| With the continuous development and progress of industrial production,chlorophenol and biphenyl compounds are widely used in industrial production,but they are a kind of pollutants with wide range of pollution,large toxicity and difficult to degrade.Scholars at home and abroad found chlorinated aromatic compounds biodegradation is a non-toxic to the environment with less investment,and no secondary pollution,the method of this method includes two processes: the initial fracture reaction of hydroxyl and reaction of aromatic ring hydroxylation process.One single oxygenase(MO)hydroxyl of catalytic degradation reaction is one of the most critical step in the process of aromatic compounds,however,there are some shortcomings in the application of monooxygenase in the degradation of aromatic compounds,which are not suitable for industrial treatment of these pollutants.And we study TfdB-JLU novel hydroxylase compared with the traditional single oxygenase compared with wider aromatic substrates spectrum,hence TfdB-JLU this more complex substrate promiscuity characteristics of aromatic pollutants bioremediation is very important.At present,TfdB-JLU,as a novel hydroxylase,is far from the TfdB family enzyme,and is a kind of monooxygenase which is extracted and expressed by macrogenomic method.While the enzyme more competitive compared with other existing environmental restoration using enzymes,but in the industrial production of catalytic activity of enzyme and the promiscuity is more demanding,so need to by protein engineering on the wild type TfdB-JLU half a rational design renovation,expects to receive a higher dynamic TfdB-JLU,and study its unique promiscuity,half a rational design to improve the specificity of the enzyme.This thesis mainly carries on the following research.(1)First of all,we made a preliminary exploration on the structure of TfdB-JLU enzyme and the non-specificity of catalytic chlorophenol and biphenyl pollutants.Due to haven't got the crystal structure of the enzyme,we in recent years,some with TfdB-JLU sequence and structure similarity is higher enzyme sequence and structure analysis,and studies the structure and function of the enzyme,we used the SWISS-MODEL by structural homology search,found TfdB-JLU with a kind of structure is parsed and only 2-hydroxy diphenyl-3-monooxygenase the structure of single oxygenase homology of 49.1%.We selected 2-hydroxy diphenyl-3-monooxygenase(PDB: 5BRT)with the binding site of FAD and substrate to form the template homologous modeling,and obtained the three-dimensional structure of TfdB-JLU.In addition,three evaluation methods of Ramachandran plot,Profiles-3D and z-score values of Discovery Studio 3.5 were used to confirm that the 3D model of TfdB-JLU,we obtained was reasonable and credible.(2)Enzyme active center is the key part of enzyme catalysis area,its specific conformation configurations on the substrate combination way and the enzyme catalytic efficiency has a great impact,the enzyme active center area of the structure of the small renovation will have a significant impact on the nature of enzymes.We use the bioinformatics methods such as molecular docking and molecular dynamics of TfdB-JLU and substrate complex structure model are studied,based on the mechanism of enzyme and substrate reaction screening docking results,analysis the selection of the substrate after entering TfdB-JLU activity centers,2,4-DCP substrate and TfdB-JLU interaction of the active center area after the key amino acids,and through the analysis of interaction between the substrate and TfdB-JLU docking method,interaction between free energy and so on,to determine the amino acid sites,Ile48,Trp222,Pro316,Phe424 these hydrophobic amino acids in the substrate the combination of 2,4-DCP is advantageous,through reasonable semi-rationality mutation design strategy and efficient enzyme activity detection method,screening of the mutant strain TfdB-P316 Q of the substrate 2,4-DCP,the mutant strain compared with wild type TfdB-JLU,on the substrate a 191.26% increase over the vitality of2,4-DCP,and the mutant enzyme of Km and significantly lower compared with thewild type,that we get mutation enzyme screening for 2,4-DCP more affinity to the substrate.(3)Conformation of enzyme activity center specific configuration of the substrate binding site,the selection of the substrate binding site has very important influence,and the substrate binding site is very important to influence the promiscuity of enzymes,at the same time,the substrate in combination with the entrance of the pocket size and shape,as well as the substrate in the pockets of hydrophobicity of the substrate into has very important influence.We found that p316-bit amino acids play a key role in the promiscuity of TfdB-JLU,and the promiscuity of the mutant enzyme is stronger than that of the wild type enzyme.It is also proved that the two amino acids,His47 and Asn116,play a key role in the promiscuity of the substrate.At the same time,we found the inlet of the bottom pocket of M251,and confirmed that the size and hydrophobicity of the substrate were significantly affected by the promiscuity of the substrate.The regulation of enzyme promiscuity was achieved by the mutation design of key amino acids(TfdB-M251 A and M251G)related to the size and hydrophobicity of the substrate of the enzyme activity center.This thesis explores the active center area half a rational design modification of enzyme activity,the influence of the promiscuity,and combined with the means of computer simulation analysis of the active center area evolution effect on enzyme activity,the promiscuity of possible reasons.The research strategy of enzyme activity and promiscuity is explored.This study is expected to do a deeper and broader study on the activity of TfdB-JLU and the modification and regulation of activity and promiscuity. |
PDF Full Text Request