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Ultra-stable phosphoglucose isomerase through immobilization of cellulose-binding module-tagged thermophilic enzyme on low-cost high-capacity cellulosic adsorbent

Authors

  • Suwan Myung,

    1. Dept. of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 210-A Seitz Hall, Blacksburg, VA 24061
    2. Institute for Critical Technology and Applied Science (ICTAS), Virginia Polytechnic Institute and State University, Blacksburg, VA 24061
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  • Xiao-Zhou Zhang,

    1. Dept. of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 210-A Seitz Hall, Blacksburg, VA 24061
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  • Y.-H. Percival Zhang

    Corresponding author
    1. Dept. of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 210-A Seitz Hall, Blacksburg, VA 24061
    2. Institute for Critical Technology and Applied Science (ICTAS), Virginia Polytechnic Institute and State University, Blacksburg, VA 24061
    3. DOE BioEnergy Science Center (BESC), Oak Ridge, TN 37831
    • Dept. of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 210-A Seitz Hall, Blacksburg, VA 24061
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Abstract

One-step enzyme purification and immobilization were developed based on simple adsorption of a family 3 cellulose-binding module (CBM)-tagged protein on the external surface of high-capacity regenerated amorphous cellulose (RAC). An open reading frame (ORF) Cthe0217 encoding a putative phosphoglucose isomerase (PGI, EC 5.3.1.9) from a thermophilic bacterium Clostridium thermocellum was cloned and the recombinant proteins with or without CBM were over-expressed in Escherichia coli. The rate constant (kcat) and Michaelis–Menten constant (Km) of CBM-free PGI at 60°C were 2,765 s−1 and 2.89 mM, respectively. PGI was stable at a high protein concentration of 0.1 g/L but deactivated rapidly at low concentrations. Immobilized CBM (iCBM)-PGI on RAC was extremely stable at ∼60°C, nearly independent of its mass concentration in bulk solution, because its local concentration on the solid support was constant. iCBM-PGI at a low concentration of 0.001 g/L had a half-life time of 190 h, approximately 80-fold of that of free PGI. Total turn-over number of iCBM-PGI was as high as 1.1 × 109 mole of product per mole of enzyme at 60°C. These results suggest that a combination of low-cost enzyme immobilization and thermoenzyme led to an ultra-stable enzyme building block suitable for cell-free synthetic pathway biotransformation that can implement complicated biochemical reactions in vitro. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.

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