Study On Selective Oxidation Of Aromatic Compounds Catalyzed By Unspecific Peroxygenases In Natural Deep Eutectic Solution

Unspecific peroxygenases?UPO?is a kind of oxidoreductase which can catalyze multiple types of oxidation reactions with heme as catalytic activity center.UPO can efficiently catalyze the reactions of C-H bond hydroxylation and C=C double bond epoxidation in aromatic compounds under mild conditions by using H₂O₂without expensive coenzyme factors.The oxidation products of UPO catalytic reactions have great potential in the fields of flavors and fragrances,health care products.Nevertheless,the oxidant H₂O₂ poses a threat to the efficiency of UPO catalysis.It is necessary to develop a reaction system for the efficient catalytic application of UPO.This study established a Choline Oxidase from Arthrobacter nicotianae?AnChOx?catalyzed online generation of hydrogen peroxide?H₂O₂?in natural deep eutectic solution?NADES?,which aimed to maintain the concentration of H₂O₂ at a stable level which can catalyze efficiently without destroying the enzyme activity.On this basis,recombinant Agrocybe aegerita Unspecific Peroxygenases?rAaeUPO?was used to catalyze the oxidation of aromatic compounds.Furthermore,the protection mechanism of NADES solution to rAaeUPO was studied by molecular simulation analysis.Main research contents and results are showed as follows:1.Establishment of AnChOx catalyzed H₂O₂ generation in NADES solution.E.coli BL21?DE3?was used as the expression host for the highly efficient recombinant expression of AnChOx,and the H₂O₂generation catalyzed by AnChOx in NADES solution was constructed.The result showed that AnChOx can catalyze choline chloride to provide stable low concentration H₂O₂ in NADES solution,the catalytic rate of H₂O₂ generation by AnChOx was0.75-1.70 m M·h^-1 and the cumulative yield was 6.41-11.36 m M after the reaction equilibrium.2.Establishment of rAae UPO catalyzed selective oxidation of aromatic compounds cascaded with AnChOx in NADES solution.In the above-mentioned system,rAaeUPO was used to catalyze the selective oxidation of organic compounds.The effects of the key factors such as NADES solution,enzyme dosage on the reaction were investigated.For the hydroxylation of ethyl benzene,22.30±1.12 m M product was obtained under the optimal reaction condition of 25%?v/v?Ch Cl-Urea-Gly solution as the reaction solvent,and the conversion rate was 89.20±4.48%.For the epoxidation of cis-?-methylstyrene,8.68±0.12 m M product was obtained under the optimal reaction condition of 50%?v/v?Ch Cl-Pro-H₂O solution as the reaction solvent,and the conversion rate was 34.72±0.48%.For the sulfoxidation of methyl phenyl sulfide,15.09±0.01 m M product was obtained under the optimal reaction condition of 50%?v/v?Ch Cl-Urea solution as the reaction solvent,and the conversion rate was60.36±0.04%.The result showed that the online generation of H₂O₂ provides a positive impact on the efficient catalysis of rAaeUPO.3.Study on the protection mechanism of NADES solution to rAaeUPO.The effect of different solvents on rAaeUPO enzyme activity was investigated,and the mechanism of NADES solution on rAaeUPO stability was explored by molecular simulation analysis technology.The results showed that NADES solution had a significant stabilizing effect on rAaeUPO,and the half-life time of enzyme activity is about 6 times more than that in buffer.Molecular simulation results showed that because of the smoother filling amount,smaller solvent accessible surface area and more stable surface fluctuation area,rAaeUPO was more stable in NADES solution than that in buffer.The above coordination played a key role in the stabilization of rAaeUPO by NADES solution.In conclusion,the study has established an AnChOx catalyzed online generation of H₂O₂in NADES solution.The rAaeUPO-catalyzed selective oxidation of aromatic compounds cascaded with AnChOx in NADES solution was further established.The study provided a new solution for the problem of the H₂O₂injury in catalyzing reaction of hydrogen peroxide driven enzymes like UPO,and provides a new way to synthesize complex functional components in food and drugs.