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Applied Microbiology and Biotechnology

, Volume 93, Issue 3, pp 1109–1117 | Cite as

One-step purification and immobilization of thermophilic polyphosphate glucokinase from Thermobifida fusca YX: glucose-6-phosphate generation without ATP

  • Hehuan Liao
  • Suwan Myung
  • Y.-H. Percival Zhang
Biotechnologically Relevant Enzymes and Proteins

Abstract

The discovery of stable and active polyphosphate glucokinase (PPGK, EC 2.7.1.63) would be vital to cascade enzyme biocatalysis that does not require a costly ATP input. An open reading frame Tfu_1811 from Thermobifida fusca YX encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK) was overexpressed in Escherichia coli. Mg2+ was an indispensible activator. This enzyme exhibited the highest activity in the presence of 4 mM Mg2+ at 55°C and pH 9.0. Under its suboptimal conditions (pH 7.5), the k cat and K m values of CBM-PPGK on glucose were 96.9 and 39.7 s−1 as well as 0.77 and 0.45 mM at 37°C and 50°C respectively. The thermoinactivation of CBM-PPGK was independent of its mass concentration. Through one-step enzyme purification and immobilization on a high-capacity regenerated amorphous cellulose, immobilized CBM-PPGK had an approximately eightfold half lifetime enhancement (i.e., t 1/2 = 120 min) as compared to free enzyme at 50°C. To our limited knowledge, this enzyme was the first thermostable PPGK reported. Free PPGK and immobilized CBM-PPGK had total turnover number values of 126,000 and 961,000 mol product per mol enzyme, respectively, suggesting their great potential in glucose-6-phosphate generation based on low-cost polyphosphate.

Keywords

Enzymatic building block Cascade enzyme reaction One-step purification and immobilization Polyphosphate glucokinase Synthetic pathway biotransformation (SyPaB) Thermobifida fusca 

Notes

Acknowledgments

This work was not possible without support from the Biological Systems Engineering Department of Virginia Tech, the Air Force Office of Scientific Research (FA9550-08-1-0145), the USDA Biodesign and Bioprocess Center, and DOE BESC to YPZ. HL was partially supported by the China Scholarship Council. SM was partially supported by ICTAS through ICTAS Scholarship. We were grateful for the genomic DNA sample provided by Dr. David Wilson at Cornell University.

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Hehuan Liao
    • 1
  • Suwan Myung
    • 1
    • 2
  • Y.-H. Percival Zhang
    • 1
    • 2
    • 3
    • 4
  1. 1.Biological Systems Engineering DepartmentVirginia Polytechnic Institute and State University (Virginia Tech)BlacksburgUSA
  2. 2.Institute for Critical Technology and Applied Science (ICTAS)Virginia Polytechnic Institute and State UniversityBlacksburgUSA
  3. 3.DOE BioEnergy Science Center (BESC)Oak RidgeUSA
  4. 4.Gate Fuels Inc.BlacksburgUSA

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