Screening,Reducing Property,and NosZ Gene Cloning And Expression From Pseudomonas Citronellolis WXP-4

High concentrations of nitrate can cause environmental problems such as eutrophication of rivers,deterioration of water sources,and endanger human health.Therefore,denitrification of polluted water is the key to environmental pollution control.Biological nitrogen removal is considered to be the most effective way to reduce nitrogen pollution because it can convert oxidized nitrogen compounds?nitrates and nitrites?into N₂.A high-efficiency denitrifying bacteria screened from the wastewater of Qige Sewage Treatment Plant in Hangzhou City,Zhejiang Province was reported in this paper.In addition,the entire genome sequence of the strain was obtained by bacterial De Novo sequencing.The nos Z gene was successfully cloned and a prokaryotic recombinant expression strain E.coli BL21?DE3?-p ET28a-nos Z containing the nos Z gene sequence was successfully constructed in E.coli by IPTG induction.The expression of N₂O and the reduction performance of N₂O were investigated.The main conclusions are as follows:A strain of heterotrophic denitrifying bacteria was isolated,and the strain was identified as Pseudomonas citronellolis and named WXP-4 through morphological observation,physiological and biochemical experiments and 16S r DNA gene sequence analysis.Studies on the denitrification characteristics of strain WXP-4 showed that sodium succinate was used as the carbon source,the C/N ratio was 7,the p H was 7.0,the temperature was 40°C,the removal rate of nitrate and nitrite can reach 100%under aerobic and anaerobic conditions The gas detection results showed that the strain mainly produces N₂ and no N₂O emissions under anaerobic conditions.The whole genome sequence of Pseudomonas citronellolis WXP-4 was obtained by De Novo sequencing,with a total length of 6672752 bp and a GC content of 67.40%.There were four types of denitrification genes,the nar LQKKGHJI,nor BC,and nos RZDFYL genes exist in clusters,the nir functional genes is only nir K,and no nap-related gene clusters were found.Based on sequence homology comparison,Swiss model was used to model the three-dimensional structure of N₂OR protein,and nos Z protein was edited with discovery studio4.5 software to learn that the protein is 4CU:2S structure and CUA has only five amino acid ligands.The gene engineering strain E.coli BL21?DE3?-p ET28a-nos Z was successfully constructed,and the nos Z gene was successfully transcribed and expressed as an active N₂OR in E.coli BL21?DE3?,which can independently play the N₂O reduction function.The N₂O reduction rate of WXP-4 is about 2 times of that of the engineering strain,which may be due to the absence of one or several genes in the nos RFDYL gene in the engineering strain,unable to encode the corresponding auxiliary protein,resulting in the decrease of N₂OR activity or the total enzyme amount.