Isolation,Identification,Characteristics And Preliminary Application Of A Chloramphenicol-degrading Strain LMS-CY

Chloramphenicol(CAP),a broad-spectrum antibiotic,has effective inhibition on lots of Gram positive and negative bacteria.Chloramphenicol was widely used in prevention and treatment of infectious disease of animals due to its good antibacterial effect.However,chloramphenicol could not be completely absorbed by animals,and most of the original antibiotic and metabolites were excreted into the environment through urine and feces.Chloramphenicol was stable in the environment and many papers showed that residual chloramphenicol existed in water and animal-derived food,which might induce cancers and a variety of damages in human hematopoietic and digestive systems.Microbial degradation is an effective way in the elimination of chloramphenicol residues in the environment.Several investigations were available for isolation of chloramphenicol-degrading strains,only one strain Pseudomonas sp.CSFO-3 was reported to degrade chloramphenicol completely but with minimal degradation efficiency.Therefore,it's necessary to isolate strains that degrade chloramphenicol more efficiently,which will provide materials and resources for the remediation of chloramphenicol-contaminated environment.Strain LMS-CY,which was capable of degrading chloramphenicol,was isolated from activated sludge in the wastewater treatment system of a pharmaceutical factory in Anhui Province.Strain LMS-CY was identified as Nocardioides sp.based on physiological and biochemical characters and the similarity analysis of its 16S rRNA gene sequence.It was the first strain discovered in the genus of Nocardioides which could degrade chloramphenicol.For its growth,the optimum temperature and pH were 30? and 7.0,respectively.Strain LMS-CY grew normally with the concentration of NaCl lower than 30 g/L.Strain LMS-CY could use chloramphenicol as the sole carbon source for growth.With the inoculation of 3%,strain LMS-CY was able to degrade 100 mg/L chloramphenicol within 24 hours.The optimum temperature and pH for the degradation of chloramphenicol were 30? and 7.0 respectively.Within a certain range,the chloramphenicol degradation rate was positively associated with the inoculation amount of bacteria.Strain LMS-CY degraded chloramphenicol quickly while the initial concentration of chloramphenicol was lower than 150 mg/L,and the degradation rate decreased with the increase of initial concentration of chloramphenicol.Besides,heavy metal ions,including Hg2+,Cu2+,Fe2+,Ni2+,Co2+and Zn2+,inhibited the degradation rate.Two intermediate metabolites in the degradation of chloramphenicol by strain LMS-CY were identified as 2-amino-3-hydroxy-3-(4-nitrophenyl)propionic acid and p-nitrobenzoic acid by MS/MS.Based on the results,it was deduced that there was a new degradation pathway of chloramphenicol by strain LMS-CY.Firstly,amide bond of chloramphenicol was broken to produce 2-amino-3-hydroxy-3-(4-nitrophenyl)propionic acid,which was degraded into p-nitrobenzoic acid.Finally,p-nitrobenzoic acid was degraded totally by strain LMS-CY.The genome sequence of strain LMS-CY was sequenced and the genes were analyzed.The bacteria were crushed and the crude enzyme was obtained by grinding the strain LMS-CY with liquid nitrogen.The optimum enzymatic temperature was 30? and the optimum pH was 7.0.Heavy metal ions,including Cu2+,Fe2+,Ni2+,Hg2+,Co2+and Zn2+inhibited the activity of the crude enzyme.While the temperature was higher than 30?,the stability of enzyme activity decreased gradually with the increase of the temperature.When it was 60?,the enzyme was completely inactivated.Antibiotic degradation of bacteria flowing into the environment might cause drug-resistant gene diffusion,and free enzyme was unstable in the environment.Therefore,in this study,immobilized enzyme was used to remediate the environment polluted by chloramphenicol.In this paper,the conventional sodium alginate was used as an embedding agent.The best embedding scheme determined by orthogonal test was as follows:3%sodium alginate(m/v),2%calcium chloride solution(m/v),embedding ratio of 6:1,and cross linking time for 4h.The degradation characteristics of immobilized enzyme were studied.The results showed that the optimal enzymatic temperature and pH was30? and 7.0 respectively.Heavy metal ions Hg2+,Co2+,Zn2+,Ni2+ and Cu2+inhibited the degradation at varying degrees.Compared with free enzyme,immobilized enzyme had better thermal stability and better adaptability to pH and lower sensitivity to heavy metal ions.The degradation effect of immobilized enzyme in water was determined by simulating the water environment polluted by chloramphenicol.The results showed that the immobilized enzyme could effectively degrade chloramphenicol residues in water environment.