Effect Of Carboxyl Cleavage Enzyme Gene On Ring Cleavage Of Phenol By Pseudomonas Stutzeri N2

Phenol is a common pollutant in the environment, and it has been separated from a lot of strains which can use phenol as the sole carbon source and energy source. The current research on phenol hydroxylase, catechol ortho and meta ortho ring cleavage enzyme in bacteria is fully. Phenol carboxylation way is one of the main ways to start the metabolism of phenol. From the present literature reports, the phenol carboxylic acid pathway is only under anaerobic conditions. However, we obtained a strain of Pseudomonas sp. N2, which was able to rapidly degrade phenol in the early stage of laboratory. The results of the study found that there was also a product of the formation of carboxylic acid under aerobic condition. In this paper, we studied the gene and its function of the carboxyl group of the strain, and it is important to reveal the mechanism of phenol degradation by Pseudomonas sp. N2. In order to more fully study the function of the bacteria cells carboxy lyase gene, preliminary laboratory obtained two carboxy lyase gene ubiX and ubiD from the N2 strain and we have been successfully constructed single mutant strains N2ubiX- bacteria and N2ubiD- bacteria and the double gene deletion mutant strains of bacteria N2ubiX-ubiD-. This paper studied N2 bacteria and mutation degrading phenol process of phenol toxicity response characteristics, ring cleavage characteristics and four strains of bacteria generate differences in phenol downstream products. And produced by different carboxy lyase gene in the degradation process of phenol were studied.In this paper the wild strain of Pseudomonas stutzeri N2 as the research object,using molecular biology technology construction a carboxy lyase gene mutant strain,and by liquid chromatography, gas chromatography-mass spectrometry hyphenated instrument and other analytical methods, through the study of differences in the metabolism of phenol between the wild strain and the mutant strains, revealing thefunction of gene ubiX and ubiD in the process of Pseudomonas stutzeri N2 degradation phenol, the following results were obtained:(1) The function of ubi D and ubiX genes in Pseudomonas sp. N2 was not clear at present.We retrieved ubi X and ubiD genes from Pseudomonas stutzeri N2 cells successfully, and constructed the single mutation strains N2ubiX-, strain N2ubiD- and double mutant strain N2ubiX-ubiD-.(2) It was found that the loss of ubiD and ubi X genes decreased the ability of the strain to resist the phenol toxicity and phenol degradation, especially the lack of ubiX gene,which significantly decreased the ability of phenol degradation in the early stage of the strain. The loss of ubiD gene is not conducive to the degradation of phenol by the way of the ring opening, and the loss of ubiX gene is not easy to ortho ring cleavage.Despite the wild strain and the mutant strain with phenol as the sole carbon source for growth can through phenol hydroxylation and carboxylation of metabolism, and the production open ring cleavage intermediates of "2-hydroxy muconic acid semi aldehyde(2-HMS)", but only wild strains and single mutant strains N2ubiX- metabolism of phenol can easily detected carboxylation and reduction reaction of ring cleavage product that ubiD in cells easily in cells produce reduction ability.(3) Carbon sources such as ethanol and acetone can increase the ability of resistance to phenol toxicity and phenol metabolism by the double mutant strain N2ubiX-ubiD-. And the growth rate is accelerated in the late stage. The coexistence of ethanol could accelerate the degradation and transformation of N2 strain and its mutant strain.The coexistence of ethanol can also induce the strain "catechol 1, 2- double plus oxygen enzyme" and "catechol 2- double plus oxygen enzyme". And lacking of ubi X gene mutant strain, due to the coexistence of ethanol can significantly reduce the degradation of liquid, because of the coexistence of ethanol can significantly reduce the degradation liquid accumulation of CIS, CIS muconic acid and 2-HMS.It showed that the two intermediate products were further degraded and transformed by the strain.