title: One-step purification and immobilization of thermophilic polyphosphate glucokinase from Thermobifida fusca YX: glucose-6-phosphate generation without ATP.
abstract: The discovery of stable and active polyphosphate glucokinase (PPGK, EC 2.7.1.63) would be vital to cascade enzyme biocatalysis that does not require a costly ATP input. An open reading frame Tfu_1811 from Thermobifida fusca YX encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK) was overexpressed in Escherichia coli. Mg²⁺ was an indispensible activator. This enzyme exhibited the highest activity in the presence of 4 mM Mg²⁺ at 55°C and pH 9.0. Under its suboptimal conditions (pH 7.5), the k (cat) and K(m) values of CBM-PPGK on glucose were 96.9 and 39.7 s⁻¹ as well as 0.77 and 0.45 mM at 37°C and 50°C respectively. The thermoinactivation of CBM-PPGK was independent of its mass concentration. Through one-step enzyme purification and immobilization on a high-capacity regenerated amorphous cellulose, immobilized CBM-PPGK had an approximately eightfold half lifetime enhancement (i.e., t(1/2) = 120 min) as compared to free enzyme at 50°C. To our limited knowledge, this enzyme was the first thermostable PPGK reported. Free PPGK and immobilized CBM-PPGK had total turnover number values of 126,000 and 961,000 mol product per mol enzyme, respectively, suggesting their great potential in glucose-6-phosphate generation based on low-cost polyphosphate.
authors.affiliation: Biological Systems Engineering Department, Virginia Polytechnic Institute and State University (Virginia Tech), 210-A Seitz Hall, Blacksburg, VA 24061, USA.
authors.forename: Hehuan
authors.initials: H
authors.lastname: Liao
authors.forename: Suwan
authors.initials: S
authors.lastname: Myung
authors.forename: Y-H Percival
authors.initials: YH
authors.lastname: Zhang
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001709:Biotechnology / Q000379:methods; D002482:Cellulose / Q000378:metabolism; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000096:biosynthesis; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D019298:Glucose-6-Phosphate; D000255:Adenosine Triphosphate; D002482:Cellulose; D010770:Phosphotransferases; C024812:polyphosphate-glucose phosphotransferase