Phosphatases are diverse groups of enzymes that deserve special attention because of their significant roles in organic phosphorus (OP) mineralization to inorganic available forms (Pi). This work 1) compared the catalytic potentials of commercially acid phosphatase from wheat germ, sweet potato, and potato, and alkaline phosphatase from E. coli; 2) demonstrated that the rate of hydrolysis, catalytic efficiency, thermal stability, and optimal pH of these enzymes depended on enzyme sources and the stereochemical or stereoisomeric structures of the substrates; 3) revealed that both acid and alkaline phosphatases exhibited broad range of substrate hydrolysis with high affinity for p-nitrophenyl phosphate bis (cyclohexylammonium) than the widely used p-nitrophenyl phosphate disodium hexahydrate for phosphatase assay. Sweet potato had relatively higher reaction kinetics (Vmax, Km, Kcat, Kcat/Km) values with most substrates tested. The order of catalytic activity was in the order: sweet potato > wheat germ > potato, while the order of substrate hydrolyzed was: PNPBC > PNP > PNP2A2E > DG6P2Na > DG6PNa > Bis-PNP > phytate. The optimum pH for the acid phosphatase was observed to be 5.0. Generally, the activity of alkaline phosphatase was similar to that of acid phosphatase with optimal pH between 10 and 13, depending on the substrates. Knowledge derived from this work would be helpful in enzyme catalysis in soils and water environments.
Phosphatases are diverse groups of enzymes that deserve special attention because of their significant roles in organic phosphorus (OP) mineralization to inorganic available forms [
While phosphatases generally hydrolyze phosphoric (H3PO4) esters and anhydrides to release phosphate, they differ in their pH optima, metal ion requirements, substrate specificities, and reaction mechanisms. Acid and alkaline phosphatases have been used to mineralize specific OP compounds in animal manure, soils, water, and sediments [
Low P availability in soils may trigger phosphatase secretions to the rhizosphere; however, soil phosphatases are sometimes derived from 1) intracellular enzymes or enzymes bound to cell components and 2) abiotic or extracellular enzymes leaking from intact cells or released from dead or lysed cells that originate from the cell membrane [
A distinctive feature of alkaline phosphatase is the presence of two Zn2+ and one Mg2+ ions per sub unit [
P-nitrophenyl phosphate disodium hexahydrate (>97%) (PNP), p-nitrophenyl phosphate di (2-amino-2-ethyl-1, 3 propanediol) (PNP2A2E), P-nitrophenyl phosphate bis (cyclohexylammonium) (PNPBC), Bis p-nitrophenyl phosphate sodium (Bis-PNP), D-glucose 6-phosphate sodium salt (98%) (DG6PNa), D-glucose 6-phosphate disodium hydrate (98% - 100%) (DG6P2Na), and inositol hexakisphosphate (phytic acid sodium salt) substrates are shown in
The optimal pH for the phosphatases as reported by the supplier is 4.8 for acid phosphatase, and 10.4 for alkaline phosphatase, while the optimal temperature is 37˚C for both acid and alkaline phosphatase. One unit (U) of the enzyme is reported to liberate 1.0 μmol orthophosphate with the appropriate substrate at the appropriate pH and temperature. With acid and alkaline phosphatases, concentrations of 0.013 - 0.166 U∙mL−1 were used to hydrolyze the substrate. The effects of pH, temperature, and time on each enzyme was determined by exposure to a wide range of temperatures ranging from 10˚C to 80˚C; pH ranging from 2 to 9, and time ranging from 1 to 10 hours using a substrate concentration of 5 times the Km [
To determine the kinetic parameters of each enzyme, each substrate (phosphate compound) analog was hydrolyzed at increasing concentrations with each enzyme at the supplier’s reported pH and temperature for 1 hour. The substrate was dissolved in 100 mM acetate buffer (pH 4.8) and a final enzyme concentration of 0.066, 0.013, and 0.166 U∙mL−1 for wheat germ, sweet potato, and potato respectively used for acid phosphatase hydrolysis. For alkaline phosphatase hydrolysis, each substrate was dissolved in 87 mM glycine buffer, (pH 10.4) and a final enzyme concentration of 0.166 U∙mL−1 used. All reaction mixtures were carried out in a total volume of 3 mL and the reaction stopped using 10% sodium dodecyl sulfate at the end of the incubation period. The controls were set up by incubating the substrate without enzyme to correct for the Pi released due to chemical hydrolysis. The amount of Pi released was determined colorimetrically [
Chemical reaction rates generally double with every 10˚C increase in temperature and is also known as temperature coefficient (Q10). The calculations [phosphatase activity at T(˚C)/phosphatase activity at T(˚C) − 10˚C] to determine the temperature coefficients (Q10) were at 10˚C intervals between 0 and 70˚C. When an enzyme reaction obeys the Arrhenius equation [k = A. exp (−Ea/RT)], the activation energy (Ea) or slope can be estimated from the logarithmic transformed equation [log k = (−Ea/2.303RT) + log A], where k is the rate constant, A is the Arrhenius constant, R is the gas constant (8.314 mol−1∙k−1), and T is the temperature on the Kelvin scale.
The Arrhenius plots were linear between 10˚C and 50˚C for acid phosphatase from wheat germ (
The kinetic parameters calculated for the hydrolyzed substrates by acid phosphatase are shown in
Substrate | Acid phosphatase | Alkaline phosphatase | ||
---|---|---|---|---|
Wheat germ | Sweet potato | Potato | E. coli | |
kJ∙mol−1 | ||||
PNP | 36.1 | 30.5 | 36.8 | 28.3 |
PNP2A2E | 35.9 | 29.5 | 34.5 | 29.3 |
PNPBC | 34.7 | 30.1 | 47.4 | 27.2 |
Bis-PNP | 18.0 | 22.8 | 19.2 | 15.7 |
DG6PNa | 25.0 | 19.6 | 48.5 | 30.6 |
DG6P2Na | 27.8 | 22.3 | 49.6 | 41.2 |
PNP (p-nitrophenyl phosphate disodium hexahydrate); PNP2A2E (p-nitrophenyl phosphate di [2-amino-2-ethyl-1, 3 propanediol]); PNPBC (P-ni- trophenyl phosphate biscyclohexylammonium); Bis-PNP (Bis p-nitrophenyl phosphate sodium); DG6PNa (D-glucose 6-phosphate sodium salt); DG6P2Na (D-glucose 6-phosphate disodium hydrate).
Substrate | 20 | 30 | 40 | 50 | 60 | 70 | Mean |
---|---|---|---|---|---|---|---|
˚C | |||||||
PNP | 2.20 | 1.65 | 1.51 | 1.21 | 0.75 | 0.74 | 1.34 |
PNP2A2E | 2.28 | 1.58 | 1.54 | 1.19 | 0.76 | 0.88 | 1.37 |
PNPBC | 2.37 | 1.48 | 1.58 | 1.14 | 0.73 | 0.80 | 1.35 |
Bis-PNP | 1.37 | 1.41 | 1.21 | 1.10 | 0.92 | 0.69 | 1.12 |
DG6PNa | 2.13 | 1.22 | 1.35 | 1.13 | 0.49 | 0.42 | 1.12 |
DG6P2Na | 2.19 | 1.45 | 1.33 | 1.04 | 0.53 | 0.33 | 1.14 |
Q10 = phosphatase activity at T(˚C)/phosphatase activity at T(˚C) − 10˚C, PNP (p-nitrophenyl phosphate disodium hexahydrate); PNP2A2E (p-nitro- phenyl phosphate di [2-amino-2-ethyl-1,3propanediol]); PNPBC (P-nitrophenyl phosphate biscyclohexylammonium); Bis-PNP (Bis p-nitrophenyl phosphate sodium); DG6PNa (D-glucose 6-phosphate sodium salt); DG6P2Na (D-glucose 6-phosphate disodium hydrate).
Substrate | 20 | 30 | 40 | 50 | 60 | 70 | Mean |
---|---|---|---|---|---|---|---|
˚C | |||||||
PNP | 2.19 | 1.38 | 1.61 | 1.65 | 1.25 | 0.42 | 1.42 |
PNP2A2E | 2.09 | 1.43 | 1.70 | 1.54 | 1.10 | 0.44 | 1.38 |
PNPBC | 2.56 | 1.86 | 1.72 | 1.51 | 0.67 | 0.50 | 1.47 |
Bis-PNP | 1.42 | 1.29 | 1.25 | 1.22 | 0.77 | 0.63 | 1.10 |
DG6PNa | 2.32 | 2.78 | 1.92 | 1.41 | 1.07 | 0.20 | 1.62 |
DG6P2Na | 2.2 | 1.05 | 3.95 | 1.43 | 0.43 | 0.57 | 1.61 |
Q10 = phosphatase activity at T(˚C)/phosphatase activity at T(˚C) − 10˚C; PNP (p-nitrophenyl phosphate disodium hexahydrate); PNP2A2E (p-ni- trophenyl phosphate di [2-amino-2-ethyl-1,3propanediol]); PNPBC (P-nitrophenyl phosphate biscyclohexylammonium); Bis-PNP (Bis p-nitrophenyl phosphate sodium); DG6PNa (D-glucose 6-phosphate sodium salt); DG6P2Na (D-glucose 6-phosphate disodium hydrate).
Substrate | 20 | 30 | 40 | 50 | 60 | 70 | 80 | 90 | Mean |
---|---|---|---|---|---|---|---|---|---|
˚C | |||||||||
PNP | 1.40 | 1.34 | 1.67 | 1.96 | 1.19 | 1.02 | 0.61 | 0.300 | 1.19 |
PNP2A2E | 1.46 | 1.83 | 1.59 | 1.25 | 1.26 | 1.25 | 0.39 | 0.47 | 1.19 |
PNPBC | 1.29 | 1.52 | 1.50 | 1.63 | 1.48 | 1.09 | 0.40 | 0.13 | 1.13 |
Bis-PNP | 1.00 | 1.12 | 1.10 | 1.22 | 2.44 | 1.52 | 0.42 | 0.40 | 1.15 |
DG6PNa | 1.31 | 2.27 | 1.35 | 1.50 | 0.91 | 0.27 | 0.85 | 0.77 | 1.03 |
DG6P2Na | 1.28 | 1.41 | 1.30 | 1.36 | 0.96 | 0.93 | 0.04 | 1.00 | 1.03 |
Q10 = phosphatase activity at T(˚C)/phosphatase activity at T(˚C) − 10˚C.
Substrate | 20 | 30 | 40 | 50 | 60 | 70 | 80 | 90 | Mean |
---|---|---|---|---|---|---|---|---|---|
˚C | |||||||||
PNP | 1.50 | 1.53 | 1.98 | 1.17 | 1.37 | 1.24 | 1.10 | 0.92 | 1.35 |
PNP2A2E | 1.93 | 1.30 | 1.92 | 1.46 | 1.32 | 1.40 | 1.10 | 1.04 | 1.43 |
PNPBC | 2.55 | 1.25 | 1.58 | 1.78 | 1.20 | 1.12 | 1.04 | 1.02 | 1.44 |
Bis-PNP | 2.20 | 1.02 | 1.03 | 1.12 | 1.27 | 1.45 | 1.53 | 1.15 | 1.21 |
DG6PNa | 1.57 | 1.35 | 1.73 | 1.49 | 1.31 | 1.76 | 1.04 | 1.10 | 1.42 |
DG6P2Na | 3.36 | 1.31 | 2.08 | 1.46 | 1.57 | 1.58 | 1.04 | 0.67 | 1.63 |
Q10 = phosphatase activity at T(˚C)/phosphatase activity at T(˚C) − 10˚C; PNP (p-nitrophenyl phosphate disodium hexahydrate); PNP2A2E (p-ni- trophenyl phosphate di [2-amino-2-ethyl-1, 3 propanediol]); PNPBC (P-nitrophenyl phosphate biscyclohexylammonium); Bis-PNP (Bis p-nitrophenyl phosphate sodium); DG6PNa (D-glucose 6-phosphate sodium salt); DG6P2Na (D-glucose 6-phosphate disodium hydrate).
Wheat germ | Sweet potato | Potato | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Vmax (mg∙L−1・hr−1) | Km (mM) | Kcat (hr−1) | Kcat/Km (hr−1・mM−1) | Vmax (mg∙L−1∙hr−1) | Km (mM) | Kcat (hr−1) | Kcat/Km (hr−1∙mM−1) | Vmax (mg∙L−1∙hr−1) | Km (mM) | Kcat (hr−1) | Kcat/Km (hr−1・mM−1) | |||
Phytate | 65 | 11.8 | 984 | 83 | 72.1 | 10.4 | 1803 | 173 | 165 | 14.0 | 330 | 24 | ||
PNP | 152 | 3.65 | 2303 | 631 | 135 | 2.69 | 10 385 | 3861 | 143 | 4.53 | 861 | 190 | ||
PNP2A2E | 154 | 4.34 | 2333 | 538 | 130 | 3.32 | 10 000 | 3012 | 162 | 4.58 | 976 | 213 | ||
PNPBC | 145 | 3.47 | 2197 | 633 | 126 | 2.25 | 9692 | 4308 | 166 | 5.00 | 1000 | 200 | ||
Bis-PNP | 5.21 | 2.20 | 78.9 | 36 | 5.35 | 6.92 | 409 | 59.0 | 2.73 | 2.26 | 17 | 7.50 | ||
DG6PNa | 38.8 | 5.16 | 588 | 114 | 223* | 8.86 | 1343 | 152 | 44.61 | 3.20 | 269 | 84.0 | ||
DG6P2Na | 31.6 | 1.70 | 479 | 282 | 251* | 11.13 | 1512 | 136 | 43.00 | 1.90 | 259 | 136 | ||
PNP (p-nitrophenyl phosphate disodium hexahydrate); PNP2A2E (p-nitrophenyl phosphate di [-2-amino-2-ethyl−1, 3 propanediol]); PNPBC (P-ni- trophenyl phosphate bis (cyclohexylammonium); Bis-PNP (Bis p-nitrophenyl phosphate sodium); DG6PNa (D-glucose 6-phosphate sodium salt); DG6P2Na (D-glucose 6-phosphate disodium hydrate); * (0.166 U/ml of enzyme used).
Substrate | Vmax (mg∙L−1∙hr−1) | Km (mM) | Kcat (hr−1) | Kcat/Km (hr−1∙mM−1) |
---|---|---|---|---|
PNP | 216 | 6.55 | 1301 | 199 |
PNP2A2E | 204 | 6.40 | 1229 | 192 |
PNPBC | 198 | 5.79 | 1195 | 206 |
Bis-PNP | nd | nd | nd | nd |
DG6PNa | 88.5 | 4.87 | 533 | 110 |
DG6P2Na | 82.8 | 5.50 | 499 | 91 |
PNP (p-nitrophenyl phosphate disodium hexahydrate); PNP2A2E (p-nitrophenyl phosphate di [-2-amino-2-ethyl−1, 3 propanediol]); PNPBC (p-nitro- phenyl phosphate bis (cyclohexylammonium); Bis-PNP (Bis p-nitrophenyl phosphate sodium); DG6PNa (D-glucose 6-phosphate sodium salt); DG6P2Na (D-glucose 6-phosphate disodium hydrate); nd: not determined.
The Km value for acid phosphatase from sweet potato was the lowest (2.25 - 3.32 mM) for the specific group while that of potato was the highest (4.53 - 5.00 mM). For the specific group, a relatively low Km value of 2.25 mM and 3.47 mM were obtained for sweet potato and wheat germ respectively using PNPBC. For the specific group, the Kcat/Km value for acid phosphatase from sweet potato was the highest (3012 - 4308 h−1∙mM−1) while that of potato was the lowest (190 - 213 h−1∙mM−1). The Kcat/Km value of acid phosphatase from all species using PNPBC was greater than when PNP and PNP2A2E were used respectively. Acid phosphatase from sweet potato had the highest Kcat values (9692 - 10,385 h−1) while acid phosphatase from potato had the lowest values (861 - 1000 h−1) for the specific group (
For the less specific group, acid phosphatase from sweet potato had the highest Km values ranging from 8.86 - 11.13 mM, and Kcat values ranging from 1343 - 1512 h−1, than acid phosphatase from wheat germ and potato. Acid phosphatase from wheat germ and potato had low Km values (1.7 and 1.9 mM, and high values of Kcat/Km of 282 and 84 h−1∙mM−1 respectively) with DG6P2Na than DG6PNa as substrate. The non-specific group showed little activity with acid phosphatase from all three species. Wyss et al. [
Results showing the enzyme kinetics for alkaline phosphatase from E. coli are presented in
While other investigators [
Hydrolysis of these substrates by E. coli alkaline phosphatase (
PNa, and DG6P2Na were linear up to 3 hours while PNPBC and Bis-PNP were linear up to 10 hours. Nigam et al. [
The optimum temperature for the acid phosphatase is shown in
with PNPBC and DG6P2Na, 60˚C with PNP, PNP2A2E, and DG6PNa (
The activity of each enzyme was also compared at various pH values (range, 2 - 9) using a concentration 5 times the Km [
Our study indicates that the rate of hydrolysis, catalytic efficiency, thermal stability, and optimal pH of these enzymes may depend on enzyme sources and the stereochemical or stereoisomeric structures of the substrates. This study also reveals that acid and alkaline phosphatases do exhibit a broad range of substrate hydrolysis with high affinity for PNPBC than the commonly used PNP. Sweet potato had relatively higher reaction kinetics (Vmax, Km, Kcat, Kcat/Km) values with most substrates tested. The order of catalytic activity for the acid phosphatase was in the order: sweet potato > wheat germ > potato, while the order of substrate hydrolyzed was: PNPBC > PNP > PNP2A2E > DG6P2Na > DG6PNa > Bis-PNP > phytate. The optimum pH for the acid phosphatase was observed to be 5.0. Alkaline phosphatase activity was similar to that of the acid phosphatase except that there was more alkaline phosphatase activity with DG6PNa than DG6P2Na.
This work is a contribution from the Winfred Thomas Agricultural Research Station, Alabama A&M University, Normal, AL. Trade or manufacturers’ names mentioned in the paper are for information only and do not constitute endorsement, recommendation, or exclusion by either Alabama A&M University or USDA-ARS. The research was supported in part by USDA-NIFA, Evans-Allen Grant # ALAX 011.