多酶级联反应合成能够促进肠道益生菌生长的纤维寡糖

Synthesis of cello-oligosaccharides which promotes the growth of intestinal probiotics by multi-enzyme cascade reaction

摘要:

聚合度2–6的可溶性纤维寡糖是一种具有多种生物功能的低聚糖，它能够促进双歧杆菌(Bifidobacteria)、副干酪乳杆菌(Lactobacillus paracei)等肠道益生菌的增殖，因此对人体肠道微生态具有调节作用。本研究通过在大肠杆菌中表达纤维寡糖磷酸化酶(cellodextrin phosphorylase,CDP)，构建Cc 01菌株，并与之前构建的COS 01菌株联合使用，建立了基于COS 01、Cc 01的三酶级联反应催化底物葡萄糖和蔗糖合成纤维寡糖反应体系。经过优化后，最终可溶性纤维寡糖的产量达到97 g/L，纯度约为97%，其中含有纤维二糖(16.8 wt%)、纤维三糖(49.8 wt%)、纤维四糖(16.4 wt%)、纤维五糖(11.5 wt%)和纤维六糖(5.5 wt%)。在纤维寡糖对益生菌株生长促进作用的测试中，以菊粉、低聚木糖、低聚果糖为基准，干酪乳杆菌(WSH 004)、副干酪乳杆菌(WSH 005)以及嗜酸乳杆菌(WSH 006)利用纤维寡糖(聚合度2–6)为碳源进行生长后，益生菌的生物量(OD₆₀₀)相比对照增加约2倍。该研究证明了三酶级联反应能够高效合成纤维寡糖，并表明聚合度2–6的纤维寡糖是一类具有促进肠道微生物增殖的功能性碳水化合物。

关键词:纤维寡糖;全细胞催化;磷酸化酶;功能性碳水化合物;肠道益生菌

Abstract:

Soluble cello-oligosaccharide with 2–6 oligosaccharide units is a kind of oligosaccharide with various biological functions, which can promote the proliferation of intestinal probiotics such as Bifidobacteria and Lactobacillus paracei. Therefore, it has a regulatory effect on human intestinal microbiota. In this study, a Cc 01 strain was constructed by expressing cellodextrin phosphorylase (CDP) in Escherichia coli. By combining with a previously constructed COS 01 strain, a three-enzyme cascade reaction system based on strains COS 01 and Cc 01 was developed, which can convert glucose and sucrose into cello-oligosaccharide. After optimization, the final titer of soluble cello-oligosaccharides with 2–6 oligosaccharide units reached 97 g/L, with a purity of about 97%. It contained cellobiose (16.8 wt%), cellotriose (49.8 wt%), cellotetrose (16.4 wt%), cellopentaose (11.5 wt%) and cellohexose (5.5 wt%). When using inulin, xylo-oligosaccharide and fructooligosaccharide as the control substrate, the biomass (OD₆₀₀) of Lactobacillus casei (WSH 004), Lactobacillus paracei (WSH 005) and Lactobacillus acidophilus (WSH 006) on cello-oligosaccharides was about 2 folds higher than that of the control. This study demonstrated the efficient synthesis of cello-oligosaccharides by a three-enzyme cascade reaction and demonstrated that the synthesized cello-oligosaccharides was capable of promoting intestinal microbial proliferation.

Key words:cello-oligosaccharides;whole-cell catalysis;phosphorylase;functional carbohydrates;intestinal probiotics