Abstract：
2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is a stable derivative of L-ascorbic acid (L-AA), which has been widely used in food and cosmetics industries. Sugar molecules, such as glucose and maltose produced by cyclodextrin glycosyltransferase (CGTase) during AA-2G synthesis may compete with L-AA as the acceptors, resulting in low AA-2G yield. Multiple sequence alignment combined with structural simulation analysis indicated that residues at positions 191 and 255 of CGTase may be responsible for the difference in substrate specificity. To investigate the effect of these two residues on the acceptor preference and the AA-2G yield, five single mutants Bs F191Y, Bs F255Y, Bc Y195F, Pm Y195F and Pm Y260F of three CGTases from Bacillus stearothermophilus NO2 (Bs), Bacillus circulans 251 (Bc) and Paenibacillus macerans (Pm) were designed for AA-2G synthesis. Under optimal conditions, the AA-2G yields of the mutants Bs F191Y and Bs F255Y AA-2G were 34.3% and 7.9% lower than that of Bs CGTase, respectively. The AA-2G yields of mutant Bc Y195F, Pm Y195F and Pm Y260F were 45.8%, 36.9% and 12.6% higher than those of wild-type CGTases, respectively. Kinetic studies revealed that the residues at positions 191 and 255 of the three CGTases were F, which decreased glucose and maltose specificity and increased L-AA specificity. This study not only proposes for the first time that the AA-2G yield can be improved by weakening the acceptor specificity of CGTase toward sugar byproducts, but also provides new insight on the modification of CGTase that catalyze the double-substrate transglycosylation reaction.
摘要：
2-O-α-D-吡喃葡萄糖基-L-抗坏血酸（AA-2G）是L-抗坏血酸（L-AA）的稳定衍生物，已广泛应用于食品和化妆品行业。糖分子,例如在AA-2G合成过程中由环糊精糖基转移酶（CGTase）产生的葡萄糖和麦芽糖可能与L-AA竞争作为受体，导致低AA-2G产量。多序列比对结合结构模拟分析表明CGTase位置191和255处的残基可能是底物特异性差异的原因。为了研究这两个残基对受体偏好和AA-2G产率的影响，五个单突变体BsF191Y,BsF255Y,BcY195F,来自嗜热脂肪芽孢杆菌NO2（Bs）的三种CGTase的PmY195F和PmY260F，设计了用于AA-2G合成的环状芽孢杆菌251（Bc）和拟状芽孢杆菌（Pm）。在最优条件下,突变体BsF191Y和BsF255YAA-2G的AA-2G产量比BsCGTase低34.3%和7.9%，分别。BcY195F突变体的AA-2G产量，PmY195F和PmY260F为45.8%，比野生型CGTases高36.9%和12.6%，分别。动力学研究表明，在位置191和255的三个CGTase的残基是F，这降低了葡萄糖和麦芽糖的特异性，增加了L-AA的特异性。这项研究不仅首次提出AA-2G产量可以通过削弱CGTase对糖副产物的受体特异性来提高。同时也为催化双底物转糖基反应的CGTase修饰提供了新的见解。
