The Functional Characteristis Involve Degradation Pathway Genes In Ibuprofen And PVA Degradable Strains

Compared with any other PPCPs drugs,Ibuprofen attributed to antipyretic,analgesia and anti-inflammatory drug was widely used because of its low toxicity and side effects.However,the large number of production and widely application of ibuprofen brings much risks to ecological environment,especially the water environment.Some researchers proved that the behavior and ecological toxicity of ibuprofen produce much disadvantages to aquatic organisms.In order to solve these environmental problems,the study of ibuprofen-degrading has important significance.Based on the previous research in our laboratory,three key function genes of ibuprofen metabolism pathway in Raoultella ornithinolytica Yangling I-2(G~-)were knocked out,and then validated their functions,which will provide information for the construction of engineering strain and the practical application.The main results of this thesis were as followed.There are many ibuprofen metabolic pathways in I2,4-hydroxyphenylacetate 3-monooxygenase,hemolysin secretion protein D and NADPH-dependent FMN reductase family protein/NAD(P)H dehydrogenase are all participated in the ibuprofen metabolism.Polyvinyl alcohol(PVA)is a kind of water-soluble polymer material with good performance,which has been widely used in innumerable areas including industry and real life.The wide production and application of PVA leads to produce a large number of Industrial wastes,associated environmental pollution has reached serious proportions.Now,PVA industrial degradation methods mainly include physical-chemical degradation and biodegradation.Due to the high efficiency,low cost and no secondary pollution,biodegradation is becoming the most applied foreground pathway of the degradation of PVA.Associated with the complete genome information of a novel effective PVA-degrader,the eight PVA/OVA-degrading related genes were analyzed their enzyme kinetic parameter by expressed in E.coli BL21(DE3).The function and PVA-degrading ability of these genes were tested.It is also found that there is a novel effective PVA-degrading enzyme,which will give an important data support for the study of the PVA-degrading bacteria agent in the future.The main results of this thesis were as followed.(1)Based on sequence alignment with genes reportedly involved in PVA degradation,we identified eight genes participated in PVA metabolic pathway.Among them,BAY15＿1712,BAY15＿2325 and BAY15＿0291 work together to implement the oxidation of PVA in S.rhizophila QL-P4,BAY15＿1712 and BAY15＿2325 have the similar function to PVADH,BAY15＿0291 is a primary electron acceptor for the above enzymes in PVA oxidation;BAY15＿0976,BAY15＿3123 and BAY15＿3143 function in the oxidation of OVA in this strain;BAY15＿0160 participates in cleavage of the C-C bond of the β-diketone of oxi PVA and BAY15＿3292 is identified as a novel PVA-degrading enzyme.(2)The five of the eight PVA/OVA-degrading related genes were expressed and purified in BL21(DE3).According to the enzyme kinetic analysis of these genes,we found that BAY15＿1712 displayed high catalytic efficiency towards PVA and OVA,BAY15＿0976、BAY15＿3123、BAY15＿3143 displayed high catalytic efficiency towards PVA.(3)Different from classical PVADH and BAY15＿1712,BAY15＿3292 has much higher PVA degradation efficiency and PQQ-independent.In addition,it can be induced in supernatant and also has exocrine function,which has important industrial application value.