The Influence Of Dynamic High Pressure Microfluidization On Activation And Conformational Changes Of Lipase

Lipase caught more and more attentions because of its widely use. Changes of activity and stability are the hotspot. But the dynamic high-pressure technology was seldom used in research. Our thesis focuses on changes of lipase activity and conformation due to different treatments. The effect of DHPM on the activity of lipase was studied through the determination of enzyme activity, CD spectra, UV absorption section and SH amount. And collaborative chemical modification research of lipase by adding the polymer PEG and organic solvent isopropanol, and to explore the relationship between the conformation and activity of lipase.It is the first time attempt to use DHPM on catalytic activity among the non-aqueous phase.CRL was used as material to study the effect of DHPM on the activity and the conformation. The results showed:CRL treated under 50MPa and100MPa through DHPM showed no significant effect on the relative activity of CRL. The activity of CRL was increased by up to 11.33% after treated under 150MPaå'Œ200MPa. And the relative activity was increased to the highest point after 1h storage and inactivated after 8h. The secondary structure of CRL was changed by DHPM. The content ofβ-sheet changed with the relative activity. The two was positively related. While the content of a-helix changed nonlinear. The 3D structure was changed after DHPM treated, the structure of CRL was unfolding.The CRL treated by DHPM in different system was studied, and the activity and conformation change was observed. The PEG was added into the buffer solution of CRL, and DHPM treatment was conducted. The results showed:the relative activity of CRL was up to 105.1% when added PEG8000 4%(w/v). Yet the relative activity of CRL was decreased after treatment under DHPM with PEG8000 4%(w/v). The activity of CRL was enhanced after storage. The most activated transit state (43.73%) was appeared in 6h after 100MPa. The effect of PEG on the Tm of CRL was insignificant. While the UV absorption was changed after PEG added. The effect of adding isopropanol was also studied. The relative activity of CRL was highest with 20%(v/v) isopropanol, the CRL activity was increased while the treatment pressure was increased form 100MPa to 200MPa. And the activity increased around 20% after 200MPa treated twice. The content ofβ-sheet showed positively related to the activity in the two system.The lipase was used widely in non-aqueous phase catalysis, thus the activity, solubility and stability was critical to lipase. Lipase in hexane system was studied, the catalytic synthesis rate of CRL on oleic acid and lauryl alcohol was higher after DHPM treatment. The relative esterification activity was increased with the treatment pressure to 103.7% under 150MPa, and then decreased with the pressure. The solubility of CRL in hexane was increased after DHPM treatment, maximum to 16.4% after 150MPa treatment. The stability of CRL in hexane after DHPM was changed, while the thermal stability was little affected.