Enzymatic Synthesis Of 2-o-α-d-glucopyranosyl-l-ascorbic Acid

L-Ascorbic acid (VC) is well known as vitamin C. VC plays key roles in many biological processes, such as collagen formation, carnitine synthesis, and iron absorption.VC is widely used as a nutrient in the pharmaceutical and cosmetic industries as well as a preservative with its excellent antioxidant activity. However, VC can be easily oxidized into dehydroascorbic acid, especially in the presence of air, light, heat, and metal ions, and the produced dehydroascorbic acid was easily degraded. The instability of VC imposed a great limitation to its application, and therefore, how to enhance the stability of VC is a major challenge in the related research field. One of the prominent VC derivatives is 2-O-a-D-glucopyranosyl-L-ascorbic acid has largely improved stability while still keep their physiological activities. The main content is about its synthesis of AA-2GThis thesis aimed to produce 2-O-a-D-Glucopyranosyl-L-Ascorbic acid (AA-2G) via the transglycosylation reaction by cyclodextrin glucanotransferase (CGTase) from recombinant Escherichia coli with ascorbic acid (VC) and P-cyclodextrin (β-CDs) as the substrates. The main research of this thesis follows:1. The product of the enzymatic conversion by LCMS/MS and identified as AA-2G. Through comparative study, a-CGTase from recombinant Escherichia coli which transform AA-2G was chosed as the best enzyme source. Study indicated that the enzyme has a broad substrate role and function of site specificity. Test results showed that the best substrate was a-CDs. However, considering the cost of industrialization, we choose P-CDs as substrate to continue to optimize. The glucoamylase treatment can effectively enhance the AA-2G production via the catalytic action in the presence of AA-2Gs.The experiment shows that the optimal glucoamylase concentration, temperature and reaction time for AA-2G synthesis were 10 U/mL and 6 h, respectively.2. By single factor experiments, the best conditions for the conversion range was determined preliminarily, under the optimal condition,160 U/mL was found to be the optimal enzyme amount for transformation. Taking a step forward through the response surface experiments, the conversion of ascorbic acid glycosylation conditions were studied, and the best conditions follows:temperature 36.5℃, pH 5.4, time 25 h, VC concentration 72 g/L, P-cyclodextrin concentration 55 g/L.3. The conditions of immobilized cyclodextrin glucosyltransferase were studied. The best immobilization conditions were glutaraldehyde (GA, cross-linker) 0.01%(v/v), SBA-15 (adsorbent) 2 g/L, CaCl2 3 g/L, alginate 20 g/L, adsorption time 3 h, and immobilization time 1 h. In comparison with free CGTase, immobilized CGTase had a similar optimal pH (5.5) and a higher temperature (45℃). The continuous production of AA-2G from ascorbic acid and P-cyclodextrin in the presence of immobilized a-CGTase was carried out, and the highest AA-2G production reached 21 g/L, which was 2-fold of that with free CGTase.