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

✍ Scribed by Suwan Myung; Xiao-Zhou Zhang; Y.-H. Percival Zhang

• Publisher: American Institute of Chemical Engineers
• Year: 2011
• Tongue: English
• Weight: 802 KB
• Volume: 27
• Category: Article
• ISSN: 8756-7938
• DOI: 10.1002/btpr.606

✦ Synopsis

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 (k~cat~) and Michaelis–Menten constant (K~m~) 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. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.