A low-temperature-adaptable carbonic anhydrase mutant hpca-m3 and application thereof

Abstract

The application discloses a low-temperature adaptive carbonic anhydrase mutant HpCA-M3 and application thereof, and relates to the technical field of enzyme engineering. The application screens a low-temperature adaptive carbonic anhydrase mutant HpCA-M3, the amino acid sequence of which is obtained by mutating the serine at the 33rd position of the amino acid sequence of wild-type carbonic anhydrase HpCA into aspartic acid, mutating the serine at the 84th position into glycine, and mutating the proline at the 108th position into serine; the amino acid sequence of the wild-type carbonic anhydrase HpCA is shown as SEQ ID NO. 1. Compared with the wild enzyme HpCA, the carbonic anhydrase mutant HpCA-M3 has higher catalytic activity at low temperature, the esterase activity is increased by 75% at 20 DEG C, and the hydration activity is increased by 23%. The application is helpful to reducing the massive dissolution of CO2 caused by high temperature during industrial carbon sequestration, and can accelerate the enzymatic repair of limestone articles (for example, limestone cultural relics) in high-cold regions, and has important value for promoting carbon capture and limestone cultural relic repair.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a low-temperature-adapted carbonic anhydrase mutant and its applications. Background Technology

[0002] The use of fossil fuels such as coal, oil, and natural gas emits large amounts of CO2 into the atmosphere, which is one of the main causes of phenomena such as rising temperatures, sea-level changes, and extreme weather events. CO2 capture, utilization, and storage (CCUS) technology can reduce carbon emissions while converting CO2 into valuable products, playing an important role in controlling global climate change and maintaining sustainable economic development.

[0003] Carbonic anhydrase (CA, EC 4.2.1.1) is a zinc-containing metalloenzyme that efficiently catalyzes the reaction of CO2 and H2O to produce HCO3. - With H + This is a reversible reaction. Carbonic anhydrase (CA) is widely found in plants, animals, and microorganisms, playing an important role in many biological processes, such as CO2 transfer, photosynthesis, and ion transport. While traditional methods, such as physical adsorption, catalytic combustion, and chemical absorption, can effectively capture CO2, they suffer from problems such as byproduct generation, high energy consumption, and secondary pollution. Using CA to capture CO2 offers a milder, more specific, efficient, and less polluting enzymatic reaction, making it an environmentally friendly green process. The catalytic products of CA can be used as raw materials for other industrial productions or, through mineralization, to generate CaCO3, achieving the final geological fixation of CO2. Using CaCO3 crystals generated through enzymatic deposition and mineralization to reinforce and protect the surface of weathered stone artifacts is also a key research direction in the field of stone artifact conservation and restoration both domestically and internationally in recent years.

[0004] Enzymes with strong low-temperature adaptability are advantageous for reducing enzyme dosage or shortening reaction time during low-temperature reactions, and their susceptibility to thermal denaturation allows for control of the enzyme reaction process through heat treatment. In industrial applications of calcium carbonate (CA) for CO2 capture, relatively low-temperature conditions are typically chosen to avoid excessive CO2 dissolution due to high temperatures. Western China and the Qinghai-Tibet Plateau, among other high-altitude and cold regions, preserve a large number of stone artifacts. In these cold and humid environments, the weathering rate of rocks is higher than in hot and dry environments, while the deposition rate of calcium carbonate is relatively slow. Therefore, improving the low-temperature catalytic performance of CA is of great significance.

[0005] Although a large number of catechins (CAs) have been screened from nature, they still cannot effectively meet the needs of low-temperature applications or industrial production. The development of protein engineering technology allows researchers to adjust and design the structure and function of proteins as desired. For example, Alvizo et al. (Proc Natl Acad Sci USA 2014, 111(46):16436-16441.) directed the evolution of β-CAs from *Deulfovibrio. Vuglaris* to increase their thermal stability. The screened CAs showed a 4 million-fold increase in thermal stability and alkali tolerance compared to wild-type enzymes, along with a 24-fold increase in hydration capacity. Warden et al. (Nat Commun. 2015, 6:10278) rationally designed bCAII, increasing the catalytic activity and thermal stability of CAs at high salt concentrations. However, there are few reports on low-temperature adaptability modifications of CAs. This lack of research limits the industrial and environmental application potential of CAs in low-temperature environments. Therefore, developing low-temperature adaptable CAs is particularly important. Summary of the Invention

[0006] This invention provides a low-temperature-adapted carbonic anhydrase mutant, HpCA-M3, and its applications. The carbonic anhydrase mutant HpCA-M3 is a semi-rational modification of the carbonic anhydrase HpCA from Helicobacter pylori 26695. A mutant library containing 23 point mutations was constructed, and the final selected hybrid mutant strain (S33N / S84G / P108S) was selected. Compared with wild-type carbonic anhydrase HpCA, the carbonic anhydrase mutant HpCA-M3 exhibits significantly enhanced esterase activity and hydration activity at low temperatures (e.g., 20°C). This is achieved through the following techniques.

[0007] In a first aspect, the present invention provides a low-temperature adapted carbonic anhydrase mutant HpCA-M3, the amino acid sequence of which is obtained by mutating serine at position 33 to aspartic acid (i.e., S33N), serine at position 84 to glycine (i.e., S84G), and proline at position 108 to serine (i.e., P108S); the amino acid sequence of the wild-type carbonic anhydrase HpCA is as shown in SEQ ID NO.1.

[0008] Furthermore, the nucleotide sequence encoding the wild-type carbonic anhydrase HpCA is shown in SEQ ID NO.2.

[0009] Furthermore, the amino acid sequence of the carbonic anhydrase mutant HpCA-M3 is shown in SEQ ID NO.3.

[0010] Furthermore, the nucleotide sequence encoding the carbonic anhydrase mutant HpCA-M3 is shown in SEQ ID NO.4.

[0011] This invention first screened for a carbonic anhydrase from Helicobacter pylori 26695, which exhibited the highest enzyme activity at pH 9.0. Within the pH range of 8.0-9.0, the carbonic anhydrase displayed relatively high enzyme activity; however, its activity decreased rapidly below pH 7.0. Therefore, this enzyme is suitable for weakly alkaline environments. Through semi-rational modification, this invention subsequently constructed a mutant library with 23 point mutations and screened for four mutants (V63G, S33N, S84G, and P108S) showing improved enzyme activity. Ultimately, the combined mutant strain (S33N / S84G / P108S) was found to have a 75% increase in esterase activity and a 23% increase in hydration activity at low temperatures.

[0012] A second aspect of the present invention provides a biomaterial, said biomaterial being any one of the following:

[0013] (1) A nucleic acid molecule that encodes any of the above-mentioned carbonic anhydrase mutants HpCA-M3;

[0014] (2) A recombinant vector comprising the nucleic acid molecule;

[0015] (3) A recombinant engineered cell comprising the nucleic acid molecule or the recombinant vector.

[0016] Optionally, the base vector selected for the above recombinant vector is a commonly used vector such as pET-28a(+).

[0017] Optionally, the host cell selected for the above-mentioned recombinant engineered cells is Escherichia coli cells.

[0018] In a third aspect, the present invention provides an application of any of the above-mentioned carbonic anhydrase mutants HpCA-M3 for capturing carbon dioxide or for repairing limestone materials.

[0019] In a fourth aspect, the present invention provides a method for capturing carbon dioxide, comprising preparing a first mixed solution by dispersing any one of the above-mentioned carbonic anhydrase mutants HpCA-M3, NaHCO3, and CaCl2; wherein the concentration of the carbonic anhydrase mutant HpCA-M3 in the first mixed solution is 0.5-20 g / L, and the concentrations of NaHCO3 and CaCl2 are 10-500 mM and 10-500 mM, respectively.

[0020] Furthermore, the carbon dioxide capture reaction was carried out at 5-40°C and 80-180 rpm.

[0021] A fifth aspect of the present invention provides a method for repairing limestone articles, comprising the following steps:

[0022] The limestone material is washed and dried several times until the mass difference before and after drying does not exceed 0.001g;

[0023] Prepare a second mixed solution by taking any one of the above-mentioned carbonic anhydrase mutants HpCA-M3, as well as CaCl2 and Na2CO3; in the second mixed solution, the concentration of the carbonic anhydrase mutant HpCA-M3 is 0.5-20 g / L, and the concentrations of CaCl2 and Na2CO3 are 10-500 mM and 10-500 mM, respectively; spray the mixed solution onto the surface of the limestone object to complete the mineralization repair.

[0024] Furthermore, the second mixed solution is sprayed onto the limestone material every 2-12 hours to carry out mineralization repair under static conditions. During mineralization repair, the pH of the second mixed solution is 8.0-10.0, the temperature is 5-40℃, and the repair time is 12-48 hours.

[0025] Compared with existing technologies, the advantages of this invention are: This invention screened and obtained a low-temperature adapted carbonic anhydrase mutant, HpCA-M3. Compared with the wild-type enzyme HpCA, the carbonic anhydrase mutant HpCA-M3 (i.e., the mutant enzyme S33N / S84G / P108S) exhibits higher catalytic activity at low temperatures, with esterase activity increased by 75% and hydration activity increased by 23% at 20°C. This invention helps reduce the large-scale dissolution of CO2 caused by high temperatures during industrial carbon fixation and can also accelerate the enzymatic restoration of limestone artifacts (such as limestone cultural relics) in cold regions, which is of great value for promoting carbon capture and the restoration of limestone cultural relics. Attached Figure Description

[0026] Figure 1 The figure shows the dissolution curves of wild-type carbonic anhydrase HpCA and each iterative mutant.

[0027] Figure 2 The specific enzyme activities of purified wild-type carbonic anhydrase HpCA and carbonic anhydrase mutant HpCA-M3 at 20℃ and 40℃ are plotted.

[0028] Figure 3 This is a graph showing the changes in the amount of mineralized layer growth in the cultural relic restoration system.

[0029] Figure 4 SEM images of calcium carbonate generated on marble surfaces after 24 hours of soaking under different treatments. Figure 4 (a) corresponds to the marble (CT) in the blank group. Figure 4(b) corresponds to the marble (HpCA-M3) in the enzyme solution treatment group. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] The main experimental materials and reagents used in the following specific embodiments of the present invention are as follows:

[0032] (1) Strains and vectors: Escherichia coli BL21(DE3) and expression vector pET-28a were selected, both of which were preserved in the laboratory of Huazhong University of Science and Technology;

[0033] (2) Enzymes and other biochemical reagents: Restriction endonucleases, DNA ligases, and purification kits were purchased from Nanjing Vazyme. All other chemicals and reagents used were analytical grade.

[0034] (3) Culture media. LB medium (Peptone 10g, Yeast extract 5g, NaCl 10g, distilled water to 1000mL, pH about 7), and solid culture medium (LB medium with 1.5% (w / v) agar added).

[0035] Example 1: Construction, screening, and enzyme activity assay of a carbonic anhydrase mutant library

[0036] 1. Construction of a carbonic anhydrase mutant library

[0037] (1) Use Alphafold 2 to model the three-level structure of HpCA.

[0038] To identify rigid regions of the enzyme, the conservation of the amino acid sequence of HpCA was first assessed using Consurfserver (https: / / consurf.tau.ac.il / consurf-old.php). When selecting mutation sites, amino acid residues with low conservation were preferred.

[0039] Then, the B-factor (temperature factor) of HpCA was analyzed. The B-factor describes the dispersion of electron density and indicates the static or dynamic flexibility of the atom. Based on the temperature factor of different residue sites in HpCA, the flexibility and rigidity of its structure can be analyzed. Sites with smaller temperature factors, i.e., smaller B-factors, were preferentially selected for mutation.

[0040] Finally, HoTMuSiCv1.0 (http: / / dezyme.com / ) was used to measure the ΔG (folding free energy change) ΔT of HpCA. m (Changes in dissolution temperature) and analysis are performed. ΔG describes the overall entropy and enthalpy changes, ΔT m The thermodynamic stability of the residue sites was described. Based on ΔG and ΔT at different residue sites in HpCA... m The flexibility and rigidity of its structure can be analyzed. Priority is given to structures with larger changes in melting temperature and smaller changes in folding free energy, i.e., smaller ΔG and ΔT. m Mutations occur at larger sites.

[0041] Based on the above points, 23 probable mutation sites were finally selected (19D, 24D, 26E, 27V, 29K, 30S, 33S, 34Q, 44H, 63V, 84S, 90D, 95H, 96A, 97P, 108P, 109L, 170N, 171G, 180E, 212R, 214V, 217D), resulting in a mutant library of 23 point mutations.

[0042] 2. Screening process of mutant library

[0043] Approximately 100 transformants were selected for each mutation site, with >95% coverage of the NNK degenerate codons used (where N = A / C / G / T and K = G / T, 32 codons / 20 amino acids).

[0044] First, add 400 μL of LB medium (kanamycin resistant) to a 96-well plate and incubate overnight at 37°C. The purpose is to make the bacterial concentration far exceed that of the culture. This plate is called the mother plate.

[0045] Add 1 mL of LB medium (kanamycin resistant) to a 96-well plate, and add 50 μL of the mother plate to this plate (referred to as the daughter plate). Add 250 μL of 50% sterile glycerol to the mother plate for preservation. Incubate the daughter plate at 37°C for 4 hours.

[0046] Add 40 μL of IPTG (isopropyl-β-D-thiogalactopyranoside, final concentration 50 mM) to the plate, incubate at 16 °C for 18 h, then add 400 μL of 20 mM Tris-HCl buffer (pH = 9.0), vortex thoroughly, centrifuge again, and discard the supernatant. This step is for bacterial washing.

[0047] Add 200 μL of Tris-HCl buffer again, mix thoroughly, and then begin liquid nitrogen freeze-thaw cycles three times. Next, add 200 μL of Lysis buffer (2 mg / mL lysozyme + 0.02 mg / mL DNase dissolved in the above Tris-HCl buffer), vortex thoroughly, and dissolve at 37°C and 200 rpm for 1 hour. Then add 400 μL of Tris-HCl buffer again and centrifuge. Use 200 μL of enzyme solution three times for three parallel experiments. The reaction system is (200 μL supernatant enzyme solution + 50 μL 8 mM p-NPA), the reaction time is 10 min, and the reaction temperature is 20°C.

[0048] 3. Enzyme activity assay of combined carbonic anhydrase mutants and wild-type pure carbonic anhydrase HpCA

[0049] The single-point mutant was subjected to iterative saturation mutagenesis, with the site S33N (46% higher than wild-type carbonic anhydrase HpCA) serving as the starting point for combined mutagenesis, resulting in the double-combination mutant S33N / S84G (58% higher than wild-type HpCA). Although the V63G single-point mutation increased enzyme activity, the increase in enzyme activity after combination with other sites was actually lower. Therefore, V63G was no longer combined with other sites.

[0050] This semi-rational modification resulted in three mutant combinations, S33N / S84G / P108S, which exhibited a 75% increase in enzyme activity at low temperatures. The optimal pH, temperature, and kinetic parameters of the purified enzymes from these three mutant strains (S33N, S33N / S84G, and S33N / S84G / P108S (HpCA-M3)) were measured. The optimal pH and temperature showed minimal variation. The optimal temperature of HpCA-M3 was the same as that of the wild-type carbonic anhydrase HpCA, at 40℃; the optimal pH was 9.0 for both.

[0051] like Figure 1 As shown, HpCA-M3 has lower tolerance to high temperatures, but the activity of HpCA-M3 at low temperature (20℃) is 58% of its activity at its optimum temperature (40℃), while HpCA at low temperature (20℃) is only 47% of its activity at its optimum temperature. Figure 2 ).

[0052] The hydration activities of wild-type carbonic anhydrases HpCA and HpCA-M3 were measured using an enzyme concentration of 0.05 mg / mL. The hydration activity of HpCA-M3 was 1.85 U, while that of wild-type carbonic anhydrase HpCA was 1.5 U. Therefore, the hydration activity of the carbonic anhydrase mutant HpCA-M3 was increased by 23%.

[0053] In addition, the kinetic parameters of HpCA and HpCA-M3 mutants were measured at low temperature (20℃) and optimum temperature (40℃) at pH=9.0. The Michaelis constants of wild-type carbonic anhydrase HpCA at 20℃ and 40℃ are shown in Table 1. It can be seen that the activity of HpCA-M3 is more significantly enhanced under low temperature conditions.

[0054] Table 1

[0055]

[0056]

[0057] Example 2: Application of recombinant carbonic anhydrase mutant HpCA-M3 in carbon capture I

[0058] The mutant enzyme solution HpCA-M3 obtained in Example 1 was added to a mixed solution of 10 mM NaHCO3 and 10 mM CaCl2 to make the enzyme concentration 20 g / L, with a total volume of 100 L; the reaction was carried out at 5 °C and 80 rpm. The control group had the enzyme solution replaced with the same volume of pH 9.0 Tris-HCl buffer.

[0059] After reacting for 48 hours, centrifuge at 5000 rpm for 5 minutes. Discard the supernatant, add pure water to wash the precipitate, and remove Cl. - Na + and Ca 2+ Repeat this process three times, then thoroughly dry the washed precipitate in a 105℃ oven, and weigh the total mass of the precipitate using a balance.

[0060] Add excess concentrated hydrochloric acid to react fully with the calcium carbonate in the precipitate, then wash the precipitate 2-3 times with pure water to remove Cl. - and Ca 2+ Finally, the precipitate was dried and its mass was measured to determine the amount of calcium carbonate formed.

[0061] The results showed that, compared with the control group, the enzyme-treated group showed obvious calcium carbonate precipitation. The average calcium carbonate production in the treated group (59.02 g) was 251.4% of the average calcium carbonate production in the control group (23.48 g).

[0062] Example 3: Application of recombinant carbonic anhydrase mutant HpCA-M3 in carbon capture II

[0063] The mutant enzyme solution HpCA-M3 obtained in Example 1 was added to a mixed solution of 500 mM NaHCO3 and 500 mM CaCl2 to make the enzyme concentration 0.5 g / L, with a total volume of 100 L; the reaction was carried out at 40 °C and 180 rpm. The control group had the enzyme solution replaced with the same volume of pH 9.0 Tris-HCl buffer.

[0064] After reacting for 48 hours, centrifuge at 5000 rpm for 5 minutes. Discard the supernatant, add pure water to wash the precipitate, and remove Cl. - Na + and Ca 2+ Repeat this process three times, then thoroughly dry the washed precipitate in a 105℃ oven, and weigh the total mass of the precipitate using a balance.

[0065] Add excess concentrated hydrochloric acid to react fully with the calcium carbonate in the precipitate, then wash the precipitate 2-3 times with pure water to remove Cl. - and Ca 2+ Finally, the precipitate was dried and its mass was measured to determine the amount of calcium carbonate formed.

[0066] The results showed that, compared with the control group, the enzyme-treated group showed obvious calcium carbonate precipitation. The average calcium carbonate production in the treated group (2080.84g) was 170.7% of the average calcium carbonate production in the control group (1220.21g).

[0067] Example 4: Application of recombinant carbonic anhydrase mutant HpCA-M3 in the restoration of limestone cultural relics I

[0068] The mutant enzyme solution obtained in Example 1 was added to a spray bottle, followed by 10 mM Na2CO3 solution and 10 mM CaCl2 solution, to achieve an enzyme concentration of 20 g / L. The pH of the mixed solution was adjusted to 8.0. In the control group, the enzyme solution was replaced with an equal volume of pH 8.0 Tris-HCl buffer. The treated limestone specimens were sprayed every 2 hours and placed under static conditions for mineralization remediation. All experimental treatment systems were repeated three times. Samples were taken at 5°C for 12, 24, and 48 hours. After the experiment, the test stones were removed, rinsed, and dried to constant weight.

[0069] The relationship between the amount of mineralized layer formed on the specimen surface and time is as follows: Figure 3 As shown, significant calcium carbonate deposition occurred 12 hours before deposition, and stabilized after 24 hours. The growth rate of the control group remained almost unchanged, consistently at a low level. The average mass growth rate of the enzyme solution-immersed deposition group after 24 hours was 2.72 times that of the control group.

[0070] Morphological characterization of calcium carbonate formed on marble surfaces after 24 hours of immersion and deposition using SEM images. Figure 4 As shown. It can be seen that, compared to the blank marble in the control group ( Figure 4 (a)), enzyme solution soaking treatment group ( Figure 4 (b) Large areas of nepheline calcium carbonate were formed in almost all samples. This indicates that the enzyme solution induced the formation of a more protective calcium carbonate deposition layer on the sample surface.

[0071] Example 5: Application of recombinant carbonic anhydrase mutant HpCA-M3 in the restoration of limestone cultural relics II

[0072] The mutant enzyme solution obtained in Example 1 was added to a spray bottle, followed by 500 mM Na2CO3 solution and 500 mM CaCl2 solution, to achieve an enzyme concentration of 0.5 g / L. The pH of the mixed solution was adjusted to 10.0. In the control group, the enzyme solution was replaced with an equal volume of pH 10.0 Tris-HCl buffer. The treated limestone specimens were sprayed every 12 hours and placed under static conditions for mineralization remediation. All experimental treatment systems were repeated three times. Samples were taken at 40°C for 12 hours, 24 hours, and 48 hours. After the experiment, the test stones were removed, rinsed, and dried to constant weight.

[0073] The change in the amount of mineralized layer formed on the specimen surface over time was consistent with the trend in Example 4. Significant calcium carbonate deposition appeared 12 hours before deposition and stabilized after 24 hours. The growth rate of the control group showed almost no change and remained stable at a low level. The average mass growth rate of the enzyme solution immersion deposition for 24 hours was 1.84 times that of the control group.

[0074] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A low-temperature adapted carbonic anhydrase mutant, HpCA-M3, characterized in that, Its amino acid sequence is obtained by mutating serine at position 33 to aspartic acid, serine at position 84 to glycine, and proline at position 108 to serine; the amino acid sequence of the wild-type carbonic anhydrase HpCA is as shown in SEQ ID NO.

1.

2. The low-temperature adapted carbonic anhydrase mutant HpCA-M3 according to claim 1, characterized in that, The nucleotide sequence encoding the wild-type carbonic anhydrase HpCA is shown in SEQ ID NO.

2.

3. The low-temperature adapted carbonic anhydrase mutant HpCA-M3 according to claim 1, characterized in that, The amino acid sequence of the carbonic anhydrase mutant HpCA-M3 is shown in SEQ ID NO.

3.

4. The low-temperature adapted carbonic anhydrase mutant HpCA-M3 according to claim 3, characterized in that, The nucleotide sequence encoding the carbonic anhydrase mutant HpCA-M3 is shown in SEQ ID NO.

4.

5. A biomaterial, characterized in that, The biomaterial is any one of the following: (1) A nucleic acid molecule for encoding the carbonic anhydrase mutant HpCA-M3 according to any one of claims 1-4; (2) A recombinant vector comprising the nucleic acid molecule; (3) A recombinant engineered cell comprising the nucleic acid molecule or the recombinant vector.

6. An application of a carbonic anhydrase mutant HpCA-M3, characterized in that, Used to capture carbon dioxide, or to repair limestone items.

7. A method for capturing carbon dioxide, characterized in that, A first mixed solution is prepared by preparing the carbonic anhydrase mutant HpCA-M3 according to any one of claims 1-4, NaHCO3 and CaCl2; in the first mixed solution, the concentration of the carbonic anhydrase mutant HpCA-M3 is 0.5-20 g / L, and the concentrations of NaHCO3 and CaCl2 are 10-500 mM and 10-500 mM, respectively.

8. The method for capturing carbon dioxide according to claim 7, characterized in that, The carbon dioxide capture reaction was carried out at 5-40°C and 80-180 rpm.

9. A method for repairing limestone artifacts, characterized in that, Includes the following steps: The limestone material is washed and dried several times until the mass difference before and after drying does not exceed 0.001g; A second mixed solution is prepared by preparing the carbonic anhydrase mutant HpCA-M3 according to any one of claims 1-4, along with CaCl2 and Na2CO3; in the second mixed solution, the concentration of the carbonic anhydrase mutant HpCA-M3 is 0.5-20 g / L, and the concentrations of CaCl2 and Na2CO3 are 10-500 mM and 10-500 mM, respectively; the mixed solution is sprayed onto the surface of the limestone object to complete the mineralization repair.

10. The method for repairing limestone articles according to claim 9, characterized in that, The second mixed solution is sprayed onto the limestone material every 2-12 hours for mineralization repair under static conditions. During mineralization repair, the pH of the second mixed solution is 8.0-10.0, the temperature is 5-40℃, and the repair time is 12-48 hours.

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