Study On The Immobilization Of Lipase And The Method For Screening Of Lipase Inhibitors

The absorption of dietary fat requires their enzymatic conversion into the fatty acids and monoglycerides which resynthesized fat by lipase in human digestive system. The exces fat accumulation eventually results in obesity. Lipase inhibitor is capable of losing weight by inhibiting the activity of lipase, which makes it a wide-use clinical anti-obesity drug. Orlistat, the only currently available lipase inhibitor on markets, has been proved the great beneficial effects in the treatment of obesity. However, it also exhibits some adverse side effects. Recently, many studied have been carried out to discover new lipase inhibitors with fewer adverse side effects, high security and more up to obesity patients'expectation. So, it is necessary to develop a simple, rapid and reliable method for lipase determination of inhibitory activity and lipase inhibitor screening.Several studies are included in the thesis:1. A rapid gas chromatography method was developed for determination of lipase inhibitory activity and lipase inhibitor preliminary screening. It was applied to test several extracts of traditional Chinese medicines. Among them, there were 3 kinds of extracts found to have inhibiting effect.2. Lipase was immobilized on surface modified silica gel by two different methods:via the reaction between silica gel-bound thiolsulfonate groups and lipase's native thiol-groups (LSH); and via the reaction between aldehyde-silica gel and lipase's amino groups (LGAL).The retention activity of LSH was twice higher than LGSL. Based on the morphological rough surface structure, LSH provides a good distribution and higher accessibility of the active site of the immobilized enzyme to the substrate. Both the two immobilized lipase have shown the good thermal stabilities and storage stabilities.3. A valid method for high throughput screening of lipase inhibitors was introduced based on the affinity effect of enzyme and inhibitors. The feasibility of the methodology was demonstrated by the positive lipase inhibitor comparison-certilistat and baicalin and it was concluded that higher activity led to higher amount of affinity absorption. By this screening method, two lipase inhibiting ingredients were obtained from Baikal skullcap root extract.