Precise Immobilization,cross-linking And Simultaneous Purification Of Aldehyde Ketone Reductase With Nonstandard Amino Acids

In the enzyme immobilization technology,covalent linkage can prevent the enzyme from leaking from the support while catalyzing some reactions,significantly enhance the stability and enhance the rigidity of the enzyme.However,in traditional covalent method,enzymes often lose most of their activities due to the destroy or cover of active sites resulting from the unwanted and random chemical linkage,which elevates the cost of the immobilized enzymes.Thus,it is especially important how to tackle this barrier in covalent immobilization and achieve the efficient,precise and site-specific immobilization.First,to avoid random and unwanted chemical linkage between the support and the enzyme protein,the target enzyme is modified by incorporating nonstandard amino acids?NSAAs?,and then attached on the functionalized resin support by click reaction.To this end,aldehyde ketone reductase?AKR?was used as a model enzyme,and the sites such as 110Y,114Y,143Y,162Q,and 189Q were replaced with p-azide-L-phenylalanine?p Az F?,respectively.The obtained AKR mutants was then covalently attached onto a cyclooctyne functionalized support by strain-promoted alkyne-azide cycloaddition reaction?SPAAC?.The effects of the number and location of nonstandard amino acids?NSAAs?on the loading rate and thermal stability of immobilized AKR were also investigated.The results showed that the mutant-type AKR generally had better specific activity than the wild-type AKR,and the activity of AKR-114Y was 1.16times higher than that of the wild-type AKR.Moreover,the half-life(t_1/2)of the five-point immobilized AKR reached 106 h and 45 h at 30?and 60?,respectively,13 and7 times that of free enzymes,respectively.Comparison of these three different number of site?one,three,and five-point?enzyme mutants shows that multi-point immobilization can increase loading rate and thermal stability and facilitate one-step purification and immobilization.Secondly,based on the above multi-point precise immobilization,and to achieve green,support-free immobilization,we develop a method for preparing rapidly and precisely crosslinked enzymes?RP-CLEs?using bio-orthogonal chemistry.Nonstandard amino acids?NSAAs?are used for multi-site insertions to modify the target enzyme proteins,and microwave irradiation is used to accelerate site-specific cross-linking by copper-free click reactions.In this method,sym-dibenzo-1,5-cyclooctadiene-3,7-diyne is used as bi-functional crosslinker to achieve the intermolecular crosslinking of the AKR multi-point mutants through the cycloaddition reaction.The effects of the position and number of mutation sites in the AKR base sequences on the immobilization rate and thermal stability were also examined.Under microwave irradiation at 10?for 4 minutes,AKR multi-point mutants were cross-linked,and the immobilization yield of five-point mutant could reach 90%,and rapidly and precisely crosslinked enzymes?RP-CLEs?are formed from cell lysate supernatant.The specific enzyme activity of RP-CLEs was 2.06 U·mg^-1,which was twice of the free AKR five-point mutant.RP-CLEs prepared under microwave radiation also have elevated thermal stability,and the half-life(t_1/2)at 60?is 26.76 h,which is about 4.5times(t_1/2)that of the AKR five-point mutant.When the RP-CLEs are used to catalyze the chiral synthesis of crizotinib intermediate?S?-1-?2,6-dichloro-3-fluorophenyl?ethanol,the product yield reaches 90%and the ee value?enantiomeric excess?exceeds99%.Most of all,RP-CLEs also achieves reach 80%of the initial cycle after 6 cycles in a 12-hour cycle of each reaction.As a result,based on the analysis of the enzyme structure,different numbers of NSAAs can be reasonably inserted into the base sequence to accurately guide and control the covalent cross-linking of the target enzyme.Unmodified enzymes are eluted by the elution of high concentration because they cannot be cross-linked without nonstandard amino acids,thereby precise cross-linking and simultaneous purification occur only to the target enzyme protein.In summary,based on the precise immobilization of aldehyde ketone reductase incorporated with multiple nonstandard amino acids,we develop a new method of enzyme immobilization on support in which the immobilization sites and covalent linkage can be accurately located and identified,respectively.In addition,we prepare the rapidly and precisely crosslinked enzymes?RP-CLEs?under microwave irradiation at low temperature.This RP-CLEs present good reuse stability in the enzymatic synthesis of the crizotinib intermediate?S?-2,6-dichloro-3-fluorophenylethanol.In this method,no organic solvents and?NH₄?₂SO₄ are used as the traditional method,and the RP-CLEs can be prepared efficiently,energy-cost,and environmentally friendly,and may be further expanded to other enzyme for industrial biocatalysis.This work was expected to promote a basic understanding of precisely oriented and controllably covalent immobilization and enable the biomanufacturing paradigm for fine chemicals and pharmaceuticals.