Screening and characterization of an α-L-fucosidase from Bacteroides fragilis NCTC9343 for synthesis of fucosyl-N-acetylglucosamine disaccharides
Screening and characterization of an α-L-fucosidase from Bacteroides fragilis NCTC9343 for synthesis of fucosyl-N-acetylglucosamine disaccharides
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DOI：
10.1007/s00253-020-10759-w
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发表时间：
2020-07
影响因子：
5
通讯作者：
Peng Liu;Huaqin Zhang;Yuying Wang;Xiaodi Chen;Lan Jin;Li Xu;M. Xiao
中科院分区：
文献类型：
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作者：
Peng Liu;Huaqin Zhang;Yuying Wang;Xiaodi Chen;Lan Jin;Li Xu;M. Xiao
岩藻糖基-N-乙酰葡糖胺二糖存在于许多生物学上重要的寡糖中，如人乳寡糖、刘易斯糖抗原和细胞表面糖缀合物受体上的聚糖，因此在婴儿配方食品、益生元和药物应用中具有巨大的潜力。为了筛选岩藻糖基-N-乙酰氨基葡萄糖二糖的酶促合成催化剂，我们对脆弱拟杆菌（Bacteroides fragilis）NCTC 9343的12种推定的和1种已知的α-L-岩藻糖苷酶进行了序列分析，并构建了9种GH 29 α-L-岩藻糖苷酶的系统发育树。在此基础上，克隆了5个GH 29 A α-L-岩藻糖苷酶，并对其中4个进行了异源表达和转糖基化活性筛选，鉴定了一个GH 29 A α-L-岩藻糖苷酶（BF 3242），该酶以pNP αFuc为供体，GlcNAc为受体，合成Fuc-α-1，3/1，6-GlcNAc二糖混合物。研究了底物初始浓度、pH、温度和反应时间对转糖基化活性的影响。在0.05 U/mL酶、20 mMpNPαFuc和500 mM GlcNAc的钠缓冲液（pH 7.5）中，37 °C反应45 min，BF 3242高效合成Fuc-α-1，3/1，6-GlcNAc，最大产率为79.0%，1，3/1，6的比例为0.48。分子动力学模拟分析表明，BF 3242三维模型中的Loop-4（His 220-Ser 245）在整个热模拟过程中发生了显著的变化，可能是导致两种区域异构体比例在不同温度下发生变化的原因。本工作不仅为岩藻糖基-N-乙酰氨基葡萄糖二糖的酶促合成提供了潜在的合成工具，而且为糖苷酶催化转糖基化反应中区域异构产物的形成提供了可能性。关键点·脆弱拟杆菌NCTC 9343 α-L-岩藻糖苷酶的序列分析·获得具有高转糖基化活性的α-L-岩藻糖苷酶·解释温度影响两种区域异构产物比例的原因
AbstractFucosyl-N-acetylglucosamine disaccharides are present in many biologically important oligosaccharides, such as human milk oligosaccharides, Lewis carbohydrate antigens, and glycans on cell-surface glycoconjugate receptors, and thus have vast potential for infant formulas, prebiotics, and pharmaceutical applications. In this work, in order to screen biocatalysts for enzymatic synthesis of fucosyl-N-acetylglucosamine disaccharides, we performed sequence analysis of 12 putative and one known α-L-fucosidases ofBacteroides fragilisNCTC9343 and constructed a phylogenetic tree of the nine GH29 α-L-fucosidases. After that, five GH29A α-L-fucosidases were cloned, and four of them were successfully heterogeneous expressed and screened for transglycosylation activity, and a GH29A α-L-fucosidase (BF3242) that synthesized a mix of Fuc-α-1,3/1,6-GlcNAc disaccharides usingpNPαFuc as donor and GlcNAc as acceptor was characterized. The effects of initial substrate concentration, pH, temperature, and reaction time on its transglycosylation activity were studied in detail. Under the optimum conditions of 0.05 U/mL enzyme, 20 mMpNPαFuc, and 500 mM GlcNAc in sodium buffer (pH 7.5) at 37 °C for 45 min, BF3242 efficiently synthesized Fuc-α-1,3/1,6-GlcNAc at a maximum yield of 79.0% with the ratio of 0.48 for 1,3/1,6. The molecular dynamics simulation analysis revealed that Loop-4 (His220-Ser245) in the putative 3D model of BF3242 displayed significant changes throughout the thermal simulations, might being responsible for the changes in the ratio of two regioisomeric products at different temperatures. This work provided not only a potential synthetic tool for enzymatic synthesis of fucosyl-N-acetylglucosamine disaccharides but also a possibility for the formation of regioisomeric products in glycosidase-catalyzed transglycosylation.Key points•Sequence analysis of α-L-fucosidases of Bacteroides fragilis NCTC9343•Obtainment of an α-L-fucosidase with high transglycosylation activity•Explanation why temperature affected the ratio of two regioisomeric products