CGtase Immobilization For AA-2G Synthesis And Biotransformation Process Optimization

Vitamine C(VC, or L-ascorbic acid) is a kind of essential elements for human beings. However, due to its extremely instable molecule structure, the application of VC has been greatly limited. AA-2G(2-O-α-D-glucopyranosyl-L-aseorbic acid) as a kind of VC glycosyl derivatives is more stable than VC and still keep the activities of VC. Biotransformation method is the main method to synthesis AA-2G in the industry. And cyclodextrin glucanotransferase(CGT) is the most widely used enzyme which catalyze various saccharides as glycosyl donors.In this dissertation, AA-2G synthesis was catalyzed by CGT-SL(provided by Amano), with soluble starch and VC as glycosyl donors and acceptor, respectively. The yield of AA-2G was improved 4.06-fold by optimizing the synthesis condition. The main results were as follows:(1) The condition of AA-2G synthetic reaction was optimized by signal factor and orthogonal experiment. The product of the enzymatic conversion was identified as AA-2G by LCMS and HPLC. Among β-cyclodextrin, maltose, soluble starch, dextrin, sucrose and glucose, soluble starch is optimal substrate for AA-2G synthesis. The optimal conditions for AA-2G production were as follows: the enzyme concentration 79 U·m L-1, VC 16 g·L-1, VC and maltodextrin with ratio of 2:4, 15℃, pH 4.0 and 32 h of incubation. Under optimal conditions, the AA-2G yield was 6.23 g·L-1, and VC conversion rate was 19.93%. The yield of AA-2G improved 4.06-fold compared to original state.(2) Different immobilized methods(embedding, adsorption and carrier bonding) were studied to find the appropriate one and the immobilized conditions were optimized. The CGT-SL immobilized by GDX-101 has high mechanical strength and adsorption rate. And the optimal conditions is as following: adsorption time 3 h, enzyme concentration 119 U·m L-1. After 5 times consecutive enzyme reaction, immobilized enzyme still kept 39%-58% of the initial activity. But the CGT-SL immobilized by align kept under 10% of the initial activity after 3 times.(3) The enzymatic property of immobilized enzyme and free enzyme were studied, including temperature and pH. Then use immobilized enzyme to synthetized AA-2G at the optimal condition. The optimal temperature of immobilized enzyme is 20℃-30℃, higher than free enzyme. The optimal pH of immobilized enzyme by GDX-101 is about 5.0, which is higher than free enzyme. Use SA immobilized enzyme to synthesize AA-2G, the highest yield of AA-2G is 17.18 g·L-1.