Lipase-catalyzed Synthesis Of (S)-indoprofen And The Interaction Mechanism Of Micromolecule To Lipase

2-Arylpropionic acid (2-APA) is an important group of non-steroidal anti-inflammatory drugs and they are widely used as efficient anti-inflammatory and analgesic drugs. They possess an asymmetric carbon atom and it is well known that the anti-inflammatory activity of these compounds is mainly due to the active (S)-enantiomer. Indoprofen is one of the most representative drugs of the group. Therefore, the research to chiral resolution of indoprofen is of important practical significance. Microbial lipases are the most important source of lipases used in many industrial fields for higher enzyme activity and stability. Lipases catalyze a variety of reactions such as hydrolysis, esterification, and transesterification. They have an extensive application prospect and high commercial value in the industrial fields of oil processing, food and pharmaceuticals. Lipase from Candida rugosa (CRL), one of the most widely used lipase, plays an indispensable role in the industrial applications, especially in the enzymatic resolution of chiral compounds. In this dissertation, CRL-catalyzed enantioselective hydrolysis of indoprofen vinyl ester was the main line, the best2-APA substrate was virtual screened by molecular docking, reaction conditions were optimized by response surface method, the interaction mechanism between acetaldehyde or metal ions and CRL were studied and the regulation of lipase enantioselectivity through the addition of these micromolecule were investigated. The kinetics model of CRL-catalyzed hydrolysis of racemic2-APA ester was established, which could be a theoretical and technological support for lipase application in industry.Based on recognition and interaction between macromolecular receptor and ligand, molecular docking was employed to predict and screen the optimal binding modes of2-APA substrate and CRL, analyzed the three-dimension conformations and interactions of CRL-2-APA ester complexes visually by the AutoDock4.2.5software. And the best binding mode filtered based on the clustering results and binding energy scores, could provide a theoretical basis for the catalytic mechanism of lipase family. The rational energy ordering results were in good agreement with the experimental results of the CRL-catalyzed hydrolysis resolution. The two sets of results were complementary and synergy study the interaction between the2-APA ester substrate and CRL and indoprofen ethyl ester was choosed for the future resolution. The virtual screening of the best substrate of lipase by molecular docking method is rarely reported until now.The primary task of this research is to investigate the basic pattern of CRL-catalyzed enantioselective hydrolysis of indoprofen ester as the model compounds. The indoprofen vinyl ester (IVE) and the mixed solvent of isoctane and toluene (90/10) were screened as the most effective substrate and the best reaction solvent for the model reaction. And then the optimum reaction conditions were achieved using the single factor test and further optimization of Box-Behnken design of response surface method. After the experimental verification, the optimal reaction conditions were as follows: water content0.16%, Tween-801.2%, pH7.5,5g/L indoprofen vinyl ester, CRL175mg/L, the reaction mixtures were placed in a water bath shaker under39Co for39h. The conversion and e.e.p value were47.51%and98.57%, and the E value was reached to421.27, which was improved by11.91%comparing the result before optimization. Compared to the predicted values, the relative error were1.17%(less than5%), indicated that the predicting effects were effective and they can used in the prediction of the (S)-indoprofen synthesis by CRL.Acetaldehyde is the by-product generated in CRL-catalyzed hydrolysis of racemic indoprofen vinyl ester. The effect of acetaldehyde on CRL catalytic properties and the interaction mechanism were studied. Acetaldehyde showed hormesis phenomenon on CRL activity and the maximal activation concentration was0.2215mmol/L. Combined study of molecular docking, ATR-FTIR, fluorescence spectroscopies and kinetic analysis indicated that there were hydrogen bonds formed in the CRL-acetaldehyde complexes with different binding affinities. CRL exposed in0.2215mmol/L acetaldehyde medium had increase in α-helix and β-sheet structures and decrease in intermolecular β-Sheet aggregate and random coil structures, but there was little effect of acetaldehyde at0.2215mmol/L acetaldehyde on β-turn structure. This leaded to a distortion of the secondary structure of CRL and the hydrophobicity in microenvironment around CRL catalytic center decreasing, appeared as the enhancement of CRL activity. Molecular docking result indicated that acetaldehyde entered into hydrophobic pocket of CRL and there were some hydrogen bonds formed between acetaldehyde and Ser209, Ser389, Trp188, Gly124or Gly342located in the CRL active pocket, and there was a hydrogen bond between Gln338in the active pocket and Glu341in the catalytic triad. Acetaldehyde affected the secondary structure of CRL molecule probably for these hydrogen bonds, which could induce the transform of secondary structure transform of CRL. Trace concentrations of acetaldehyde exhibited almost no effect on CRL enantioselectivity in the open system.The effect of Different kinds of metal ions showed various influences on CRL catalytic properties. The alkaline-earth metals (Mg2+、Ca2+、Sr2+and Ba2+) showed a similar influence trend on CRL and showed a low-dose stimulation and high-dose inhibition, i. e. hormesis. For transition metal, like Cu2+and Zn2+, mild promotion took place at low metal concentrations and serious inhibition at high concentrations, while Mn2+played as inhibitor in all tested concentrations.0.01mol/L Mg2+could significantly improve CRL enantioselectivity. Spectral analysis showed that0.01mol/L Mg2+was conducive to the mild increasing of α-helix/β-sheet contents and CRL transformed to a more flexible conformation whilst maintaining its basic secondary structure. Mg2+increased the contact area of substrate onto CRL and the probability of enter into CRL active site, resulting in the enhancement of CRL activity. The caculated data from Tsou‘s model were well fitted to the experiment data.0.01mol/L Mg2+was an efficacious activator and could be classified as the noncompetitive type, while the effect of1mol/L Mg2+on CRL was found to be noncompetitive irreversible inhibition.The mechanism of the CRL-catalyzed indoprofen vinyl ester hydrolysis in the nonaqueous organic system was proposed as irreversible and mixed reaction mechanism Ping-Pong Bi-Bi reaction with substrate inhibition and product inhibition. A universal rate equation for the reversible Ping-Pong Bi-Bi reaction with substrate inhibition and product inhibition was obtained by modification of the kinetic model under the ideal conditions which was derived from the diagrams of King and Altman. When the kinetic model was used in the reaction system of CRL-catalyzed irreversible hydrolysis of indoprofen vinyl ester in organic solvent system, the simplified rate equation based on the actual conditions was obtained. The kinetic parameters of the obtained kinetic model of the actual CRL-catalyzed hydrolysis investigated in the research were simulated from the experimental data through fitting and debugging, the simulated values were well fitted to the experiment values, which indicated that the kinetic model was suitable to explain the process of CRL-catalyzed hydrolysis of indoprofen vinyl ester in organic solvent.