Sophoricoside glycosylated derivatives, especially long-chain glycosylated sophoricosides (LCGS), have greatly improved water solubility compared with sophoricoside. Here, cyclodextrin glycosyltransferase from Paenibacillus macerans (PmCGTase) was employed for sophoricoside glycosylation. Saturation mutagenesis of alanine 156, alanine 166, glycine 173, and leucine 174 was performed due to their nonconservative properties among α-, β-, and -CGTases with different product specificities. Variants L174P, A156V/L174P, and A156V/L174P/A166Y greatly improved the product specificity for LCGS. pH significantly affected the extent of glycosylation catalyzed by the variants. Further investigations revealed that the pH-regulated mechanism for LCGS synthesis mainly depends on a disproportionation route at a lower pH (pH 4) and a cyclization-coupling route at a higher pH (pH 8) and equivalent effects of cyclization-coupling and disproportionation routes at pH 5. Whereas short-chain glycosylated sophoricosides (SCGS) are primarily produced via disproportionation of maltodextrin at pH 4 and secondary disproportionation of LCGS at pH 8. At pH 5, SCGS synthesis mainly depends on a hydrolysis route by the wild type (WT) and a secondary disproportionation route by variant A156V/L174P/A166Y. Kinetics analysis showed a decreased K_m value of variant A156V/L174P/A166Y. Dynamics simulation results demonstrated that the improved LCGS specificity of the variant is possibly attributed to the enhanced affinity to long-chain substrates, which may be caused by the changes of hydrogen bond interactions at the –5, –6, and –7 subsites. Our results reveal a pH-regulated mechanism for product specificity of CGTase and provide guidance for engineering CGTase toward products with different sugar chain lengths.

IMPORTANCE The low water solubility of sophoricoside seriously limits its applications in the food and pharmaceutical industries. Long-chain glycosylated sophoricosides show greatly improved water solubility. Here, the product specificity of cyclodextrin glycosyltransferase (CGTase) for long-chain glycosylated sophoricosides was significantly affected by pH. Our results reveal the pH-regulated mechanism of the glycosylated product specificity of CGTase. This work adds to our understanding of the synthesis of long-chain glycosylated sophoricosides and provides guidance for exploring related product specificity of CGTase based on pH regulation.

与槐糖苷相比，槐糖苷糖基化衍生物，尤其是长链糖基化槐糖苷（LCGS），具有大大改善的水溶性。在这里，Macerans（Pm）中的环糊精糖基转移酶将CGTase）用于槐糖苷糖基化。由于丙氨酸156，丙氨酸166，甘氨酸173和亮氨酸174的非保守特性在具有不同产物特异性的α-，β-和-CGTase中进行了饱和诱变。L174P，A156V / L174P和A156V / L174P / A166Y变体大大提高了对LCGS的产品特异性。pH值显着影响变体催化的糖基化程度。进一步的研究表明，LCGS合成的pH调节机制主要取决于较低pH（pH 4）的歧化途径和较高pH（pH 8）的环化偶联途径以及环化偶联和歧化途径的等效作用在pH 5。而短链糖基化槐糖甙（SCGS）主要是通过在pH 4时麦芽糖糊精歧化和在pH 8中LCGS继发歧化而产生的。在pH 5时，SCGS的合成主要取决于野生型（WT）的水解途径和第二步变体A156V / L174P / A166Y的歧化途径。动力学分析显示下降变型A156V / L174P / A166Y的K _m值。动力学模拟结果表明，改进的LCGS特异性可能归因于对长链底物的亲和力增强，这可能是由–5，–6和–7亚位处氢键相互作用的变化引起的。我们的结果揭示了CGTase产品特异性的pH调节机制，并为针对不同糖链长度的产品工程化CGTase提供了指导。

重要信息槐槐糖苷的低水溶性严重限制了其在食品和制药行业中的应用。长链糖基化槐糖甙显示出大大改善的水溶性。在这里，环糊精糖基转移酶（CGTase）对长链糖基化槐糖苷的产物特异性受pH值的显着影响。我们的结果揭示了CGTase糖基化产物特异性的pH调节机制。这项工作增加了我们对长链糖基化槐糖苷合成的理解，并为基于pH调节探索CGTase相关产品特异性提供了指导。