Screening Of Carbazole-degrading Bacteria Strain And Characterization Of Degradation Genes

Carbazole is a kind of nitrogen-containing heterocyclic aromatic compounds and important raw materials in modern chemical industries,which is widely used.Carbazole has teratogenicity,mutagenicity,and potential carcinogenicity.It is difficult to degrade due to its stable structure,and has strong environmental toxicological effects.Therefore,the screening of efficient cabazole-degrading strains and the cloning of related degradation genes have become one of the hotspots in the field of bioremediation.Carbazole-degrading bacterium B1 was isolated from sludge in the oil wellhead of Qinghai by enriched culture.Physiological properties and 16S rRNA gene sequence analysis were performed to identify the isolated strain B1,and it was identified to be Sphingosinicella sp.stain and gram-negative bacteria.The results of degradation characteristics showed that 100 mg/L carbazole was degraded over98%by B1 in 72 h under the optimum condition?30°C and pH 7.0?;the strain could tolerate carbazole up to 700 mg/L;the degradation rate of carbazole was more than 95%within 96 h when pH was among7.0⁹.0,while it was higher when the salt concentration was less than 10 g/L.In the meanwhile,the degradation rate was significantly improved by adding 0.1 g/L glucose and ammonium sulfate.Soil remediation in situ simulation experiments showed that the degradation rate of carbazole at 100 mg/kg was about 90%within 28 days by strain B1 and soil indigenous microorganisms.In this study,carAa gene?560 bp?,carBa gene?201 bp?,carBb gene?793 bp?and carC gene?392bp?were successfully cloned from the isolated strain B1 according to the conservativeness of the car gene cluster.The RT-PCR results of the degradation genes in B1 strain grown in different media confirmed that the car gene cluster of strain B1 was inductively expressed,and as same as the expression pattern of genes in most strains.The expression of carAa gene,carBb gene and carC gene were significantly different under different pH by qRT-PCR.At pH 9.0,the relative expression levels of the three genes were the highest.Bioinformatics analysis showed the presence of overlapping sequences of eight bases?ATGGCTGA?when the carBa and carBb genes made up the carB gene.Meanwhile,the key catalytic sites for carB were found in carBb subunit?His12,His53 and Glu230?.The expression vectors of carBa,carBb and carB were successfully constructed within pET-32a?+?vector and were inductively expressed the target proteins.Enzyme activity results indicated that CarB had a cleavage function while a single subunit did not.The enzymatic properties of CarB were analyzed,and the results showed that the optimal pH value and temperature of CarB were 7.4 and 30°C respectively.The enzyme activity would be sifnificantly reduced if pH was too high or too low?pH<6.0 or pH>9.0?,and high and low temperature also had a great impact on the enzyme activity.Different metal ions had different effects on the enzyme activity as Fe²⁺could particularly improve the catalytic activity.Conclusively,in this study,the carbazole degradation strain of Sphingosinicella sp.was obtained at home and abroad for the first time,which enriched the microbial resources of carbazole-degrading bacteria.The theoretical research on the degradation of the cloning and enzymatic properties of the key enzyme would provide theoretical basis and technical support for carbazole degradation and environmental remediation.