Study Of Enzymatic Synthesis Of L-ascorbyl Pro-drug Of Aryl Propionic Acid Of Nsaids

In this paper, we have used the ketoprofen and L-ascorbic acid as raw materials, lipase Novozym 435 as the main catalyst to study the enzymatic synthesis. The result showed that: the ketoprofen could react with L-ascorbic acid and get L-ascorbyl ketoprofen ester (L-AK) product. Here in our synthesis process, we used a method to determine the L-AK quantitatively. High Performance Liquid Chromatography with UV Detector and Iodine titration was tested and optimized. This method is simple, rapid, and accurate and can be used for the quantitative determination of L-AK.The technology of synthesis of the ibuprofen and its derivatives and L-ascorbic acid is studied, to optimize the reaction conditions. First, we screen three kinds of lipases. Second, we established a method to determine the primary reaction velocity. And finally we separately screen the effect of rotary speed, temperature, different lipase concentration, different substrate concentration and different organic solvents on primary reaction velocity. The result showed that:Novozym 435 and TLIM can be carried out to synthesis the target products. By comparing, in unit time the yield of products which is catalyzed by Novozym 435 is higher by TLIM. Finally, we determine the optimum conditions of reaction as the standard on primary reaction velocity.For the particular enzymatic reaction system, it is important to reaserch the kinetics of the synthesis L-ascorbyl aryl propionic acid ester catalyzed by immobilized lipase. In this paper, we studied the substrate concentration to obtain the parameters of primary reaction velocity, further to determine the kinetic parameters of enzymatic reaction. The result showed that:in three substrates, the affinity of ibuprofen and enzyme is maximum, ketoprofen takes second palce, and fluprofen is minimum. And there is the phenomenon of the substrate inhibitionin in the enzymatic reaction which is catalyzed by immobilized lipase from ibuprofen and ketoprofen. Through establishing the single substrate inhibition model, we obtained the inhibition constants and kinetic constants of the substrate inhibitionin.In the view of the thermodynamics, because of the complex compound formed by enzyme and reactant, the activation energy is reduced to promote the reaction process. Enzyme can contribute to the rate of biochemical reactions, not alter the direction of the reaction and thermodynamic equilibrium. In this paper, we studied reaction time of the enzymatic reaction, the product concentration in equilibrium, the thermodynamic equilibrium constant of enzymatic reaction and the stability of Novozym 435. The result showed that:The reaction conditions of ibuprofen have been optimized:200 r/min,65℃,5% for adding enzyme quantity,1000 mmol/L of substrate concentration, reaction time 66 h,19.07% of concentration of products; and ketoprofen:200 r/min,60℃,7.5% for adding enzyme quantity, 600 mmol/L of substrate concentration,132 h,10.63%; and fluprofen:200 r/min,65℃,5% for adding enzyme quantity,400 mmol/L of substrate concentration,144 h,6.76%. The results have been modeled through Lineweaver-Burk equation. The parameters of kinetics and thermodynamics are solved. Ibuprofen: Michaelis constant Km=0.101μmol/L, vmax=32.68μmol/(min·g), equilibrium constant K=0.166; ketoprofen:Km=0.144μmol/L, vmax=12.97 μmol/(min·g), K=0.094; fluprofen:Km=0.185μmol/L, vmax=9.35μmol/(min·g), K=0.055.