胃肠道微生物群成员产生α-1-岩藻糖苷酶,在粘膜中起关键作用,人乳,和膳食寡糖同化。这里,通过对母乳喂养的婴儿粪便微生物组的宏基因组分析,鉴定了36个编码属于糖基水解酶家族29(GH29)的推定α-1-岩藻糖苷酶的开放阅读框(ORF)。这些ORF中有22个显示出完整的编码序列,其推导的氨基酸序列与来自拟杆菌的α-1-岩藻糖苷酶具有最高的同一性,拟杆菌,Pocaeicolavulgatus,Phocaeicoladorei,gnavusRuminococcus,和副血链球菌.基于序列同源性,选择10个α-1-岩藻糖苷酶基因用于底物特异性表征。α-l-岩藻糖苷酶Fuc18,Fuc19A,Fuc35B,Fuc39和Fuc1584对存在于Lewis血液抗原和人乳寡糖(HMO)3-岩藻糖基乳糖中的α1,3/4连接的岩藻糖显示出水解活性。此外,Fuc1584还水解岩藻糖基-α-1,6-N-乙酰葡糖胺(6FN),N-聚糖的核心岩藻糖基化的组分。Fuc35A和Fuc193对来自H-2型Lewis血液抗原的α1,2/3/4/6键显示出活性,HMO和6FN。Fuc30仅在α1,6-连接的l-岩藻糖上显示活性,和Fuc5372显示对α1,2键的偏好。Fuc2358表现出广泛的底物特异性,从所有测试的游离组织血型抗原中释放l-岩藻糖,HMO,6FN。这种最新的酶还在携带乳-N-岩藻五糖II(Lea)和乳-N-岩藻五糖III(Lex)的糖缀合物中以及糖蛋白粘蛋白中显示出活性。Fuc18,Fuc19A,和Fuc39还从新糖蛋白和人α-1酸糖蛋白中去除l-岩藻糖。这些结果提供了对来自婴儿肠道微生物群的α-l-岩藻糖苷酶的巨大多样性的见解。因此支持岩藻糖基化聚糖对于塑造新生儿微生物群组成至关重要的假设。重要性α-1-岩藻糖基残基经常存在于许多相关的聚糖中,例如人乳寡糖(HMO),组织血型抗原(HBGA),和细胞表面糖缀合物受体上的表位。这些岩藻糖基化的聚糖参与许多哺乳动物的生理过程,包括病原体的粘附和免疫反应。在这些过程中调节l-岩藻糖含量可以提供关于分子相互作用的新见解和知识,并且可以帮助设计新的治疗策略。微生物α-1-岩藻糖苷酶是从游离寡糖和糖缀合物中去除α-1-岩藻糖基残基的外切糖苷酶,并且还可用于转糖基化反应以合成寡糖。在这项工作中,从母乳喂养婴儿的粪便样品的宏基因组中鉴定并表征了来自GH29家族的α-1-岩藻糖苷酶。这些酶对HMO显示出不同的底物特异性,HBGA,天然存在的糖蛋白,和新糖蛋白。这些来自母乳喂养婴儿肠道微生物群的新型糖苷酶,这导致了α-l-岩藻糖苷酶的良好来源,具有巨大的生物技术潜力。
The gastrointestinal microbiota members produce α-l-fucosidases that play key roles in mucosal, human milk, and dietary oligosaccharide assimilation. Here, 36 open reading frames (ORFs) coding for putative α-l-fucosidases belonging to glycosyl hydrolase family 29 (GH29) were identified through metagenome analysis of breast-fed infant fecal microbiome. Twenty-two of those ORFs showed a complete coding sequence with deduced amino acid sequences displaying the highest degree of identity with α-l-fucosidases from Bacteroides thetaiotaomicron, Bacteroides caccae, Phocaeicola vulgatus, Phocaeicola dorei, Ruminococcus gnavus, and Streptococcus parasanguinis. Based on sequence homology, 10 α-l-fucosidase genes were selected for substrate specificity characterization. The α-l-fucosidases Fuc18, Fuc19A, Fuc35B, Fuc39, and Fuc1584 showed hydrolytic activity on α1,3/4-linked fucose present in Lewis blood antigens and the human milk oligosaccharide (HMO) 3-fucosyllactose. In addition, Fuc1584 also hydrolyzed fucosyl-α-1,6-N-acetylglucosamine (6FN), a component of the core fucosylation of N-glycans. Fuc35A and Fuc193 showed activity on α1,2/3/4/6 linkages from H type-2, Lewis blood antigens, HMOs and 6FN. Fuc30 displayed activity only on α1,6-linked l-fucose, and Fuc5372 showed a preference for α1,2 linkages. Fuc2358 exhibited a broad substrate specificity releasing l-fucose from all the tested free histo-blood group antigens, HMOs, and 6FN. This latest enzyme also displayed activity in glycoconjugates carrying lacto-N-fucopentaose II (Lea) and lacto-N-fucopentaose III (Lex) and in the glycoprotein mucin. Fuc18, Fuc19A, and Fuc39 also removed l-fucose from neoglycoproteins and human α-1 acid glycoprotein. These results give insight into the great diversity of α-l-fucosidases from the infant gut microbiota, thus supporting the hypothesis that fucosylated glycans are crucial for shaping the newborn microbiota composition. IMPORTANCE α-l-Fucosyl residues are frequently present in many relevant glycans, such as human milk oligosaccharides (HMOs), histo-blood group antigens (HBGAs), and epitopes on cell surface glycoconjugate receptors. These fucosylated glycans are involved in a number of mammalian physiological processes, including adhesion of pathogens and immune responses. The modulation of l-fucose content in such processes may provide new insights and knowledge regarding molecular interactions and may help to devise new therapeutic strategies. Microbial α-l-fucosidases are exoglycosidases that remove α-l-fucosyl residues from free oligosaccharides and glycoconjugates and can be also used in transglycosylation reactions to synthesize oligosaccharides. In this work, α-l-fucosidases from the GH29 family were identified and characterized from the metagenome of fecal samples of breastfed infants. These enzymes showed different substrate specificities toward HMOs, HBGAs, naturally occurring glycoproteins, and neoglycoproteins. These novel glycosidase enzymes from the breast-fed infant gut microbiota, which resulted in a good source of α-l-fucosidases, have great biotechnological potential.