The Study Of 2-O-α-D-glucopyranosyl-L-ascorbic Acid Synthesis By Cyclodextrin Glycosyltransferase

2-O-α-D-glucopyranosyl- L-ascorbic acid(AA-2G), an important VC derivative, has attracted attention in recent years due to its excellent resistance to oxidation, high temperature, light and extreme p H conditions. In addition, AA-2G can be hydrolyzed by α- glucosidase in vivo or skin environment, slowly and continuously releasing the healthful benefits of VC, which has been widely used in food, medicine, cosmetics and husbandry industry. AA-2G is synthesized by glycosyltransferases via transferring a glycosyl residue from a glycosyl donor to the C-2 position of VC. In comparison, CGTase is considered to be suitably emp loyed in large-scale production of AA-2G owing to its high substrate specificity.In the present study, the optimized gene of CGTase from Bacillus stearothermophilus NO2 was cloned and expressed in Escherichia coli BL21(DE3). The cases of AA-2G synthesized by CGTases from various sources were investigated in detail. Multiple sequence alignment and site-directed mutagenesis were performed to study the key amino acid residues of C GTase for AA-2G production. At last, CGTase was displayed on the cell surface of saccharomyces cerevisiae to improve AA-2G production. The main results were listed as follows:(1) The B. stearothermophilus NO2 CGTase gene(cgtori) was optimized and subcloned into E. coli BL21(DE3), obtaining E. coli BL21(DE3)/p ET-20b(+)-cgtopt. In shake flask, the extracellular enzyme activity was 50 U×m L-1 after cultivation of 60 h in TB medium. The recombinant CGTase was purified and characterized in detail. The optimum temperature and p H of recombinant enzyme was 70°C and 6.0. The half- life of recombinant C GTase was 10 d and 2 h at 50°C and 60°C, respectively. The enzyme showed more than 60% of its initial activity after incubation at 4°C for 21 d. Among several commonions, the appearance of Co2+ and Ba2+ could active the recombinant enzyme.(2) Using β-CD as donor and VC as receptor, the optimal conditions for AA-2G production by CGTases from B. stearothermophilus NO2, Bacillus circulans 251, Anaerobranca gottschalkii and Paenibacillus macerans were studied as follows. The optimal temperature, p H and the ratio of donor(β-CD) and acceptor(VC) for AA-2G production was 35°C, 5.0 and 1:1 for all enzymes. After 24 h of incubation, the reaction reached equilibrium. The production of AA-2G was 41.2, 35.7, 28.9 and 19.5 g×L-1 with enzyme concentration of 250, 500, 500, 1500 U per gram β-CD respectively produced by CGTases from B. stearothermophilus NO2, B. circulans 251, A. gottschalkii and P. macerans.(3) In order to study the effect of key amino acid residues of CGTase on AA-2G production, amino acid sequences of various CGTases were aligned and the Phe228 in P. macerans CGTase, near to the key catalytic amino acid Asp229, was found to be remarkably discriminated with that of other CGTases. Site-directed mutagenesis of Phe228 was performed to obtain two mutant F228 M and F228 V. Under their individual optimal conditions, the production of AA-2G was 24.8 and 24.0 g×L-1 for F228 M and F228 V, which was 27.2% and 23.1% higher than that of the wild-type CGTase(19.5 g×L-1). Compared with that of wildtype CGTase, the Km value of mutant F228 M and F228 V towards VC decreased by 13.9% and 10.5%, whereas the kcat/Km value increased by 61.7% and 52.9%; the Km value of mutant F228 M and F228 V with β-CD were approximately the same as that of the wild-type CGTase, while the kcat/Km value increased by 34.7% and 40.8%. The transglycosylation activities of CGTases were studied in detail. Mutant F228 M and F228 V showed disproportionation activity increased by 19.4% and 21.2%.(4) CGTase was displayed on the cell surface of S. cerevisiae EBY100, obtaining S.cerevisiae EBY100/p YD1-cgt. The activity of displayed C GTase reached 0.5 U×m L-1 in 48 h fermentation using YPG culture medium with 20% galactose as sole carbon source and inducer at 25°C. The displayed CGTase showed more than 75% of its initial activity after incubation at 40°C for 24 h and showed more than 80% of its initial activity after incubation at the p H range of 6.0-8.0 for 24 h. The concentration of AA-2G produced by the surface displayed CGTase was 2.9 g×L-1 at its optimal transformation conditions of 30°C and p H 4.5, which was 37% higher than that produced by free C GTase(2.1 g×L-1).