National Glycoengineering Research Center, Shandong University, B604 Luneng Keji Dasha, 29-1 Shanda Nanlu, Jinan, 250100 Shandong, People's Republic of China.
Biotechnol Lett. 2012 Jul;34(7):1321-6. doi: 10.1007/s10529-012-0910-y. Epub 2012 Mar 29.
The availability of uridine 5'-diphosphate N-acetylglucosamine (UDP-GlcNAc) is a prerequisite for the GlcNAc-transferase-catalyzed glycosylation reaction. UDP-GlcNAc has already been synthesized using an N-acetylhexosamine 1-kinase (NahK) and a GlcNAc-1-P uridyltransferase (truncated GlmU) and here, a fusion enzyme was constructed with truncated GlmU and NahK. After determination of the optimum catalytic condition (pH 8.0 at 40 °C), the fusion enzyme was used to synthesize UDP-GlcNAc in a single step with a yield of 88 % from GlcNAc, ATP and UTP. Furthermore, a simplified purification method was demonstrated using separation by gel filtration after by-product digestion with alkaline phosphatase. An overall yield of 77 % and a purity of over 90 % were achieved.
尿苷二磷酸 N-乙酰葡萄糖胺 (UDP-GlcNAc) 的可用性是 GlcNAc-转移酶催化的糖基化反应的前提条件。已经使用 N-乙酰己糖胺 1-激酶 (NahK) 和 GlcNAc-1-P 尿苷酰转移酶 (截短的 GlmU) 合成了 UDP-GlcNAc，此处构建了带有截短的 GlmU 和 NahK 的融合酶。在确定最佳催化条件（40°C 时 pH8.0）后，该融合酶用于一步合成 UDP-GlcNAc，产率为 88%，从 GlcNAc、ATP 和 UTP 开始。此外，通过碱性磷酸酶消化副产物后进行凝胶过滤分离，展示了一种简化的纯化方法。总产率为 77%，纯度超过 90%。
