Applied Basic Research Concerning Stereoselective Lipase On Resolution Of Chiral Compounds

Property or bioactivity of organic compounds is determined by their structure, and stereoselectivity had been one of the key factors in chiral catalysis process. Because of the mild reaction condition, high enantioselectivity, less side reactions and environment pollution of enzyme-catalyzed reactions, utilization of enzymes or microorganisms is of great interest both in economical and social benefits. In this dissertation, the screen of high-yield stereoselective lipase producing strain, production and character of the lipase, immobilization and application of the lipase in the enzymatic resolution of (R, S)-allethrolone and (R, S)-2-octanol, control of the enantioselectivity, kinetics of the enzymatic resolution and bioreactor for the immobilized lipase was investigated.A new system for quick directed screening of stereoselective lipase producing strain and the lipase by this strain was established on the base of Rhodamine B / ester assimilation plate. By this method, Alcaligenes sp and P. expansion PED-03 for the resolution of (R, S)-allethrolone and P. expansum PED-03 for (R, S)-2-octanol were screened out from the lipase producing strains deposited in our laboratory. The result showed that the lipases produced by this screened strains had very high enantioselectivity in the resolution reaction.Production and properties of the stereoselective lipase from the screened P. expansum PED-03 (PEL) were investigated. It was found that PEL was an alkaline lipase, whose optimum pH value was 9.5 and was stable within the range of pH 6.0¹0.0. The maximal lipase activity of production reached 850 U/ml under the optimized fermentation conditions.A new method for lipase immobilization using modified Ultrastable-Y molecular sieve as support was established. PEL immobilized in Ultrastable-Y molecular sieve exhibited obvious activity within a wider pH value range and at a much higher temperature, and showed a marked enhancement of stability against thermal inactivation. In addition, ions such as Fe3+, Cu²⁺ and Mn²⁺, which had an inhibitory effect on the free PEL, were enhancers of activity for the immobilized PEL. By this method, the immobilized PEL was not easy to agglomerate in nonaqueous media, and the required microenvironment of PEL for the resolution reaction could be well maintained. Moreover, the enclosure of the protein in a well-defined space may also help prevent denaturing of the protein and enhance enzyme stability. Additionally, immobilization using modified Ultrastable-Y molecular sieve as support make it easy for lipase to be reclaimed and the process is simple and easy to popularize in industry.The resolution of racemic allethrolone was carried out in a constant temperature bioreactor by PEL immobilized on modified Ultrastable-Y molecular sieve. The suitable concentration of substrate and PEL, rotation speed, the optimum reaction temperature and pH value were 0.12 g/mL, 200 U/g, 200 r/min, 35 ℃ and 9.5, respectively. Under this condition, the conversion of the reaction reached 50% and the ee_p reached 99% after 36 h. Combination of enzymatic catalysis and chemical transformation for the resolution of (R, S)-allethrolone was studied. Sulfonate, organicmedia, alkaline additive and temperature played an important role in the sulfonation reaction, and it was found that the suitable Sulfonate, organic media, alkaline additive and temperature were benzenesulfonyl choride, methylene dichloride, triethylamine and 20 °C, respectively. Addition method of triethylamine was of great importance, and the proper addition method was dropwise titration.The resolution of (R, S)-2-octanol was performed in microaqueous media by PEL immobilized on modified Ultrastable-Y molecular sieve. 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 modified USY molecular sieve reached 97.68% of the theoretical value with excellent enantioselectivity in n-hexane with 0.8% (v/v) water at "memorial" pH 9.5, 50°C for 24 h. Suitable Surfactants such as Tween-80 was an enhancer of enantioselectivity for immobilized PEL at low concentration but an inhibitor at high concentration. To explain the impact of immobilization on the effects of ions, we brought forward a hypothesis of "conditional activation". It is supposed that the catalytic center of enzyme has a "gate". This "gate" is open or closed according to the microenvironment affected by external conditions such as ions or salts.According to the mechanism of enzymatic transesterification during the resolution of (R, S)-2-octanol 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)-2-octanol catalyzed by PEL immobilized in modified Ultrastable-Y molecular successfully.For industrial application, immobilized PEL reactor was investigated. The enzymatic resolution of (R, S)-2-octanol in laboratory-scale was carried out in Batch Stirred Tank Reactor (BSTR), Continuous Stirred Tank Reactor (CSTR) and Packed-bed Reactor (PBR), respectively. It was found that by semi cycle operation in PBR, the conversion of the reaction catalyzed by PEL immobilized on modified USY molecular sieve nearly reached 50% and the eep was more than 94% with excellent enantioselectivity (Average E of 8 batches > 400) when the enzymatic resolution reached equilibrium after 12 h, which showed that the immobilized PEL PBR was a reliable bioreactor with fine operational stability, suggesting a good application potential in industrial application.The characteristics and innovations in this dissertation were obvious in the following aspects: directed screening of stereoselective microbial lipases and their producing strains, method for lipase immobilization and enzymatic resolution by PEL immobilized in modified Ultrastable-Y molecular in nonaqueous media, and so on. The results were of meaningful value both in academic research and in practical applications.