3.5 Formation of AA-2G catalyzed by rAGL
In the present, rAGL from O. sativa catalyzed the glycosylation
of AA·Na to produce AA-2G, a stable L-ascorbic acid derivative. The
proceeding of AA-2G synthesis was monitored by HPLC. As is shown in Fig.
6, using maltose as the glycosyl donor, the influences of pH,
temperature, substrate ratio and enzyme concentration on AA-2G
production catalyzed by rAGL were studied.
Among the pH range of 3.0 to 5.5 (Figure 6a), the product concentration
in the reaction mixture increased obviously in the first 3 h, and then
declined possibly because of the hydrolytic activity of rAGL. At pH 4.0,
the highest yield of AA-2G (6.73±0.02 g/L) was obtained within 3 h, and
after 3 h, the concentration of AA-2G decreased faster at pH 3.0 than at
other pH values.
As is indicated in Fig. 6b, the optimum temperature of glycosylation
reaction appears to be 37 °C, because the concentration of AA-2G remains
higher than other conditions within 6 h. Among the different reaction
temperatures, decrease of the AA-2G concentration was observed from the
beginning (reaction of 1 h) at 50 °C, probably degradation of the
substate AA·Na at high temperature is a factor that can not be ignored.
At 30 °C, AA-2G increased in 2 h, and then remained basically unchanged
within the next 4 h.
As for the influence of the molar ratio of maltose/AA·Na on the
synthesis of AA-2G (Fig. 6c), when maltose is kept at 267 mM in the
initial reaction system, the highest product concentration can increase
with the rise of AA·Na concentration (from 89 mM to 267 mM). Excessive
maltose increased the reaction rate and affected the yield from AA·Na.
In the case that the maltose/AA·Na molar ratio was 1:1, the AA-2G
concentration in the reaction mixture topped 8.7±0.4 g/L after reaction
for 3 h, and the yield from AA·Na was 9.7%. The higher yield (10.6%)
was achieved in 2 h at the maltose/AA·Na ratio of 3:2, although the
AA-2G concentration was only 6.4±0.1 g/L.
The enzyme concentration has an effect on the reaction rate as well, as
was presented in Fig. 6d. The formation patterns of AA-2G are similar
that the product increased firstly until it approaches the high summit,
and then the reaction decreased, but with the increase of enzyme amount,
the time to reach the highest concentration was shortened. Under the
condition of 4 U/mL rAGL, 7.5±0.3 g/L of AA-2G can be produced within 2
h, but its hydrolysis rate was also higher than other reactions with
lower enzyme concentration. The highest yield of AA-2G (8.3±0.7 g/L) was
reached in 5 h of the reaction with 2 U/mL of rAGL.
Figure 6