# Improving the Phosphatase-Catalyzed Synthesis of 5'-Nucleotides: A Reaction Engineering Approach
## 3 Results and Discussion  
PhoC-_Mn_ was expressed in the heterologous host _E. coli_ BL21(DE3) and subsequently purified to homogeneity by using His-tag affinity techniques (see Supplementary Materials, Figure S1) in high yield (76 mg/L) [26]. As a first approach, the prepared enzyme was used for the synthesis of inosine 5'-monophosphate (IMP) in experimental conditions similar to those previously described by [PERSON] et al. [24; 25], i.e., 10 mg/mL of wild-type PhoC-_Mn_ and 40 mM inosine (I) in 100 mM NaOAc buffer (pH 5.0) at 30 \({}^{\circ}\)C. Upon monitoring the reaction progress by HPLC, a 31% conversion of I to IMP after 6 h (Scheme 2) was observed. Figure 2: Semi-preparative synthesis of IMP by PhoC-_Mn_-catalyzed phosphorylation of I. (**a**) Addition of 10 mg/mL PhoC-_Mn_ and 250 mM PPI at t\({}_{0}\), 6 h, and 24 h; (**b**) addition of 20 mg/mL PhoC-_Mn_ and 250 mM PPI at t\({}_{0}\), 4 h, and 24 h.  
Interestingly, unlike the results reported by [PERSON] et al. [24; 25], such a value remained unchanged after 24 h (see Supplementary Materials, Table S1). This result, indicative of an optimal balance between the phosphorylating and hydrolytic activity toward inosine and IMP, respectively, could be attributed to the reported inhibitory action of inorganic phosphate [33], accumulating at a rate fast enough to prevent the hydrolysis reaction to occur after 6 h in the selected conditions. Starting from this result and keeping the enzyme amount constant, we proceeded to optimize the reaction conditions by varying the type of buffer and amount of pyrophosphate (PPi) together with the mode/time of its addition and inosine concentration (Tables 1-3 and Supplementary Materials, Paragraph S3.1). The data of Table 1 indicate that the substitution of NaOAc buffer with either MES (2-(\(N\)-morpholino)ethanesulfonic acid) or sodium citrate at the same concentrations and similar pH did not yield better results with respect to the reference reaction (Table 1, Entries 2 and 3 vs. 1), as well as lowering the concentration of PPi to 200 mM or increasing it to 400 mM (Table 2, Entries 4 and 5). As for the mode/time of phosphate donor addition to the reaction mixture, a fed-batch approach was adopted. Using from 250 to 750 mM PPi in 3-30 h intervals (Table 2, Entries 6-8), a significant increase in the conversion up to 44% was observed (vs. 41% reported by [PERSON] [25]) only when the highest final concentration of PPi (750 mM) was used by portionwise additions, in the presence of 80 mM of I. The evidence that the improved conversion was related to the mode of addition/concentration of PPi was inferred by investigating the influence of the concentration of I (Table 3, Entries 9-12). The effects of the increasing inosine concentration up to 200 mM were proved to be unsuccessful in most cases, with the worst results obtained when a suspension was formed at 200 mM inosine (Table 3, Entry 10). These results can be attributed to the scarce solubility of the substrate, which could be completely dissolved in the reaction medium up to 100 mM at 30 \({}^{\circ}\)C.  
With these results in hand, we moved to investigate the influence of single or multiple additions of increasing amounts of PhoC-_Mn_ and/or PPi (Table 4), keeping all the remaining parameters constant (80 mM substrate concentration, 100 mM NaOAc (pH 5.0) buffer, 30 \({}^{\circ}\)C)  
A comparison between the extent of phosphorylations with 10, 20, and 30 \(\mathrm{\SIUnitSymbolMicro}\)/mL PhoC-_Mn_ at 250 mM PPi (Entries 1, 2, and 3 and Figure 1a) indicates that an increase in the enzyme amount results in the increase in the conversion of I into IMP (from 31 to 40%).