Thermostable continuous coupled assay for measuring glucose using glucokinase and glucose-6-phosphate dehydrogenase from the marine hyperthermophile Thermotoga maritima.
A novel, thermostable adaptation of the coupled-enzyme assay for monitoring glucose concentrations was developed for an optimal temperature of 85 degrees C. This is the first report of a thermostable glucostat from a marine hyperthermophile. The continuous assay, using glucokinase (Glk) and glucose-6-phosphate dehydrogenase (Gpd) from Thermotoga maritima, demonstrated robust activity over a range of temperatures (75-90 degrees C) and pH values (6.8- 8.5). Purified glucokinase had a monomeric molecular mass of 33.8kDa while that of glucose-6-phosphate dehydrogenase (D-glucose 6-phosphate:NADP oxidoreductase) was 57.5kDa. The high-temperature assay provided a method for directly assaying the activity of another hyperthermophilic enzyme, 1,4-beta-D-glucan glucohydrolase (GghA) from Thermotoga neapolitana. To provide a benchmark for protein-engineering experiments involving GghA, a three-enzyme continuous assay (performed at 85 degrees C), linking wild-type GghA, Glk, and Gpd, measured glucose produced from GghA's hydrolysis of cellobiose, one of GghA's secondary substrates. The assay established the kinetic behavior of wild-type GghA toward cellobiose and was used to screen for changes in the catalytic efficiency of variant GghA(s) induced by random mutagenesis. The assay's development will allow high-throughput screening of other thermostable glucose-producing enzymes, including those applicable to commercial biomass conversion.
McCarthy
James K
JK
Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, NJ 08901, USA.
O'Brien
Charles E
CE
Eveleigh
Douglas E
DE
D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005941:Glucokinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005947:Glucose / Q000032:analysis*; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020124:Thermotoga maritima / Q000201:enzymology*
D011994:Recombinant Proteins; D005954:Glucosephosphate Dehydrogenase; D005941:Glucokinase; D005947:Glucose