National Glycoengineering Research Center, State Key Laboratory of Microbial Technology, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan, People's Republic of China.
National Glycoengineering Research Center, State Key Laboratory of Microbial Technology, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Jinan, People's Republic of China
Appl Environ Microbiol. 2018 Jun 18;84(13). doi: 10.1128/AEM.00071-18. Print 2018 Jul 1.
Gut bacteria provide a rich source of glycosidases that can recognize and/or hydrolyze glycans for nutrition. Interestingly, some glycosidases have also been found to catalyze transglycosylation reactions and thus can be used for oligosaccharide synthesis. In this work, six putative and one known -α-sialidase genes-three from NCTC9343, three from ATCC 13124, and one known from JCM1254-were subjected to gene cloning and heterogeneous expression in The recombinant enzymes were purified, characterized for substrate specificity, and screened for transglycosylation activity. A sialidase, named BfGH33C, from NCTC9343 was found to possess excellent transglycosylation activity for the synthesis of sialylated human milk oligosaccharide. The native BfGH33C was a homodimer with a molecular weight of 113.6 kDa. The and values for 4-methylumbelliferyl -acetyl-α-d-neuraminic acid and sialic acid dimer were determined to be 0.06 mM and 283.2 s, and 0.75 mM and 329.6 s, respectively. The enzyme was able to transfer sialyl from sialic acid dimer or oligomer to lactose with high efficiency and strict α2-6 regioselectivity. The influences of the initial substrate concentration, pH, temperature, and reaction time on transglycosylation were investigated in detail. Using 40 mM sialic acid dimer (or 40 mg/ml oligomer) and 1 M lactose (pH 6.5) at 50°C for 10 min, BfGH33C could specifically produce 6'-sialyllactose, a dominant sialylated human milk oligosaccharide, at a maximal conversion ratio above 20%. It provides a promising alternative to the current chemical and enzymatic methods for obtaining sialylated oligosaccharides. Sialylated human milk oligosaccharides are significantly beneficial to the neonate, as they play important roles in supporting resistance to pathogens, gut maturation, immune function, and brain and cognitive development. Therefore, access to the sialylated oligosaccharides has attracted increasing attention both for the study of saccharide functions and for the development of infant formulas that could mimic the nutritional value of human milk. Nevertheless, nine-carbon sialic acids are rather complicated for the traditional chemical modifications, which require multiple protection and deprotection steps to achieve a specific glycosidic bond. Here, the -α-sialidase BfGH33C synthesized 6'-sialyllactose in a simple step with high transglycosylation activity and strict regioselectivity. Additionally, it could utilize oligosialic acid, which was newly prepared in an easy, economical way to reduce the substrate cost, as a glycosyl donor. All the studies laid a foundation for the practical use of BfGH33C in large-scale synthesis of sialylated oligosaccharides in the future.
肠道细菌为糖苷酶提供了丰富的来源，这些糖苷酶可以识别和/或水解聚糖以获取营养。有趣的是，一些糖苷酶也被发现能催化转糖基反应，因此可用于寡糖合成。在这项工作中，对来自 NCTC9343 的三个、来自 ATCC 13124 的三个和一个已知的 -α-唾液酸酶基因进行了基因克隆和异源表达。重组酶被纯化、鉴定为具有底物特异性，并筛选其转糖基活性。来自 NCTC9343 的唾液酸酶 BfGH33C 被发现具有极好的转糖基活性，可用于合成唾液酸化的人乳寡糖。天然的 BfGH33C 是一个分子量为 113.6 kDa 的同源二聚体。4-甲基伞形酮 -N-乙酰基-α-D-神经氨酸和唾液酸二聚体的 和 值分别为 0.06 mM 和 283.2 s，以及 0.75 mM 和 329.6 s。该酶能够高效且严格地以 α2-6 键特异性地将唾液酸从唾液酸二聚体或低聚糖转移至乳糖上。详细研究了初始底物浓度、pH 值、温度和反应时间对转糖基的影响。在 50°C 下，使用 40 mM 唾液酸二聚体（或 40 mg/ml 低聚糖）和 1 M 乳糖（pH 6.5）反应 10 min，BfGH33C 可特异性产生 6'-唾液酸乳糖，一种主要的唾液酸化人乳寡糖，转化率超过 20%。这为获得唾液酸化寡糖提供了一种有前途的替代当前化学和酶法的方法。唾液酸化的人乳寡糖对新生儿有显著的益处，因为它们在支持抵抗病原体、肠道成熟、免疫功能以及大脑和认知发育方面发挥着重要作用。因此，获得唾液酸化寡糖不仅引起了对糖功能的研究的关注，也引起了对能够模拟人乳营养价值的婴儿配方的关注。然而，九碳唾液酸对于传统的化学修饰来说相当复杂，这需要多个保护和脱保护步骤才能实现特定的糖苷键。在这里，-α-唾液酸酶 BfGH33C 以简单的一步合成 6'-唾液酸乳糖，具有高转糖基活性和严格的区域选择性。此外，它可以利用新以简单、经济的方式制备的寡唾液酸作为糖基供体。所有的研究都为 BfGH33C 在未来大规模合成唾液酸化寡糖的实际应用奠定了基础。
