title: Ultra-stable phosphoglucose isomerase through immobilization of cellulose-binding module-tagged thermophilic enzyme on low-cost high-capacity cellulosic adsorbent.
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 1.1×10(9) 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.
authors.affiliation: Dept. of Biological Systems Engineering, Virginia Polytechnic Institute and State University, 210-A Seitz Hall, Blacksburg, VA 24061, USA.
authors.forename: Suwan
authors.initials: S
authors.lastname: Myung
authors.forename: Xiao-Zhou
authors.initials: XZ
authors.lastname: Zhang
authors.forename: Y-H Percival
authors.initials: YH
authors.lastname: Zhang
mesh_terms: D002482:Cellulose / Q000737:chemistry*; D003001:Cloning, Molecular; D048013:Acetivibrio thermocellus / Q000201:enzymology; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005956:Glucose-6-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D002482:Cellulose; D005956:Glucose-6-Phosphate Isomerase