Study On Ester Synthesis By Immobilized Lipase In Non-Aqueous Media

Over the past decade non-aqueous enzymology has emerged as a major area of research and development in biotechnology. Research work in this area, up to now, however, has been mostly focused on experimental study. In order to fully exploit non-aqueous enzymology, the work described this dissertation, with the ester synthesis by immobilized lipase in organic solvents as model reactions, and a systematic study of the various factors which affect the reaction equilibrium and lipase activity of ester synthesis reaction in non-aqueous solvents both experimentally and theoretically. The main contents are as follows:The method of preparing immobilized lipase entrapped within an inorganic matrix by a sol-gel process has been improved. Compared with the previous method, the improved route can provide directly a spheroid form of immobilized lipase particles of which the size can be controlled by adding non-ionic surfactants during the preparation process so as to increase the recovery rate of enzyme as well as decrease the external diffusional restriction which can diminish the enzyme activity. The improved method can also alter the hydrophobicity and microstructure of the immobilized enzyme matrix by changing the molar ratios of different alkoxyl silanes so as to enhance the enzyme activity by a "surface active effect" and eliminating the internal diffusional restriction.With ester synthesis from octanol and octanoic acid by free or immobilized porcine pancreas lipase in organic solvents as a model reaction, the effects of water content and water activity of the system on the reaction equilibrium were studied both experimentally and theoretically. The results show that water activity is a more reliable measurement than water content as a reflection the mass effect of water on the reaction equilibrium. The effect of water activity on lipase activity when varying the amount of added lipase, the nature of the organic solvent and the molar ratio of alkoxyl silanes was examined experimentally. The results show that these factors affect the lipase activity through altering the partition equilibrium of water in different phases of the non-aqueous system. Based on experimental results and theoretical analysis, a more simple and convenient method called "Water ActivityMethod" has been proposed to determine whether the catalytic conformation of the enzymes have been changed. Based on the theory that organic solvents can affect the enzyme activity by stripping water molecules bound to protein from the enzyme, two semi-empirical equations were deduced to predict the effects of water content or water activity of the non-aqueous system as well as the kind of organic solvents on enzyme activity. By using these equations, most of the experimental results can be fully explained.With the ester synthesis from octanol and octanoic acid by free or immobilized porcine pancreas lipase in organic solvents as a model reaction, the changes of enzyme activity in non-aqueous system were correlated with the property parameters of the organic solvents. The results show that the dielectric coefficient and dipole moments of the organic solvents almost have no relation to these changes, while parameters such as log S log S,logP and ET +DN are good indexes to reflect the effect of organic solvents on enzyme activity. Suggestions of how to use these parameters in distinct circumstances have been provided by a thorough theoretical analysis. Based on a theoretical analysis of the partition process in different phases or solvation process in different organic solvents of the substrates, equations were deduced to predict the effects of organic solvents on the dynamic parameters of the enzyme catalysis reactions.Based on the theoretical analysis of the partition process in different phases or solvation process in different organic solvents of the substrates, methods called "Partition Coefficient Method" and "Activity Coefficient Method" have been developed to predict the effect of organic solvents on the ester synthesis reaction equilibrium. Using these two methods,...