Engineering of Cyclodextrin Glycosyltransferase through a Size/Polarity Guided Triple-Code Strategy with Enhanced α-Glycosyl Hesperidin Synthesis Ability
Engineering of Cyclodextrin Glycosyltransferase through a Size/Polarity Guided Triple-Code Strategy with Enhanced α-Glycosyl Hesperidin Synthesis Ability
复制标题
通过大小/极性引导的三代码策略工程环糊精糖基转移酶，具有增强的α-糖基橙皮苷合成能力。
DOI：
10.1128/aem.01027-22
复制
发表时间：
2022-08-11
影响因子：
4.4
通讯作者：
Chen, Xiaolong
中科院分区：
文献类型：
--
作者：
Chen, Hanchi;Liu, Yi;Chen, Xiaolong
橙皮苷是一种富含于柑橘皮中的黄酮类化合物，可以用CGTase酶促糖基化，并显著改善其水溶性。然而，由野生型CGTase催化的反应是相当低效的，反映在差的生产速率和产率上。通过关注糖苷配基攻击步骤，选择七个残基进行诱变，以提高转糖基化效率。由于缺乏高通量筛选技术，关于所研究的反应，我们开发了一种大小/极性指导的三重编码策略，以减少库的大小。选择的残基被替换为三个合理选择的氨基酸，改变大小或极性，导致一个非常浓缩的文库，只有32个突变体进行筛选。25%的突变体被证明是阳性的，表明所构建的文库的质量高。最佳突变体Y217 F的转糖基化活性为935.7U/g，其k（cat）/K-mA是野生型的6.43倍。同源建模和对接计算表明，催化效率显着提高的来源是配体转移和结合效应的结合。本研究发展了一种新的半理性突变策略--大小/极性指导的三重编码策略，并将其应用于CGTase糖苷配基攻击位点的基因工程。筛选压力设定为提高橙皮苷糖苷合成能力，仅筛选32个突变体就获得8个阳性突变体。设计的文库的高质量证实了所开发策略的有效性，对未来的诱变研究具有潜在价值。并对积极作用的机制进行了解释。优化后的突变体的k（cat）/K-mA值提高了6.43倍，是一种上级全细胞催化剂，在橙皮苷合成中具有潜在的应用价值。筛选压力设定为提高橙皮苷糖苷合成能力，仅筛选32个突变体就获得8个阳性突变体。
Hesperidin, a flavonoid enriched in citrus peel, can be enzymatically glycosylated using CGTase with significantly improved water solubility. However, the reaction catalyzed by wild-type CGTase is rather inefficient, reflected in the poor production rate and yield. By focusing on the aglycon attacking step, seven residues were selected for mutagenesis in order to improve the transglycosylation efficiency. Due to the lack of high-throughput screening technology regarding to the studied reaction, we developed a size/polarity guided triple-code strategy in order to reduce the library size. The selected residues were replaced by three rationally chosen amino acids with either changed size or polarity, leading to an extremely condensed library with only 32 mutants to be screened. Twenty-five percent of the constructed mutants were proved to be positive, suggesting the high quality of the constructed library. Specific transglycosylation activity of the best mutant Y217F was assayed to be 935.7 U/g, and its k(cat)/K-mA is 6.43 times greater than that of the wild type. Homology modeling and docking computation suggest the source of notably enhanced catalytic efficiency is resulted from the combination of ligand transfer and binding effect. IMPORTANCE Size/polarity guided triple-code strategy, a novel semirational mutagenesis strategy, was developed in this study and employed to engineer the aglycon attacking site of CGTase. Screening pressure was set as improved hesperidin glucoside synthesis ability, and eight positive mutants were obtained by screening only 32 mutants. The high quality of the designed library confirms the effectiveness of the developed strategy is potentially valuable to future mutagenesis studies. Mechanisms of positive effect were explained. The best mutant exhibits 6.43 times enhanced k(cat)/K-mA value and confirmed to be a superior whole-cell catalyst with potential application value in synthesizing hesperidin glucosides.Size/polarity guided triple-code strategy, a novel semirational mutagenesis strategy, was developed in this study and employed to engineer the aglycon attacking site of CGTase. Screening pressure was set as improved hesperidin glucoside synthesis ability, and eight positive mutants were obtained by screening only 32 mutants.