Heterologous Expression And Immobilization Of Unspecific Peroxidase From Agrocybe Aegerita (AaeUPO)

Selective oxygen functionalization of inert hydrocarbon bonds is one of the main challenges in organic synthesis.Chemical catalysis is often carried out under extreme conditions,while enzyme catalysis can be realized under mild conditions.Heme dependent monooxygenase is an exploratory catalyst for selective oxygen functionalization.P450 has attracted much attention,but its complex electron transfer process and"oxygen dilemma"limits its application to a certain extent.Unspecific peroxygenase（Aae UPO）from Agrocybe aegerita has become a practical substitute for P450 monooxygenase because of its simple electron transfer process and higher economic benefits.However,Aae UPO uses H₂O₂ as the co-substrate to catalyze the monooxygenation reaction,and the heme group of its active center is easy to be inactivated under high concentration H₂O₂.Therefore,the construction of a circulating system for in-situ generation of H₂O₂ is the key to realize the catalytic production of hydroxylation products by Aae UPO.Based on this,in this paper,the cross-linked enzyme aggregates of unspecific peroxidase were prepared by carrier free immobilization to improve the stability and reusability of free enzyme,and the cascade system of Aae UPO and glucose oxidase（GOx）was designed to produce H₂O₂ in situ.The prepared composite cross-linked enzyme aggregates can catalyze stably.The specific research contents are as follows:（1）Expression and enzymatic properties of Aae UPO.From the NCBI library,we obtained the sequence of Pa Da-I from unspecific peroxygenase Aae UPO mutant derived from Agrocybe aegerita and constructed the inducible plasmid p PIC9K-Aae UPO.The extracellular expression of Aae UPO was achieved in Pichia pastoris,and Aae UPO with high activity was obtained.After induction of 144 h,the enzyme activity reached 108 U/mg.Through the study of its enzymatic properties,it is found that the catalytic activity of the enzyme reaches the maximum at p H 5.0 and temperature 30^oC,and the higher enzyme activity is retained in 20%acetonitrile solution,while other organic solvents are easy to cause the loss of enzyme activity.The kinetics of ABTS,DMP and NBD were studied.The K_mvalues were 0.0561 m M,0.102 m M and 0.141 m M respectively.（2）Aae UPO aggregates were prepared by cross-linked enzyme aggregate technology.Aae UPO crosslinking enzyme aggregates（Aae UPO-CLEAs）were prepared with isopropanol as precipitant,200 k Da glucan aldehyde as crosslinking agent and BSA as protective agent.The optimum p H of Aae UPO-CLEAs shifted to 6.0,and the optimum temperature was still30^oC.Its p H stability,thermal stability and storage stability were significantly better than those of free enzyme.Using ethylbenzene and 4-methoxybenzophenone as model substrates,when Aae UPO-CLEAs enzyme activity is 8 U,ethylbenzene concentration is 2 m M（4-methoxybenzophenone concentration is 1 m M）,H₂O₂ is 6 m M（3 m M）,and acetonitrile concentration is 20%,it is catalyzed for 2 h（3 h）at p H 7.0（7.5）and 30^oC（35^oC）,The yield is more than 99.9%（the conversion of 4-methoxybenzophenone is 87.4%）,and the ee value is more than 99.9%.Aae UPO-CLEAs shows good catalytic performance.In the hydroxylation reaction of ethylbenzene,the yield can still maintain 58.19%after Aae UPO-CLEAs is recycled for eight times,showing good reusability.（3）Construction of cascade system for in-situ H₂O₂ production.Using isopropanol as precipitant and 200 k Da dextran aldehyde as crosslinking agent,the composite crosslinking enzyme aggregate（combi-CLEAs）of GOx and Aae UPO was prepared.GOx catalyzed the oxidation of glucose to produce H₂O₂.Aae UPO used it as co substrate to catalyze the selective oxygen functionalization of inert hydrocarbon bonds.The preparation conditions were optimized,and combi-CLEAs with high enzyme activity was obtained.The system realized the cascade of in-situ production of H₂O₂.When combi-CLEAs takes ethylbenzene as substrate,the yield is 67.2%after repeated use for eight times,indicating that combi-CLEAs has higher operational stability than Aae UPO-CLEAs.At the same time,combi-CLEAs showed broad substrate universality in hydroxylation and demethylation.