# Improving the Phosphatase-Catalyzed Synthesis of 5'-Nucleotides: A Reaction Engineering Approach
## 3 Results and Discussion
However, a 30 \(\mathrm{\SIUnitSymbolMicro}\)/mL PhoC-_Mn_ concentration rapidly led to a decline in conversion over time, with the phosphorylated product IMP being completely hydrolyzed after only 6 h, according to the reported kinetic parameters of the wild-type enzyme (\(\mathrm{K_{m\,inosine}}\) = 122 vs. \(\mathrm{K_{m\,IMP}}\) = 0.59 mM; \(\mathrm{v_{max\,inosine}}\) = 16.24 vs. \(\mathrm{K_{m\,IMP}}\) = 76.95 \(\mathrm{\SIUnitSymbolMicro}\)/mg PhoC/min) [25]. Conversely, the reaction catalyzed by 20 \(\mathrm{\SIUnitSymbolMicro}\)/mL PhoC-_Mn_ reached a higher conversion value of IMP (40%) more rapidly (4 h) with respect to the reference reaction, despite a slight decrease to 36 and 34% after 6 and 24 h, respectively. Then, a series of experiments with successive additions of enzyme and/or PPi were carried out (Entries 4-7), resulting in a significant improvement in IMP formation (Figure 1b,c. Among them, the best result was achieved in the case of Entry 7 (20 \(\mathrm{\SIUnitSymbolMicro}\)/mL PhoC-_Mn_ at t and 4 h, together with two concomitant additions of 250 mM PPi), in which the highest conversion value was obtained (49%). Moreover, the formed IMP was found to be stable within 24 h (see Figure 1c). The unsatisfactory results obtained with a single addition of PPi (Entry 6, 37% conversion after 4 h followed by a rapid decline) may be justified by taking into account the ratio between the biocatalyst amount and the pyrophosphate ion available in the reaction medium. Indeed, in the absence of fresh PPi as an alternative substrate after 4 h, PhoC-_Mm_ would turn to processing IMP, thereby accelerating the hydrolysis reaction before being inhibited by phosphate. Driven by this positive outcome, we scaled up the phosphorylation by applying this approach based on multiple additions of enzyme and PPi to a semi-preparative scale. Thus, 80 mM of I, dissolved in 10 mL NaOAc buffer, was converted into IMP by feeding the reaction with three fixed amounts of PhoC-_Mm_ (10 or 20 \(\upmu\)g/mL) and PPi (250 mM) at different times (t0, 4 or 6 h, 24 h), as shown in Figure 2a,b. As a result, the conversion values of inosine into IMP rose up to 62 and 66% by adding 10 or 20 \(\upmu\)g/mL PhoC-_Mm_, respectively. It is worth noting that IMP was preserved from the hydrolytic activity of PhoC-_Mm_ and proved to be remarkably stable within 48 h in the selected conditions. Using these optimized reaction set-ups, starting from 215 mg of inosine, 162 mg and 173 mg of pure IMP were obtained after purification by semi-preparative HPLC, corresponding to a 58 and 62% isolated yield, respectively. In conclusion, phosphorylation of alcohols represents a key reaction both in biology and in organic synthesis due to the important role that phosphorylated molecules play in living systems and in various industrial sectors (food, pharmaceutical, cosmetic, agrochemical). In particular, purine 5'-nucleoside monophosphates, such as IMP and GMP, are of significant economic interest because they are widely used as food additives (flavor enhancers, E 631 and E 627, respectively) [6,7]. Chemical phosphorylation of nucleosides requires harsh reaction conditions, the use of toxic reagents and petroleum-based organic solvents, and the need for protection-deprotection steps, resulting frequently in poor/moderate yields associated with high energy consumption and waste disposal issues. An enzymatic approach, based on the use of phosphatases, appears to be a good alternative to introduce a phosphate group into nucleosides, overcoming the above-mentioned drawbacks and offering a much more viable and green route [17]. In this paper, the phosphotransferase activity of the wild-type phosphatase from _M. morganii_ (PhoC-_Mm_) has been successfully exploited through the combined use of an inexpensive phosphate donor (pyrophosphate) and a reaction engineering approach.