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.