Lipase Conformation Recording And Fluorescence Spectrum Analysis

The thesis mentioned that the conformation of lipase molecular can be induced by substrate to be an activated one in organic phase through constructing a water-in-oil micro-emulsion system. The activated conformation was recorded using high cross linking degree polymer. The effect of inducement on the activity of recorded lipase polymer was studied, the catalyzed property of recorded lipase polymer in water phase was investigated, and the fluorescence spectrum of lipase solution and recorded lipase polymer under different kinds of denaturalized circumstance were analyzed.Eighteen recorded lipase polymers were obtained by ultraviolet initiated polymerization, dryness and grinding, using nine kinds of monomers with different chain length and two kinds of cross-linking agents as pre-polymers. The recorded lipase polymers had high activity and activity yield when using I(PEG400-diacrylate ester) and G(PEG200-diacrylate ester) as function monomer, the activity of Ib reached up to 3.6U/g, and the yield of activity was 346.4%. The result showed four recorded lipase polymers with highest activity(Ia, Ib, Ga, Gb), maintain a majority of activity under the circumstance of strong acid(pH=3.5), strong alkaline(pH=12.1)and high temperature(80℃), while the lipase solution completely become inactive in the same circumstance, proving that the recoded method improved the stability of lipase effectively.The recorded lipase with inducement was obtained by using lauric acid as inducer which was extracted by organic solvent. The elution effect of acetone and petroleum ether as extractive solvent was similar, but the activity of recorded lipase polymers eluted with petroleum ether was higher.The activity and stability of recorded lipase with inducement were measured and analyzed, the result showed its activity was twice more than the activity of recorded lipase without inducement, and had almost the same stability.The conformation of lipase solution and recorded lipase within the polymer werestudied with fluorescence spectrum in this thesis. The fluorescence spectrum of lipase solution was according to the luminescence ability of amino acid Tip, while the lipase labeled with a fluorescence probe FITC (fluorescein isothiocyanate) was recorded and its fluorescence spectrum was measured. The fluorescence intensity grew and emission wavelength gradually increased between 331nm and 350nm under the condition of strong acid(pH=3.5), strong alkaline(pH=12.1)and high temperature(80 °C), showing that the conformation of lipase protein changed from globularity to unfolding state. The fluorescence intensity of fluorescence probe inside recoded lipase polymer indicated the distance of amino acids on the surface of protein. The result showed that the fluorescence intensity increased in a certain extent under the circumstance of strong acid(pH=3.5), strong alkaline (pH=12.1), denaturants(Guanidine Hydrochloride, urea and 95% ethanol) and high temperature(80°C), In other word, part of protein changed its conformation.