| Enzymes can be used to catalyze reactions in non-aqueous media, which can be particularly useful for the processing of materials with low water solubility. One such reaction is the lipase-catalyzed esterification between citronellol and acetic acid, in a medium of hexane. Both reaction conditions and special pretreatments on the lipase impact the lipase's catalytic performance. This project focuses on improving lipase performance by optimizing its pretreatment. The pretreatment factors studied include pH adjustment, lyophilization time, addition of buffer salts, non-buffer salts, denaturants or active site protectants to the lyophilizate, and flash-freezing in the presence of phase interfaces. The effectiveness of a particular pretreatment is presented in terms of 'relative performance' (RP), which is equal to the number of times faster the pretreated lipase catalyzes the reaction relative to untreated lipase.; The individual and interactive effects of the pretreatment factors were studied in detail and compared. Buffer salts had a much stronger performance enhancing effect than non-buffer salts; pretreatment with 90% (w/w) sodium phosphate yielded lipase with an RP of ∼64 (catalyzed the reaction ∼64 times faster that untreated lipase). A strong interaction between the treatments with sodium phosphate and pH adjustment was found. Both these treatments may mitigate the inhibitory effect of acetic acid, one of the substrates. Activating effects of phase interfaces and active site protectants are shown to be complementary to other treatments, demonstrating that they act by a distinct mechanism. An optimization process known as 'the method of steepest ascent' was used to simultaneously optimize the pH and buffer salt pretreatments, as well as the concentration of acetic acid in the reaction mixture, This process succeeded in rapidly developing pretreatment combinations that yielded lipase with RP as high as 170. Studies were also conducted to develop methods for recovery, reprocessing and reuse of lipase from completed reactions. |
