One advantage of thermostable enzymes is the possibility to use a heat-treatment step to enhance protein purification. For TmTK and TmPK heat steps at 70 °C to 80 °C for 20 to 30 min were already described . To confirm the thermostability of all expressed enzymes, we analyzed the heat denaturation of the enzymes purified by affinity chromatography in a thermal shift assay at pH values of 7, 8 and 9 in the absence and presence of DTT. Indeed, no denaturation of the enzymes was detected up to 95 °C and during an additional 20 min incubation step at 95 °C (Figure ). Since the enzymes were shown to be hyperthermostable, they were reexpressed and purified by heat-treatment and affinity chromatography.