Study On The Construction Of Enzyme@MOF Bioreactors And Its Application In Chiral Amino Acids Analysis

As a multifunctional green catalyst,enzyme has attracted great attention due to its high catalytic efficiency,fast reaction rate and strong specificity.However,the disadvantages of natural enzymes,such as low operational stability,poor reusability,and narrow optimal temperature and p H range,strictly hinder the scope of applications.Therefore,it is necessary to develop an enzyme-immobilized carrier with good stability and high recyclability.As a novel porous material,the metal-organic framework（MOF）has the advantages of large surface area,high stability,good biocompatibility,and strong affinity for enzymes,providing a broad scope for enzyme immobilization prospect.Currently,although enzyme@MOF is widely used in biocatalysis,biomedical engineering,and bioanalysis,the application research in chirality has not been reported.Therefore,based on the potential advantages of enzyme@MOF,it is of great significance and value to use MOF as the immobilization substrate of natural enzymes to expand the application of enzyme@MOF in the field of chiral analysis.Herein,we carry out the following researches aroun the design and construction of enzyme@MOF bioreactors and the application in the analysis of chiral amino acid（AAs）.The main research work is as follows:（1）The study constructed a novel cascade bioreactor for chiral sensing of multiple AAs enantiomers by coupling L-amino acid oxidase（LAAO）and horseradish peroxidase（HRP）into zeolitic imidazolate framework-8（ZIF-8）（LAAO/HRP@ZIF-8）.LAAO specifically catalyzes L-AAs toα-keto acid,ammonium and H₂O₂,and then the yielded H₂O₂ oxidizes o-phenylenediamine（OPD）to strong yellow fluorescence signal of 2,3-diaminophenazine（DAP）by wrapped HRP.ZIF-8 not only guaranteed free enzymes stability owing to the steric confinement effect of the framework,but also reinforced multi-enzyme cascade catalytic activity due to a preconcentration effect of the analyte inside the cavities.In view of this,the fabricated chiral sensor allowed highly sensitive enantioselectivity discrimination a range of chiral AAs enantiomers and achieved the quantitative determination.Additionally,the results favorably elucidated that the chiral sensor had the prominent stability and satisfactory reusability.（2）The study designed and fabricated a poly（vinylidene fluoride）（PVDF）hollow fiber membrane coated with LAAO@ZIF-8（LAAO@ZIF-8/PVDF）by the interfacial crystallization method for efficient preparation of D-AAs.Then,the biocatalytic membrane is placed in a built-up circulation device.When the L-AAs pass through the LAAO@ZIF-8/PVDF biocatalytic membrane,the L-AAs can be specifically oxidized by the catalytic membrane and obtain the corresponding imino acid.Subsequently,in the presence of reducing agent（H₃N·BH₃）,the imino acid is reduced to obtain racemic AAs.In the obtained racemic mixture,only the L-enantiomer can be further converted by the LAAO@ZIF-8/PVDF biocatalytic membrane.Finally,after multiple rounds of continuously recirculating stereoinversion,L-AAs are reacted completely in the reaction mixture,and D-AAs are accumulated and prepared.Under the optimal experimental conditions,the LAAO@ZIF-8/PVDF biocatalytic membrane exhibits satisfactory stability and recyclability.In addition,compared with uncontinuous enzymatic reduction,dynamic continuous enzymatic reduction has outstanding advantages such as high enantiomeric excess（ee）value,high conversion rate and high yield.