核苷酸糖鸟嘌呤5’-二磷酸-β-L-岩藻糖(5′-diphospho-β-L-fucose, GDP-L-岩藻糖)是岩藻糖基化寡糖植物和细菌生物合成途径的关键中间体。从脆弱拟杆菌(Bacteroides fragilis)的基因组DNA中克隆得到L-岩藻糖激酶/GDP-L-岩藻糖焦磷酸化酶(fucose pyrophosphorylase, FKP)基因,并与载体pET-22b (+)连接,在Escherichia coli BL21(DE3)中诱导表达。利用载体上组氨酸标签对重组酶分离纯化,并进行酶学性质分析。结果表明:FKP的最适温度和最适pH分别为45 ℃和7.5,该酶在pH 7.5~9.5之间很稳定,在4和12 ℃下孵育3 h,酶的初始活性保持在90％以上。最适反应条件下,重组FKP对底物岩藻糖的Km和Kcat分别为0.44 mmol/L、(31.49±2.5) s-1。45 ℃下,在底物L-岩藻糖摩尔浓度为10 mmol/L条件下反应11 h后,最终产生的GDP-L-岩藻糖为2.1 mmol/L。
Guanosine 5′-diphospho-β-L-fucose (GDP-L-Fuc) is a key intermediate for fucosylated oligosaccharides biosynthesis in plants and bacteria. In this study, a gene encoding L-fucose kinase/GDP-L-fucose pyrophosphorylase (FKP) from Bacteroides fragilis was cloned into pET-22b(+) and expressed in Escherichia coli BL21(DE3). The purified recombinant FKP had the maximum activity at 45 ℃ and 7.5. The enzyme was stable at between pH 7.5 and 9.5 and at 4 ℃ and 12 ℃. Under the optimal reaction conditions, its Km and Kcat were 0.44 mmol/L and (31.49±2.5) s-1, respectively. With FKP catalyzing at 45 ℃ for 11 h, 2.1 mmol/L of GDP-L-fucose from 10 mmol/L of L-fucose was finally synthesized.
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