title: Thermophilic Thermotoga maritima ribose-5-phosphate isomerase RpiB: optimized heat treatment purification and basic characterization.
abstract: The open reading frame TM1080 from Thermotoga maritima encoding ribose-5-phosphate isomerase type B (RpiB) was cloned and over-expressed in Escherichia coli BL21 (DE3). After optimization of cell culture conditions, more than 30% of intracellular proteins were soluble recombinant RpiB. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 70°C and pH 6.5-8.0. Under its suboptimal conditions (60°C and pH 7.0), k(cat) and K(m) values were 540s(-1) and 7.6mM, respectively; it had a half lifetime of 71h, resulting in its turn-over number of more than 2×10(8)mol of product per mol of enzyme. This study suggests that it is highly feasible to discover thermostable enzymes from exploding genomic DNA database of extremophiles with the desired stability suitable for in vitro synthetic biology projects and produce high-purity thermoenzymes at very low costs.
authors.affiliation: Biological Systems Engineering Department, 304 Seitz Hall, Virginia Tech, Blacksburg, VA 24061, USA.
authors.forename: Fangfang
authors.initials: F
authors.lastname: Sun
authors.forename: Xiao-Zhou
authors.initials: XZ
authors.lastname: Zhang
authors.forename: Suwan
authors.initials: S
authors.lastname: Myung
authors.forename: Y-H Percival
authors.initials: YH
authors.lastname: Zhang
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli; D015870:Gene Expression; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D020124:Thermotoga maritima / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D019747:Aldose-Ketose Isomerases