An ATP regeneration system is a coupled enzymatic reaction that constantly produces ATP from ADP and a secondary phosphate donor. ATP then serves as the phosphate donor for the main enzymatic reaction, e.g. the phosphorylation of a nucleoside. The incorporation of an ATP regeneration system offers several advantages:

1. The presence of a constant amount of ATP as co-substrate shifts the reaction equilibrium towards the desired product, therefore higher yields can be achieved in shorter time.
2. Less by-product ADP is present in the reaction, therefore possible enzyme inhibition effects are minimized.
3. Lower ATP concentrations are sufficient to complete the reaction, therefore purification is greatly simplified and costs can be saved.

There are several ATP regeneration systems that each consist of an enzyme and a phosphate donor, for example acetate kinase/acetyl phosphate (AcK/AcP), pyruvate kinase/phosphoenolpyruvate (PK/PEP) or polyphosphate kinase/polyphosphate (PPK/PolyP). Optimal reaction conditions, e.g. moderate or elevated reaction temperatures, depend on the organism the biocatalyst was obtained from.

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