Laboratory of Biochemistry, Institut Químic de Sarrià, University Ramon Llull, 08017 Barcelona, Spain.
Int J Mol Sci. 2021 Mar 22;22(6):3230. doi: 10.3390/ijms22063230.
The health benefits of human milk oligosaccharides (HMOs) make them attractive targets as supplements for infant formula milks. However, HMO synthesis is still challenging and only two HMOs have been marketed. Engineering glycoside hydrolases into transglycosylases may provide biocatalytic routes to the synthesis of complex oligosaccharides. Lacto--biosidase from (LnbB) is a GH20 enzyme present in the gut microbiota of breast-fed infants that hydrolyzes lacto--tetraose (LNT), the core structure of the most abundant type I HMOs. Here we report a mutational study in the donor subsites of the substrate binding cleft with the aim of reducing hydrolytic activity and conferring transglycosylation activity for the synthesis of LNT from -nitrophenyl β-lacto--bioside and lactose. As compared with the wt enzyme with negligible transglycosylation activity, mutants with residual hydrolase activity within 0.05% to 1.6% of the wild-type enzyme result in transglycosylating enzymes with LNT yields in the range of 10-30%. Mutations of Trp394, located in subsite -1 next to the catalytic residues, have a large impact on the transglycosylation/hydrolysis ratio, with W394F being the best mutant as a biocatalyst producing LNT at 32% yield. It is the first reported transglycosylating LnbB enzyme variant, amenable to further engineering for practical enzymatic synthesis of LNT.
人乳寡糖 (HMOs) 的健康益处使其成为婴儿配方奶粉的理想补充剂。然而，HMO 的合成仍然具有挑战性，目前只有两种 HMO 被推向市场。将糖苷水解酶工程化为转糖苷酶可能为合成复杂寡糖提供生物催化途径。（LnbB）来源于母乳喂养婴儿的肠道微生物群，是一种 GH20 酶，可水解乳四糖（LNT），LNT 是最丰富的 I 型 HMOs 的核心结构。在这里，我们报告了底物结合裂隙供体部位的突变研究，目的是降低水解活性并赋予转糖苷活性，用于从 -硝基苯 β-乳 - 内酰 - 苷和乳糖合成 LNT。与野生型酶几乎没有转糖苷活性的 wt 酶相比，残留水解酶活性在野生型酶的 0.05%至 1.6%范围内的突变体导致 LNT 产率在 10%至 30%范围内的转糖苷酶。位于催化残基旁边的 -1 亚基的色氨酸 394 的突变对转糖苷/水解比有很大影响，W394F 是产生 LNT 的最佳突变体，产率为 32%。这是第一个报道的转糖苷 LnbB 酶变体，可进一步工程化用于 LNT 的实际酶法合成。
