Method for improving stability of phosphotriesterase
Through the covalent cross-linking immobilization technology of inulin and chitosan, the immobilization of phosphate triesterase solves the problem of insufficient stability and significantly improves the thermal, pH and solvent stability of the enzyme.
Patent Information
- Application Number
- CN202411946567.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-04-15
AI Technical Summary
How to improve the stability of phosphate triesterase and solve its instability problem to the external environment.
The phosphotriesterase is immobilized by covalent crosslinking immobilization technology of inulin and chitosan to form an inulin-chitosan covalent crosslinking immobilized phosphotriesterase.
It significantly improves the thermal stability, pH stability and solvent stability of phosphate triesterase, and alleviates the damage to enzymes by environments such as high temperature, low pH and organic solvents.
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Figure CN119823979A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of improving the stability of phosphotriesterase, and specifically relates to a method for improving the stability of phosphotriesterase through the covalent cross-linking immobilization technology of inulin and chitosan. Background Art
[0002] Bioenzymes are highly efficient, low-corrosive, and environmentally friendly. They can degrade typical chemical agents such as G-type, V-type neurotoxic agents, and H-type erosive agents by biocatalytic means such as oxidation and hydrolysis. They are considered to be "one of the most ideal disinfectants" and meet the development needs of green disinfection. Organophosphorus degrading enzymes (OPH, EC 3.1.8.1) are currently the most studied type of enzyme in the bioenzymatic decontamination of organophosphorus nerve agents. Organophosphorus degrading enzymes can hydrolyze the PO, PS, and PF bonds of triesters of phosphates, organophosphorus thioalkyl esters, and fluorophosphates, and have strong hydrolysis and detoxification effects on organophosphorus agents GB, GD, VX and their simulants diisopropyl fluorophosphate (DFP), paraoxon, parathion, and demeton.
[0003] Compared with free enzymes, immobilized enzymes have a wider application space due to their good stability, high reuse rate, and easy separation from the reaction system. Inulin (Inu) contains a large number of hydroxyl groups and has good water solubility. Using it to covalently cross-link immobilize biological enzymes can significantly improve the stability of biological enzymes. Chitosan (Cs) is a linear polymer composed of amino-D-glucose and N-acetyl-D-glucosamine connected by β-(1,4) glycosidic bonds. There are abundant hydroxyl and amino groups in the molecule, with an average molecular weight of tens of thousands to hundreds of thousands or even millions. It has the advantages of being non-toxic and degradable. Summary of the invention
[0004] 1. Technical issues to be resolved
[0005] The invention provides a method for improving the stability of phosphotriesterase to solve the technical problem of how to improve the stability of phosphotriesterase to the external environment.
[0006] (II) Technical solution
[0007] In order to solve the above technical problems, the present invention provides a method for improving the stability of phosphotriesterase, which comprises the following steps:
[0008] S1. Expression and purification of phosphotriesterase
[0009] The glycerol bacteria expressing phosphotriesterase with a maltose binding protein tag were added to LB medium and revived overnight; then the bacteria were transferred to TB medium and cultured until OD600 = 0.6-0.8, and IPTG and zinc sulfate were added for induction; the bacteria were collected by centrifugation, crushed by an ultrasonic crusher, and then the crushed supernatant was collected by centrifugation to obtain a crude enzyme solution; the PTE in the crude enzyme solution was further purified by an affinity chromatography column to obtain purified PTE;
[0010] S2. Immobilization of phosphotriesterase by covalent cross-linking of inulin and chitosan
[0011] Weigh inulin, add acetic acid-sodium acetate buffer to fully dissolve, then add sodium periodate solution prepared with the same buffer, react at room temperature in the dark, add a small amount of ethylene glycol to terminate the reaction, and fully dialyze the reaction system to obtain an inulin derivative with an aldehyde group;
[0012] Weigh chitosan and fully dissolve it with citric acid buffer to obtain a chitosan solution; adjust the buffer of the inulin derivative to citric acid buffer with citric acid buffer, mix it with the chitosan solution, add sodium cyanoborohydride, react at room temperature, and dialyze it with phosphate buffer overnight to obtain an inulin-chitosan complex; then add the purified PTE and sodium cyanoborohydride obtained in step S1, react overnight, and dialyze to remove NaCNBH 3 , and obtained inulin-chitosan covalently cross-linked immobilized phosphotriesterase.
[0013] Further, in step S1, a glycerol bacteria expressing a phosphotriesterase with a maltose binding protein tag is taken, added to 100 ml of LB medium at a ratio of 0.5%, and revived at 37°C overnight; then transferred to TB medium at a ratio of 2%, cultured at 37°C until OD600 = 0.6-0.8, IPTG and zinc sulfate are added, and induced at 16°C for 24 hours; the bacteria are collected by centrifugation, crushed with an ultrasonic crusher, and then the crushed supernatant is collected by centrifugation to obtain a crude enzyme solution.
[0014] Further, in step S1, the culture was carried out at 37°C and 200rpm until OD600 = 0.6-0.8; IPTG and zinc sulfate were added at a final concentration of 0.1 mM; the cells were induced at 200rpm at 16°C for 24 hours; the cells were collected by centrifugation at 4000rpm, crushed with an ultrasonic crusher for 16 minutes, and then the crushed supernatant was collected by centrifugation at 10000rpm.
[0015] Furthermore, in step S1, an affinity chromatography column is used to further purify the PTE in the crude enzyme solution: first, the affinity chromatography column is equilibrated with a 20 mM Tris-HCl buffer containing 20 mM imidazole and 0.5 M NaCl for 10 to 15 column volumes; the crude enzyme solution is loaded, and then the chromatography column is equilibrated for 10 to 15 column volumes, and finally, gradient elution is performed with a Tris-HCl buffer containing imidazole and NaCl, the elution peak is collected and concentrated, and the buffer is replaced with a 20 mM phosphate buffer, and the purified PTE is obtained by freezing in a -80°C refrigerator for later use.
[0016] Furthermore, in step S1, the crude enzyme solution was loaded at a flow rate of 1 mL / min; gradient elution was performed using a 20 mM Tris-HCl buffer containing 0.5 M imidazole and 0.5 M NaCl, and the elution volume was 75 mL.
[0017] Furthermore, in step S1, the affinity chromatography column uses a HisTrap HighPerformance affinity chromatography column.
[0018] Furthermore, in step S2, 24 mg of inulin was weighed, 20 mM acetic acid-sodium acetate buffer was added to fully dissolve it, and then a sodium periodate solution prepared with the same buffer was added to make the final concentrations of inulin and sodium periodate 2.5 mg / mL and 20 mmol / L, respectively. After reacting at room temperature in the dark for 20 minutes, a small amount of ethylene glycol was added to terminate the reaction; the reaction system was fully dialyzed with a dialysis card with a molecular weight cutoff of 2 kDa to obtain an inulin derivative with an aldehyde group.
[0019] Furthermore, in step S2, when the reaction system is fully dialyzed using a dialysis card with a molecular weight cutoff of 2 kDa, the replacement buffer used is sodium chloride with a mass fraction of 0.9%.
[0020] Further, in step S2, 8 mg of chitosan was weighed and fully dissolved with 20 mM citric acid buffer to obtain a chitosan solution with a final concentration of 2.5 mg / mL; the buffer of the inulin derivative was adjusted to 20 mM citric acid buffer with 0.2 M citric acid buffer, mixed with the chitosan solution, and then 8 mg of sodium cyanoborohydride was added. After reacting at room temperature for 8 hours, it was dialyzed with phosphate buffer overnight to obtain an inulin-chitosan complex; 2 mg of the purified PTE obtained in step S1 and 8 mg of sodium cyanoborohydride were added, and the reaction was carried out at 4° C. overnight, and NaCNBH was removed by dialysis. 3 , and obtained inulin-chitosan covalently cross-linked immobilized phosphotriesterase.
[0021] Furthermore, in step S2, the pH value of the 20 mM citric acid buffer is 3.0; and the pH value of the phosphate buffer is 7.2.
[0022] (III) Beneficial effects
[0023] The present invention provides a method for improving the stability of phosphotriesterase, including the expression and purification of phosphotriesterase and the immobilization of phosphotriesterase by covalent crosslinking of inulin-chitosan. The present invention uses inulin with good water solubility to covalently modify chitosan, so that chitosan is easily soluble in water, to obtain an inulin-chitosan complex (inu-Cs), and then uses inu-Cs to covalently crosslink and immobilize phosphotriesterase (PTE) with a maltose binding protein (MBP) tag to obtain PTE-inu-Cs (PiCs), which can significantly alleviate the damage of high temperature, low pH and organic solvent environments to the secondary structure and tertiary structure of the PTE enzyme. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Purify PTE using HisTrap HP high-efficiency affinity chromatography column;
[0025] Figure 2 8% SDS-PAGE electrophoresis was used to identify the purified PTE, where lane 1 is the whole bacteria before induction, lane 2 is the whole bacteria after induction, and lane 3 is the purified PTE (P2);
[0026] Figure 3 The preparation principle of inulin-chitosan covalently cross-linked PTE;
[0027] Figure 4 The purified PTE was identified by SDS-PAGE electrophoresis, wherein lane 1 is the purified PTE without cross-linking immobilization, and lane 2 is PiCs;
[0028] Figure 5 is the relative activity of inulin-chitosan cross-linked immobilized PTE;
[0029] Figure 6 is the thermal stability of the PTE sample;
[0030] Figure 7 The pH stability of inulin-chitosan cross-linked immobilized PTE;
[0031] Figure 8 is the relative activity of inulin-chitosan cross-linked immobilized PTE at pH 3-5;
[0032] Fig. 9 The organic solvent stability of cross-linked immobilized PTE. DETAILED DESCRIPTION
[0033] In order to make the purpose, content and advantages of the present invention more clear, the specific implementation methods of the present invention are further described in detail below in conjunction with the drawings and examples.
[0034] This example provides a method for improving the stability of phosphotriesterase, which specifically comprises the following steps:
[0035] S1. Expression and purification of phosphotriesterase
[0036] Take a glycerol bacteria expressing phosphotriesterase (MBP-PTE) with a maltose binding protein tag, add it to 100 ml LB medium at a ratio of 0.5%, and resuscitate it at 37°C overnight; then transfer it to TB medium at a ratio of 2%, culture it at 200rpm at 37°C until OD600=0.6-0.8, add IPTG and zinc sulfate with a final concentration of 0.1 mM, and induce it at 200rpm at 16°C for 24 hours; collect the bacteria by centrifugation at 4000rpm, and crush them with an ultrasonic crusher for 16min (ultrasound for 2s, stop for 5s), and then collect the crushed supernatant by centrifugation at 10000rpm to obtain a crude enzyme solution.
[0037] A HisTrap High Performance affinity chromatography column (5 mL) was used to further purify the PTE in the crude enzyme solution: first, the HisTrap HP High Performance affinity chromatography column was equilibrated for 10 to 15 column volumes with 20 mM Tris-HCl buffer (Buffer A, pH 8.0) containing 20 mM imidazole and 0.5 M NaCl; the crude enzyme solution was loaded at a flow rate of 1 mL / min, and then the chromatography column was equilibrated for 10 to 15 column volumes, and finally, gradient elution was performed with 20 mM Tris-HCl buffer (Buffer B, pH 8.0) containing 0.5 M imidazole and 0.5 M NaCl, with an elution volume of 75 mL, and the elution peak was collected and concentrated.
[0038] like Figure 1 As shown, after the crude enzyme solution was loaded, the chromatography column was equilibrated with Buffer A, and a large elution peak appeared, indicating that a large amount of foreign proteins and other impurities were not adsorbed by the chromatography column. After gradient elution, two smaller elution peaks appeared, and the P2 elution peak was collected.
[0039] The purity of the eluted peak was verified by 8% SDS-PAGE electrophoresis. Figure 2 As shown, the sample in lane 3 of the electrophoresis diagram corresponds to the elution peak P2. The sample showed a single point of electrophoresis purity, and the corresponding molecular weight was about 70 kDa, which indicated that the PTE with higher purity was obtained by purification by affinity chromatography column, which was concentrated and the buffer was replaced in 20 mM phosphate buffer, and frozen in a -80 ° C refrigerator for use to obtain purified PTE.
[0040] S2. Immobilization of phosphotriesterase by covalent cross-linking of inulin and chitosan
[0041] The reaction principle of inulin-chitosan cross-linked immobilized phosphotriesterase is as follows Figure 3 shown.
[0042] Weigh 24 mg of inulin, add 20 mM acetic acid-sodium acetate buffer (pH 5.8) to fully dissolve, then add sodium periodate solution prepared with the same buffer to make the final concentrations of inulin and sodium periodate 2.5 mg / mL and 20 mmol / L, respectively. After reacting at room temperature in the dark for 20 minutes, add a small amount of ethylene glycol to terminate the reaction. The reaction system is fully dialyzed with a dialysis card with a molecular weight cutoff of 2 kDa to remove other small molecules. The replacement buffer used is sodium chloride with a mass fraction of 0.9%, and an inulin derivative (inulin-CHO) with an aldehyde group is obtained.
[0043] 8 mg of chitosan (Cs) was weighed and fully dissolved in 20 mM citric acid buffer (pH 3.0) to obtain a chitosan solution with a final concentration of 2.5 mg / mL. The buffer of the above inulin derivative was adjusted to 20 mM citric acid buffer (pH 3.0) with 0.2 M citric acid buffer, mixed with the chitosan solution, and then 8 mg of sodium cyanoborohydride (NaCNBH 3 ), reacted at room temperature for 8 h, and then dialyzed overnight with a pH 7.2 phosphate buffer to obtain an inulin-chitosan complex (inu-Cs). Then 2 mg of the purified PTE obtained in step S1 and 8 mg of sodium cyanoborohydride (NaCNBH 3 ), react at 4°C overnight, and dialyze to remove NaCNBH 3 , and obtained inulin-chitosan covalently cross-linked immobilized phosphotriesterase (PiCs).
[0044] The cross-linking immobilization effect was detected by SDS-PAGE. Figure 4 As shown in the figure, it can be seen that compared with the purified PTE (original enzyme PTE) (lane 1) that was not cross-linked and immobilized, the molecular weight of inu-Cs cross-linked immobilized PTE (lane 2) increased significantly, basically all above 200 kDa, and there was almost no residual PTE, indicating that the efficiency of using inu-Cs cross-linked immobilized PTE can almost reach 100%.
[0045] Relative activity test of covalently cross-linked immobilized PTE:
[0046] Take 10ug of the original enzyme PTE and the cross-linked immobilized sample PiCs respectively, dilute to 0.1mL with 20mM phosphate buffer, and use 20mM phosphate buffer (PB) as a blank control. Add 10mM Tris-HCl to 0.5mL, and finally add 1μL (20μg) of the substrate methyl parathion. After mixing, place at 37℃ for 20min, and immediately take 200ul and read at a wavelength of 405nm. Calculate according to the following formula:
[0047]
[0048] The results are as follows Figure 5 As shown, compared with the activity of the original enzyme PTE (set as 100%), the relative activity of PiCs is about 91.15%, which shows that although the inulin-chitosan covalent cross-linking immobilized PTE partially changed its secondary structure, it did not significantly affect its active center and could maintain the activity of the original enzyme to a large extent.
[0049] Thermal stability test of covalently cross-linked immobilized PTE:
[0050] Take appropriate amount of original enzyme PTE and cross-linked immobilized PiCs, place original enzyme PTE and PiCs in a 56°C water bath for 2 hours, take out and measure the activity respectively, taking the activity before heating as 100%, the relative biological activity of the sample is calculated according to the following formula:
[0051]
[0052] The results are as follows Figure 6 After heating in a water bath at 56°C for 2 hours, the original enzyme PTE still had 19.87% activity; the PiCs still had 87.91% activity, which was 3.42 times higher than the original enzyme PTE, indicating that the thermal stability of PTE can be greatly improved after covalent cross-linking of inulin-chitosan with immobilized PTE.
[0053] pH stability test of covalently cross-linked immobilized PTE:
[0054] Reaction buffers with a pH range of 3 to 9 were prepared (20 mM citric acid-sodium citrate buffer solution was used for pH 3.0 to 6.0, and 20 mM Tris-HCl buffer was used for pH 7.0 to 9.0). The original enzyme PTE and the covalently cross-linked immobilized product PiCs were placed in each pH buffer for 0.5 h, and the enzyme activity was defined as 100% at pH 7.0. Figure 7 and Figure 8 As shown in the figure. As can be seen from the figure, in the range of pH = 3.0-6.0, the activity of PTE decreases sharply with the decrease of pH value. At pH 5.0, the enzyme activity is still 4.61%, and it is close to inactivation at pH 4.0; while PiCs still has nearly 17% activity at pH 5.0, which is 2.69 times higher than the activity of PTE, and nearly 3% activity at pH 4.0, showing better acid resistance than the original enzyme PTE. When the pH value is greater than 7.0, the enzyme activities of both show a downward trend, but under the same pH conditions, the residual activity of PiCs is higher than that of PTE. The above results show that PiCs has a strong ability to resist pH changes, and inulin-chitosan covalent cross-linking immobilization can improve the pH stability of PTE.
[0055] Study on the organic solvent stability of covalently cross-linked immobilized PTE:
[0056] When determining the solvent stability, 40% DMSO was added to the sample and placed at room temperature for 0.5 h. The residual activity of the original enzyme PTE and the cross-linked immobilized product was defined as 100% activity in 20 mM phosphate buffer (pH 7.4). Fig. 9 As shown in the figure, after the sample was treated with 40% DMSO as an organic solvent for 0.5 h, the PTE enzyme still had 16.55% activity, while the PiCs enzyme still had 42.54% activity, and the residual activity rate was 2.57 times that of the former, indicating that the immobilization of the PTE enzyme by covalent cross-linking with inulin-chitosan can significantly improve its solvent stability.
[0057] Through the above tests, it can be seen that the method of the present invention expresses and purifies the original enzyme PTE with higher purity, and successfully prepares PiCs based on inulin-chitosan covalent cross-linking immobilization. Compared with the original enzyme PTE enzyme, the activity of PiCs remains about 91.15%, which indicates that inulin-chitosan covalent cross-linking immobilization can generally maintain the biological activity of PTE enzyme. Based on inulin-chitosan covalent cross-linking immobilization, the thermal stability, pH stability and solvent stability of PTE enzyme can also be greatly improved, wherein the thermal stability can be increased by 3.42 times under the condition of heating in a 56°C water bath for 2h, and the pH stability can be increased by 2.69 times under the condition of pH5.0; under the condition of 40% DMSO for 0.5h, the residual activity is 2.57 times that of PTE enzyme, which indicates that inulin-chitosan covalent cross-linking immobilization can significantly alleviate the damage of high temperature, low pH and organic solvent environments to the secondary structure and tertiary structure of the original enzyme PTE.
[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for improving the stability of phosphotriesterase, characterized in that: The method for improving the stability of phosphotriesterase comprises the following steps: S1. Expression and purification of phosphotriesterase The glycerol bacteria expressing phosphotriesterase with a maltose binding protein tag were added to LB medium and revived overnight; then the bacteria were transferred to TB medium and cultured until OD600 = 0.6-0.8, and IPTG and zinc sulfate were added for induction; the bacteria were collected by centrifugation, crushed by an ultrasonic crusher, and then the crushed supernatant was collected by centrifugation to obtain a crude enzyme solution; the PTE in the crude enzyme solution was further purified by an affinity chromatography column to obtain purified PTE; S2. Immobilization of phosphotriesterase by covalent cross-linking of inulin and chitosan Weigh inulin, add acetic acid-sodium acetate buffer to fully dissolve, then add sodium periodate solution prepared with the same buffer, react at room temperature in the dark, add a small amount of ethylene glycol to terminate the reaction, and fully dialyze the reaction system to obtain an inulin derivative with an aldehyde group; Weigh chitosan, and fully dissolve it with citric acid buffer to obtain a chitosan solution; adjust the buffer of the inulin derivative to citric acid buffer with citric acid buffer, mix it with the chitosan solution, add sodium cyanoborohydride, react at room temperature, and dialyze it with phosphate buffer overnight to obtain an inulin-chitosan complex; then add the purified PTE and sodium cyanoborohydride obtained in step S1, react overnight, dialyze to remove NaCNBH3, and obtain inulin-chitosan covalently cross-linked immobilized phosphotriesterase.
2. The method for improving the stability of phosphotriesterase according to claim 1, wherein In step S1, a glycerol bacteria expressing a phosphotriesterase with a maltose binding protein tag is taken, added to 100 ml LB medium at a ratio of 0.5%, and revived at 37°C overnight; then transferred to TB medium at a ratio of 2%, cultured at 37°C until OD600 = 0.6-0.8, IPTG and zinc sulfate are added, and induced at 16°C for 24 hours; the bacteria are collected by centrifugation, crushed with an ultrasonic crusher, and then the crushed supernatant is collected by centrifugation to obtain a crude enzyme solution.
3. The method for improving the stability of phosphotriesterase according to claim 2, wherein: In step S1, the cells were cultured at 37°C and 200rpm until OD600 = 0.6-0.8; IPTG and zinc sulfate were added at a final concentration of 0.1 mM; the cells were induced at 200rpm at 16°C for 24 hours; the cells were collected by centrifugation at 4000rpm, crushed with an ultrasonic crusher for 16 minutes, and then the crushed supernatant was collected by centrifugation at 10000rpm.
4. The method for improving the stability of phosphotriesterase according to claim 1, wherein In step S1, an affinity chromatography column is used to further purify the PTE in the crude enzyme solution: first, the affinity chromatography column is equilibrated with a 20 mM Tris-HCl buffer containing 20 mM imidazole and 0.5 M NaCl for 10 to 15 column volumes; the crude enzyme solution is loaded, and then the chromatography column is equilibrated for 10 to 15 column volumes, and finally, gradient elution is performed with a Tris-HCl buffer containing imidazole and NaCl, the elution peak is collected and concentrated, and the buffer is replaced with a 20 mM phosphate buffer, and the purified PTE is obtained by freezing in a -80°C refrigerator for later use.
5. The method for improving the stability of phosphotriesterase according to claim 4, wherein: In step S1, the crude enzyme solution was loaded at a flow rate of 1 mL / min; gradient elution was performed using a 20 mM Tris-HCl buffer containing 0.5 M imidazole and 0.5 M NaCl, and the elution volume was 75 mL.
6. The method for improving the stability of phosphotriesterase according to claim 4, characterized in that: In step S1, the affinity chromatography column is a HisTrap HighPerformance affinity chromatography column.
7. The method for improving the stability of phosphotriesterase according to claim 1, wherein: In step S2, 24 mg of inulin is weighed, 20 mM acetic acid-sodium acetate buffer is added to fully dissolve it, and then a sodium periodate solution prepared with the same buffer is added to make the final concentrations of inulin and sodium periodate 2.5 mg / mL and 20 mmol / L, respectively. After reacting at room temperature in the dark for 20 minutes, a small amount of ethylene glycol is added to terminate the reaction; the reaction system is fully dialyzed with a dialysis card with a molecular weight cutoff of 2 kDa to obtain an inulin derivative with an aldehyde group.
8. The method for improving the stability of phosphotriesterase according to claim 7, wherein: In step S2, when the reaction system is fully dialyzed using a dialysis card with a molecular weight cutoff of 2 kDa, the replacement buffer used is sodium chloride with a mass fraction of 0.9%.
9. The method for improving the stability of phosphotriesterase according to claim 7, characterized in that: In step S2, 8 mg of chitosan was weighed and fully dissolved with 20 mM citric acid buffer to obtain a chitosan solution with a final concentration of 2.5 mg / mL; the buffer of the inulin derivative was adjusted to 20 mM citric acid buffer with 0.2 M citric acid buffer, mixed with the chitosan solution, and then 8 mg of sodium cyanoborohydride was added. After reacting at room temperature for 8 hours, it was dialyzed with phosphate buffer overnight to obtain an inulin-chitosan complex; then 2 mg of the purified PTE obtained in step S1 and 8 mg of sodium cyanoborohydride were added, reacted at 4°C overnight, and NaCNBH3 was removed by dialysis to obtain inulin-chitosan covalently cross-linked immobilized phosphotriesterase.
10. The method for improving the stability of phosphotriesterase according to claim 9, characterized in that: In step S2, the pH value of the 20 mM citric acid buffer is 3.0; the pH value of the phosphate buffer is 7.2.