Study Of Separation, Purification Of Porcine Pancreatic Lipase And Preparation Of (s)-2-Methyl-l-Butanol Via Lipase Catalysis

The application of the biological catalysts is of greater importance in the resolution of racemic mixtures. Presently, most of the catalysts used for the resolution of racemic mixtures are hydrolases, especially lipases. With the development of non-aqueous medium in enzymology, more and more research and application focus on the lipase-catalyzed process carried out in non-aqueous medium, especially in anhydrous organic solvents. Based on the previous research achieved by our research group, the thesis tries to do a further investigation ─the free porcine pancreas lipase (PPL), the immobilized PPL and the PPL sublimed by salt-outting catalyze the transesterification of 2-methyl-1-butanol and tributyin. The results show that the porcine pancreas lipase's stereoselectivity has been transferred from catalyzing the transeterification of (R)-2-methyl-1-butanol to transeterificaton of (S)-2-methyl-1-butanol. Because the stereoselectivity of PPL has changed, the hydrolyzation of the (S)-isoamyl butyrate has to be done for final product, (S)-2-methyl-1-butanol. The experiment does a comparison between the different hydrolytic systems of (S)-isoamyl butyrate and studies the whole reaction including the transesterification catalyzed by different PPL and the gotten of the (S)-2-methyl-1-butanol by hydrolyzation the (S)-isoamyl butyrate. And the results show that the suitable technics is as follows: the PPL from the Fluka company, adding straight the sodium hydroxide solution to (S)-isoamyl butyrate, extracting by water to get the (S)-2-methyl-1-butanol. In fact, the commercial porcine pancreas lipase is mixed lipase, composed of many lipases, steady reagents, impurity, etc. Different lipases have different catalytic activities and catalytic selectivity with substrates.For further study on the mechanism of the lipase's catalytic stereoselectivity, the separation and purification of PPL were done by the means of salt-outting, electrophoresis, gelfitration chromatography, etc, and the lipases at five different molecular ranges were obtanied, then the different lipase activity of these lipases were determined and compared, which can provid a good fundament for the understanding and adjusting the lipase stereoselectivity. Whereas this experiment is just the laboratory's reseach and development, there will be few midterm or ultimate product, for example, (S)-2-methyl-1-butanol. For reducing the error which results from the dilution in the analysis of optical purity by common polariapparatus, the experiment establishs a new method about the measurement of optical purity. The method utilizes the on-line polariapparatus coupling with UV decetor. The method is very simple, and its accuracy is very high.