Fructose-1,6-bisphosphatase from a hyper-thermophilic bacterium Thermotoga maritima: Characterization, metabolite stability, and its implications

Fructose-1,6-bisphosphatase gene from a hyperthermophilic bacterium Thermotoga maritima was cloned, and the recombinant protein was produced in E. coli, purified, and characterized. The fructose-1,6-bisphosphatase (FBPase) with a molecular mass of ca. 28 kDa was purified from the fusion protein cellulose-binding module (CBM)-intein-FBPase by affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage. The substrate fructose 1,6-bisphosphate was not stable at high temperatures, especially at high pHs. The degradation constants of fructose 1,6-bisphosphate, glucose-6-phosphate, and fructose-6-phosphate were determined at different temperatures (37, 60, and 80 degrees C) and pH 7.5 or 9.0. The k(cat) and K-m values of FBPase were 8.57 s(-1) and 0.04 mM at 60 degrees C, as well as 58.7 s(-1) and 0.12 mM at 80 degrees C. This enzyme was very stable at its suboptimal temperatures, with half-life times of ca. 1330 and 55.6h at 60 and 80 degrees C, respectively. At 60 degrees C, this enzyme had an estimated total turn-over number of 20,500,000 (mol product/mol enzyme) and weight-based total turn-over umber of 192,000 (kg product/kg enzyme), respectively. These results indicated that this enzyme would be a stable building block for cell-free synthetic pathway biotransformation (SyPaB) that can implement complicated biochemical reactions. In order to obtain high-yield desired products, we suggest that over-addition or over-expression of the enzymes responsible for converting easily degraded metabolites should be important to prevent unnecessary metabolite loss for in vitro or in vivo synthetic pathway design. (C) 2010 Elsevier Ltd. All rights reserved.