Study Of Construction And Catalytical Properties Of Affinity-oriented Photo-crosslinked Immobilized Enzyme

As an efficient biocatalyst,enzyme has the advantages of high specificity and biodegradability.However,major drawbacks of enzymes are their instability and difficulty for recycling,and thus significantly restrict their industrial practices.Immobilized enzymes have been used to overcome the disadvantages of free enzymes,but the random immobilization may result a sharp decrease in enzyme activity due to masking of the active site.Oriented immobilization is a strategy to solve this problem because enzyme attachment with a specific orientation ensures active sites of enzymes are fully exposed to solution,thus preserving bioaffinity or bioactivity.The covalent attachment generally requires additional chemical crosslinking agents,incurring toxicity,high costs and complicated procedures.Photo-crosslinking reaction with high temporal and spatial resolution was widely used in physics,chemistry,and biomedicine,which allows simplicity execution for the oriented covalent immobilization.The most classical photosensitive non-natural amino acid of benzophenone is 4-benzoyl-L-phenylalanine（Bpa）,which can grab hydrogen atoms from the activated C-H bond and recombine to form covalent complexes under ultraviolet light with a wavelength of 365 nm.As a novel and natural functional sugar,isomaltulose has the excellent characteristics of non-cariogenic and low calorie,which provides a healthier option for individuals with obesity,diabetes or cardiovascular disease.Sucrose isomerase（SI）hydrolyzes theα-1,2 bond between glucose and fructose in sucrose molecule and then forms anα-1,6 bond to produce isomaltulose.In this study,sucrose isomerase was selected as the model enzyme.Firstly,molecular simulation software was used to select the region far away from the SI active site as the oriented immobilization region.Then,a short peptide ligand（H₂N-VNIGGX-COOH,VG）with high affinity to this region was rational designed,and the non-natural amino acid4-benzoyl-L-phenylalanine（Bpa）with the photosensitive group of benzophenone was introduced into its carboxyl-terminal.And then,the affinity between the ligand and the SI was validated by simulation,the photo-crosslinking reaction between VG and SI was also confirmed by the ultraviolet light experiment.Thereafter,SI was oriented immobilized on the surface of epoxy（EP）guiding by VG.Their enzymatic activity,thermostability and reusability of immobilized enzymes with different strategies（random adsorption,oriented adsorption,and oriented photo-crosslinking）were systematically studied.It is shown that the activity of sucrose isomerase immobilized by affinity adsorption（EP-VG-SI）and photo-crosslinking（EP-VG-SI hv）preserved 93.9%and 72.4%of activity with the free SI.And,the EP-VG-SI hv retained 50%of its catalytic activity after 11 cycles,and 90%of its catalytic activity after incubating at 45℃for 2 h.Affinity-oriented photo-crosslinked immobilized sucrose isomerases have obvious improvement in recycling and heat resistance,and basically does not affect the enzymatic activity.In comparison with traditional immobilized enzymes,this study provided a novel protocol for efficient immobilized enzymes with the combination of oriented adsorption and photo-crosslinking.