Five GH29B α-1,3/4-l-fucosidases (EC 3.2.1.111) were investigated for their ability to catalyze the formation of the human milk oligosaccharide lacto-N-fucopentaose II (LNFP II) from lacto-N-tetraose (LNT) and 3-fucosyllactose (3FL) via transglycosylation. We studied the effect of pH on transfucosylation and hydrolysis and explored the impact of specific mutations using molecular dynamics simulations. LNFP II yields of 91 and 65% were obtained for the wild-type SpGH29^C and CpAfc2 enzymes, respectively, being the highest LNFP II transglycosylation yields reported to date. BbAfcB and BiAfcB are highly hydrolytic enzymes. The results indicate that the effects of pH and buffer systems are enzyme-dependent yet relevant to consider when designing transglycosylation reactions. Replacing Thr284 in BiAfcB with Val resulted in increased transglycosylation yields, while the opposite replacement of Val258 in SpGH29^C and Val289 CpAfc2 with Thr decreased the transfucosylation, confirming a role of Thr and Val in controlling the flexibility of the acid/base loop in the enzymes, which in turn affects transglycosylation. The substitution of an Ala residue with His almost abolished secondary hydrolysis in CpAfc2 and BbAfcB. The results are directly applicable in the enhancement of transglycosylation and may have significant implications for manufacturing of LNFP II as a new infant formula ingredient.

中文翻译：

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使用 GH29B α-1,3/4-l-岩藻糖苷酶改进岩藻糖基化母乳低聚糖 LNFP II 的酶促生产


研究了五种 GH29B α-1,3/4-l-岩藻糖苷酶 (EC 3.2.1.111) 催化从乳-N-四糖 (LNFP II) 形成人乳寡糖乳-N-岩藻糖五糖 II (LNFP II) 的能力（ LNT) 和 3-岩藻糖基乳糖 (3FL) 通过转糖基化。我们研究了 pH 对转岩藻糖基化和水解的影响，并利用分子动力学模拟探索了特定突变的影响。野生型SpGH29 ^C 和CpAfc2酶的LNFP II产率分别为91%和65%，是迄今为止报道的最高LNFP II转糖基产率。 BbAfcB 和 BiAfcB 是高度水解酶。结果表明，pH 和缓冲系统的影响是酶依赖性的，但在设计转糖基反应时需要考虑。用Val替换BiAfcB中的Thr284导致转糖基化产量增加，而用Thr替换SpGH29 ^C 和Val289 CpAfc2中的Val258则降低了转岩藻糖基化，证实了Thr和Val在控制酸的灵活性方面的作用/ 酶中的碱基环，进而影响转糖基化。用 His 取代 Ala 残基几乎消除了 CpAfc2 和 BbAfcB 中的二次水解。结果可直接应用于糖基转移的增强，并可能对 LNFP II 作为新婴儿配方奶粉成分的生产产生重大影响。