Expression Of Biphenyl Dioxygense And The Binding Properties With Substrates

In order to study the effects of the binding ability of Enterobacter sp. LY402biphenyl dioxygenase with the substrates on its degradation, biphenyl dioxygenase was expressed by genetic engineering, and then terminal dioxygenase was purified. Then the binding energies between terminal dioxygenase and six aromatic pollutants were investigated, and compared with degradation abilities of LY402to the substrates. Finally, relation of molecular characteristics and degradation rates of the substrates degraded by L Y402was studied. The results are listed below:1. The terminal dioxygenase was successfully expressed through two different protocols. When bphAlA2was expressed successively by one expression cassette, the expression level of BphAl was higher than that of BphA2. When bphA1A2was expressed by two expression cassettes, their expression levels were similar. However, most of the BphA and BphA2were precipitated as they were expressed using two expression cassettes. Thus, expression of BphA1A2using one expression cassette was chosen for the following research.2. BphA3was successfully expressed, and was soluble in part. The expression of BphA4was much lower because BphA4contains many rare codons.3. The terminal dioxygenase was firstly purified by anion exchange chromatography (Q-column). The BphAl/A2complex was eluted at0.25M NaCl, and the recovery ratio could be up to about70%with80%of purity. Thereafter, phenyl-based hydrophobic chromatography was applied, and the BphA1/A2complex was eluted when the concentration of (NH4)2SO4was about0. The recovery ratio could be up to about70%, and the purity could be more than90%finally.4. The binding enthalpies of terminal dioxygenase with naphthalene, biphenyl,3-PCB,4-PCB, pentachlorophenol, and2-PCB decreased in turn, while the degradation rates of pentachlorophenol, biphenyl,3-PCB,4-PCB,2-PCB, and naphthalene decreased successively. Therefore, there may be other factors that could influence the function of biphenyl dioxygenase.5. The conformations of the six substrates were different when they are binding to the active centre of biphenyl dioxygenase. The two benzene rings of biphenyl were faced to active centre as biphenyl binded to the enzyme. The benzene ring without chloro-substituent was faced to active centre when monochorinated biphenyls close to the enzyme. Pentachlorophenol or naphthalene molecule has only one planar, and they were faced to the active centre. These conformation differences may affect their binding ability, and could also partly explain the results of degradation rates.6. Different molecular characteristic of the substrates after forming complexes of the substrates with terminal dioxygenase had different effect on biodegradation. Ipole moment of the substrates was positively correlated to degradation rates. Stretch-bend energiy was negatively correlated to degradation rates. Others did not have a linear relationship. However, several molecular characteristic were significantly different between different substrates. For example, for naphthalene, LUMO was the highest, gap was the largest, and gauss was lowest. However, hydrogen force was only existed between pentachlorophenol and enzyme, and the distance from molecular centroid of pentachlorophenol to the iron core of enzyme was the closest. The above special molecular characteristic might explain the reason naphthalene was difficultly degraded and pentachlorophenol was easily degraded.