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

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97g/L,纯度约为97%,其中含有纤维二糖(16.8wt%)、纤维三糖(49.8wt%)、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(WSH004)、副干酪乳杆菌(WSH005)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。

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.