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|標題:||Production of N-acetyl-D-neuraminic acid by recombinant whole cells expressing Anabaena sp CH1N-acetyl-D-glucosamine 2-epimerase and Escherichia coli N-acetyl-D-neuraminic acid lyase||作者:||Lee, Y.C.
|關鍵字:||N-acetyl-D-glucosamine 2-epimerase;N-acetyl-D-neuraminic acid lyase;sialic acid production;epimerization;Anabaena sp.;d-glucosamine 2-epimerase;renin-binding-protein;sialic-acid;acetylneuraminic acid;porcine kidney;glcnac 2-epimerase;molecular-cloning;identification;purification;motif||Project:||Journal of Biotechnology||期刊/報告no：:||Journal of Biotechnology, Volume 129, Issue 3, Page(s) 453-460.||摘要:||
N-acetyl-D-neuraminic acid (NeuAc; sialic acid) is a precursor for the manufacture of many pharmaceutical drugs, such as anti-influenza virus agents. To develop a whole cell process for NeuAc production, genes of Anabaena sp. CH1 N-acetyl-D-glucosamine 2-epimerase (bage) and Escherichia coli N-acetyl-D-neuraminic acid lyase (nanA) were cloned and expressed in E. coli BL21 (DE3). The expressed bGlcNAc 2-epimerase was purified from the crude cell extract of IPTG-induced E. coli BL21 (DE3) (pET-bage) to homogeneity by nickel-chelate chromatography. The molecular mass of the purified bGlcNAc 2-epimerase was determined to be 42 kDa by SIDS-PAGE. The pH and temperature optima of the recombinant bGlcNAc 2-epimerase were pH 7.0 and 50 degrees C, respectively, and only needs 20 mu m ATP for maximal activity. The specific activity of bGlcNAc 2-epimerase (124U mg(-1) protein) for the conversion of N-acetyl-D-glucosamine to N-acetyl-D-manosamine was about four-fold higher than that of porcine N-acetyl-D-glucosamine 2-epimerase. A stirred glass vessel containing transformed E. coli cells expressing age gene from Anabaena sp. CH1 and NeuAc lyase gene from E. coli NovaBlue separately was used for the conversion of GlcNAc and pyruvate to NeuAc. A maximal productivity of 10.2 g NeuAc l(-1) h(-1) with 33.3% conversion yield from GlcNAc could be obtained in a 12-h reaction. The recombinant E. coli cells can be reused for more than eight cycles with a productivity of > 8.0 g NeuAc L-1 h(-1). In this process, the expensive activator, ATP, necessary for maximal activity of GlcNAc 2-epimerase in free enzyme system can be omitted. (c) 2007 Elsevier B.V. All rights reserved.
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