Isolation,Identification And Degradation Genes Of ?-lactam Antibiotic Degrading Bacteria

Antibiotics are compounds that are synthesized by microorganisms or chemically synthesized to kill pathogenic microorganisms and are widely used in clinical treatment and disease prevention.Excess antibiotics in the environment will not only destroy the ecological balance,but also directly affect the human and animal health.Beta-lactam antibiotics are a class of antibiotics that contain a 4-membered ring of the ?-lactam core in their chemical structure and are the most commonly used class of antibiotics in veterinary clinics as well as in humans.Physical and chemical methods for degrading antibiotics not only have problems of incomplete degradation and high cost,but also produce more difficult to degrade intermediate products in some processes.Therefore,biological methods for degradation of antibiotics have attracted attention.A strain of beta lactam antibiotics was isolated from soil contaminated by antibiotics.The strain was identified from morphological detection,physiological experiment and the development of 16S rRNA phylogenetic tree.The strain was identified as Stenotrophomonas maltophilia and named HS-KS1.The growh characteristics of HS-KS1 and its degradation characteristics were investigated in this paper.It was found that the optimum growth temperature of strain HS-KS1 was 34 C,and the optimum growth pH was 6.5.The optimum degradation temperature was 31,and the optimum degradation rate of pH was 7.The degradation efficiency of substrate was detected by high performance liquid chromatograph.The results showed that HS-KS1 could degrade a variety of beta lactam antibiotics.The degradation efficiency of different antibiotics was different.The degradation rate of cefaclor was the highest in various substrates,the concentration of substrate was 200mg/L,the pH value was 7 and the degradation time was 28.At 24 hours,the biodegradation rate of cefaclor was 67.22%.Each 1000 mL medium was added 10 mg of different carbon sources and nitrogen sources,of which glucose and peptone significantly increased the substrate utilization efficiency.Beta-lactamases are enzymes for the hydrolysis of beta-lactam antibiotics.Herein,primer design of the antibiotic-degrading gene ?-lactamase gene of strain HS-KS1 was performed using?-lactamase gene information of Stenotrophomonas maltophilia sequenced in the NCBI database.Through gene cloning,all the nucleotide fragments of the gene were obtained,and the full-length gene was 1434 bp.Comparison of the NCBI database showed that the ?-lactamase gene in the strain HS-KS1 was more than 90%similar to the ampC type ?-lactamase gene of Stenotrophomonas maltophilia k279a and R551.The expression strain BL21/pET-ampC was constructed by connecting ampC to the pET-28a vector and transferring it into BL21 competent cells.Recombinant strains were induced to express proteins by IPTG inducer,and the protein MW was between 44.3 kD and 66.4 kD which was consistent with the software predicted 51 kD.The crude enzyme solution of the recombinant strain BL21/pET-ampC was tested by hydroxylamine method,showing that the recombinant strain can completely degrade penicillin sodium within 3 hours.The purpose of this article is to discover a strain of ?-lactam antibiotic-degrading bacteria,and to study its degradation characteristics,to provide theoretical and experimental data for potential antibiotic pollution control.