(593p) Cinnamyl Acetate Synthesis by Lipase-Catalyzed Transesterification in a Solvent-Free System

Flavor esters have various applications in
food, perfume, flavor and pharmaceuticals since their
aroma qualities. Recently, application of enzyme in the synthesis of esters has aroused widely
interests for its high efficiency, moderate reaction condition and environmental
friendly. The enzymatic synthesis of esters was mostly carried out through the
esterification between alcohols and acids. However, it usually obtained low yield
due to the severe deactivation of lipase activity under the high concentration of
acid [G¨¹venç, 2002].The transesterification route using alcohols and esters as substrates could eliminate
this problem. Vinyl esters are usually used as acyl donors in transesterification to obtain
the higher conversion since the produced vinyl alcohol can tautomerize to acetaldehyde, which could promote the synthesis
process. However, the co-product acetaldehyde has an unfavorable deactivation
effect on lipase since it would bond to lysine residues by the formation of
Schiff-base [Villeneuve, 2000]. Also, most of these lipase catalyzed reactions are carried out in the
organic solvent such as n-hexane, heptane or toluene which are toxic and
inflammable.

In this work, a commercial flavor ester, cinnamyl acetate, was synthesized by lipase (Novozym435) through transesterification route
in a solvent-free system (Fig A). Ethyl acetate was chosen as the solvent
as well as the acyl donor instead of vinyl acetate in order to avoid the deactivation
of acetaldehyde on enzyme. 90.06% of
conversion was achieved after 3 h when the transesterification was carried out
at ethyl acetate/cinnamyl alcohol=15:1, 2.67 g L^-1 of lipase
loading and 40^oC (Fig B). The excellent stability and reusability of enzyme resulted from
the moderate reaction system. The immobilized lipase can be reused at least 6
cycles with few decline of activity. Kinetic studies indicated that the reaction abode by the Ping-Pong
Bi-Bi mechanism with the inhibition of cinnamyl alcohol on lipase (Fig C) .The Michaelis
constant for ethyl acetate and cinnamyl alcohol were calculated as 2.241 mmol L^-1 and 206.81 mmol L^-1, respectively (Fig D). The inhibition
constant of cinnamyl alcohol was 0.461 mmol L^-1, and the maximum reaction rate was 41.807 mmol L^-1min^-1.

(1) G¨¹venç, A., Kapucu, N. and Mehmetoğlu, Ü. (2002) The production of
isoamyl acetate using immobilized lipases in a solvent-free system. Process. Biochem. 38, 379-386.

(2) Villeneuve, P., Muderhwa, J. M., Graille, J. and Haas, M. J.
(2000) Customizing lipases for biocatalysis a survey of chemical, physical and
molecular biological approaches. J. Mol.Catal.B-Enzym.9113-148.

This work was supported by the Program for
New Century Excellent Talents in Chinese University (NCET-08-0386; NCET-11-0372), the 863
Program of China (2008AA10Z318), the Natural Science Foundation of China
(20976125; 31071509; 51173128) and Tianjin (10JCYBJC05100), and the Program of Introducing
Talents of Discipline to Universities of China (No. B06006).

Figure(A)Reaction equation, (B) Reaction curve, (C) Transesterification mechanism, (D) Kinetic curve for synthesis of cinnamyl acetate