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Ultra‐stable phosphoglucose isomerase through immobilization of cellulose‐binding module‐tagged thermophilic enzyme on low‐cost high‐capacity cellulosic adsorbent
Author(s) -
Myung Suwan,
Zhang XiaoZhou,
Percival Zhang Y.H.
Publication year - 2011
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.606
Subject(s) - glucose 6 phosphate isomerase , clostridium thermocellum , thermophile , chemistry , cellulose , enzyme , michaelis–menten kinetics , biotransformation , adsorption , bioreactor , biochemistry , immobilized enzyme , escherichia coli , chromatography , enzyme assay , organic chemistry , cellulase , gene
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.