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Chemical Modification of Lipase and its Immobilization on Polymer Beads for Use in Organic Synthesis
Dissertation Abstract:
A simple and effective method to produce a more active, stable, and practical lipase preparation was identified. Soluble lipase from Candida rugosa was modified with different types of hydrophobic chemical modifying reagents
The esterification activities of the modified lipases were enhanced following their modification. The degree of activity enhancement depends on the type and molecular weight of the modifiers used and the degree of modification of the enzyme. A lower degree of enzyme derivatization was required for modification with the high molecular weight modifiers to attain maximal activities. In the case of monothoxypolyethylene glycol (PEG), however, maximal activity was attained only after exhaustive modification. The optimum esterification temperature and preference of fatty acids as acyl donors of the modified lipases were very similar to those of the native enzyme. Both were more active in non-polar solvents than in polar solvents. The modified lipases showed higher thermostability, solvent stabi lity, and storage stability compared to the native lipase.
The lipase modified with PEG 1900 was the most thermostable, and that modified with methy14-phenylbutyrimidate (imidoester VI) was the most stable when incubated in benzene for 10 days. The best storage condition was at low temperature and in the lyophilized form.
When porous polymer beads were added to a solution of the modified lipase at room temperature and stirred gently for 0.5 to 2 hours, the enzyme was strongly adsorbed onto them. Native lipase was only weakly adsorbed onto the supports.
The immobilized activity of modified lipases was higher compared with the native enzyme. The magnitude of the difference depended on the type of modifiers, the degree of modification of the enzyme, and the type of polymers used for immobilization. Lipase that was modified with the more hydrophobic modifiers showed higher immobilized activity than those modified with the less hydrophobic modifiers (except for PEGSOOO). The highest immobilized activity was exhibited by lipase with PEG 1900 (95% modification). Amberlite XAD7, XAD8, and Polycarboxylic acid beads (RCOOH) were good supports for enzyme immobilization by the said methods.
The optimum esterification temperature of lipase immobilized in this manner was not changed. However, its preference for certain fatty acids as acyl donors was altered. Medium length chain fatty acids were favored over longer chain fatty acids when the enzymes were immobi lized. Their activities were increased even in the presence of polar so lvents. The immobilized lipases also showed higher thermostability, solvent stability, and storage stability compared with native and modified lipases.