Enhancing lactose recognition of a key enzyme in 2′-fucosyllactose synthesis: α-1,2-fucosyltransferase
Enhancing lactose recognition of a key enzyme in 2′-fucosyllactose synthesis: α-1,2-fucosyltransferase
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增强 2-2-岩藻糖基乳糖合成中关键酶的乳糖识别：α-1,2-岩藻糖基转移酶
DOI：
10.1002/jsfa.12224
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发表时间：
2022-10-04
影响因子：
4.1
通讯作者：
Zhou，Hongbo
中科院分区：
文献类型：
--
作者：
Liu，Wenxian;Tang，Shizhe;Zhou，Hongbo
背景2 ′-岩藻糖基乳糖是人乳中的代表性低聚糖，由于其强大的健康益处，如参与免疫调节、调节肠道植物群等，是新兴的和有前途的食品和药物成分。为了使2′-岩藻糖基乳糖的生产经济可行，已经开发了使用前体和途径酶的不同生物合成策略。α-1，2-岩藻糖基转移酶是这些策略中必不可少的一部分，但其严格的底物选择性和不理想的底物耐受性是限制生物合成的关键障碍之一。与野生型相比，突变体的催化效率增加了100倍。在体外酶催化条件下，2′-岩藻糖基乳糖的最高产量可达0.65 mol mol− 1乳糖，生产率为2.56 g mL−1h− 1。进一步的分析表明，突变体和底物之间的相互作用减少。结论α-1，2-岩藻糖基转移酶基因工程可用于缓解2′-岩藻糖基乳糖生物合成的限制，为阐明岩藻糖基转移酶的底物识别机制奠定理论基础。© 2022化学工业学会。
BACKGROUND2′‐Fucosyllactose, a representative oligosaccharide in human milk, is an emerging and promising food and pharmaceutical ingredient due to its powerful health benefits, such as participating in immune regulation, regulation of intestinal flora, etc.To enable economically viable production of 2′‐fucosyllactose, different biosynthesis strategies using precursors and pathway enzymes have been developed. The α‐1,2‐fucosyltransferases are an essential part involved in these strategies, but their strict substrate selectivity and unsatisfactory substrate tolerance are one of the key roadblocks limiting biosynthesis.RESULTSTo tackle this issue, a semi‐rational manipulation combining computer‐aided designing and screening with biochemical experiments were adopted. The mutant had a 100‐fold increase in catalytic efficiency compared to the wild‐type. The highest 2′‐fucosyllactose yield was up to 0.65 mol mol−1lactose with a productivity of 2.56 g mL−1h−1performed by enzymatic catalysisin vitro. Further analysis revealed that the interactions between the mutant and substrates were reduced. The crucial contributions of wild‐type and mutant to substrate recognition ability were closely related to their distinct phylotypes in terms of amino acid preference.CONCLUSIONIt is envisioned that the engineered α‐1,2‐fucosyltransferase could be harnessed to relieve constraints imposed on the bioproduction of 2′‐fucosyllactose and lay a theoretical foundation for elucidating the substrate recognition mechanisms of fucosyltransferases. © 2022 Society of Chemical Industry.