Heterologous Expression Of An Unspecific Peroxygenase From Agaricus Bisporus Var.bisporus And Its Application In Catalytic Oxidation

Unspecific Peroxygenases?UPO?are a type of peroxidase derived from fungi and belong to heme-thiolate peroxidase?HTP?superfamily.UPOs are generally present in Dikarya,higher fungi that includes the two phylum:Ascomycota and Basidiomycota of the fungal kingdom.UPO can catalyze different types of catalytic reactions such as the activation of C-H-bonds,epoxidation of C=C-bonds and sulfonation of sulfide,it is considered as a new generation of biocatalyst for the selective oxidation of organic compounds.However,the difficult large-scale preparation of UPO by fungi is limiting the application for biotechnology.Finding a suitable heterologous expression host for UPO is critical to solving the above problems.In this study,the unspecific peroxygenase derived from Agaricus bisporus var.bisporus?Abvb?was heterologously expressed in different microbial hosts,and the enzymatic properties will be explored.This study provides a reference for UPO heterologous expression and catalysis research.Main research contents and results are showed as follows:?1?Bioinformatic analysis of Abvb UPO.Ex PASy,Swiss website and Py MOL software were used to predict the structure and properties of Abvb UPO?Genbank ID:XP?006457061.1?.The results showed that the theoretical molecular mass was 35.56 k Da,the predicted isoelectric point was 6.27,the predicted stability coefficient was 21.81,and the predicted half-life of Abvb UPO expressed in Pichia pastoris,E.coli,and mammalian cells are 20 h and 10 h and1.90 h,respectively.Homology modeling results showed that Abvb UPO contained 11?helices,5?sheets and a pair of disulfide bonds?Cys277-Cys318?.Mg²⁺in the structure was coordinated by a heme propionate and two water molecules and by three oxygen atoms of Glu-122,Gly-123and Ser-126.?2?Heterologous expression and enzymatic properties of Abvb UPO.The gene sequence of Abvb UPO was obtained by whole gene synthesis technology,and a series of recombinant expression plasmids such as p ET22b-MBP-Abvb UPO,p MA5-Abvb UPO,p BC-Abvb UPO and p PIC9K-Abvb UPO were constructed,and the recombinant plasmids were transformed into E.coli Origami B?DE3?,Bacillus subtilis 1A751,Aspergillus niger G1 and Pichia pastoris GS115for heterologous expression research.The results showed that Abvb UPO expressed heterologously as active enzymes in Pichia pastoris GS115 heterologous expression system,with a molecular mass of about 35 k Da;under the induction temperature of 25?and p H 6.0,the unit fermentation activity of the 5 L fermenter was 45399.28±153.67 U/L.Abvb UPO was incubated with different concentrations of hydrogen peroxide?H₂O₂?for 5 min,and the residual enzyme activity of Abvb UPO was determined.The results showed that the H₂O₂-tolerance of Abvb UPO was low.After 30-minute incubation of Abvb UPO with 2 m M H₂O₂,the enzyme activity decreased to 95.52±2.00%of initial activity.And the enzyme activity of Abvb UPO decreased with the increase of H₂O₂ concentration.After incubation at 30?for 5 min with Abvb UPO and 5 m M H₂O₂,the enzyme activity was only 54.98±5.74%of the initial activity.However,the recombinant Abvb UPO appeared inactive form in E.coli and B.subtilis,while there is no expression in Aspergillus niger G1.?3?Preliminary study on the catalytic oxidation of Abvb UPO.To avoid the excessive H₂O₂-mediated inactivation of Abvb UPO.This study constructed an enzymatic cascade reaction system of choline oxidase-Abvb UPO for in situ production of H₂O₂ by choline oxidase to reduce H₂O₂-mediated inactivation of Abvb UPO.Further,the effect of p H and temperature on enzymatic cascade reaction were investigated.Ethylbenzene,cyclohexane,styrene,cyclohexene,methyl phenyl sulfide and phenyl vinyl sulfide were used as substrates to carry out enzymatic cascade reaction.GC-MS method was used to monitor products of?-phenylethanol,cyclohexanol,styrene oxide,cyclohexene oxide,methylphenyl sulfoxide and phenylvinyl sulfoxide.The catalytic results showed that the optimal reaction conditions for the cascade reaction system were p H 8.0,reaction temperature 30?;under these conditions,Abvb UPO had the highest activity for cyclohexene epoxidation,and the product concentration of cyclohexene oxide was 2.91 m M.In addition,the products of Abvb UPO enzymatic cascade reaction were all R enantiomeric structure,except for styrene oxide.This indicates that Abvb UPO has preference for R enantiomeric structure.