Supporting Information. Phosphatase mediated hydrolysis of linear polyphosphates. Rixiang Huang, Biao Wan, Margot Hultz, Julia M. Diaz, Yuanzhi Tang

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1 Supporting Information Phosphatase mediated hydrolysis of linear polyphosphates Rixiang Huang, Biao Wan, Margot Hultz, Julia M. Diaz, Yuanzhi Tang Total 1pages 9 figures S1

2 PolyP_end PolyP_middle PyroP Chemical shift (ppm) Figure S1. 31 P liquid NMR spectrum of the soda polyphosphate sample with a broad range of chain lengths (obtained from ICL Performance Products, MO, USA, with 76% of the total P as polyphosphates with 4 12 P units, 7% with >12 P units). As shown, multiple middle group peaks at about -2 ppm were present due to the broad distribution of chain lengths. S2

3 15P hydrolysis 88.3% h.17% 11.5% 87.5% 15 min 2.4% 1.1% 6 min 6.7% 7.1% 86.2% Chemical shift (ppm) Figure S2. 31 P liquid NMR spectra of 15P hydrolysis experiments with a slower hydrolysis rate than that of Figure 1A (using double the concentration of 15P and half the concentration of alkaline phosphatase, compared to experimental conditions for the data shown in Figure 1A). Spectra showed decreasing intensity of middle groups (chemical shift around -22 ppm) and end groups (around -5 ppm) and the increasing intensity of orthophosphate (around 4 ppm) over time. No split peaks similar to the mixed chain length soda polyphosphate sample (Figure S1) were observed, supporting the terminal-only hydrolysis mechanism. Percentages of the total integrated peak area are provided. S3

4 6P-.5h 6P-24h Chemical shift (ppm) Figure S3. 31 P liquid NMR spectra of 6P polyphosphate in the presence of pyrophosphatase after.5 and 24 h reaction. No orthophosphate formation was observed even after 24 h, indicating little to no hydrolysis occurred within this time frame. S4

5 PyroP-h PyroP-1h Chemical shift (ppm) Figure S4. 31 P liquid NMR spectra of pyrophosphate (chemical shift of about -6 ppm) in the presence of pyrophosphatase, showing the complete hydrolysis of pyrophosphate (disappearance of its signal at -6 ppm) into orthophosphate (appearance of chemical shift at 3 ppm) within 1 h. S5

6 Phosphate concentration (µm) % of orthophosphate to total P µm 98 µm 196 µm 49 µm C-6P A-6P Phosphate concentration (µm) % of orthophosphate to total P µm 49 µm 98 µm 196 µm 49 µm 2 D-13P B-13P Figure S5. Effects of initial polyphosphate concentration on the rates of enzymatic hydrolysis (quantified by orthophosphate production) of 6P (A and C) and 13P (B and D) by alkaline phosphatase. The hydrolysis rates were expressed in both orthophosphate concentration (upper panels) and the percentage of produced orthophosphate to total P (lower panels). A range of initial polyphosphate concentrations were used: 196, 49, 98, 196, and 49 µm P. Concentration of alkaline phosphatase =.5 unit per ml, ph = 8.. S6

7 Phosphate Con. (µm) A - Alkaline mM 4mM 1mM 2 mm 1mM-C 4mM-C 1mM-C 2 mm-c Phosphate Con. (µm) B - Acid mM 4mM 1mM 2 mm 1mM-C 4mM-C 1mM-C 2 mm-c Figure S6. (A) Alkaline phosphatase and (B) acid phosphatase mediated hydrolysis of 6P in the presence of different Ca 2+ concentrations. Empty symbols are control experiments (no enzyme addition). Tris (ph 8.) and HEPES (ph 7.) were used for the alkaline and acid phosphatases, respectively. S7

8 45 36 Count rate (kps) ASW, acid ASW, alkaline Ca 1 mm, alkaline Ca 4 mm, alkaline Ca 1 mm, alkaline Ca 2 mm, alkaline Figure S7. Dynamic light scattering (DLS) measurement (in term of scattering intensity) for phosphatase-mediated hydrolysis of 6P in different solution chemistries. Precipitation only occurred in the buffered 1 and 2 mm Ca 2+, but not in low Ca and ASW conditions during 4 hours of observation. S8

9 A B Normalized absorption (offset) ACP_1mg ACP SodaP_Ca SodaP_Na 6P 15P Normalized absorption (offset) ACP ACP_1Mg OctaCaP HAP Energy (ev) Energy (ev) Figure S8. (A) Normalized P K-edge XANES spectra of different polyphosphates (15P and 6P), Na and Ca salts of soda polyphosphate (SodaP_Ca and SodaP_Na; the Ca salt was produced by reacting dissolved Na-soda polyphosphate with excess amount of CaCl 2 solution, followed by filtration, DI rinsing, and airdrying), amorphous calcium phosphate (ACP), and Mg substituted ACP with 1% Mg/(Ca+Mg) ratio (ACP_1Mg); (B) Normalized P K-edge XANES spectra of different Ca-phosphate phases: ACP, ACP_1Mg, octacalcium phosphate (OctaCaP) 1, and hydroxylapatite (HAP) 2. S9

10 A O B P Ca Mg Ca Ca Figure S9. (A) SEM image of the precipitates from the hydrolysis of 15P in the presence of alkaline phosphatase enzyme and Ca, scale bar represents 1 µm. (B) Corresponding EDX spectrum, indicating an atomic composition of 13.2% Ca, 1.4% Mg, 14.5% P, and 58.%O (in molar basis). References 1. Shober, A. L.; Hesterberg, D. L.; Sims, J. T.; Gardner, S., Characterization of phosphorus species in biosolids and manures using XANES spectroscopy. J Environ Qual 26,35, (6), Werner, F.; Prietzel, J., Standard Protocol and Quality Assessment of Soil Phosphorus Speciation by P K-Edge XANES Spectroscopy. Environ Sci Technol 215,49, (17), S1