Diversity Of Aromatic Oxygenases And Characterization Of Catechol 1,2-dioxygenase From Fecal Microbiome Of Nycticebus Pygmaeus

Aromatic oxygenases play key roles in the metabolism of various aromatic compounds by microorganism.At present,most study focus on aromatic oxygenases from environment that have rich organic pollutants,while there are few reports on the study of aromatic oxygenases from animal gastrointestinal tract.Animals particularly herbivores eat all kinds of plants that lignin and aromatic compounds can be metabolism in gastrointestinal tract.In the previous study,we found many metabolism pathway and enzymes related to aromatic compounds through metagenomic analysis of N.pygmaeus fecal microbiome.Phenol hydroxylase,catechol 1,2-dioxygenase and catechol 2,3-dioxygenase are important aromatic oxygenases in phenol aerobic degradation.In this study,degenerate primers were used to amplify phenol hydroxylase,catechol1,2-dioxygenase and catechol 2,3-dioxygenase gene fragments from metagenomic DNA of N.pygmaeus fecal microbiome,and diversity of phenol hydroxylase,catechol 1,2-dioxygenase was analysis.cat PL12 was got through TAIL-PCR that was inserted into p EASY-E2 vector and expressed successfully in Escherichia coli BL21?DE3?.The recombinant enzymes cat PL12 were purified with electrophoretic homogeneity by Ni²⁺-NTA metal chelating affinity chromatography,and characterizations of cat PL12 was analysis.The detailed results and conclusions of this study were as follows:?1?The BLAST analysis of phenol hydroxylase gene sequences from N.pygmaeus fecal microbiome showed 92%-100% identity to the known phenol hydroxylase sequences in Gen Bank;phylogenetic analysis showed that phenol hydroxylase gene sequences have high similarity with phenol hydroxylase sequences from Neisseria,Burkholderia,Alcaligenes,Acinetobacter;sequence alignment showed two DEXRH motifs that have in Lm PH were detected in phenol hydroxylase sequences.?2?The BLAST analysis of catechol 1,2-dioxygenase gene sequences from N.pygmaeus fecal microbiome showed 87%-100% identity to the known catechol1,2-dioxygenase sequences in Gen Bank;phylogenetic analysis showed that catechol1,2-dioxygenase gene sequences have high similarity with catechol 1,2-dioxygenase from Acinetobacter;sequence alignment showed the conserved cysteine was detected in catechol 1,2-dioxygenase sequences which was inhibited by Ag+ and Hg²⁺.?3?Optimal activity of purified recombinant cat PL12 at p H 8.0 and 25 °C.The enzyme activity was stable at p H 7.0-9.0;cat PL12 retained 30% of the maximum activity at 0 °C;cat PL12 has 65% and 19% activity after incubation at 25 ? and35 ? for 26 h.Hg²⁺,Co²⁺,Zn²⁺ and SDS has a strong inhibitory effect for cat PL12,while Pb²⁺,Fe3+ and ?-Mercaptoethanol has activation effect for cat PL12.Recombinant cat PL12 have activity for catechol,3-methylcatechol,4-methylcatechol and 4-chlorocatechol,and no activity for hydroquinone.The result of analysis gene diversity of phenol hydroxylase and catechol 1,2-dioxygenase of N.pygmaeus fecal microbiome show phenol hydroxylase was multicomponent phenol hydroxylase,and catechol that middle production of phenol aerobic degradation can be cleaved by catechol 1,2-dioxygenase through ortho-pathway.Study characterization of recombinant cat PL12 show cat PL12 have high activity at room temperature and alkaline environmen.It retained 31% of the maximum activity at 0 °C.This study provides a foundation for research metabolism of aromatic compounds in animals gastrointestinal tract,also provide a foundation for exploring and exploiting microorganism in N.pygmaeus gastrointestinal tract.