Enhanced 2- O-α-d-Glucopyranosyl-l-ascorbic Acid Synthesis through Iterative Saturation Mutagenesis of Acceptor Subsite Residues in Bacillus stearothermophilus NO2 Cyclodextrin Glycosyltransferase.
Enhanced 2- O-α-d-Glucopyranosyl-l-ascorbic Acid Synthesis through Iterative Saturation Mutagenesis of Acceptor Subsite Residues in Bacillus stearothermophilus NO2 Cyclodextrin Glycosyltransferase.
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DOI：
10.1021/acs.jafc.8b03080
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发表时间：
2018-08
影响因子：
6.1
通讯作者：
Xiumei Tao;Tianmu Wang;Lingqia Su;Jing Wu
中科院分区：
文献类型：
--
作者：
Xiumei Tao;Tianmu Wang;Lingqia Su;Jing Wu
L-抗坏血酸（l-AA）衍生物2- O-α-d-吡喃葡萄糖基-L-抗坏血酸（AA-2G）的合成收率低，限制了其在食品工业中的应用。本研究提高了嗜热脂肪芽孢杆菌NO2环糊精糖基转移酶（CGTase）合成AA-2G的产率。选择在催化残基Glu 253的10 nm内显示≤30%保守性并位于受体亚位点的9个残基进行迭代饱和诱变。最好的突变体K228 R/M230 L在麦芽糖糊精作为葡萄糖基供体的情况下产生的AA-2G产量比其亲本野生型产生的产量更高。突变体K228 R/M230 L的l-AA Km值降低了35%，而kcat/ Km值增加了2.69倍。动力学分析表明，K228 R/M230 L对L-AA的专一性增强。这些结果表明受体亚位点残基在受体底物特异性中起重要作用。突变体K228 R/M230 L提供了最高的AA-2G浓度（211 g L-1，624 mM），迄今为止报道的反应条件优化后。
Low synthesis yields of the l-ascorbic acid (l-AA) derivative 2- O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) limit its application in the food industry. In this work, the AA-2G synthesis yield of Bacillus stearothermophilus NO2 cyclodextrin glycosyltransferase (CGTase) was improved. Nine residues within 10 Å of the catalytic residue Glu253 displaying ≤30% conservation and located in the acceptor subsite were selected for iterative saturation mutagenesis. The best mutant, K228R/M230L, produced a higher AA-2G yield with maltodextrin as the glucosyl donor than that produced by its parent wild-type. The l-AA Km values of the mutant K228R/M230L decreased by 35%, whereas the kcat/ Km increased by 2.69-fold. Kinetic analysis indicated that K228R/M230L displayed enhanced l-AA specificity. These results demonstrate that acceptor subsite residues play an important role in acceptor substrate specificity. Mutant K228R/M230L afforded the highest AA-2G concentration (211 g L-1, 624 mM) reported to date after optimization of the reaction conditions.