| 摘要 | 第4-7页 |
| Abstract | 第7-9页 |
| Chapter 1 Review on Enzyme Engineering and Divergence Evolution | 第16-47页 |
| 1.1 Glimpse on enzyme engineering approaches | 第16-25页 |
| 1.1.1 Directed evolution of enzymes | 第16-20页 |
| 1.1.1.1 Random mutagenesis | 第17-18页 |
| 1.1.1.2 Random insertion and deletion | 第18-19页 |
| 1.1.1.3 Homologous genetic recombination by DNA shuffling | 第19-20页 |
| 1.1.2 Rational design of the enzymes | 第20-24页 |
| 1.1.2.1 Site-directed mutagenesis | 第21-22页 |
| 1.1.2.2 Iterative site saturation mutagenesis (ISM) | 第22-23页 |
| 1.1.2.3 Enzyme molecular structure grafting or loop switching | 第23-24页 |
| 1.1.3 Semi-rational design of enzymes | 第24-25页 |
| 1.1.3.1 Computer-guided semi-rational design | 第24-25页 |
| 1.2 Divergence of enzyme evolution | 第25-37页 |
| 1.2.1 The role of functional promiscuity in developing new enzymes | 第25-26页 |
| 1.2.2 The important role of loop conformation in the acquisition of new functions | 第26-28页 |
| 1.2.3 The amidohydrolase superfamily is an example of divergent evolution | 第28-29页 |
| 1.2.4 Environmental factor for the divergence of enzyme evolution | 第29-34页 |
| 1.2.4.1 Synthetic organphosphate compounds (OPs) | 第29-30页 |
| 1.2.4.2 Basic structure of organophosphorus compounds | 第30-31页 |
| 1.2.4.3 Neurotoxicity of OP Compounds | 第31-34页 |
| 1.2.5 Organophosphate compounds degradation by bacterial enzymes | 第34页 |
| 1.2.6 X-ray structure of phosphotriesterase (PTE) from Pseudomonas diminuta | 第34-36页 |
| 1.2.7 Catalytic mechanism of phosphotriesterase | 第36-37页 |
| 1.3 Other organic phosphorus hydrolases | 第37-40页 |
| 1.3.1 Di-isopropylfluorophosphatase (DFPases) | 第37页 |
| 1.3.2 Serum paraoxonase (PON) | 第37-38页 |
| 1.3.3 Organophosphorus acid anhydride enzyme | 第38-39页 |
| 1.3.4 Methyl parathion hydrolase | 第39-40页 |
| 1.4 Phosphotriesterase like lactonases (PLLs); a link to evolutionary origin of PTE’s. | 第40-44页 |
| 1.4.1 Phylogenetic analysis of the PTE and its related PLL family | 第41-42页 |
| 1.4.2 Phosphotriesterase like lactonase from Geobacillus kaustophilus HTA426 | 第42-43页 |
| 1.4.3 Molecular evolution of the PLL to the PTE | 第43-44页 |
| 1.5 Experiment Design | 第44-46页 |
| Note | 第46-47页 |
| Chapter 2 Recombinant Expression and Characterization of the Thermostable Lactonasefrom Geobacillus kaustophilus HTA426 | 第47-60页 |
| 2.1 Introduction | 第47页 |
| 2.2 Materials | 第47-49页 |
| 2.2.1 Strains and plasmids | 第47页 |
| 2.2.2 Main reagents | 第47-48页 |
| 2.2.3 Main instrument | 第48页 |
| 2.2.4 Medium | 第48页 |
| 2.2.5 The use of the database and software | 第48-49页 |
| 2.3 Methods | 第49-54页 |
| 2.3.1 Resuscitation and culture of Geobacillus kaustophilus HTA426 | 第49-52页 |
| 2.3.1.1 Primers design | 第49-50页 |
| 2.3.1.2 PCR reaction system | 第50页 |
| 2.3.1.3 The PCR reaction condition | 第50页 |
| 2.3.1.4 Preparation of E. coli BL21 (DE3) competent cells competent cells | 第50-51页 |
| 2.3.1.5 Transformation of plasmids into competent cells | 第51页 |
| 2.3.1.6 Sequencing verification | 第51-52页 |
| 2.3.2 Expression and purification of recombinant Gka P-PLL protein | 第52页 |
| 2.3.3 Purification of Gka P-PLL HTA426 | 第52-53页 |
| 2.3.4 SDS-PAGE analysis of the purified Gka P-PLL | 第53页 |
| 2.3.5 Catalytic Kinetics of Gka P-PLL to various OP compounds and lactone substrates | 第53-54页 |
| 2.4 Results and discussion | 第54-59页 |
| 2.4.1 Homology sequence alignment in amidohydrolase superfamily | 第54-56页 |
| 2.4.2 Identification of positive clones | 第56页 |
| 2.4.3 Expression and purification of Gka P-PLL | 第56-57页 |
| 2.4.4 Kinetic properties of Gka P-PLL | 第57-59页 |
| 2.5 Summary | 第59-60页 |
| Chapter 3 Active site Loop Remodeling by Stepwise Loop Insertion Strategy (St Lois) onthe Gka P-PLL | 第60-92页 |
| 3.1 Introduction | 第60-61页 |
| 3.2 Methods and Materials | 第61-67页 |
| 3.2.1 Strains, Plasmids, and Chemicals | 第61页 |
| 3.2.2 Cloning and library construction at a defined region of the Gka P-PLL loop7 | 第61-62页 |
| 3.2.3 PCR reaction and digestion | 第62-63页 |
| 3.2.4 Mutant library screening | 第63页 |
| 3.2.5 Protein expression and purification | 第63-64页 |
| 3.2.6 Enzyme kinetic and specific activity | 第64页 |
| 3.2.7 Determination of optimum p H | 第64-65页 |
| 3.2.8 Optimum temperature and thermal stability test | 第65页 |
| 3.2.9 Crystallizations of Gka P wild-type and L7-A2B2 mutant | 第65-66页 |
| 3.2.10 Substrate docking | 第66页 |
| 3.2.11 Molecular dynamics simulations | 第66-67页 |
| 3.3 Results | 第67-89页 |
| 3.3.1 Sequence alignment analysis between Gka P-PLL and pd PTE | 第67-68页 |
| 3.3.2 Identifying the enzyme active site loops differences between pd PTE and Gka P-PLL | 第68-69页 |
| 3.3.3 Comparison and dissection of the active-site loop7 of both Gka P-PLL and pd PTE | 第69-70页 |
| 3.3.4 Designing stepwise loop insertion strategy (St Lois) for active site loop remodeling | 第70-72页 |
| 3.3.5 Generating loop 7 insertion mutation library in Gka P-PLL and screening it for higherPTE activity | 第72-73页 |
| 3.3.6 Generating Gka P-PLL loop7 insertion mutations library in advanced template in ML8and ML7 | 第73-74页 |
| 3.3.7 Kinetic analysis of the mutants derived from Gka P-PLL | 第74-75页 |
| 3.3.8 Kinetic analysis of the evolved variant from advanced template ML7 | 第75-78页 |
| 3.3.9 Broad substrate spectra of Gka P variants for organophosphates hydrolysis | 第78-80页 |
| 3.3.10 Thermostability test of the evolved variant | 第80-81页 |
| 3.3.11 Optimum temperature and p H analysis of the evolved variant | 第81-82页 |
| 3.3.12 X-ray structures analysis of the wild-type Gka P-PLL and the mutant L7-A2B2 | 第82-85页 |
| 3.3.13 Molecular docking for the Gka P-PLL and mutant L7-A2B2 with ethyl paraoxon | 第85-87页 |
| 3.3.14 Molecular docking for the Gka P and mutant L7-A2B2 with δ-decanolactone | 第87页 |
| 3.3.15 Molecular simulation analysis of the Gka P and the mutant L7-A2B2 | 第87-89页 |
| 3.4 Discussions | 第89-92页 |
| Chapter 4 Novelty, Conclusion and Prospects | 第92-95页 |
| References | 第95-112页 |
| Acknowledgement | 第112-113页 |
| Publications | 第113-115页 |
