To Investigate The Degradation Mechanism Of Ochrobactrum To Doxycycline

The use of microbial elimination of antibiotic residues in the environment is one of the current research hot spots,but the current research focused on the screening of degradable bacteria and its ability of degradation of antibiotics,less attention to the degradation mechanism of antibiotics and its application may cause drug resistance gene contamination.Therefore,the degradation mechanism of doxycycline was studied in this paper.The degradation mechanism of doxycycline was analyzed,and the possibility of degrading enzyme instead of degrading bacteria was discussed.This paper can provide a reference for the removal of veterinary antibiotics in the environment,and provide the theoretical basis for the environmental application of veterinary antibiotic degrading enzymes.The crude enzymes,such as an intracellular enzyme,extracellular enzyme and whole enzyme of doxycycline-degrading bacteria,were added into PBS solution containing 10 mg/L doxycycline and shaken at 37?,140 r/min for 2 days to determine the degradation of doxycycline from different sources.The tetX was transfected into the competent cells DH5? and BL21,and the expression of tetX was observed at 37?,140 r/min for 2 days to determine the degradation of doxycycline.The intracellular crude enzyme and tetX expression enzyme obtained from the above-mentioned test were added to PBS solution containing 10 mg/L doxycycline and reacted at 37 °C and 140 r/min for 10 days.The degradation of doxycycline and the drug resistance genes in the reaction system were determined.The intracellular crude enzyme and tetX expression enzyme were graded with 10% ~ 30%,30% ~ 50%,and 50% ~ 80% saturated ammonium sulfate.The corresponding enzyme was extracted and the enzymatic 35% ~ 40%,40% ~ 45%,45% ~ 50%,50% ~ 55%,55% ~ 35%,55% ~ 50%,55% ~ 50% 60%,60% ~ 70%,70% ~ 80% ammonium sulfate saturation of the intracellular enzyme and tetX expression enzyme,and further strength doxorubicin degradation test to determine the best saturated ammonium sulfate concentration.The main results of this paper are as follows:(1)The degradation efficiency of 10 mg/L doxycycline for whole enzyme intracellular enzyme and the extracellular enzyme was 77.21±0.93%,66.62±1.27%,51.62±3.72%,respectively.The degradation efficiency of the whole enzyme was higher than the other groups(P<0.05).The degradation efficiency of the intracellular enzyme crude enzyme was significantly higher than that of extracellular enzyme crude enzyme(P<0.05).The intracellular enzyme crude enzyme accounted for 86.28±1.6% of the enzymatic degradation performance.(2)The tetX expression of tetracycline,which can effectively degrade doxycycline,was obtained and expressed in the tetX gene,and the degradation rate of 10 mg/L doxycycline was 86.17 ± 0.97% for 5 days.(3)The degradation rates of 10 mg/L doxycycline for tetX expression enzyme,intracellular enzyme,and Ochrobactrum were 86.17 ± 0.97%,80.41 ± 1.47% and 85.49 ± 1.20%,respectively(P <0.05).The degradation rate of intracellular enzyme coenzyme was significantly lower than that of the two groups(P <0.05).The number of the tetA and tetM genes copies in Ochrobactrum group was significantly higher than that of the other two groups(P <0.05).The copy number of tetX gene in tet X expression group was significantly higher than that in the other two groups(P <0.05).(4)Grading enzyme obtained by fractionating precipitation of intracellular crude enzyme of Ochrobactrum in 45% to 50%,50% to 55%,55% to 60%,70% to 80%.The degradation rates of doxycycline were 60.01 ± 3.11%,59.72 ± 1.01%,58.07 ± 4.01% and 59.35 ± 2.84%,respectively,which were significantly higher than those of other groups(P <0.05)The degradation rate of 10 mg/L doxycycline for the tetX expression enzyme seprated by 50%~55% and 55%~60% ammonium sulfate saturation was 55.41 ± 5.75% and 59.31 ± 4.52%,respectively,which was significantly higher than that of other treatments(P <0.05).In summary,the degradation of doxycycline on Ochrobactrum is mainly enzymatic degradation.Degrading enzymes are mainly from intracellular enzymes,in which passivation enzyme-resistant gene tetX expression enzyme may play an important role.However,when tetX expression enzyme is used to degrade the doxycycline in the environment,the tetracycule resistance gene should be reduced.