Biomedical Engineering Reference
In-Depth Information
was carried out at 60
o
C with vigorous shaking. At appropriate intervals, a portion of the
reaction mixture was sampled and used for HPLC analysis. Among the lipases tested,
Chirazyme
®
L-2 C2 produced a sufficient amount of product and lipase PS made a trace
amount of product during 24h-reaction. The other lipases did not produce any product. The
two methods to synthesize acyl ascorbate are condensation and transfer reaction. For the
transfer reaction, methyl laurate was used as the acyl donor, and the conversion was
calculated based on the amount of ascorbic acid added, which was the limiting reactant. The
conversion during condensation was much higher than that for the transfer reaction. For the
synthesis of 6-
O
-palmitoyl ascorbate in 2-methyl-2-butanol by Chirazyme
®
L-2 C2, the
transfer reaction was superior to condensation [18]. Although the reason for the difference is
not clear, a possible one might be a difference in water content of medium between their
studies and ours. The gradual decrease in conversion was observed at prolonged reaction
times for both the condensation and the transfer reaction. This would be due to the
degradation of the product to the compounds corresponding to dehydroascorbic acid and 2,3-
diketo-gulonic acid [38]. Therefore, too long a reaction time would be undesirable for the
production of acyl ascorbate. The condensation of ascorbic acid and lauric acid was carried
out at various temperatures. The reaction rate was faster at higher temperature. The
conversion leveled off at lower value at 70
o
C than at 55 and 60
o
C. This low conversion
seemed ascribable to denaturation of the enzyme at that temperature. Therefore, condensation
at 60
o
C was adopted.
The transient changes in the conversion of conjugated linoleoyl ascorbate in various
organic solvents, such as acetone, acetonitrile, 2-methyl-2-propanol and 2-methyl-2-butanol,
were measured [37]. The conversion was high in acetonitrile, acetone and 2-methyl-2-
propanol, and that in 2-methyl-2-butanol followed. It seemed that the conversion was in
proportion to the polarity of the solvent used. We studied the equilibrium constant based on
the concentrations,
K
C
, for lipase-catalyzed condensation of mannose and lauric acid in water-
miscible solvents [17, 39]. The
K
C
value correlated with the relative dielectric constant of the
solvent, because the solubility of hydrophilic substrate such as ascorbic acid was higher in the
solvent with the higher relative dielectric constant. Furthermore, the equilibrium constant for
the formation of fatty acid butyl esters by the lipase-catalyzed condensation depended on the
type of solvent polar group [40]. Both the polarity and polar group of the solvent would also
affect the productivity for the lipase-catalyzed condensation of conjugated linoleic acid with
ascorbic acid, because of the stronger interaction between conjugated linoleic acid and the
solvent than that between conjugated linoleoyl ascorbate and the solvent.
The condensation of eicosapentaenoic acid and ascorbic acid was carried out at the
various molar ratios in acetone dehydrated with molecular sieves 5A, the initial water content
of which was 0.011% (v/v) [25]. The molar ratios were varied in two ways: 1) the amount of
eicosapentaenoic acid was fixed at 0.124 mmol, and 2) the amount of ascorbic acid was fixed
at 0.125 mmol. Figure 3(a) shows the transient changes in the conversion of the desired
product. The experimental points were empirically connected to form a curve for each run, as
shown by the solid curve in Figure 3(a). The initial reaction rate and the apparent maximum
conversion were read from the curve, and are plotted against the molar ratio of
eicosapentaenoic acid to ascorbic acid in Figure 3(b). The apparent maximum conversion was
higher at the higher molar ratio. When the amount of ascorbic acid was fixed and that of
eicosapentaenoic acid was changed (open circle), the initial reaction rate increased as the ratio
increased. In another case where the amount of eicosapentaenoic acid was fixed and that of
