High Level Expression of XMP Aminase in Escherichia coli and Its Application for the Industrial Production of 5′-Guanylic Acid

Article abstract of DOI:10.1271/bbb.61.840

To improve the efficiency of the enzymatic conversion of 5′-xanthylic acid (XMP) to 5′-guanylic acid (GMP), we attempted to increase the activity of the conversion enzyme, XMP aminase (GMP synthetase) encoded by the guaA gene in Escherichia coli. By connecting the P_L promoter of λ phage, the SD sequence of trpL of E. coli, and ATG, at a suitable position upstream of the guaA gene, we obtained plasmid pPLA66. Sequencing of the nucleotides of the upstream region of the guaA gene on pPLA66 showed that the C-terminal region of the guaB gene, which encodes IMP dehydrogenase, was conserved and a short peptide consisted of 14 amino acids was coded. E. coli MP347/pPLA66 showed an increase in the activity of approximately 370 times when compared with that of the strain MM294, and the amount of the enzyme protein represented approx. 34% of the total cellular protein. Strain MP347/pPLA66 was cultivated in a 5-liter jar fermentor using a medium which contained mainly corn steep liquor. The culture broth had high XMP aminase activity. In the conversion reaction using mixed broths consisted of 600ml of XMP-fermentation broth of Corynebacterium ammoniagenes KY13203 and 30 ml of cultured broth of E. coli MP347/pPLA66, a surfactant, Nymeen S-215 and xylene were added to the reaction mixture to make the cell membrane permeable to nucleotides. After 23 h of the reaction, 70mg/ml (131 mM) of GMP·Na₂·7H₂O was accumulated from 83 mg/ml (155 mM) of XMP·Na₃ ·7H₂O, without addition of ATP. The molar conversion yield was approx. 85%. The facts that the cell membrane was treated to allow nucleotides to permeate and that the conversion reaction proceeded well enough in spite of a small amount of E. coli cells indicate ATP was regenerated from AMP by C. ammoniagenes cells and supplied to E. coli cells. Therefore, it was considered that the coupling reaction between these two kind of strains was established.

Full text of DOI:10.1271/bbb.61.840

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High Level Expression of XMP Aminase in Escherichia
coli and Its Application for the Industrial Production
of 5′-Guanylic Acid
a
ac
ad
ab
Tatsuro Fujio , Tatsunari Nishi , Seiga Ito & Akihiko Maruyama
a
Planning & Development Department/Food, Liquor & Food Division, 6-1-1 Ohtemachi,
Chiyoda-ku, Tokyo 100, Japan
b
Fine Chemicals/Biochemicals Dept., Medicals Research and Development Center,
Kyowa Hakko Kogyo Co., Ltd., 6-1-1 Ohtemachi, Chiyoda-ku, Tokyo 100, Japan
c
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahi-machi,
Machida-shi, Tokyo 194, Japan
d
Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 1188
Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka-ken, 411, Japan
Published online: 12 Jun 2014.
To cite this article: Tatsuro Fujio, Tatsunari Nishi, Seiga Ito & Akihiko Maruyama (1997) High Level Expression of
XMP Aminase in Escherichia coli and Its Application for the Industrial Production of 5′-Guanylic Acid, Bioscience,
Biotechnology, and Biochemistry, 61:5, 840-845, DOI: 10.1271/bbb.61.840
To link to this article: http://dx.doi.org/10.1271/bbb.61.840
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