UNLABELLED β-N-Acetylhexosaminidases have attracted interest particularly for oligosaccharide synthesis, but their use remains limited by the rarity of enzyme sources , low efficiency, and relaxed regioselectivity of transglycosylation. In this work, genes of 13 β-N-acetylhexosaminidases, including 5 from Bacteroides fragilis ATCC 25285, 5 from Clostridium perfringens ATCC 13124, and 3 from Bifidobacterium bifidum JCM 1254, were cloned and heterogeneously expressed in Escherichia coli The resulting recombinant enzymes were purified and screened for transglycosylation activity. A β-N-acetylhexosaminidase named BbhI, which belongs to glycoside hydrolase family 20 and was obtained from B. bifidum JCM 1254, possesses the bifunctional property of efficiently transferring both GalNAc and GlcNAc residues through β1-3 linkage to the Gal residue of lactose. The effects of initial substrate concentration, pH, temperature, and reaction time on transglycosylation activities of BbhI were studied in detail. With the use of 10 mM pNP-β-GalNAc or 20 mM pNP-β-GlcNAc as the donor and 400 mM lactose as the acceptor in phosphate buffer (pH 5.8), BbhI synthesized GalNAcβ1-3Galβ1-4Glc and GlcNAcβ1-3Galβ1-4Glc at maximal yields of 55.4% at 45°C and 4 h and 44.9% at 55°C and 1.5 h, respectively. The model docking of BbhI with lactose showed the possible molecular basis of strict regioselectivity of β1-3 linkage in β-N-acetylhexosaminyl lactose synthesis. IMPORTANCE Oligosaccharides play a crucial role in many biological events and therefore are promising potential therapeutic agents. However, their use is limited because large-scale production of oligosaccharides is difficult. The chemical synthesis requires multiple protecting group manipulations to control the regio- and stereoselectivity of glycosidic bonds. In comparison, enzymatic synthesis can produce oligosaccharides in one step by using glycosyltransferases and glycosidases. Given the lower price of their glycosyl donor and their broader acceptor specificity, glycosidases are more advantageous than glycosyltransferases for large-scale synthesis. β-N-Acetylhexosaminidases have attracted interest particularly for β-N-acetylhexosaminyl oligosaccharide synthesis, but their application is affected by having few enzyme sources, low efficiency, and relaxed regioselectivity of transglycosylation. In this work, we describe a microbial β-N-acetylhexosaminidase that exhibited strong transglycosylation activity and strict regioselectivity for β-N-acetylhexosaminyl lactose synthesis and thus provides a powerful synthetic tool to obtain biologically important GalNAcβ1-3Lac and GlcNAcβ1-3Lac.

中文翻译：

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通过双功能双歧杆菌的β-N-乙酰己糖胺酶的双功能转糖基化，高效，区域选择性地合成β-GalNAc/ GlcNAc-乳糖。

未贴标签的β-N-乙酰己糖胺酶已引起人们的特别是寡糖合成的兴趣，但其使用仍然受到酶源稀少，效率低和转糖基化的区域选择性宽松的限制。在这项工作中，克隆了13种β-N-乙酰基己糖胺酶的基因，其中包括来自脆弱拟杆菌（Bacteroides fragilis ATCC 25285）的5个，产气荚膜梭状芽胞杆菌（Clostridium perfringens）ATCC 13124的5个，以及双歧双歧杆菌JCM 1254的3个在大肠杆菌中的异源表达。并筛选转糖基化活性。得自双歧杆菌JCM 1254的属于糖苷水解酶家族20的β-N-乙酰基己糖胺酶BbhI具有通过β1-3键与乳糖的Gal残基有效转移GalNAc和GlcNAc残基的双功能性质。详细研究了初始底物浓度，pH，温度和反应时间对BbhI转糖基化活性的影响。通过在磷酸盐缓冲液（pH 5.8）中使用10 mMpNP-β-GalNAc或20 mMpNP-β-GlcNAc作为供体，400 mM乳糖作为受体，BbhI合成了GalNAcβ1-3Galβ1-4Glc和GlcNAcβ1-3Galβ1-4Glc在45°C和4 h时最大产率分别为55.4％和55°C和1.5 h时最大产率为44.9％。BbhI与乳糖的模型对接显示了β-N-乙酰基己糖胺乳糖合成中严格的β1-3键区域选择性的分子基础。重要信息寡糖在许多生物学事件中起着至关重要的作用，因此是有前途的潜在治疗剂。但是，由于难以大规模生产寡糖，因此其使用受到限制。化学合成需要多个保护基操纵以控制糖苷键的区域选择性和立体选择性。相比之下，酶促合成可以通过使用糖基转移酶和糖苷酶在一步骤中产生寡糖。鉴于其糖基供体的价格较低且其受体特异性更广，对于大规模合成而言，糖苷酶比糖基转移酶更具优势。β-N-乙酰基己糖胺酶特别是对于β-N-乙酰基己糖胺寡糖合成引起了兴趣，但是其应用受到酶源少，效率低和转糖基化的区域选择性放宽的影响。在这项工作中，