The Characteristic Research Of Pseudomonas Putida Dll-E4P-Nitrophenol Degeneration Key Enzyme Pnpc And Pnpa

Microorganisms play important roles in bioremediation of polluted environment, their degradation of pollutant is carried out by a series of enzymes. Researches on the key enzymes involved in pollutant degradation may help us understand the degradation mechamisms of pollutants, and make contributions in bioremediation.p-Nitrophenol (PNP) is an important intermediate in chemical industry and is the metabolite of some pesticides like methyl parathion, it residues in the environment for a long time. It is harmful to animal, plants and human beings. PnpC and PnpA are two key enzymes involved in the degradation of p-nitrophenol by a methyl parathion degradation strain Pseudomonas putida DLL-E4.Hydroxyquinol1,2-dioxygenase (PnpC) catalyzes ring cleavage of hydroxyquinol and be converted to maleyacetate. It is composed of6a helixes and7β sheets and strands, and be devided into2domains:N terminal a helix domain (NTD) and C terminal catalysis domain (CCD).3a helixe in NTD are far from CCD, the two monomers were linked together by them. Away from catalytic site, reports on affecting on activity of enzyme are less.p-nitrophenol4-monooxygenase (PnpA) was a flavin adenine dinucleotide-dependent single-component PNP4-monooxygenase which converts PNP to para-benzoquinone in the presence of NADH and FAD.We constructed expression vectors for cutting23,10and5aas from the NTD of PnpC with pET expression system respectively, and expressed them in E. coli BL21(DE3) pLysS. The expression product of cutting23aas resulted in loss of activity, which means the helix is necessary for the activity of PnpC. But the expression product of cutting5aas resulted in activity, so the5aas is not necessary for the activity of PnpC.PnpA was purified by Ni-NTA and dialysis. As of substrate inhibition, PNP, FAD,NADH affected on acivity of PnpA. We studied them, and got the optimal reaction system:0.3mM PNP+0.8mM NADH+0.1mM FAD+20mM Tris-HCl. Before adding PNP, the system adding PnpA put into4℃, and made FAD and enzyme combining40min. Then adding into PNP, they reacted20min in20℃. Then decteted the concetation of NO2" by nitrite-Griess method. PnpC1C2b/PnpAb and PnpC1C2/PnpA in Pseudomonas putida DLL-E4share a high identity, we constructed expression vectors for PnpC1C2b/PnpAb with pET expression system respectively, and expressed them in E. coli BL21(DE3) pLysS. PnpC1C2b resulted in the similar activity with PnpC1C2, but PnpAb resulted in non-activity.