Effect Of Additive On The Enzymatic Reaction

Chirality is one of the essential attributes of compounds existing in nature. Biomacromolecules playing essential roles in life activities, such as proteins, polysaccharides, nucleic acids and enzymes are almost chiral and have important physiological functions in vivo. The active ingredients of most pharmaceuticals, pesticides and herbicides are also chiral molecules. Since the chiral match is related to the ability of molecular recognition, the biological activity of two chiral molecules may be significantly different. Single chiral glycidol is widely used in the synthesis of protease inhibitor in AIDS drugs andβ-blockers in cardiovascular disease drugs, so it has great pharmaceutical value. Because of the structural stability and representative chirality of Single chiral 2-octanol, it has been used as one of the most important materials of liquid crystal.Lipase, as a kind of biocatalyst, has been widely used in the field of chiral resolution because of its high stereoselectivity to chiral alcohols ,mild reaction conditions and free from pollution.It is well known that in an enzymatic reaction system , the catalytic activity, stereoselectivity and stability of enzyme can be improved through changing the amount of substrate and product and reaction medium. In addition, adding some additives intentionally can greatly improve the catalytic activity, stereoselectivity and stability of enzyme. The diversity of additives to be used and simple operation offer an alternative and simple way to control and improve the enzymatic reaction efficiently. So adding certain additives to an enzymatic reaction system in an appropriate way needs further study. In this thesis, enzymatic resolution systems of glycidol butyrate and 2 - octanol were studied respectively through adding different additives.We synthesized racemic glycidol butyrate through chemical method, and investigated the optimum reaction conditions to resolve glycidol butyrate through hydrolysis catalyzed by Bacillus subtilis lipase (BSL2) in aqueous system. More attention were paid on the effects of different organic additives on the catalytic activity and enantioselectivity of BSL2 catalyzing the hydrolysis of glycidol butyrate. It was found that adding different organic additives caused different impacts on the enantioselectivity and catalytic activity of BSL2. Most additives could improve the catalytic activity and enantioselectivity of BSL2, but the effect of dioxane is more obvious. In addition, pH, temperature, lipase and amount of additives were also investigated. We found that the optimum conditions of enzymatic resolution of glycidol butyrate through hydrolysis are: pH of phosphate buffer is 7.8,temperature is 5℃, BSL2 as enzyme and dioxane as additive (18%, V/V).Under the optimum conditions, BSL2 showed the highest enantioselectivity(E=108),and the remained (R)- glycidol butyrate with high enantiometic purity(ee﹥98%) and yield(Y=36%) could be obtained when the conversion was about 52.3%.Ionic liquid wins the reputation of'green solvent'because of its advantages of non-smell, non- pollution, non-flammability, easy separation from the product, easy recovery, recycling use and convenience. Since the use of ionic liquid meets the requests of technology without population and sustainable development, it has been widely accepted. In the second part of this thesis, we studied the effects of different ionic liquids as additives on the enzymatic resolution of 2 - octanol in non-aqueous system. Effects of different ionic liquids used as additives on the catalytic activity and enantioselectivity of enzyme were investigated. The results showed that the enantioselectivity of enzyme could be significantly improved by selecting appropriate ionic liquids as additives. Besides, effects of reaction conditions, such as acyl donor, ionic liquid content, temperature, pH and water activity on the resolution of 2– octanol catalyzed by lipase through transesterification and on the catalytic activity and enantioselectivity of lipase were investigated. The results observed show the optimum reaction conditions are: ionic additive: [C8MIM][PF6], acyl donor: vinyl acetate, amount of additive : 80μmol , temperature: 40℃, pH: 7.5, water activity: 0.28. Based on the above experiment, enzymatic resolution of Racemic 2 - octanol through esterification under solvent-free system were performed. Under the optimum conditions, comparing with the system without ionic additives, the activity of the lipase was enhanced 1.7-fold and the enantioselectivity of the enzyme increased 3.1-fold. Although previous researchers have also improved the catalytic activity and enantioselectivity of the enzyme in enzymatic resolution of Racemic 2-octanol through esterification, ionic liquids have not been used as additives so far.