Heterologous Expression Of An Unspecific Peroxygenase From Galerina Marginata And Its Application In Catalytic Oxidation

Unspecific Peroxygenases(UPOs)are a kind of oxidase derived from fungi,which can catalyze a variety of oxidation reactions,such as sulfonation,epoxidation,hydroxylation,dealkylation,oxidation of organic heteroatoms and inorganic halides,etc.It is widely used in environmental governance,organic synthesis and biotechnology.At present,thousands of putative UPO genes have been found in the genetic database,so it is very important to find the suitable heterogenous expression system and explore the heterogenous expression conditions of different UPOs.In this study,the unspecific peroxygenase GmaUPO,which originated from Galerina marginata(Gma),was studied in bioinformatics,heterogenous expression in different hosts and its catalytic performance in catalytic oxidation were studied,which provided a certain research foundation for the catalytic application of GmaUPO.The specific contents and results are as follows:(1)Bioinformatics analysis of GmaUPO.The structure and properties of GmaUPO were analyzed by using DNAMAN and PyMOL software,Multalin,Protscale,Expasy and Swiss model websites.The results showed that there was 15.49-67.23% sequence identity between GmaUPO and UPO from different sources.The theoretical molecular weight was 38.27 kDa,the isoelectric point was 5.09,the predicted stability coefficient was 24.96,and the total average hydrophilicity coefficient of the whole polypeptide chain was-0.244.The secondary structure prediction analysis showed that GmaUPO contained 12 ? helices,2 ? folds and a pair of disulfide bonds(Cys61-Cys323).Three dimensional structure analysis of homologous modeling showed that GmaUPO had UPO catalytic conserved sequences of Pro60-Cys61-Pro62? Glu147-Gly148-Asp149 and Arg190-Glu197,which suggested that GmaUPO might have UPO catalytic properties.(2)The recombinant expression of GmaUPO.The GmaUPO gene was obtained by the whole gene synthesis technology,and cloned into the expression vectors of Escherichia coli,Bacillus subtilis and Pichia pastoris,and the recombinant expression was studied.The results showed that GmaUPO existed as an inactive inclusion body in E.coli,but not in B.subtilis.The active recombinant expression was only obtained in P.pastoris X33,with a molecular weight of about 40 kDa.In addition,the highest fermentation activity was 6 U / L when the fermentation temperature was 22 ? and the concentration of ALA(?-aminolevulinic acid)was 45 ?M.(3)Study on the catalytic oxidation of GmaUPO.The effects of p H,temperature and substrate on the catalytic efficiency of GmaUPO were studied.The results showed that the optimum reaction temperature and p H of GmaUPO were 35 ? and 9.0,respectively.The products were R-phenylethanol,S-phenylethanol and acetophenone,naphthenic ketone and methylphenylsulfoxide by GC-MS.The results show that GmaUPO can catalyze the reactions of aromatic compounds,aliphatic compounds and ethers.Among them,the oxidation product of ethylbenzene is R-isomer,which indicates that GmaUPO may have R-isomer substrate preference.