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.