以CotC为分子载体在枯草芽孢杆菌表面展示海藻糖合酶

Surface Display of Tres Using Cot C as a Molecular Vector on Bacillus subtilis Spores

海藻糖是自然界中普遍存在的一种非还原性双糖,是一种极好的天然干燥剂和保鲜剂。海藻糖合酶能够催化α,α-1,4-糖苷键连接的麦芽糖直接转化为α,α-1,1-糖苷键连接的海藻糖,是生产海藻糖的首选。为获得具有良好展示效果的海藻糖合酶,将其高效稳定的展示于枯草芽孢杆菌芽孢表面,实验同时分别选取增强型绿色荧光蛋白(EGFP)和海藻糖合酶(Tres)作为模型蛋白,以来自枯草芽孢杆菌的芽孢衣壳蛋白Cot C作为枯草芽杆菌表面展示的锚定蛋白进行表面展示研究。利用流式细胞仪分析EGFP在芽孢表面展示的情况,结果表明芽孢衣壳蛋白Cot C可以将EGFP固定在芽孢的表面。然后将荧光蛋白基因egfp通过酶切替换为海藻糖合酶基因tres,将重组菌株使用p H7.5的缓冲液清洗并重悬,与底物浓度为30%的麦芽糖在50℃水浴条件下作用2h,反应产物利用HPLC检测,能够检测到海藻糖峰,通过计算得到的酶活为252U/ml。说明海藻糖合酶基因通过与芽孢衣壳蛋白Cot C融合后可被展示在芽孢的表面。

Trehalose is a ubiquitous non-reducing disaccharide in nature,and is an excellent natural drying agent and preservative. Trehalose synthase capable of catalyzing the maltose directly into trehalose,and is the preferred production of trehalose. To obtain trehalose synthase having good catalytic surface,which is displayed in a highly efficient and stable surface of Bacillus subtilis,at the same experiments were selected enhanced green fluorescent protein（ EGFP） and trehalose synthase（ Tres） as a model protein,to come from Bacillus subtilis spore coat protein Cot C as Bacillus subtilis anchored proteins displayed on the surface. Flow cytometry analysis of the situation in the spore surface display EGFP,the results showed that the capsid protein of Bacillus Cot C can EGFP fixed spore surface. Then replace fluorescent protein gene egfp and trehalose synthase gene tres. The recombinant strains was hang up using p H 7. 5 buffer suspension and the concentration of substrate for 30% of the maltose in 50℃ water bath roling 2h. Reaction products were analyzed by HPLC and the enzymatic activity can be detected,the enzyme activity of trehalose by calculating reached 252 U / ml. This suggests that Cot C is associated with the outer part of the coat. Cot C can therefore be used as a molecular vehicle for spore surface display of exogenous proteins.