Efficient Hydrogen Production Using Enzymes of the Pentose Phosphate Pathway

Abstract

Combination of the enzymes of the pentose phosphate pathway with hydrogenase has been demonstrated to increase the hydrogen yield from glucose to nearly theoretical yield (12 H2/1 glucose). The instability of the commercially available mesophilic enzymes as well as that of the cofactor NADP+ is an obstacle for practical application of the method to hydrogen production. Improvement of enzyme and cofactor stability was pursued by employment of thermophilic enzymes and encapsulation of the enzymes in a gas-permeable matrix. To obtain thermophilic enzymes, the pentose phosphate pathway enzymes from the thermophile Thermotoga maritima are being cloned and expressed in Escherichia coli. Primers were designed for the cloning of the target genes. The genes encoding the two NADP-dependent dehydrogenases, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, were cloned into the vector PCR2.1 and expressed in E. coli. Production of hydrogen by enzymes in cell-free extracts of T. maritima, alone or in combination with Pyrococcus furiosus hydrogenase, was demonstrated. Studies were initiated on a novel method for practical implementation of enzymes for production of hydrogen, the incorporation of enzymes and cofactors into stable liposomes that will function as nanobioreactors. Hydrogenase and glucose dehydrogenase were encapsulated in liposomes and subsequently demonstrated to produce hydrogen.