| Enzymatic catalysis, with the advantages of high effectiveness and specificity, has attracted more and more attentions from the synthetic communities. With the development of the technique, an effective strategy of biocatalyst modification was developed by using of random mutation, directed evolution and high-through screening techniques, and thus led the enzymatic catalysis to be with higher efficiency and specificity. Recently, there is an obvious trend for the directed evolution of the enzyme, which transforms from completely random mutation to site-directed mutation with clear target. Thus, deeper understandings toward the microstructures of different enzymes and other proteins were obtained via random substitution of amino acids in enzymes, even for the structures and catalytic mechanisms of enzymes. These techniques and findings attract great interest from the both biological and chemical communities.The study identified the hotspot "Ile12" of lipase A by the probe technique, and the Ile 12 of lipase A was substituted by the other natural amino acids successively via saturation mutation. Then the activity and stereoselectivity of the mutants and WT of lipase A were investigated. Combine docking and energy evaluation approaches were applied to predict the stereoselectivity of lipase A. The reverse QASR method was proposed to parameterize the chemical properties of the active site of enzyme and then construct a model between the enantioselectity and the parameters.The hotspot "Ile 12" of lipase A was identified by the molecular probe technique, Combined with the docking and ONIOM methods were applied to explore the binding models between substrate (pNPP) and the five mutants and WT of B.sub lipase A. The results showed that the docking method can generally describe the orientation of the pNPP in the active site of mutants and WT of B.sub lipase A. In order to verify the reliability of the predicted binding model of enzyme-substrate complex, site-directed mutation was applied to mutate the Ile12, and quantify the corresponding Km values which standed for the affinity of enzyme-substrate complex. It showed that there was a good linear relation in binding energies between theoretic prediction and experiments. And thus these results verified the models of the enzyme-substrate complexes which were obtained from the theoretical prediction.The relationships between the structure of the amino acids and the hydrolysis activities of the mutants of lipase A in the position 12 via saturation mutation were systemically studied by the molecular dynamic simulation, and the regularity understandings were obtained. The amino acid in the position 12 of the lipase A was substituted by three kinds of natural amino acids respectively, and the corresponding hydrolysis activities of Wild-type and mutants of B.sub lipase A toward pNPP were verified. The results showed that, when the Ile12 was substituted by the nonpolar amino acids, the hydrolysis activity of I12M mutant was 130% of the Wild-type lipase A, while the activities of the I12L and I12P mutants significantly decreased. When the Ile 12 was substituted by the polar amino acids, the experimental results suggested that the polarity of amino acids in the position 12 of lipase A didn't have obvious effects on the activity of lipase A, but the activity of the I12T mutant was 3% of the WT. when the Ile 12 was substituted by the charged amino acids, the negative charged amino acids in the position 12 of lipase A resulted in the decrease of the activity, while the positive charged amino acids had no effect. Moreover, deep analyses were performed to elucidate the reasons of activity loss of some mutants by using of molecular dynamic simulation. For the I12T mutant, there was a hydrogen bond between the Thrl2 and catalytic residue Ser77, the energy of the hydrogen bond was almost 5.5 kcal/mol, and might lower the catalytic ability of I12T mutant. And also, while the Ile 12 was substituted by the residues which had negative charges, such as Asp and Glu, there also existed a hydrogen bond between Asp12/Glu12 and Ser77, which resulted in loss of activity of corresponding mutants of B.sub lipase A. For the I12L mutant, the substitution of Ile 12 to Leu resulted in that the loop region turn close to the active site of enzyme; this might hamper the substrate close to the active site of enzyme, led to the loss of activity of I12L mutant. For the I12P mutant, the loop region where Pro 12 located turned outside against the active site of enzyme, which led the active site over-expose to the solvent, and thus may influence the activity of enzyme. Combined with docking and QM/MM method was applied to predict the stereoselectivity of B.sub lipase A toward the substrate ketoprofen vinyl ester, moreover, elucidate the stereoselevtivity of B.sub lipase A by the binding energy between enzyme and enantiomer of substrate. The results showed that, the theoretical simulation successfully predicted and evaluated the stereoselectivity of B.sub lipase A. The results showed that it is considerably reliable to apply the theoretical simulation to predict the stereoselectivity of enzyme.Reverse QSAR technology was used to parameter the chemical properties of the active site of WT and mutants of B.sub lipase A which were optimized by the molecular dynamic with 2ns, the active site of lipase A was divided into 11×11×11 grids and the CoSFA was applied to parameterize the distribution of the three non-bond potential energies, and construct the model between the stereoselectivity and the parameters. The model exhibited a strong fitting power, stability, and generalization ability (r2=0.956 and q2=0.929), and could find the direct factors which related to the stereoselectivity of lipase A...
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