Screening Of Highly Efficient Biphenyl Degrading Bacteria And Cloning,Expression Of The Related Degradation Genes

In this paper,a highly effective degrading strain CL-2 was isolated from the activated sludge collected from a pesticide factory in Jiangsu.Through the morphological physiological and biochemical characteristics,and combined with the sequence analysis of 16S rRNA sequence,the strain CL-2 was identified to be the genus Sphingobium.The growth characteristics of strain CL-2 in LB liquid medium were studied,0-10h was the lag phase,10-23h was the logarithmic growth phase and after 23 h,the growth of the strain tends to be stable.When the concentration of NaCl was lower than 15 g·L-1,the strains grew well.However the concentration of NaCl was higher than 15 g·L-1,the growth of the strain was inhibited.The optimum growth temperature was 30 ?,and the optimum pH value was 7.0-8.0.The more the amount of liquid,the smaller the dissolved oxygen,leding to the lower growth of strain.The strain grew best with the glucose and fructose as the carbon source.When the nitrogen source was peptone,the strain grew best.The degradation characteristics of strain CL-2 in inorganic salt liquid medium indicate that under the condition of 30 degrees Celsius and pH value of 7,with the inoculation of the strain of 5%,the strain could be completely degraded the concentration of 100 mg·L-1 biphenyl by 28h;When the substrate concentration was increased,the degradation time of the strain was prolonged,and the degradation rate of substrate was slow.When the substrate concentration increased to 500 mg·L-1,the degradation of the strain was inhibited;The Lao Mai equations was to investigate the substrate degradation kinetics,the fitting results showed that the maximum degradation rate Vmax was 19.57 mg·L-1·H-1,the half rate constant Ks was 32.89 mg·L-1.HPLC-MS was used to analyze the intermediates of biphenyl degradation,and three metabolites were identified:2,3-dihydroxybiphenyl,2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid and benzoic.Therefore,it is inferred that the pathway of metabolic biphenyl is:one molecular oxygen to introduce into the biphenyl molecule at the 2,3 position to obtain a 2,3-dihydro-2,3-diol,which is then dehydrogenated to 2,3-dihydroxybiphenyl,then the 2,3-dihydroxylated ring cleaves between carbon atoms 1 and 2 to produce 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid(HOPDA,the ring meta-cleavage product),which is then hydrolyzed to benzoic acid and 2-hydroxypenta-2,4-dienoate.The whole genome sequence of strain CL-2 was sequenced,and the degradation gene cluster of metabolic biphenyl was analyzed by comparative genomics.Comparative analysis shows that the gene structure of biphenyl is bphC(orfl)bphD(orf2)(orf3)bphAlbphA2bphA3bphA4bphBbphH.There are some differences in the arrangement and gene composition to the four typical biphenyl metabolism genes.Molecular biology technology was used to cloning and expression of 2,3-dihydroxybiphenyl-1,2-dioxygenase.Provide a theoretical basis for the remediation of biphenyl and its engineering application.