Format

Send to

Choose Destination
See comment in PubMed Commons below
J Biotechnol. 2007 May 1;129(3):453-60. Epub 2007 Feb 9.

Production of N-acetyl-D-neuraminic acid by recombinant whole cells expressing Anabaena sp. CH1 N-acetyl-D-glucosamine 2-epimerase and Escherichia coli N-acetyl-D-neuraminic acid lyase.

Author information

1
Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan.

Abstract

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 42kDa by SDS-PAGE. The pH and temperature optima of the recombinant bGlcNAc 2-epimerase were pH 7.0 and 50 degrees C, respectively, and only needs 20mum ATP for maximal activity. The specific activity of bGlcNAc 2-epimerase (124Umg(-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 10.2gNeuAcl(-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 >8.0gNeuAcL(-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.

PMID:
17349707
DOI:
10.1016/j.jbiotec.2007.01.027
[Indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science
    Loading ...
    Support Center