期刊
BIOTECHNOLOGY LETTERS
卷 34, 期 7, 页码 1321-1326出版社
SPRINGER
DOI: 10.1007/s10529-012-0910-y
关键词
N-Acetylglucosamine-1-phosphate uridyltransferase; N-Acetylhexosamine 1-kinase; Alkaline phosphatase; Uridine 5 '-diphosphate N-acetylglucosamine
资金
- National Natural Science Foundation of China [31000369]
- China Postdoctoral Science Foundation [20090461238]
- Independent Innovation Foundation of Shandong University (IIFSDU) [2009GN038]
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 A degrees 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.
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