Kinetic Study on the Hydrolysis of p-nitrophenyl picolinate Catalyzed by g-C 3 N 4 /LaNiO 3 -Perovskite-type Oxides

LaNiO 3 -Perovskite-type oxides, as a novel hydrolytic catalyst, were synthesized using a sol-gel-calcination method, and were characterized by employing x-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), and Brunauer–Emmett–Teller (BET). It was revealed that LaNiO 3 and 2% g-C 3 N 4 /LaNiO 3 -Perovskite-type oxides are nanoparticles about 12 nm and 10nm easily agglomerated in large amounts, and the specific surface area of LaNiO 3 is 21.277 m 2 /g and 2% g-C 3 N 4 /LaNiO 3 is 26.645 m 2 /g, respectively. The results also indicate that there are irregular microporoes in the material. In the catalytic activity test, the absorption spectra were collected when hydrolysis of p-nitrophenyl picolinate (PNPP) (C PNPP =1-5×10 -5 mol/L) was catalyzed by LaNiO 3 (0.5g/L) and 2% g-C 3 N 4 /LaNiO 3 (0.5g/L) in buffer solution at different pH values (6.5-7.8), respectively. Based on the absorption spectrum data, the pseudo first-order rate constant is estimated to be 0.59min -1 and 1.14min -1 , respectively. In addition, the proposed kinetics model of this reaction was confirmed by the results of the spectrum and the calculations.


Introduction
Enzymes are known for their efficient catalysis of reactions in the body.
However, the limitations of natural enzymes such as limited source, instability, complex structure and short life span have restricted their applications in research.
People are concerned about the mechanism of carboxylesterase catalysis in vivo and how to achieve catalysis in vitro, How to simulate the synthesis of simple artificial enzymes and construct a chemical model for natural enzyme simulation has aroused widespread interests. [1,2] In recent decades, the decomposition of carboxylic acid esters by metallomicelles based on the polymetallic active sites of natural enzymes has rapidly developed. The PNPP (p-nitrophenyl picolinate) is often used as a model of carboxylesterase to study the kinetic process of catalytic hydrolysis. Bin Xu et al. [3] reported that the catalytic constant kcat (1111 s −1 ) for the Cu2L(OAc)-induced PNPP hydrolysis is about 1.8 times greater than that for the CuHL-promoted hydrolysis of PNPP(625 s −1 ). At the same time, the binding between PNPP and the binuclear Cu2L(OAc) is much easier than that of PNPP to CuHL. In the above two metal mimetic models, copper ions were used to mimic the active center structure of the enzyme. Although metal micelles have high catalytic activity, their application is limited because they cannot be reused. Dr. Xiao-lan Huang provided us with a new idea --inorganic enzyme [4]. Huang previously reported that inorganic iron ion solutions, in which iron oxide nanoparticles are adsorbed in dialysis membrane tubes, significantly promote the hydrolysis of various phosphate esters, including inorganic 4 polyphosphates, with the same kinetics as enzymes. The results of the study on iron oxide, vanadium pentoxide and molybdenum trioxide nanoparticles showed that they simulated peroxidase, bromine peroxidase and sulfite oxidase, respectively. All the above results indicated that the oxygen-metal bond in the oxide nanoparticles could simulate the functions of these corresponding natural metalloproteins. Inorganic biocatalysts no longer follow the traditional approach of replicating enzymes, supporting the hypothesis of metabolic precedence. As biocatalysts, these inorganic nanoparticles with enzyme-like activity may have acted in the natural terrestrial environment, and may have also acted in the early earth environment. They may have played an important role in the metabolic pathways of C, H, O, S and P in the emergence and early evolution of life. Can we find an inorganic enzyme like this?
Perovskite has become an important new material in the mixed oxide family because of its excellent thermal stability, mechanical stability, electronic structure, ionic conductivity, electron mobility and redox ability, as well as its low cost [5]. It has good performance in eliminating toxic compounds under photocatalysis.
As known for their multimetal ions, perovskite material is a kind of composite metal oxide with natural mineral CaTiO3 structure, which is represented by the general formula ABO3. In this structure, A-site ions on the corners of the lattice, and B-site ions on the center of the lattice, and oxygen ions form 12 coordination or 6 coordination. Usually, A is mostly rare earth, alkaline earth or alkali metal ion, and B is a transition metal of 3d, 4d or 5d configuration [6]. In recent years, perovskites have been widely used for solar cells [7,8], photocatalysis [9,10]and other fields [11][12][13]. 5 The B-site metal cation in the perovskite structure has a significant effect on the catalytic activity. However, its application to catalyze the hydrolysis of esters has not been reported. Jin Luo et al. [14]designed and constructed a z-type hybrid composed of LaNiO3 nanoparticles and g-C3N4 nanometers by a simple heat treatment method.
As expected, compounds significantly improved tetracycline (TC) degradation in aqueous solutions under visible light irradiation (> 420nm).The TC degradation rate was up to 0.00282 min -1 , 3.8 and 3.9 times higher than pure g-C3N4 and pure LaNiO3.
Due to the synergistic effect between LaNiO3 and g-C3N4, the enhancement of photocatalytic activity is mainly attributed to the formation of z-LaNiO3/ g-C3N4 heterojunction, which not only promotes the interfacial charge transfer efficiency, but also preserves the redox ability of strong light generating electrons and holes. I.
Rossetti et al. [15]synthesized La1-xAgxMnO3±δ，x= 0；0.05；0.10; The activity of the catalysts prepared by FP is always higher than that of the catalysts prepared by SG with the same nominal composition in the flame spray (FP) and citrate sol-gel (SG) reactions for the flameless combustion of methane. In addition, Ag's partial substitution of La led to a significant increase in the activity of the catalysts prepared for SG and FP, and the catalytic activity increased with the increase of Ag substitution.
Haman Tavakkoli et al. [16] prepared the perovskite-type LaFe0.9Co0.1O3 (LFCO1) and LaFe0.1Co0.9O3 (LFCO2) nanoparticles (NPs). The average particle size is 20-40nm, to evaluate the ability of the two NPs to remove the pesticide nilium from the aqueous solution; dynamic studies were performed on LFCOs NPs with different exposure time, temperature, initial pesticide concentration and adsorbent dose to find the 6 optimal conditions. Results showed the percentage of removal of vitavax by LFCO2 NPs is less than that for LFCO1 NPs under the same conditions. Shahzad Afzal et al. [12] synthesized mesoporous nanocast perovskites (NC-LaMnO3 and NC-LaFeO3) and applied them in catalytic ozonation of 2-chlorophenol. NC-LaMnO3 showed good performance in terms of catalytic activity and stability. A stronger interaction between NC-LaMnO3 and O3 as well as an interfacial electron transfer resulted in enhanced catalytic activity. The B-site metal ion has a great influence on the catalysis of organic compound decomposition.
Based on the researches in the above literatures, the metal ion in B-site of perovskite can be analogous to the active central structure of the metal hydrolase.
Therefore, in this study, we have synthesized LaNiO3 and 2% g-C3N4/LaNiO3 by sol-gel method, Perovskite composite oxide was used to simulate the active center structure of the enzyme, carried out LaNiO3 and 2% g-C3N4/LaNiO3 catalyzed PNPP hydrolysis experiment. The model of carboxylesterase (PNPP) was used to study the kinetics of catalytic hydrolysis. We have established the reaction intermediate state complex dynamic model, and calculated the pH-independent rate constant of the pseudo first-order reaction by a reasonable way. Finally we have proposed the reaction mechanism and the proposed mechanism are proved to be correct.

Preparation of LaNiO 3 and 2% g-C 3 N 4 /LaNiO 3
LaNiO3 was synthesized by the citric acid sol-gel method [18]. La(NO3)3·6H2O, Ni(NO3)2·2H2O and citric acid were accurately weighed according to the stoichiometric ratio 1:1:2. Firstly, citric acid was dissolved into deionized water as complexing agent, and then La(NO3)3·6H2O and Ni (NO3)2·2H2O were added. At the same time, ammonia was added dropwise until pH was 1. The mixed solution was heated at 70 °C in a water bath for 10h for the formation of sol. After that, the sol was placed in a blast oven at 120 °C for 10 h to obtain the precursor. The precursor was calcined at 400 °C for 4h and then at 700 °C for 4h in the muffle furnace under air.
The g-C3N4 was synthesized by calcination. A certain amount of urea was taken and dried at 80℃ for 24 hours. The samples were placed in the muffle furnace with programmed temperature rise, and the temperature was set at 200℃ for 4h, 400℃ for 2h, and 550℃ for 2h. The temperature rise rate was 5℃/min. When the crucible is removed, let it cool to room temperature. The light yellow g-C3N4 powder can be prepared in the crucible.
The total mass was 0.01g. A certain amount of g-C3N4 and a certain amount of LaNiO3 were weighed and dispersed in 10mL anhydrous ethanol, respectively. LaNiO3/ethanol dispersion system was dripped into g-C3N4/ethanol dispersion system while stirring. 8 The mixed system was by ultrasound 30 minutes and by stirred at room temperature for 12 hours then transferred to the reaction kettle carefully and placed in the blast drying oven at 120℃ for 6h. It was cooled down to room temperature. After centrifugation, it was dried for 12 hours and weighed.

Preparation of Tris-HCl buffer solution
A certain amount of Tris was dissolved in deionized water, and then 0.01 mol/L KNO3 or HCl were added until pH value is 6.5, 7, 7.3,7.5, 7.8. 0.01mol·L -1 Tris-HCl buffer solution was obtained as a stock solution.

Characterization
In order to characterize the structures, morphologies, and compositions of the samples, the following measurements were conducted. The structures of catalysts were analyzed using X-ray Diffraction (XRD) with Cu Kα at λ = 0.154 nm. The morphologies of the catalysts were examined using Scanning Electron Microscopy (SEM, Hitachi S-4800).
The surface areas were determined by Brunauer-Emmett-Teller (BET).

Catalytic activity test
Catalytic experiments by LaNiO3 were conducted in Tris-HCl buffer solution at different pH (6.5, 7, 7.3,7.5, 7.8,). 0.01g catalyst was added in 20 ml Tris-HCl buffer solution. Then it was mixed with PNPP solution (concentration is 1×10 -5 -5×10 -5 mol/L) prepared with acetonitrile. The suspension was filtered every 10 minutes, injected into a 3mL catalyst-containing cuvette with a micro-syringe, and analyzed by the dual-beam ultraviolet-visible (UV-Vis) spectrophotometer with a thermostatic device (TU-1901). 9 The reaction rates were followed by monitoring the variation of p-nitrophenol at the wavelength of 400 nm with time under the conditions of excess of catalyst over substrate (PNPP). The apparent first-order rate constants were obtained on fitting an equation (ln (At-A∞)-ln (A0-A∞) = -kobsdt) by linear least squares treatment [19] by repeating three times. The standard deviation of the reaction rate is less than 3%.  In figure (a), LaNiO3 is spherical with uniform size, and the particle size is significantly larger than that of the XRD pattern. This is due to the large agglomeration of nanoparticles. During the preparation of LaNiO3, the solution had a high pH and was not so acidic, which helped perovskite grains to disperse and form a planar and loose network structure, which was conducive to the increase of specific surface area and the increase of contact between PNPP and the catalyst. As shown in figure (b), a large number of holes are distributed on the material plane, with small (a) (b) (b) 11 and regular pore sizes, but different pore sizes. These holes can increase the contact between PNPP and the catalyst, adsorb PNPP, and accelerate the catalytic reaction rate.

Structures and morphologies of the catalysts
The N2 isotherm in Fig. 3 (a) corresponds to a type IV isotherm in the IUPAC classification. At moderate relative pressure, the type IV isotherm rises rapidly due to the occurrence of capillary condensation. The characteristic of these curves is their hysteresis loops, and they correspond to a system in which the porous adsorbent exhibiting capillary condensation. According to the IUPAC classification, the loop observed is ascribed to type H3 loops [22], which does not exhibit limiting adsorption at high relative pressures and has no obvious saturated adsorption platform, indicating the presence of irregular microporoes in the material. The surface area was calculated to be 21.277 m 2 /g.   Fig. 3 (b) shows the pore size distribution. The pore size was derived from desorption data and calculated from the isotherm using the BJH model. The main size 12 of pores ranges from 2 to 10 nm and the average pore size is 1.839 nm by calculation, which consists with the results of SEM in Fig. 2.
Many literatures reported that g-C3N4 and perovskite oxides were doped with each other to increase the specific surface area of the catalyst, so as to improve the catalytic efficiency. Can this improve the efficiency of ester hydrolysis? Wt1 %-10% g-C3N4/ LaNiO3 composite catalyst was prepared by combining simple mechanical stirring with hydrothermal method. We found that the 2% g-C3N4/ LaNiO3 composite catalyst had good catalytic effect. So we studied the 2% g-C3N4/ LaNiO3 composite catalyst.
In  N2 adsorption and desorption of 2% g-C3N4 / LaNiO3 catalyst are shown in Fig.   5. Fig. 5 (a) is 2% g -C3N4 LaNiO3 / N2 adsorption -stripping isotherm is similar to (a) (b) 13 the isotherm LaFeO3, according to the IUPAC classification, corresponding Ⅳ type isotherm, the relative pressure (P/P0) value in the middle section, depending on the presence of isotherm rise suddenly, exist this kind of circumstance is the type IV isotherm, is the result of the occurrence of capillary condensation. Another prominent feature of the type IV isotherm is the presence of adsorption hysteresis in the intermediate segment, which proves to be a porous adsorbent system. H3 type hysteresis ring has no obvious saturated adsorption platform, and shows no adsorption saturation in the area of higher relative pressure, which is a fracture hole material.
Through the above analysis, it is shown that 2% g-C3N4 / LaNiO3 perovskite composite oxide is a porous adsorption material with irregular pore structure. It is a fissure pore material, and the measured specific surface area is 26.645 m 2 /g.

Apparent rate constants for hydrolysis of PNPP (pH=6.5-8, T=298K)
Catalytic reaction proceeds as described in the experimental section. In the catalytic reaction system, the concentration of PNPP substrate is much larger than the catalyst concentration, so the catalyzing reaction for the decomposition of PNPP can be regarded as a pseudo first-order reaction. Thus, the pseudo first-order apparent rate constant can be calculated according to the rate equation. Table 1 shows the pseudo-first-order rate constants obtained at different pH values and 298K. Although the rate of PNPP spontaneous hydrolysis in buffer is small, the effects of PNPP spontaneous hydrolysis on the catalytic reaction still need to rule out. As the buffered pH and concentration increases, the catalytic effect of LaNiO3 increases obviously as shown in Table 1. From this table, the apparent rate constants of LaNiO3, kobsd, is 0.5726 min -1 at pH=7.8, C=5×10 -5 mol/L and T=298K. As mentioned above, the optimal catalyst, 2% g-C3N4/ LaNiO3, was selected to further study its catalytic kinetics. In the tris-HCl buffer system under different pH conditions, CPNPP=1×10 -5 , 2×10 -5 , 3×10 -5 , 4×10 -5 , 4×10 -5 , 5×10 -5 mol/L, 298K, CCat =0.5g/L, 2% g-C3N4/ LaNiO3 catalyst catalyzed the hydrolysis of PNPP at the pseudo first order apparent rate constant is shown in table2. The self-hydrolysis rate of PNPP 16 in buffer solution is very small. However, the effect of self-hydrolysis rate needs to be excluded to ensure the accuracy of experimental results. With the increase of buffer pH and PNPP concentration, the catalytic efficiency of 2% g-C3N4/ LaNiO3 increased.

Reaction mechanism for the catalytic hydrolysis of PNPP
As shown in Table 1, apparent rate constant of PNPP catalytic hydrolysis by LaNiO3 is greater than the rate of PNPP spontaneous hydrolysis in buffer. It has been reported in the literature that complexes can bind to water molecules and form hydrates [23]. The N atom on the pyridine ring in the PNPP molecule can coordinate with the metal ion to form a complex that promotes the decomposition of the PNPP molecule [24]. On the basis of the references and the experimental results, the mechanism of the hydrolysis of PNPP catalyzed by perovskite-type oxide catalysts LaNiO3 was proposed in this work.
In order to prove the existence of the complex, the spectral scanning 18 (200nm-700nm) of the PNPP-added tris-HCl buffer system was carried out by ultraviolet-visible spectrophotometer. The maximum absorption wavelength of PNPP molecule at 272nm and the maximum absorption wavelength of the catalyst was 246nm. After the catalyst was added, the same wavelength spectrum scan was performed at the same time, and the maximum absorption wavelength was blue-shifted.
The results are shown in Fig. 6.  [25]. Therefore, LaNiO3 can catalyze the decomposition of PNPP efficiently.
Metal ions in this catalyst play an important role in this process. One is to disperse the negative charge of the complex, and the other is to activate the water molecules by metal ions, leading to the reactions perform much more easily. PNPP spontaneous hydrolysis can be ignored because of its relatively low reaction rate. From fig. 7, the rate equation can be expressed:

Kinetic model of first-order rate constant
(1) 21 According to the law of chemical equilibrium: (2) Km [26] from the steady state hypothesis can be determined as follows: (3) Combination of equations (2) and (3), (4) According to the relationship between the concentrations of the catalyst species during the reaction, the following formula can be obtained: Substituting the formula (7) into the formula (1), Reciprocal on both sides of the equation, So the final formula we get is 22 (11) In this equation, k2 is the pseudo first-order rate constant in dependent of pH. [S] is the initial concentration of PNPP. kobsd is the observed rate constant of production.
According to the equation (11), 1/kobsd and 1/[S] are linear, and k2 can be obtained by the slope. The results are shown in Fig. 8(a) and (b), and the results show that 1/ kobsd and 1/[S] does exhibit a good linear relationship, and the correlation coefficient r 2 >0.99, which proves that the proposed catalytic mechanism and kinetic model are reasonable.   Table 3 indicates that the acidity of the buffer solution has an effect on the pseudo first-order reaction rate constant kH. The possible reason is shown in Fig. 9.
When pH is 7.8, the water molecules are easily dissociated to form a negatively charged intermediate state, which will speed up the removal of the product p-nitrophenol, and then accelerate the hydrolytic reaction. In Figure 10, Ka represents the acid ionization equilibrium constant, and kH is the pseudo first-order reaction rate constant independent of the hydrogen ion concentration. According to Table 3 and Fig. 10, kH and Ka are calculated to be 0.59 (min -1 ) and 6.67×10 -8 , respectively.  Table 3 and Fig. 11, kH and Ka are calculated to be 1.14 (min -1 ) and 1.08×10 -7 , respectively. The stability of LaNiO3 is investigated in Fig. 12. After 3 continuous hydrolysis cycles, the catalytic activity of LaNiO3 remained essentially unchanged. After the test, it was slightly lowered. However, it has no effect on LaNiO3-catalyst stabilization.

Conclusion
In this work, we have studied hydrolysis of PNPP catalyzed by LaNiO3 and 2% g-C3N4/LaNiO3 and the conclusions are summarized as follows: (1) perovskite-type oxides LaNiO3 and 2% g-C3N4/LaNiO3 nanoparticles were synthesized using a citric acid sol-gel-calcination method; (2) the proposed reaction mechanism in this work has been confirmed by spectrum and kinetic calculations; (3) perovskite-type oxides LaNiO3 exhibited good catalytic activity and stability to catalyze the hydrolysis of PNPP; (4) the pseudo first-order reaction rate constant of LaNiO3 and 2% g-C3N4/LaNiO3 independent of pH is 0.59 and 1.14 min -1 , respectively. This result indicates that we have not found the ideal doped ratio, which will be the goal of our future research.
In this paper, inorganic enzyme was used to simulate carboxypeptidase to catalyze PNPP. Although the reaction rate could not be compared with the decomposition rate of micelle system, the decomposition rate was much higher than the self-hydrolysis of PNPP. In recent years, the inorganic enzymes proposed by scientists have given us a lot of enlightenment and confidence, and we believe that other methods can be used to further improve the reaction rate in future studies.

Availability of data and materials
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Competing interests
The authors declare that they have no competing interests.

Funding
Not applicable.

Authors' contributions
Changyu Ye was the main author of the work, performed syntheses, and coordinated all characterization and catalytic studies. Ruiwang was responsible for electron microscopy, Haoyu Wang for BET test, Huixing Zhang and Fubin Jiang assisted with manuscript writing. All authors read and approved the final manuscript.