Study On The Function Of Copper Oxidase In Anaerobic Ammonium Oxidation Bacteria Resistant To Phenol Stress

Anammox bacteria are a class of microorganisms of great significance in the biological nitrogen cycle of the earth.The anammox process developed by its denitrification performance is currently one of the most promising sewage treatment methods.However,the sensitivity of anammox bacteria to environmental changes limits the application of practical engineering,and the research on the stress mechanism of anammox bacteria is still in the early stage.Encapsulin（c Enc）and copper oxidase（Cop）are both participants in the cell stress response in bacteria,which can help bacteria resist the damage to cells caused by many types of factors（such as peroxides,metal ions,etc.）.Phenol is a typical toxic phenolic compound and a common organic pollutant in nitrogen-rich wastewater.In this study,phenol was used as an inhibitor to study the characteristic changes of anammox granular sludge under the stress of toxic organic matter.A class of functional proteins in the cell stress process of anammox bacteria has been further studied.The Encapsulin（c Enc）and copper oxidase（Cop）of anammox bacteria have been studied through the heterologous expression method of prokaryotes.Functional analysis and enzymatic properties research.A more in-depth exploration of the effect of anammox bacteria on the expression levels of Encapsulin and copper oxidase after phenol poisoning,which provides the possible functions of Encapsulin and copper oxidase in the cell stress process of anammox bacteria New clues.The specific research results are as follows:（1）The anammox granular sludge was subjected to long-term stress with phenol as an inhibitor,and the denitrification performance,apparent morphology and microbial community changes of the anammox granular sludge under different concentrations of phenol were studied.difference.After 270 days of enrichment culture,the relative abundance of anammox bacteria in the anammox granular sludge increased from 13.24%to 56.87%,which provided good conditions for subsequent experiments.Phenol will inhibit the NH₄⁺-N removal rate of anammox granular sludge.The higher the concentration,the more obvious the inhibition,while the NO₂^--N removal rate is not affected.The apparent color of the inhibited anammox granular sludge will gradually change to a dull reddish brown.Phenol stimulates the secretion of microbial EPS in the granular sludge to make the granular sludge denser.In addition,due to the long-term stress of phenol,the microbial community in the granular sludge has also changed.Compared with the control group,the anaerobic ammonia oxidizing bacteria in the granular sludge belong to the phylum of Planctomycetes at 50 mg/L and 150 mg/L phenol The relative abundance was reduced by 2.49%and 7.17%,respectively.In addition,through the prediction of PICRUSt,the differences in the functional metabolic pathways of the microbial community were analyzed,and it was found that the nitrogen metabolism of the key metabolic pathways related to anammox bacteria was inhibited.（2）Before performing heterologous expression,comprehensive analysis of the bioinformatics predicted by Encapsulin and copper oxidase in various protein databases was carried out to provide scientific guidance for subsequent protein expression and function research.Through the identification of protein conserved domains,it is clear that the amino acid sequence of the Encapsulin retains the Linocin＿M18（PF04454）domain,and the amino acid sequence of the copper oxidase fusion protein contains Cu-oxidase＿3（PF07732）,Cu-oxidase＿2（PF07731）and Multi-haem＿cyto（PF13447）Functional domain.By analyzing the primary structure and physical and chemical properties of the protein,it is determined that the theoretical molecular weight of the Encapsulin is about40 k Da,and the theoretical molecular weight of the Encapsulin is about 34 k Da.Through the analysis of homology modeling to predict the three-dimensional structure of the protein,it is speculated that copper oxidase may be a two-domain laccase,which forms a complete quaternary structure of copper oxidase in the form of a homotrimer,with a total of 9 coordinated Cu²⁺Three catalytic active centers of copper ions are formed.（3）The Encapsulin and copper oxidase were expressed heterologously through the prokaryotic expression system,and their functions and enzymatic properties were studied.The copper oxidase expression vector p ET-28a-Cop of four freshwater anammox bacteria and the recombinant expression vector p ET-28a-c Enc-Cop co-expressed with Encapsulin and copper oxidase were successfully constructed through modern molecular biology technology.The Cop and c Enc-Cop proteins were successfully expressed through the E.coli BL21（DE3）expression system.In order to obtain the recombinant protein efficiently,the expression conditions were optimized.The final concentration of IPTG was 0.2mmol/L and the temperature was 30.The expression effect is best when the temperature is℃and the induction culture time is 8 h.By optimizing the purification process of recombinant protein inclusion bodies,the purification rates of Cop and c Enc-Cop proteins were increased from 72.57%and 64.89%to 95.61%and 84.37%,respectively.Finally,Cop and c Enc-Cop with laccase activity were obtained by gradient dialysis and refolding.For c Enc-Cop protein,the laccase activity of 4 kinds of c Enc-Cop is lower than Cop through enzyme activity test comparison.Enzyme kinetics tests were carried out under the optimal conditions of the two recombinant proteins.The comparison of the k_cat/K_mvalues of Cop and c Enc-Cop shows that the copper oxidase that is not encapsulated by the Encapsulin has a greater affinity for the laccase substrate ABTS,and the catalytic rate is also faster.（4）Taking the toxic phenolic compound phenol as the stress condition,the potential functions of Encapsulin and copper oxidase in the stress process of anammox bacteria were further studied.Under the short-term stimulation of different concentrations of phenol,real-time fluorescent PCR experiments found that with the increase of phenol concentration,the relative expression of hydrazine synthase HZS gene m RNA gradually decreased,and the toxic organic substance phenol increased the metabolic level of anammox bacteria.Has changed.The relative m RNA expression of copper oxidase（Cop）and Encapsulin（c Enc）genes varies with phenol concentration and phenol exposure time.The expression of these two types of proteins is regulated by the stress response of anammox cells.The analysis of the functional properties of Encapsulin and copper oxidase suggests that they may be involved in the process of cells resisting the toxic pressure of phenol.