of heat intolerance,acidity and alkalinity,metal ions and chemical reagents.Notably,Ca²⁺and Fe³⁺ions had an obvious positive impact on the activity of the free enzyme and respectively increasing it by 58%and 21%of the original activity.However,these two ions caused less enhancement on the activities on the embedded QpeH in composites.Thus,the ZIF framework provides protection against enzyme conformational changes without regard to the positive or negative effects on the enzymatic activity.Of note,in the formation process of enzyme@MOF crystals,the addition of metal ions was found to significantly increase enzymatic activity resulting from the interaction between metal ions and the enzyme.QpeH@ZIF composites displayed excellent reusability over 10 cycles.After 5 times of reuse,QpeH@ZIF-10showed a higher catalytic activity compared to QpeH@ZIF-8,which is formed following continuous growth based on QpeH@ZIF-8 in the presence of excessive Zn²⁺ions,and more stable than the QpeH@ZIF-8.When two structural enzyme preparations were used to remove herbicide that added containing quizalofop-p-ethyl to soil,the effect of QpeH@ZIF-10 was more obvious than QpeH@ZIF-10 in the long term.Magnetic nanoparticles（MNPs）display have high catalytic activity and can be considered as co-catalysts for ZIF-8 in most chemical reactions.In addition,It is an economical,simple and effective method to separate magnetic enzyme preparation from solution using external magnets.On the basis of the above synthesis,the magnetic double enzyme system（QpeH/AOx@m ZIF-8）was synthesized by one-step synthesis Fe₃O₄ MNPs,which can be detected AOPP herbicides and alcohols.Moreover,as a peroxidase mimic,and it has strong affinity.QpeH and AOx were embedded in the m ZIF-8 also indicated better catalytic activity and stability and good recyclability in the detection.QpeH/AOx@m ZIF-8 was proved to be a colorimetric sensor with high stability and low cost detection AOPP herbicides,which improves the stability of the enzyme,achieves the convesion of intermediate metabolites,and simulates catalase activity.the QpeH/AOx@m ZIF-8 system appears to be a multifunctional colorimetric biosensor with the performance of rapidly achieving the visible detection of various AOPP herbicides,which can be provided as a reference for the development of other multi-enzyme systems.