Facile synthesis of glucose-1-phosphate from starch by Thermus caldophilus GK24 α-glucan phosphorylase

Jungdon Bae, DuckHee Lee, Dooil Kim, Soo Jin Cho, Eun Park Jung, Sukhoon Koh, Joongsu Kim, Bo Hyun Park, Yongseok Choi, Hyun Jae Shin, Suk In Hong, Dae Sil Lee

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11 Citations (Scopus)

Abstract

The glgP gene encoding α-glucan phosphorylase (α-GP) from the thermopile Thermus caldophilus GK24 has been identified, cloned, and overexpressed in Escherichia coli and used to synthesize d-glucose-1-phospate (G1P) from an inexpensive starch. The enzyme, purified 6.5-fold, was isolated in 31% yield from the transformed E. coli, and gave a single band. The purified enzyme may exist as a homohexamer with an apparent molecular mass of a 550 kDa molecule, consisting of 90 kDa per subunit. The optimal pH and temperature were 7.0 and 70°C in the α-GP reaction with starch producing G1P. Soluble starch (amylopectin, amylose) turned out to be a better substrate giving a higher yield of G1P than α-1,6-branched α-1,4-glucans (glycogen, potato starch, etc.). As a result, G1P was obtained in a good yield (47%, w/w) from the reaction containing 5% (w/v) soluble starch in 0.7 M potassium phosphate at pH 7.0. T. caldophilus α-GP shows a high tolerance (up to 0.7 M) of potassium phosphate and plays a critical role in shifting the reaction equilibrium in favor of G1P synthesis. The G1P product can be purified simply by ethanol precipitation, after removing the unreacted starch and inorganic phosphate by activated charcoal and magnesium acetate precipitation. It is concluded that T. caldophilus α-GP readily utilized in large scale synthesis of G1P.

Original languageEnglish
Pages (from-to)3707-3713
Number of pages7
JournalProcess Biochemistry
Volume40
Issue number12
DOIs
Publication statusPublished - 2005 Dec 1
Externally publishedYes

Keywords

  • α-Glucan phosphorylase
  • Enzymatic synthesis
  • Glucose-1-phosphate
  • Glycogen
  • Starch
  • Thermus caldophilus

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry
  • Engineering (miscellaneous)
  • Industrial and Manufacturing Engineering

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  • Cite this

    Bae, J., Lee, D., Kim, D., Cho, S. J., Jung, E. P., Koh, S., Kim, J., Park, B. H., Choi, Y., Shin, H. J., Hong, S. I., & Lee, D. S. (2005). Facile synthesis of glucose-1-phosphate from starch by Thermus caldophilus GK24 α-glucan phosphorylase. Process Biochemistry, 40(12), 3707-3713. https://doi.org/10.1016/j.procbio.2005.05.007