Table 5.
Conditions for performing the poly-enzymatic cascade with recombinant thermophilic and mesophilic enzymes with various heterocyclic bases.
| Nucleoside | D-Rib Conc., mM | ATP Conc., mM | Base, 0.5 mM |
Enzyme Ratio, Units | Max. Nucleoside Content, % (LC MS) | ||
|---|---|---|---|---|---|---|---|
| RK | PPM | PNP | |||||
| Thermus thermophilus | |||||||
| Ribo-8azaGua (8) | 2 | 2.5 | 8-Aza-Gua | 0.4 * | 0.1 | 0.4 ** | 81 (24 h) |
| Ribo-Allop (9) | Allop | 0.4 * | 0.1 | 0.4 ** | 73 (8 h) | ||
| Escherichia coli | |||||||
| Ribo-8azaGua (8) | 2 | 2.5 | 8-Aza-Gua | 0.2 | 0.1 | 0.4 | 86 (24 h) |
| Ribo-Allop (9) | Allop | 0.2 | 0.1 | 0.4 | 96 (24 h) | ||
* Ribokinase from Thermus species 2.9 (TspRK, gene QT17_05185) [23] was used. ** Purine nucleoside phosphorylase I (TthPNPI) was used.