Thermophilic inorganic pyrophosphatase mutant and preparation method thereof

Abstract

The invention discloses a thermophilic inorganic pyrophosphatase mutant and a preparation method thereof. The thermophilic inorganic pyrophosphatase mutant disclosed by the invention is derived from a PPase gene of Thermus burkeri, the mutation sites are as follows: Asp55Ile, Leu70Tyr, Met96Val, Gly102Tyr, Glu112Val and Ala142 Leu, and the amino acid sequence of the mutant is as shown in SEQ ID NO. 1. The invention also discloses a preparation method of the thermophilic inorganic pyrophosphatase mutant. The invention also discloses an optimized gene sequence of the thermophilic inorganic pyrophosphatase mutant after codon optimization of escherichia coli, wherein the optimized gene sequence is as shown in SEQ ID NO. 2. The invention provides a thermophilic inorganic pyrophosphatase mutant protein. The thermophilic inorganic pyrophosphatase mutant can be expressed in escherichia coli in a soluble manner, and is low in production cost, good in thermal stability and high in activity.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically relates to a thermophilic inorganic pyrophosphatase mutant and a preparation method thereof. Background Art

[0002] Inorganic pyrophosphatase (PPase) is a metal-dependent hydrolase that catalyzes the conversion of one molecule of pyrophosphate into two molecules of phosphate ions under the action of divalent metal ions such as Mg2+ and Mn2+. It plays an important role in controlling the concentration of inorganic pyrophosphate in organisms. Inorganic pyrophosphate (PPi) is a common by-product in metabolic processes, participating in the biosynthesis of nucleic acids, proteins, lipids, peptidoglycans, starch, etc., and also playing an important role in the post-translational modification process of proteins. Most importantly, inorganic pyrophosphate is produced in large quantities during processes such as PCR reactions, in vitro transcription reactions, gene sequencing, and industrial sugar production, and affects these biochemical reactions. Therefore, inorganic pyrophosphatase has a wide range of applications in these biochemical reactions. For example, it can improve the efficiency of PCR reactions and increase DNA yield; optimize in vitro transcription reactions of RNA and increase RNA yield; be applied to aminoacylation quantification; and remove PPi contaminants in SNP gene sequencing genotyping reactions.

[0003] The rapid development of mRNA vaccines has urgently promoted the demand and quality improvement of the upstream raw material PPase for mRNA production. Currently, commercially available inorganic pyrophosphatases mainly include PPase derived from Escherichia coli expressed by an Escherichia coli expression system and the PPase gene derived from Saccharomyces cerevisiae. And modifying proteins through site-directed mutagenesis technology to obtain mutants with better functionality is one of the main means for the research and development of related enzyme products. For example, Patent Document CN 106834249A discloses the mutation of two sites of mutant inorganic pyrophosphatase T6S / E11K / L72I / D124A / A160Q and V73I / D124A / A160K to obtain two mutants with improved amplification efficiency and thermal stability. Patent Document CN 116555218A discloses the mutation of I91T and G150D of yeast-derived inorganic pyrophosphatase to obtain a mutant with a catalytic activity more than twice that of the wild-type inorganic pyrophosphatase.

[0004] Thermophilic inorganic pyrophosphatase has extremely high thermal stability and high catalytic performance, and has a broader application field. Therefore, it is of great significance and market value to perform site-directed mutagenesis on the PPase gene of Thermus brockianus (Genebank number: WP_071677022, amino acid sequence shown in SEQ ID NO.5) to screen mutants with high functional activity.

[0005] At present, there is a lack of a highly active thermophilic inorganic pyrophosphatase mutant and a method for preparing the same. SUMMARY OF THE INVENTION

[0006] In view of the above-mentioned prior art, the object of the present invention is to provide a highly active thermophilic inorganic pyrophosphatase mutant and a method for preparing the same.

[0007] To achieve the above object, the present invention provides the following technical solutions: A thermophilic inorganic pyrophosphatase mutant of the present invention, the amino acid sequence of the thermophilic inorganic pyrophosphatase mutant is shown in SEQ ID NO.1. Mutations were made to the PPase gene derived from Thermus brockianus, and the mutation positions are as follows: Asp55Ile, Leu70Tyr, Met96Val, Gly102Tyr, Glu112Val, Ala142Leu.

[0008] The gene encoding the thermophilic inorganic pyrophosphatase mutant (PPa-mut) of the present invention. To improve the expression level of PPa-mut during prokaryotic expression, the present invention also discloses an optimized gene sequence of PPa-mut optimized for Escherichia coli codons. The nucleotide sequence of the thermophilic inorganic pyrophosphatase mutant gene is shown in SEQ ID NO.2.

[0009] Furthermore, the gene sequence is optimized according to the codon bias of Escherichia coli. The expressed protein mostly exists in the form of soluble protein, which can improve the yield of purified protein, and its expression effect is as Figure 3 shown, in which the expression level of the soluble inorganic pyrophosphatase mutant protein in the supernatant fraction is much higher than that in the precipitate fraction.

[0010] A recombinant expression vector containing the coding sequence of the thermophilic inorganic pyrophosphatase mutant protein of the present invention.

[0011] A recombinant strain containing the recombinant expression vector of the present invention.

[0012] A recombinant cell of a recombinant expression vector of the present invention.

[0013] Furthermore, the host cell of the recombinant cell is Escherichia coli BL21(DE3) and is transformed by the recombinant expression vector.

[0014] A method for preparing a thermophilic inorganic pyrophosphatase mutant protein of the present invention, comprising the following steps:

[0015] (1) Synthesize the gene sequence encoding the thermophilic inorganic pyrophosphatase mutant;

[0016] (2) Construct a highly efficient recombinant expression vector to obtain the recombinant plasmid pCold-PPa-mut;

[0017] (3) Transform the host bacterium BL21(DE3) with the expression vector to construct a genetically engineered bacterium (recombinant cell);

[0018] (4) Cultivate and express the mutant protein of thermophilic inorganic pyrophosphatase using the obtained recombinant cell;

[0019] (5) Isolate and purify the expression product obtained in step (4) to prepare the mutant protein of thermophilic inorganic pyrophosphatase.

[0020] Further, in step (2), the synthesized gene is assembled into the cloning plasmid pUC18 to obtain the plasmid pUC18-PPa-mut, and transform the cloning Escherichia coli DH5α.

[0021] Further, in step (2), the gene encoding the mutant protein of thermophilic inorganic pyrophosphatase is amplified by the standard PCR technique, and restriction enzyme cutting sites (Kpn I, Xba I) are added at both ends. After restriction enzyme digestion, it is loaded between the corresponding restriction enzyme cutting sites of the pCold III vector, and transform the cloning host, screen and sequence to prepare the plasmid pCold-PPa-mut;

[0022] The specific primer pair for the coding gene is primer PPa-mut-F and primer PPa-mut-R; primer PPa-mut-F has the nucleotide sequence shown in SEQ ID NO.3; primer PPa-mut-R has the nucleotide sequence shown in SEQ ID NO.4.

[0023] Introduce appropriate restriction enzyme cutting sites at the head and tail of the fragment by the standard PCR technique, assemble it into the plasmid pColdIII and transform the host cell to complete the construction of the engineered bacterium.

[0024] Ferment the constructed engineered bacterium, centrifuge to obtain the bacterial cells, break the cells after washing, centrifuge to collect the supernatant, and then purify the protein by nickel particle affinity chromatography. The purified protein is stored at low temperature after adding a stabilizer.

[0025] Beneficial effects: The present invention provides a mutant protein of thermophilic inorganic pyrophosphatase, and this mutant of thermophilic inorganic pyrophosphatase can be solubly expressed in Escherichia coli, with low production cost, good thermal stability and high activity.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] (1) The enzyme activity of the mutant of thermophilic inorganic pyrophosphatase is significantly higher than that of the wild type.

[0028] (2) The thermostability of the mutant of thermophilic inorganic pyrophosphatase is significantly higher than that of the wild type. It has significantly improved the expression level and yield of thermophilic inorganic pyrophosphatase in Escherichia coli. Compared with the wild-type thermophilic inorganic pyrophosphatase, the catalytic activity of this mutant has increased by more than 5 times.

[0029] (3) In the prokaryotic expression system, the mutant protein of thermophilic inorganic pyrophosphatase mostly exists in the form of soluble protein, which can improve the yield of purified protein. The expression level of the soluble mutant protein of thermophilic inorganic pyrophosphatase in the supernatant fraction is much higher than that in the precipitate fraction. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0031] Figure 1 Structural prediction and analysis of the mutant protein of thermophilic inorganic pyrophosphatase of the present invention;

[0032] Figure 2 Plasmid map of the pCold-PPa-mut recombinant plasmid of the present invention;

[0033] Figure 3 Electrophoresis diagram of the expression of the mutant protein of thermophilic inorganic pyrophosphatase of the present invention;

[0034] Figure 4 Standard curve graph of pyrophosphate concentration and absorbance of the present invention;

[0035] Figure 5 Thermostability detection graph of the mutant enzyme of thermophilic inorganic pyrophosphatase of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] In order to make the technical problems to be solved, technical solutions and beneficial effects of the present application clearer, the following further details the present application in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0037] In the present application, the term "and / or" describes the association relationship of associated objects and indicates that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after.

[0038] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or a similar expression means any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can each be single or multiple.

[0039] It should be understood that in various embodiments of this application, the magnitudes of the sequence numbers of the above processes do not imply the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of this application.

[0040] The terms "first" and "second" are only used for descriptive purposes to distinguish objects such as substances from each other, and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. For example, without departing from the scope of the embodiments of this application, the first XX can also be referred to as the second XX, and similarly, the second XX can also be referred to as the first XX. Thus, the features defined with "first" and "second" can explicitly or implicitly include one or more of such features.

[0041] In the first aspect of the embodiments of this application, a thermophilic inorganic pyrophosphatase mutant protein is provided, the amino acid sequence of which is shown in SEQ ID NO.1. The PPase gene derived from Thermus brockianus is mutated, and the mutation positions are as follows: Asp55Ile, Leu70Tyr, Met96Val, Gly102Tyr, Glu112Val, Ala142Leu.

[0042] In the second aspect of the embodiments of this application, a gene encoding a thermophilic inorganic pyrophosphatase mutant protein is provided.

[0043] The nucleotide sequence encoding the thermophilic inorganic pyrophosphatase mutant protein is shown in SEQ ID NO.2. This gene sequence is optimized and designed according to the codon bias of Escherichia coli.

[0044] In the third aspect of the embodiments of this application, a recombinant expression vector containing the coding sequence of the thermophilic inorganic pyrophosphatase mutant protein is provided.

[0045] In the fourth aspect of the embodiments of this application, a recombinant strain containing the recombinant expression vector is provided.

[0046] In the fifth aspect of the embodiments of this application, a recombinant cell of the recombinant expression vector is provided.

[0047] The host cell of the recombinant cell is Escherichia coli BL21(DE3), and it is transformed with a recombinant expression vector.

[0048] The sixth aspect of the embodiments of the present application provides a method for preparing a mutant protein of thermophilic inorganic pyrophosphatase, including the following steps:

[0049] (1) Synthesize the gene sequence encoding the mutant protein of thermophilic inorganic pyrophosphatase;

[0050] (2) Construct a highly efficient recombinant expression vector to obtain the recombinant plasmid pCold-PPa-mut;

[0051] (3) Transform the host bacterium BL21(DE3) with the expression vector to construct a genetically engineered bacterium (recombinant cell);

[0052] (4) Cultivate and express the mutant protein of thermophilic inorganic pyrophosphatase using the obtained recombinant cell;

[0053] (5) Isolate and purify the expression product obtained in step (4) to obtain the mutant protein of thermophilic inorganic pyrophosphatase;

[0054] In step (2), the synthesized gene is assembled into the cloning plasmid pUC18 to obtain the plasmid pUC18-PPa-mut, and then transformed into the cloning Escherichia coli DH5α;

[0055] In step (2), the gene encoding the mutant protein of thermophilic inorganic pyrophosphatase is amplified by standard PCR technology, and restriction enzyme cutting sites (Kpn I, Xba I) are added at both ends. After restriction enzyme digestion, it is inserted between the corresponding restriction enzyme cutting sites of the pCold III vector, and then transformed into the cloning host, screened and sequenced to prepare the plasmid pCold-PPa-mut;

[0056] The specific primer pair for the coding gene is PPa-F / R; the primer PPa-F has the nucleotide sequence shown in SEQ ID NO.3; the primer PPa-R has the nucleotide sequence shown in SEQ ID NO.4.

[0057] Appropriate restriction enzyme cutting sites are introduced at the head and tail of the fragment by standard PCR technology, assembled into the plasmid pColdIII and transformed into the host cell to complete the construction of the genetically engineered bacterium.

[0058] Ferment the constructed genetically engineered bacterium, centrifuge to obtain the bacterial cells, break the cells after washing, centrifuge to collect the supernatant, and then purify the protein by nickel particle affinity chromatography. The purified protein is stored at low temperature after adding a stabilizer.

[0059] The following is illustrated with specific examples.

[0060] Example 1

[0061] Design and chemical synthesis of the mutant protein gene of thermophilic inorganic pyrophosphatase

[0062] First, in order to improve the enzyme activity of thermophilic inorganic pyrophosphatase, the present invention mutated the wild-type PPase gene from Thermus brockianus published in the Genbank database. The mutation sites are as follows: Asp55Ile, Leu70Tyr, Met96Val, Gly102Tyr, Glu112Val, Ala142Leu, to obtain a mutant of thermophilic inorganic pyrophosphatase (the amino acid sequence is SEQ ID NO.1), and the predicted protein structure is as Figure 1 shown

[0063] Subsequently, the present invention optimized the codons of the mutant protein of thermophilic inorganic pyrophosphatase according to the codon bias of Escherichia coli to obtain the optimized gene sequence (SEQ ID NO.2) of the mutant of thermophilic inorganic pyrophosphatase;

[0064] Finally, by chemical methods, the optimized gene (SEQ ID NO.2) of the mutant of thermophilic inorganic pyrophosphatase was artificially synthesized and assembled into the cloning plasmid pUC18 to obtain the plasmid pUC18-PPa-mut, which was then transformed into the cloning Escherichia coli DH5α.

[0065] Example 2

[0066] Construction of the plasmid pCold-PPa-mut expressing the mutant protein of thermophilic inorganic pyrophosphatase

[0067] The gene encoding the mutant protein of thermophilic inorganic pyrophosphatase was amplified by standard PCR technology, and restriction enzyme cleavage sites (Kpn I, Xba I) were added at both ends. After restriction enzyme digestion, it was inserted between the corresponding restriction enzyme cleavage sites of the pCold III vector, and the cloning host was transformed and screened for sequencing to prepare the plasmid pCold-PPa-mut, and its gene structure is as Figure 2 shown

[0068] The specific operations are as follows:

[0069] (1) Design of PCR primers

[0070] PPa-F (introducing the Kpn I restriction enzyme cleavage site GGTACC at the 5' end), and the primer PPa-F has the nucleotide sequence shown in SEQ ID NO.3;

[0071] 5’-GGTACCATGGCTAATTTAAAAACACTACCCGTA-3’

[0072] PPa-R (introduce Xba I restriction site TCTAGA at the 5'-end), the primer PPa-R has the nucleotide sequence shown in SEQ ID NO.4:

[0073] 5’-TCTAGACTTACCATAACGCGCAATGCACGC-3’,

[0074] (2) Prepare the sample reaction

[0075]

[0076] (3) PCR cycling conditions:

[0077] 3) PCR cycling conditions:

[0078]

[0079] 4) Store at 4°C or -20°C.

[0080] After the reaction, 10 μl can be taken for electrophoresis detection.

[0081] (4) PCR product recovery:

[0082] Use the PCR Purification Kit from QIAGEN to recover the above PCR product.

[0083] (5) Double-digest the recovered PCR product and pCold III vector with Kpn I and Xba I, and recover the digested products for ligation.

[0084] (8) Ligate the gene fragment of the mutant protein of thermophilic inorganic pyrophosphatase prepared in (5) to the pCold III vector after double-digestion with T 4 DNA ligase, transform the cloned Escherichia coli DH5α, and screen and sequence.

[0085] (9) If the sequencing is correct, complete the construction of the pCold-PPa-mut plasmid, extract the plasmid and transform the BL21(DE3) competent cells, and then prepare the cells containing the pCold-PPa-mut plasmid into competent cells BL21(DE3)pCold-PPa-mut.

[0086] Example 3

[0087] Expression of mutant protein of thermophilic inorganic pyrophosphatase

[0088] Take the seed solution identified by sequencing and inoculate it into a sterilized LB medium containing 100 μg / ml ampicillin. Incubate overnight at 250 rpm at 37°C. Inoculate into a sterilized LB medium containing 100 μg / ml ampicillin at an inoculation amount of 1%, and culture at 37°C. When the A 600 value reaches 0.6, add IPTG to make its final concentration reach 0.25 mmol / L, and induce expression at 16°C for 20 h. Centrifuge (at 4°C, 5000×g, 10 min) to collect the bacterial cells, then suspend the bacterial cells with PBS (pH 8.0), centrifuge (at 4°C, 5000×g, 10 min) to collect the bacterial cells, and store at -20°C or directly purify.

[0089] Example 4

[0090] Purification of Thermophilic Inorganic Pyrophosphatase Mutant Protein

[0091] (1) Cell disruption:

[0092] Thaw the bacterial cells, add cell disruption solution at a wet weight ratio of 1:10, suspend the bacterial cells, disrupt the cells with a high-pressure homogenizer, centrifuge, and collect the supernatant.

[0093] (2) Perform electrophoresis using SDS-PAGE protein gel, followed by Coomassie Brilliant Blue staining to analyze the expression levels in the supernatant and precipitate. Figure 3 This is the electrophoresis diagram of the expression and protein purification of the thermophilic inorganic pyrophosphatase mutant protein of the present invention.

[0094] (3) Load onto a nickel ion affinity column:

[0095] ① Column pretreatment:

[0096] a) The column bed height is 5 cm, and at least 2 BV of distilled water is used to wash the column bed.

[0097] b) Load 0.2 BV of 0.2 M NiSO 4 solution onto the column to bind Ni 2+ .

[0098] c) Wash the column bed with 5 BV of distilled water to remove excess Ni 2+ .

[0099] ② Equilibration:

[0100] Equilibrate with at least 2 BV of the increasing solution to make the pH of the equilibrium effluent the same as that of the increasing solution.

[0101] ③ Loading:

[0102] ④ Washing:

[0103] Wash with at least 2 BV of the increasing solution to stabilize the A 280 baseline.

[0104] ⑤ Elution:

[0105] Elute with the eluent and collect the effluent of peak A 280 from the elution peak.

[0106] ⑥ Regeneration, cleaning and storage of the column bed:

[0107] a) Use 0.5 BV of 0.2 M EDTA solution and 0.5 M NaCl solution to remove metal ions.

[0108] b) Load 1 BV of 2 M NaCl solution onto the column and hold for 15 min.

[0109] c) Load 1 BV of 1 M NaOH solution onto the column and hold for 1 - 2 h.

[0110] d) Wash the column bed with distilled water until the pH is around 7.0.

[0111] e) Store the column bed in 20% ethanol or 0.01 M NaOH solution.

[0112] (5) Perform electrophoresis on an SDS-PAGE protein gel, followed by Coomassie Brilliant Blue staining to analyze its purity. As Figure 3 shown, its purity is as high as over 90%.

[0113] (6) Sub-packaging:

[0114] Add 100% glycerol to make the final concentration of glycerol reach 50%, sub-package as needed, and store at -20°C.

[0115] Unless otherwise specified, the above operations are all required to be carried out in a 4°C environment.

[0116] Test Example 1

[0117] Determination of the specific activity of the inorganic pyrophosphatase mutant:

[0118] Use the colorimetric phosphomolybdate assay to determine the activity of PPase. The principle is that PPase hydrolyzes sodium pyrophosphate (Na 4 P 2 O 7 ) to produce phosphate ions (HPO 4 2- ). Under acidic conditions, the phosphate ions react with molybdate to form phosphomolybdate, which can be reduced by ferrous chloride to blue phosphomolybdenum blue. The depth of its blue color can be used to reflect the concentration of inorganic phosphorus. Read the absorbance value at 620 - 660 nm to calculate the concentration of phosphate ions, and then reflect the hydrolysis activity of PPase.

[0119] The enzyme reaction system is shown in Table 1:

[0120] Table 1

[0121]

[0122] After mixing evenly, water bath at 37 °C for 10 min, and add 20 μL of 1 M citric acid.

[0123] 2. Plotting of the standard curve

[0124] Select Na 2 HPO 4 as the standard product, and prepare a 5 mM Na 2 HPO 4 standard product solution, which is diluted to 0, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5 mM respectively. Take 20 μL of phosphorus standard solutions with different concentrations and add 180 μL of the chromogenic solution, vortex and mix evenly, let stand for 3 min, measure the absorbance at 620 nm using a microplate reader, and plot the standard curve of Pi concentration vs. absorbance, as Figure 4 shown.

[0125] 3. Detection of the reaction solution

[0126] According to the detection method of the reaction solution in the standard curve, measure OD 415 .

[0127] Substitute the measured data into the standard curve formula: U / mL = (the detected Pi μM value * df) / (10 * the added enzyme solution volume), where: df - dilution factor; 10 - enzyme reaction time, 10 min; the added enzyme solution volume - mL, and obtain the enzyme activity ratio, as shown in Table 2.

[0128] Table 2

[0129]

[0130] As shown in Table 2, the specific activity of the mutant enzyme of thermophilic inorganic pyrophosphatase is increased by more than 5 times compared with the wild type.

[0131] Experimental Example 2

[0132] Determination of the thermal stability of the mutant of thermophilic inorganic pyrophosphatase:

[0133] In 50 mM Tris-HCl pH 9.0, 1.5 mM Mg 2+, Under the reaction conditions of 1.5 mM PPi, 10 ng of PPa enzyme was added to 1 mL of the reaction solution, and the reaction was carried out at 0, 25, 35, 45, 55, 65, 75, 80, 85, 90, and 95 °C for 5 min. After the reaction ended, it was quickly placed on ice and allowed to stand for 3 min. Then, 20 μL of the reaction solution was added to 180 μL of the chromogenic solution, vortexed and mixed well, and allowed to stand for 3 min. The absorbance was measured at 620 nm using a microplate reader. According to the standard curve of Pi concentration versus absorbance, the thermal stabilities of different PPases were compared. As Figure 5 shown, the thermal stability of the thermophilic inorganic pyrophosphatase mutant was significantly higher than that of the wild-type thermophilic inorganic pyrophosphatase.

[0134] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements. The scope of protection required by the present invention is defined by the appended claims, the specification, and their equivalents.

Claims

1. A thermophilic inorganic pyrophosphatase mutant, characterized in that: The amino acid sequence of the thermophilic inorganic pyrophosphatase mutant is shown in SEQ ID NO.

1.

2. A gene encoding the thermophilic inorganic pyrophosphatase mutant according to claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the thermophilic inorganic pyrophosphatase mutant is shown in SEQ ID NO.

2.

4. A recombinant expression vector comprising the thermophilic inorganic pyrophosphatase mutant coding sequence according to claim 2.

5. A recombinant strain comprising the recombinant expression vector according to claim 4.

6. A recombinant cell containing the recombinant expression vector according to claim 4.

7. The recombinant cell according to claim 6, characterized in that: The host cell of the recombinant cell is Escherichia coli BL21 (DE3).

8. A method for preparing the thermophilic inorganic pyrophosphatase mutant according to claim 1, characterized in that The steps include: (1) synthesizing a gene sequence encoding the thermophilic inorganic pyrophosphatase mutant according to claim 2; (2) constructing an efficient recombinant expression vector to obtain the recombinant plasmid pCold-PPa-mut; (3) transforming the host bacteria BL21 (DE3) with the recombinant expression vector to construct a recombinant expression bacterium; (4) expressing the thermophilic inorganic pyrophosphatase mutant by culturing the obtained recombinant cells; (5) isolating and purifying the expression product obtained in step (4) to obtain a thermophilic inorganic pyrophosphatase mutant.

9. The method for preparing a thermophilic inorganic pyrophosphatase mutant according to claim 8, characterized in that: In step (2), the synthesized gene is assembled into the cloning plasmid pUC18 to obtain the plasmid pUC18-PPa-mut, which is then transformed into the cloning Escherichia coli DH5α.

10. The method for preparing a thermophilic inorganic pyrophosphatase mutant according to claim 9, characterized in that: In step (2), the mutant gene of thermophilic inorganic pyrophosphatase is amplified by standard PCR technology, and restriction enzyme sites (Kpn I, Xba I) are added at both ends. After restriction enzyme digestion, it is inserted between the corresponding restriction enzyme sites of pCold III vector, transformed into a cloning host, screened and sequenced, and the plasmid pCold-PPa-mut is prepared; The specific primer pair for the coding gene is primer PPa-mut-F and primer PPa-mut-R; primer PPa-mut-F has the nucleotide sequence shown in SEQ ID NO.3; primer PPa-mut-R has the nucleotide sequence shown in SEQ ID NO.4.

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