右旋糖酐是由α-1,6键连接的葡萄糖聚合物,不同分子质量的右旋糖酐有不同的应用价值。右旋糖酐T2000是重均分子质量为2 MDa左右的葡聚糖,可以作为食品添加剂使用。作为介质可以分离血液中的各类蛋白,用于医学检测,是一种高端多糖产品。目前采用酶法定向合成右旋糖酐T2000的研究报道甚少。该文将来源于肠膜状明串珠菌0326的右旋糖酐蔗糖酶和来源于短杆菌的右旋糖酐酶通过连接肽进行连接,设计构建特异性融合酶,旨在达到一步法定向制备右旋糖酐T2000。并成功在大肠杆菌中得到表达,构建具有双酶活性的融合酶。研究结果得到,融合酶中右旋糖酐蔗糖酶的最适pH值是5.5、最适温度是30 ℃;融合酶中右旋糖酐酶的最适pH值是9.0、最适温度是50 ℃。以0.1 g/mL的蔗糖溶液作为底物,添加终酶活力为5 U/mL的融合酶,在25 ℃,120~180 r/min,反应24 h,分离纯化后成功得到重均分子质量为2 MDa左右的右旋糖酐,产率为32.6%。研究结果为高分子多糖的酶法定向制备提供了新的途径。
Dextran is a glucose polymer linked by α-1,6 bonds.Different molecular weight dextrans have different applications.Dextran T2000 is a dextran with a heavy average molecular weight of around 2 MDa.It can be used as a food additive.T2000 is a high-end polysaccharide product.It can be used in medical testing to separate various types of proteins in blood.Currently, there are few reports on the enzymatic directed synthesis of dextran T2000.In this paper, the dextransucrase derived from Leuconostoc mesenteroides 0326 and the dextranase BMdex derived from Brevibacterium fuscum were linked by a linker.This study designed and constructed the specific fusion enzyme, achieving a one-step method for the direct preparation of dextran T2000.A fusion enzyme with dual enzyme activity was constructed and successfully expressed in Escherichia coli.The optimal pH and temperature for dextransucrase in the fusion enzyme were 5.5 and 30 ℃.The optimal pH and temperature for dextranase in the fusion enzyme were 9.0 and 50 ℃.0.1 g/mL sucrose solution was used as substrate and fusion enzyme with a final enzyme activity of 5 U/mL was added.The reaction was carried out at 25 ℃ for 24 hours at 120-180 r/min.The reaction solution was successfully isolated and purified to obtain dextran with a heavy average molecular mass of about 2 MDa.And the reaction yield could reach 32.6%.The results of the study provided a new pathway for the enzyme-directed preparation of high-molecular polysaccharides.
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