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