Applied Basic Research Concerning Lipases: Production Of The Lipases And Enzymatic Resolution Of Chiral Ibuprofen

Bioactivity of chifal compounds such as ibuprofen is determined by their structure. For ibuprofen,there is a chiral β-carbon atom in the molecule.so a pair of enantiomers((R)-enantiomer and (S)-enantiomer) exists.The bioactivity of ibuprofen is mainly due to the (S)-enantiomer, though (R)-enantiomer has side-effects and low bioactivity. Recently, utilization of lipase in the resolution of ibuprofen receives greater interest because of the mild reaction condition, high enantioselectivity, less side reactions and environment pollution of enzyme-catalyzed reactions. In this dissertation, the screen of high-yield stereoselective lipase producing strain, production,purification and characterization of the lipase, immobilization and application of the lipase in the enzymatic resolution of (R, S)-ibuprofen, control of the enantioselectivity, kinetics of the enzymatic resolution and bioreactor for the immobilized lipase was investigated.Among the lipase producing strains deposited in our laboratory, Bacillus coagulans ZJU318 and P. expansum TS414 for the resolution of (R, S)-ibuprofen were screened out .The result showed that the lipases produced by these screened strains had very high enantioselectivity in the resolution reaction.Uniform desigh was used for optimizing the fermentation conditions of Bacillus coagulans ZJU318 lipase and P. expansum TS414 lipase on the basis of single-factor experiments.After optimization,the productivities of Bacillus coagulans ZJU318 and P. expansum TS414 were enhanced for about 3 and 2 times,respectively. The maximal lipase activity produced by P. expansum TS414 could reach 315 U/ml under the optimized fermentation conditions. .Extracellular lipases were purified from the fermentation broth of Bacillus coagulans ZJU318 and Penicillium expansum TS414 by two-step chromatography. The enzymes were purified 14.7 and 81.8-fold, respectively. The molecular weight of the homogeneous enzymes were about 32 kDa and 28.8 kDa, determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis:Also,properties of the lipases were investigated. It was found that both were alkaline lipase, whose optimum pH value was 9.0 and were stable within the range of pH 8-10.Purification could improve the enantioselectivity of the both enzymes to varied extend. The purified Upases preferred to catalyze the esterification of (S)-ibuprofen. And, PEL showed higher activity and enantioselectivity than BCL under the same conditions. The reaction conversion catalyzed by PEL could reach 46-47%, the eeP reached 99% and the ee_s reached 90-92%. PEL was determined for the first time as the best enzyme for the resolution of the racematic ibuprofen.Water content, "memorial"pH, reaction temperature, acyl receptor and organic solvent played an important role in the enatioselectivity of free PEL. The suitable concentration of free PEL, water content, the optimum reactipn temperature and pH value, acyl receptor and organic solvent were 1000U/mL, 0.5%, 60℃, 9.0, 1-propanol and Isooctane, respectively. Under this condition, theconversion of the reaction reached 47% (E reached 99%) when the enzymatic resolution. reached equilibrium after 48 h.A new method for PEL immobilization using macrosporous resin AB-8 as support was established. PEL immobilized in macrosporous resin AB-8 exhibited higher activity and enatioselectivity than others. And the immobilized PEL in the resolution reaction were stable and could be well maintained. It was found that the conversion of the reaction catalyzed by immobilized PEL reached 47% and the eep was more than 98% with excellent enantioselectivity (Average E of 10 batches>380) when the enzymatic resolution reached equilibrium after 30 h.The resolution of (R,S)-ibuprofen was performed in microaqueous media by PEL immobilized in macrosporous resin AB-8. It was found that media type, "memorial" pH value and water content were of great importance, and the conversion of the reaction catalyzed by PEL immobilized on macrosporous resin AB-8 reached 47% with excellent eep(98.75%) in isooctane with 0% (v/v) water at "memorial" pH 9.0, 40°C for 30 h. Immobilization By this method affected the enzymatic properties of PEL in nonaqueous reaction media. The immobilized PEL was then not easy to agglomerate in nonaqueous media.And its activity in the resolution reaction was enhanced accordingly.According to the mechanism of enzymatic esterification during the resolution of (R, S)-ibuprofen in nonaqueous media, the kinetic model of this enzymatic resolution was established and the equation was simplified. There was no notable difference between measured data and predicted values -by cross-validation, suggesting that this model could describe the resolution process of (R,S)-ibuprofen catalyzed by PEL immobilized in macrosporous resin AB-8 successfully.The resolution of racemic ibuprofen was carried out in a temperature constant bioreactor(BSTR) by PEL immobilized on macrosporous resin AB-8. Uniform desigh was used for optimizing the reaction conditions of enzymatic resolution in BSTR. The result showed that the suitable concentration of ibuprofen, PEL and 1-propanol were 200 mg,6g and 0.383mL, respectively. Under this condition, the conversion of the reaction nearly reached 50% ,the eep reached more than 98% and the ee_s reached more than 94% after 12h. It was also found that in BSTR, the conversion of the reaction catalyzed by PEL immobilized on macrosporous resin AB-8 and the eep remained high with excellent enantioselectivity (Average E of 10 batches>380) when the enzymatic resolution reached equilibrium after 12 h, which showed that BSTR was a reliable bioreactor for the immobilized PEL with excellent operational stability, suggesting a good application potential in industrial application.The innovations and characteristics of the dissertation were obvious in the following: screening of stereoselective microbial lipases and their producing strains, optimization of the fermentation conditions of the lipases and reaction conditions in BSTR by the method of uniform design, method for lipase immobilization and enzymatic resolution by PEL immobilized in macrosporous resin AB-8 in nonaqueous media and so on. The results were of important value both in research and in industrial applications.