3. Kinetics of hydrolysis phosphate esters

As presented in Table 2, the hydrolysis reaction rate constant at different initial concentrations of phosphate esters in these aged inorganic iron salt solutions or inorganic iron oxides solutions were not constant. Surprisingly, the k from 5 to 250 μM G6P in the 16.5 nM Fe(NO3)3 solution aged for 14 months at room temperature


#### Iron Oxide Nanoparticles: An Inorganic Phosphatase DOI: http://dx.doi.org/10.5772/intechopen.82650

#### Table 2.

Phosphate ester hydrolysis in the different inorganic iron solutions.

3. Kinetics of hydrolysis phosphate esters

Hydrolysis rate constant of 20 μM G6P in inorganic iron solutions.<sup>a</sup>

The hydrolysis rate constant of 20 <sup>μ</sup>M G6P in the DIW is 1.84 <sup>10</sup><sup>8</sup> <sup>s</sup>

Fe (NH4)2(SO4)2

Fe source Manufacturer Aged

Fe(NO3)3 Riedel-de

FeCl3 Riedel-de

Fe(NO3)3 Riedel-de

FeCl3 Riedel-de

Iron standard solution (metal Fe in 0.3 M HNO3) Haën

Haën

Haën

Haën

Riedel-de Haën

Riedel-de Haën

Iron oxide nanoparticles (IO)

Nanocatalysts

Aged acidic forced hydrolysis inorganic Fe solution

a

100

Table 1.

time (mo.) Aged temperature (°C)

Fe(NO3)3 JT Baker 0.25 5 A 16.9 11 Fe(NO3)3 JT Baker 0.25 22 B 70.3 2.7 Fe(NO3)3 JT Baker 0.25 50 C 59.5 3.2 Fe(NO3)3 JT Baker 0.25 80 D 106 1.8 FeCl3 JT Baker 0.25 25 E 13.8 14

Fe(NO3)3 JT Baker 14 22 16.5 30.17 6.4

FeCl3 JT Baker 16 22 2 6.59 29.2

FeClO4 Aldrich 4 22 10 2.46 78.4

1

EM Science 16 22 16.5 9.49 20.3

IO nanoparticles or total Fe concentration (nM)

0.25 50 H 18.5 4.0

0.25 50 F 27.4 7.0

0.25 80 L 80.7 2.4

0.25 80 G 54.1 3.6

4 22 16.5 5.08 37.8

6 22 1000 18.77 10.3

4 22 10 3.28 58.6

, and the corresponding half time is 10,450 h.

JT Baker 4 22 1 2.38 81.0

22 100 2.96 65.6

2.5 4.85 39.7 7.5 7.22 26.7 6.38 30.2 6.55 29.4 6.58 29.2 8.14 23.6 5.86 32.8

10 4.95 38.9

100 0.83 231.9

100 0.61 318.3

20 μM G6P

) t0.5 (h)

k (10<sup>6</sup> s 1

As presented in Table 2, the hydrolysis reaction rate constant at different initial concentrations of phosphate esters in these aged inorganic iron salt solutions or inorganic iron oxides solutions were not constant. Surprisingly, the k from 5 to 250 μM G6P in the 16.5 nM Fe(NO3)3 solution aged for 14 months at room temperature

Figure 2.

Relationship between the soaked time of IO and hydrolysis rate of phosphorus in different DMT-IO solutions: (a) 100 μM G6P, and (b) 100 μM ATP (for details of IOs see Table 1).

can be further described by the Michaelis-Menten equation (Figure 5a and b), as the typical behavior of biocatalysts. This is contrast to previously reported promotion effects by metals [30–32, 40] and minerals [20, 22, 23, 41, 42]. The maximum k of G6P hydrolysis was about 1 nM s�<sup>1</sup> , or 3.6 μM h�<sup>1</sup> , and the Michaelis-Menten constant (Km) was 13.7 μM of this aged inorganic iron solution.

$$\frac{1}{\text{v}} = 9.985 \times 10^8 + \frac{1.371 \times 10^9}{[\text{G\'6P}]\_o} \left(\text{r}^2 = 0.997\right) \tag{2}$$

constant (Table 2). The catalytic activity of the different concentration of phosphorus also can be described by the typical Michaelis-Menten equations (Figure 3c). Based on the Lineweaver-Burk linear equation (1/V is a linear function of 1/[S]), the Michaelis-Menten constant (Km) and maximum velocity (Vm), as well as the range of concentration of phosphorous among these compounds, were determined (Table 3). Meanwhile, the catalysis activity was still observed even when the total phosphorus esters exceeded the range of the Michaelis-Menten equations, as

(made by Fe(NO3)3�9H2O and NaOH, aged a week at 80°C, IO-D, soaked a month).

G2P 2.0 7.0 6–200 0.99 G6P 3.2 8.3 5–100 0.99 ATP 0.9 9.2 5–50 0.99 Polyphosphate (poly-Pi) 1.1 5.5 5–25 1.00 Pyrophosphate (PPi) 2.2 1.3 5–25 0.98

Michaelis-Menten constant (Km) and maximum velocity (Vm) of different phosphorus in a DMT-IO solution

It should further be pointed out that the similar enzyme kinetics (Michaelis-Menten equations) were observed recently by many inorganic nanoparticles studies, which have been described as nanozyme [44–47]. For example, Fe3O4 [44], α-Fe2O3 [48], γ-Fe2O3 [49], γ-FeOOH [50], Co3O4 [51], MnFe2O4 [52, 53], MFe2O4 (M = Mg, Ni, Cu) [54], ZnFe2O4 [55], NiO [56], and MnO2 [57] have been observed to have peroxidase-like or catalase-like activity, whereas the vanadium pentoxide (V2O5) was demonstrated to have antioxidant enzyme-like (glutathione peroxidase) activity [58–61] and molybdenum trioxide (MoO3) nanoparticles to have

The hydrolysis of phosphorus ester was significantly inhibited when the tetrahedral oxyanions were introduced into inorganic iron oxides nanoparticle solution, e.g., G6P in a 10-month aged iron solution (Figure 4), as the natural PAPs. Both the catalytic and the inhibition behaviors of the catalysis in the presence of 5–125 μM G6P with different tetrahedral oxyanions can be described by a Michaelis-Menten

> <sup>2</sup>:<sup>001</sup> � <sup>10</sup><sup>9</sup> ½ � G6P <sup>o</sup>

<sup>3</sup>:<sup>423</sup> � 1010 ½ � G6P <sup>o</sup>

<sup>1</sup>:<sup>216</sup> � 1010 ½ � G6P <sup>o</sup>

<sup>1</sup>:<sup>044</sup> � <sup>10</sup><sup>10</sup> ½ � G6P <sup>o</sup>

<sup>8</sup>:<sup>317</sup> � 109 ½ � G6P <sup>o</sup>

r

) Km (μM) Range (μM) r<sup>2</sup>

r

r

r

r

<sup>2</sup> <sup>¼</sup> <sup>0</sup>:<sup>945</sup> (3)

<sup>2</sup> <sup>¼</sup> <sup>0</sup>:<sup>998</sup> (4)

<sup>2</sup> <sup>¼</sup> <sup>0</sup>:<sup>995</sup> (5)

<sup>2</sup> <sup>¼</sup> <sup>0</sup>:<sup>988</sup> (6)

<sup>2</sup> <sup>¼</sup> <sup>0</sup>:<sup>997</sup> (7)

with many of the natural enzymes, including the PAP [43].

Phosphorus source Vm (nM S�<sup>1</sup>

Iron Oxide Nanoparticles: An Inorganic Phosphatase DOI: http://dx.doi.org/10.5772/intechopen.82650

4. Inhabitation effects of tetrahedral oxyanions

1

<sup>v</sup> <sup>¼</sup> <sup>7</sup>:<sup>4</sup> � <sup>10</sup><sup>8</sup> <sup>þ</sup>

<sup>v</sup> <sup>¼</sup> <sup>7</sup>:<sup>4</sup> � <sup>10</sup><sup>8</sup> <sup>þ</sup>

1

with 1 <sup>μ</sup><sup>M</sup> WO4 : <sup>1</sup>

with 10 <sup>μ</sup><sup>M</sup> PO4 : <sup>1</sup>

<sup>v</sup> <sup>¼</sup> <sup>7</sup>:<sup>4</sup> � <sup>10</sup><sup>8</sup> <sup>þ</sup>

<sup>v</sup> <sup>¼</sup> <sup>7</sup>:<sup>4</sup> � <sup>10</sup><sup>8</sup> <sup>þ</sup>

<sup>v</sup> <sup>¼</sup> <sup>7</sup>:<sup>4</sup> � <sup>108</sup> <sup>þ</sup>

sulfite oxidase activity [62].

Table 3.

equation (Eqs. (3)–(7)) as follows:

without any addition :

with 1 μM MoO4 :

with 5 <sup>μ</sup><sup>M</sup> PO4 : <sup>1</sup>

103

In fact, the promotion effect of G6P hydrolysis can be extended to 2500 μM in this aged iron solution with a <sup>k</sup> of 6.53 � <sup>10</sup>�<sup>7</sup> <sup>s</sup> �1 , and t1/2 of 295 h. It should be pointed out that the concentration of total phosphorus in the solution was 10<sup>3</sup> –10<sup>5</sup> higher than that of iron in the solution (e.g., 16.5 nM Fe and 2500 μM G6P).

The same patterns were also observed in the solution bearing inorganic iron oxide nanoparticles. Like the aged inorganic iron solution, the k of various organic phosphate esters or condensed phosphates at different concentrations were not

#### Figure 3.

Kinetics of hydrolysis of phosphate esters in inorganic iron solutions at room temperature (22 � 2°C). (a) Double reciprocal (initial velocity and initial concentration of G6P) plot of G6P in the 14 month aged 16.5 nM Fe(NO3)3 solution, (b) initial velocity of G6P hydrolysis (v0) as a function of the initial concentration of G6P in the aged iron solution, and (c) Lineweaver-Burk plot of different phosphate compounds in a DMT-IO solutions (IO-D).


Iron Oxide Nanoparticles: An Inorganic Phosphatase DOI: http://dx.doi.org/10.5772/intechopen.82650

Table 3.

can be further described by the Michaelis-Menten equation (Figure 5a and b), as the typical behavior of biocatalysts. This is contrast to previously reported promotion effects by metals [30–32, 40] and minerals [20, 22, 23, 41, 42]. The maximum k

Relationship between the soaked time of IO and hydrolysis rate of phosphorus in different DMT-IO solutions:

constant (Km) was 13.7 μM of this aged inorganic iron solution.

<sup>v</sup> <sup>¼</sup> <sup>9</sup>:<sup>985</sup> � <sup>10</sup><sup>8</sup> <sup>þ</sup>

(a) 100 μM G6P, and (b) 100 μM ATP (for details of IOs see Table 1).

, or 3.6 μM h�<sup>1</sup>

<sup>1</sup>:<sup>371</sup> � 109 ½ � G6P <sup>o</sup>

�1

In fact, the promotion effect of G6P hydrolysis can be extended to 2500 μM in

pointed out that the concentration of total phosphorus in the solution was 10<sup>3</sup>

Kinetics of hydrolysis of phosphate esters in inorganic iron solutions at room temperature (22 � 2°C). (a) Double reciprocal (initial velocity and initial concentration of G6P) plot of G6P in the 14 month aged 16.5 nM Fe(NO3)3 solution, (b) initial velocity of G6P hydrolysis (v0) as a function of the initial concentration of G6P in the aged iron solution, and (c) Lineweaver-Burk plot of different phosphate compounds in a DMT-IO

higher than that of iron in the solution (e.g., 16.5 nM Fe and 2500 μM G6P). The same patterns were also observed in the solution bearing inorganic iron oxide nanoparticles. Like the aged inorganic iron solution, the k of various organic phosphate esters or condensed phosphates at different concentrations were not

r

, and the Michaelis-Menten

<sup>2</sup> <sup>¼</sup> <sup>0</sup>:<sup>997</sup> (2)

, and t1/2 of 295 h. It should be

–10<sup>5</sup>

of G6P hydrolysis was about 1 nM s�<sup>1</sup>

Figure 2.

Nanocatalysts

Figure 3.

102

solutions (IO-D).

1

this aged iron solution with a <sup>k</sup> of 6.53 � <sup>10</sup>�<sup>7</sup> <sup>s</sup>

Michaelis-Menten constant (Km) and maximum velocity (Vm) of different phosphorus in a DMT-IO solution (made by Fe(NO3)3�9H2O and NaOH, aged a week at 80°C, IO-D, soaked a month).

constant (Table 2). The catalytic activity of the different concentration of phosphorus also can be described by the typical Michaelis-Menten equations (Figure 3c). Based on the Lineweaver-Burk linear equation (1/V is a linear function of 1/[S]), the Michaelis-Menten constant (Km) and maximum velocity (Vm), as well as the range of concentration of phosphorous among these compounds, were determined (Table 3). Meanwhile, the catalysis activity was still observed even when the total phosphorus esters exceeded the range of the Michaelis-Menten equations, as with many of the natural enzymes, including the PAP [43].

It should further be pointed out that the similar enzyme kinetics (Michaelis-Menten equations) were observed recently by many inorganic nanoparticles studies, which have been described as nanozyme [44–47]. For example, Fe3O4 [44], α-Fe2O3 [48], γ-Fe2O3 [49], γ-FeOOH [50], Co3O4 [51], MnFe2O4 [52, 53], MFe2O4 (M = Mg, Ni, Cu) [54], ZnFe2O4 [55], NiO [56], and MnO2 [57] have been observed to have peroxidase-like or catalase-like activity, whereas the vanadium pentoxide (V2O5) was demonstrated to have antioxidant enzyme-like (glutathione peroxidase) activity [58–61] and molybdenum trioxide (MoO3) nanoparticles to have sulfite oxidase activity [62].

### 4. Inhabitation effects of tetrahedral oxyanions

The hydrolysis of phosphorus ester was significantly inhibited when the tetrahedral oxyanions were introduced into inorganic iron oxides nanoparticle solution, e.g., G6P in a 10-month aged iron solution (Figure 4), as the natural PAPs. Both the catalytic and the inhibition behaviors of the catalysis in the presence of 5–125 μM G6P with different tetrahedral oxyanions can be described by a Michaelis-Menten equation (Eqs. (3)–(7)) as follows:

$$\text{without any addition}: \frac{1}{v} = 7.4 \times 10^8 + \frac{2.001 \times 10^9}{\text{[G\'6P]}\_o} \quad \left(r^2 = 0.945\right) \tag{3}$$

$$\text{with } \mathbf{1} \,\mu\text{M } \text{MoO}\_4: \frac{\mathbf{1}}{v} = 7.4 \times \mathbf{10}^8 + \frac{\mathbf{1}.044 \times \mathbf{10}^{\text{10}}}{[\text{G\'}\mathbf{P}]\_o} \qquad \qquad \left(r^2 = 0.998\right) \tag{4}$$

$$\text{with } 1 \,\mu\text{M }\text{WO}\_4: \frac{1}{v} = 7.4 \times 10^8 + \frac{3.423 \times 10^{10}}{[\text{G\'6P}]\_o} \quad \left(r^2 = 0.995\right) \tag{5}$$

$$\text{with } 5 \,\mu\text{M }\text{PO}\_4: \frac{1}{v} = 7.4 \times 10^8 + \frac{8.317 \times 10^9}{[\text{G\'eP}]\_o} \qquad\qquad \left(r^2 = 0.988\right) \tag{6}$$

$$\text{with 10 }\mu\text{M }\text{PO}\_4: \frac{1}{v} = 7.4 \times 10^8 + \frac{1.216 \times 10^{10}}{[\text{G\'6P}]\_o} \quad \left(r^2 = 0.997\right) \tag{7}$$

of molybdate and tungstate inhibition are noncompetitive [65, 66, 70–74]. Only

A more significant difference between the inorganic catalyst and the natural phosphoesterase is revealed in their response to the fluoride ion. The activity of all known natural phosphoesterase is very sensitive to fluoride, even at the micromolar level [67, 72–78], while the catalytic activity of the inorganic iron oxides solutions still remain, even when the final concentration of fluoride in the solutions were up to 0.5 M.

The catalyst on the hydrolysis of phosphate ester is sensitive to temperature, as natural enzymes. The optimum temperature for the phosphate ester hydrolysis reaction by these inorganic catalysts was around 50°C (Figure 6), which is comparable to recent observations on the natural enzymes [79–82]. However, catalytic activity of the IO nanoparticles in solution was lost as the temperature was raised to 90°C for an hour or to 72°C for 16 h. This behavior is similar to the thermal denaturation of the natural enzyme. Moreover, the temperature coefficient, Q10, a measure of the hydrolysis velocity, is also decreased as a consequence of increasing the temperature by 10°C. This effect too, is comparable to the general patterns of enzyme behavior in biological systems [80, 81]. Taken together, these observations also carry the implication that moderate, i.e., 50°C, and not high temperatures, were likely favorable to the catalytic reactions from the view of efficiency and speed of the catalyst. In actuality, the catalytic activity of the nanoparticles remained high after removal from their source (IO) for days, even when stored at 18°C, demonstrated

by a storage experiment (Table 4) [29], which further suggested that IO

nanoparticles can be displaced to a considerable distance from their source and still

Relationship between environment temperature and the hydrolysis rate of phosphorus. (a) G6P, (b) ATP,

(c) polyphosphate, and (d) the temperature coefficient Q10 (for details of IOs see Table 1).

orthophosphate for natural PAPs are competitive [65, 66] in most cases.

5. Effect of temperature

Iron Oxide Nanoparticles: An Inorganic Phosphatase DOI: http://dx.doi.org/10.5772/intechopen.82650

Figure 6.

105

#### Figure 4.

Inhibiting behavior of different tetrahedral oxyanions on the hydrolysis of Glucose-6-phosphate in an aged 10-month, 1000 nM Fe(NO3)3 solution at room temperature (22 2°C). (a) Effect of initial concentration of G6P on the initial hydrolysis velocity of G6P, and (b) Lineweaver-Burk plot of aged iron solution in the absence and presence of tetrahedral oxyanions.

The results indicated that the catalysis sites from these catalyst, i.e., the inorganic iron oxide nanoparticles, may be only bound to either the tetrahedral oxyanions (PO4, MoO4, and WO4) or phosphate esters to form an intermediate, but cannot bind both of them at any given moment. The modes of tetrahedral oxyanions and G6P are competitive (Figure 5). The Km, the G6P concentration at which the reaction rate reaches one-half of maximum velocity (vmax/2), was about 2.7 μM G6P in this aged iron solutions with no inhibitors. The KMapp with addition 1 μM WO4, MoO4, and 5 and 10 μM PO4 was 46.2, 14.1, 11.1 and 17.1 μM G6P, respectively. Therefore, the Ki of WO4, MoO4, and PO4 are 0.06, 0.24 and 1.6– 1.9 μM, respectively. It is interesting to compare the catalytic behavior of these inorganic iron oxides nanoparticles solution to natural PAPs and their biomimetics, though the velocities of hydrolysis G6P in the inorganic catalyst are still lower than that of natural phosphoesterase. For natural PAPs, Km and Ki of PO4 is usually in the millimolar range [63–69], only Ki of WO4 and MoO4 is in the micromolar range [65, 66, 70–72]. The value of Km of G6P is 920 μM for PAP extracted from sweet potato [33] and 300–310 μM for those from soybean seed [73]. Besides, the modes

#### Figure 5.

Diagram of catalysis process of G6P hydrolysis in the presence of the tetrahedral anions in the aged inorganic iron solutions. (a) Reaction without tetrahedral anions; (b) Inhibition with the competitive tetrahedral anions.

#### Iron Oxide Nanoparticles: An Inorganic Phosphatase DOI: http://dx.doi.org/10.5772/intechopen.82650

of molybdate and tungstate inhibition are noncompetitive [65, 66, 70–74]. Only orthophosphate for natural PAPs are competitive [65, 66] in most cases.

A more significant difference between the inorganic catalyst and the natural phosphoesterase is revealed in their response to the fluoride ion. The activity of all known natural phosphoesterase is very sensitive to fluoride, even at the micromolar level [67, 72–78], while the catalytic activity of the inorganic iron oxides solutions still remain, even when the final concentration of fluoride in the solutions were up to 0.5 M.

#### 5. Effect of temperature

The results indicated that the catalysis sites from these catalyst, i.e., the inor-

Inhibiting behavior of different tetrahedral oxyanions on the hydrolysis of Glucose-6-phosphate in an aged 10-month, 1000 nM Fe(NO3)3 solution at room temperature (22 2°C). (a) Effect of initial concentration of G6P on the initial hydrolysis velocity of G6P, and (b) Lineweaver-Burk plot of aged iron solution in the absence

oxyanions (PO4, MoO4, and WO4) or phosphate esters to form an intermediate, but

oxyanions and G6P are competitive (Figure 5). The Km, the G6P concentration at which the reaction rate reaches one-half of maximum velocity (vmax/2), was about 2.7 μM G6P in this aged iron solutions with no inhibitors. The KMapp with addition 1 μM WO4, MoO4, and 5 and 10 μM PO4 was 46.2, 14.1, 11.1 and 17.1 μM G6P, respectively. Therefore, the Ki of WO4, MoO4, and PO4 are 0.06, 0.24 and 1.6– 1.9 μM, respectively. It is interesting to compare the catalytic behavior of these inorganic iron oxides nanoparticles solution to natural PAPs and their biomimetics, though the velocities of hydrolysis G6P in the inorganic catalyst are still lower than that of natural phosphoesterase. For natural PAPs, Km and Ki of PO4 is usually in the millimolar range [63–69], only Ki of WO4 and MoO4 is in the micromolar range [65, 66, 70–72]. The value of Km of G6P is 920 μM for PAP extracted from sweet potato [33] and 300–310 μM for those from soybean seed [73]. Besides, the modes

Diagram of catalysis process of G6P hydrolysis in the presence of the tetrahedral anions in the aged inorganic iron solutions. (a) Reaction without tetrahedral anions; (b) Inhibition with the competitive tetrahedral anions.

ganic iron oxide nanoparticles, may be only bound to either the tetrahedral

Figure 4.

Nanocatalysts

Figure 5.

104

and presence of tetrahedral oxyanions.

cannot bind both of them at any given moment. The modes of tetrahedral

The catalyst on the hydrolysis of phosphate ester is sensitive to temperature, as natural enzymes. The optimum temperature for the phosphate ester hydrolysis reaction by these inorganic catalysts was around 50°C (Figure 6), which is comparable to recent observations on the natural enzymes [79–82]. However, catalytic activity of the IO nanoparticles in solution was lost as the temperature was raised to 90°C for an hour or to 72°C for 16 h. This behavior is similar to the thermal denaturation of the natural enzyme. Moreover, the temperature coefficient, Q10, a measure of the hydrolysis velocity, is also decreased as a consequence of increasing the temperature by 10°C. This effect too, is comparable to the general patterns of enzyme behavior in biological systems [80, 81]. Taken together, these observations also carry the implication that moderate, i.e., 50°C, and not high temperatures, were likely favorable to the catalytic reactions from the view of efficiency and speed of the catalyst.

In actuality, the catalytic activity of the nanoparticles remained high after removal from their source (IO) for days, even when stored at 18°C, demonstrated by a storage experiment (Table 4) [29], which further suggested that IO nanoparticles can be displaced to a considerable distance from their source and still

#### Figure 6.

Relationship between environment temperature and the hydrolysis rate of phosphorus. (a) G6P, (b) ATP, (c) polyphosphate, and (d) the temperature coefficient Q10 (for details of IOs see Table 1).


a The Pi of the 20 μM G6P (DIW, control) after 120 h at the room temperature was changed from 1.67 to 1.83 μM. The corresponding half-life was 10,450 h. The Pi of the 20 μM ATP (DIW, control) after 120 h at the room temperature was changed from 1.89 to 2.07 μM. The corresponding half-life was 9290 h.

#### Table 4.

Effect of storage conditions on the catalysis activity of iron oxide (IO-F) nanoparticle solutions.<sup>a</sup>

maintain catalytic activities for a considerable time. Meanwhile, low temperatures, even frozen conditions, also favor the persistence of catalytic activity from these IO nanoparticles. These are important from the view of astrobiology (origin of life) [28], but also for plant acquisition, nanoengineering and the potential application for industrial production.

#### 6. Effect of pH and buffer solution

pH is another key factor for enzyme activity. The aged inorganic iron solution or the water bearing iron oxide nanoparticles, e.g., DMT-IO, are generally mildly acidic (pH 5.5–6.5). Various concentrations of bicarbonate were introduced in the DMT-IO system, but in all cases enzyme-like activity for phosphate ester hydrolysis remained quite high (Figure 7a). In general, the most favorable pH of the enzymelike activity was found to be between 6 and 7, though the phosphorus source, the concentrations of bicarbonate, and the type of DMT-IO also influenced its activity (Figure 7b and c). When pH was raised beyond 7 (e.g., pH 7, 7.2 and 8), the catalysis coefficient, k, decreased as the concentration of HCO3 increased, especially for the DMT-IO-D. When pH in solution was <7 (e.g., pH 6.2, 6.4, and 6.8), however, there were no clear patterns of k with respect to the concentration of HCO3 and both the ATP and G6P in these two nanoparticles-bearing solutions. At the same time, k at weak acidic conditions (pH 6.2–6.8) was much higher than at weak base conditions (pH 7–8). This conclusion was further supported by an additional experiment involving the hydrolysis of ATP, whereby the pH values of DMT-IO solution were extended from 4 to 9.3 units by employing four different buffer systems (1.0 M acetate buffer (pH 4.0–5.6), 0.2 M dimethylglutaric acid buffer (pH 4.2–6.8), 20 mM NaHCO3 (pH 7.6–9.3), and 40 mM NaHCO3 (pH 5.8–9.3)) (Figure 7d).

It should be pointed out that the catalysis capacity of these solutions bearing inorganic iron oxide nanoparticles is closely related to the buffer used in the system. The catalytic activity dropped precipitously after a small amount of citrate buffer (pH 4.0–6.2) or tris(hydroxymethyl)-aminomethane (TRIS) (pH 5.8–7.2) was introduced into an inorganic iron solution (Table 5). Both citrate [83, 84] and

Relationship between pH and the hydrolysis rate of phosphorus. (a) 100 μM four phosphorus in DMT-IO-G; (b) and (c) bicarbonate concentration on 20 μM G6P DMT-IO-D and F; (d) different buffers for 20 μM ATP

Figure 7.

107

in DMT-IO-F (for details of IOs see Table 1).

Iron Oxide Nanoparticles: An Inorganic Phosphatase DOI: http://dx.doi.org/10.5772/intechopen.82650

Iron Oxide Nanoparticles: An Inorganic Phosphatase DOI: http://dx.doi.org/10.5772/intechopen.82650

Figure 7.

maintain catalytic activities for a considerable time. Meanwhile, low temperatures, even frozen conditions, also favor the persistence of catalytic activity from these IO nanoparticles. These are important from the view of astrobiology (origin of life) [28], but also for plant acquisition, nanoengineering and the potential application

Effect of storage conditions on the catalysis activity of iron oxide (IO-F) nanoparticle solutions.<sup>a</sup>

The Pi of the 20 μM G6P (DIW, control) after 120 h at the room temperature was changed from 1.67 to 1.83 μM. The corresponding half-life was 10,450 h. The Pi of the 20 μM ATP (DIW, control) after 120 h at the room temperature was

Treatment 20 μM G6P 20 μM ATP

11 days at 22°C 11R 5.49 0.004 13.89 0.064 3.2 4.47 0.016 9.87 0.255 6.0 11 days at 4°C 11 L 5.51 0.010 13.64 0.042 3.3 4.36 0.005 9.71 0.066 6.3

1hPi (μM) 5 h Pi (μM) t0.5 (h) 1 h Pi (μM) 5 h Pi (μM) t0.5 (h)

5F6L 4.57 0.013 11.48 0.030 4.5 3.76 0.009 7.61 0.043 9.4

5L6R 5.85 0.011 14.10 0.211 3.1 4.51 0.018 10.30 0.123 5.7

5H6R 2.93 0.01 6.68 0.020 11 2.71 0.012 4.92 0.032 19

5H6L 2.62 0.002 5.44 0.001 16 2.48 0.002 3.72 0.04 36

9H2R 2.43 0.005 4.07 0.005 26 2.38 0.016 3.08 0.025 60

pH is another key factor for enzyme activity. The aged inorganic iron solution or

the water bearing iron oxide nanoparticles, e.g., DMT-IO, are generally mildly acidic (pH 5.5–6.5). Various concentrations of bicarbonate were introduced in the DMT-IO system, but in all cases enzyme-like activity for phosphate ester hydrolysis remained quite high (Figure 7a). In general, the most favorable pH of the enzymelike activity was found to be between 6 and 7, though the phosphorus source, the concentrations of bicarbonate, and the type of DMT-IO also influenced its activity (Figure 7b and c). When pH was raised beyond 7 (e.g., pH 7, 7.2 and 8), the catalysis coefficient, k, decreased as the concentration of HCO3 increased, especially for the DMT-IO-D. When pH in solution was <7 (e.g., pH 6.2, 6.4, and 6.8), however, there were no clear patterns of k with respect to the concentration of HCO3 and both the ATP and G6P in these two nanoparticles-bearing solutions. At the same time, k at weak acidic conditions (pH 6.2–6.8) was much higher than at weak base conditions (pH 7–8). This conclusion was further supported by an additional experiment involving the hydrolysis of ATP, whereby the pH values of DMT-IO solution were extended from 4 to 9.3 units by employing four different buffer systems (1.0 M acetate buffer (pH 4.0–5.6), 0.2 M dimethylglutaric acid buffer (pH 4.2–6.8), 20 mM NaHCO3 (pH 7.6–9.3), and 40 mM NaHCO3 (pH 5.8–9.3))

for industrial production.

5 days frozen (18°C) and 6 days at 4°C

Nanocatalysts

5 days at 4°C and 6 days at 22°C

5 days at 50°C and 6 days at 22°C

5 days at 50°C and 6 days at 4°C

9 days at 50°C and 2 days at 22°C

a

Table 4.

(Figure 7d).

106

6. Effect of pH and buffer solution

changed from 1.89 to 2.07 μM. The corresponding half-life was 9290 h.

Relationship between pH and the hydrolysis rate of phosphorus. (a) 100 μM four phosphorus in DMT-IO-G; (b) and (c) bicarbonate concentration on 20 μM G6P DMT-IO-D and F; (d) different buffers for 20 μM ATP in DMT-IO-F (for details of IOs see Table 1).

It should be pointed out that the catalysis capacity of these solutions bearing inorganic iron oxide nanoparticles is closely related to the buffer used in the system. The catalytic activity dropped precipitously after a small amount of citrate buffer (pH 4.0–6.2) or tris(hydroxymethyl)-aminomethane (TRIS) (pH 5.8–7.2) was introduced into an inorganic iron solution (Table 5). Both citrate [83, 84] and


a Buffer experiments were conducted with a 1000 nM aged (14-month) inorganic iron solution. The iron solution diluted by DIW (1:1) was used as a control. Either 4 ml of 10 mM Tris-HCl (pH 7.0) or 4 ml of DIW was mixed with a 4 ml of aged iron solution, then 0.1 ml of 1.6 or 4.0 mM G6P stock solution was added to make a final solution containing 20 or 50 μM G6P. Pi concentration was determined at 0, 0.17, 1, 2 and 6 h, respectively, after the addition of G6P. The results of Pi in the table was an average of three replications. The rate constant and half-life were calculated based on the first order kinetics.

phosphatases; bacterial exonucleases; diadenosine tetraphosphatase; 5<sup>0</sup>

can be present in aqueous solutions, as binuclear dihydroxobridging [Fe

hydroxo-bridged Fe-(OH)2-Fe dimers are the structure units in the polymetric hydroxo complex, which are dependent on pH and aging time [105, 106]. Molecular dynamics simulation further demonstrated the presence of aqueous di-iron or poly-irons, in which the Fe-Fe distance is 3.0–3.5 Å, with bonds by oxo-bridge or hydroxo-bridge [107]. Meanwhile, the (hydr)oxo-bridged Fe-Fe structure has been confirmed by experiments in the interface of iron oxide (IO, solid) to water [108–111]. The μ-oxo iron ion have been identified in-situ in the high concentrated inorganic iron solution (e.g., 0.1 M Fe(NO3)3 [101], and 0.1 M FeCl3 [112]. The solubility of IOs further indicates that the critical ferrihydrite nucleus with an equivalent diameter of �15 Å and containing only �30 Fe atoms is stable in aqueous solution [113]. The 10-angstrom discrete iron-oxo cluster (known as the Keggin ion, Fe13) is also soluble [114], as a constitute structure of ferrihydrite nanoparticles [102]. Consequently, iron oxide nanoparticles with 3.5 Å oxo-Fe bindings (e.g., doubly shared iron octahedra) such as ferrihydrite, goethite, hematite, magnetite, and even green rust (fougerite) can be presented in the natural environment (Figure 8b) [115–121]. Therefore, it is reasonable to suggest that the oxo bridged Fe-Fe structure in the aqueous IO nanoparticles contribute to the catalysis of phosphate ester hydrolysis [28]. This compares to the activity of the aged nanomolar inorganic iron ion solutions [26, 27] and mimics of the artificial phosphatases

4) cations in the hydrolysis products of cations [Fe(H2O)6]

n+ and oxobridging [Fe(H2O)5(μ-O)Fe�(H2O)5]

(H2O)4(μ-OH)2Fe(H2O)4]

(b) Fe-Fe structure in different iron oxides phases.

Iron Oxide Nanoparticles: An Inorganic Phosphatase DOI: http://dx.doi.org/10.5772/intechopen.82650

Figure 8.

109

phosphodiesterase; sphingomyelin phosphodiesterase, an enzyme involved in RNA debranching; and a phosphatase in the bacteriophage genome as well as for the family of Ser/Thr protein phosphatase (PP1, PP2A, and calcineurin) [95, 96]. Based on the μ-(hydr)oxo metal bridge structure, different artificial phosphatases have been synthesized by using different organic ligands to stabilize the metal center [19, 97, 98]. It is well known that iron speciation changes due to ion (III) hydrolysis in the solution during the aging process, diiron or polyirons oxide with the oxo-bridge or hydroxo-bridge (bond) might be formed [99–103]. Based on the quantum-chemical calculations by density-functional theory, dihydroxobridging binuclear compounds

The metal center of phosphatase. (a) The μ-(hydr)oxo-bridges in purple acid phosphatase (PAP), and


n+ (n = 2,

m+ (m = 2, 3) [104]. The

#### Table 5.

Interaction between Tris-HCl buffer and aged inorganic iron solution on the hydrolysis rate of G6P.a

TRIS [85] can react with Fe (III) in solution to form the aqueous Fe-complex, particularly at high ratios of citrate or TRIS to Fe (>>10,000:1 molar ratio). But the catalysis of these solutions does not change when the acetic acid-acetate buffer system was introduced, the final acetate concentration in the solution was up to 0.25 M. All of these responses imply that the significant change was not due to the pH itself, but to the interactions between nanoparticles in the solution and chemicals in the environment. It has been reported that some buffer systems can significantly inhibit the activity of natural enzymes, for example, citrate on special PAPs [86] and alkaline phosphatase [87] and TRIS on aminopeptidase and RimO methylthiotransferase [88, 89] due to structure changes and metal-complex formation [84, 85, 90].
