Polyphosphokinase mutant, coding gene and application thereof

Abstract

The invention discloses a polyphosphate kinase mutant, a coding gene and application of the polyphosphate kinase mutant, and belongs to the technical field of bioengineering. The polyphosphate kinase mutant disclosed by the invention is obtained by carrying out amino acid mutation on PPK2 from Sphingobium siyangense 1.6855, wherein the amino acid sequence of the PPK2 is as shown in SEQ ID NO.1, and the site of the amino acid mutation is at least one of I125F, I192P and S228A. The PPK2 mutant disclosed by the invention has excellent thermal stability, and the enzymatic activity retention rate of the mutant after thermal treatment can be up to 20 times or more that of a wild type. The PPK2 mutant can efficiently catalyze ADP (adenosine diphosphate) to generate ATP (adenosine triphosphate), the product yield is high, no by-product is generated, and the PPK2 mutant has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a method for targeting *Sphingomonas siyangensis*. Sphingobium siyangense 1.6855 The modified polyphosphokinase mutant, its encoding gene, and its application in ATP regeneration. Background Technology

[0002] Adenosine triphosphate (ATP), a key energy carrier in living organisms, releases a large amount of free energy during hydrolysis, providing energy for core physiological reactions in cellular metabolism such as the tricarboxylic acid cycle, glycolysis, and oxidative phosphorylation. Simultaneously, ATP is an indispensable energy donor for enzymatic synthesis reactions in in vitro biocatalytic systems. However, the chemical synthesis of ATP is costly and unstable; directly adding ATP exogenously significantly increases the economic cost of industrial production, becoming a key bottleneck restricting the large-scale application of ATP-dependent biocatalytic technologies. Therefore, developing efficient and low-cost ATP regeneration systems to replace the traditional method of directly adding ATP is a core requirement for reducing the cost of biocatalytic processes and promoting their industrial application.

[0003] Polyphosphate kinases (PPKs) are core enzymes in the ATP regeneration system. Based on their amino acid sequence characteristics and catalytic function differences, they can be divided into two major families: PPK1 and PPK2. The amino acid sequence similarity between the two classes of enzymes is less than 30%, and there are significant differences in their selectivity for nucleotide substrates: PPK1 can only catalyze the synthesis of polyphosphate (PolyP) using ATP as a substrate, and cannot reverse the process to generate ATP using PolyP; while PPK2 has bidirectional catalytic activity, and can synthesize PolyP using GTP or ATP as substrates, and can also use PolyP as a phosphorus source to efficiently catalyze the phosphorylation of GDP to generate GTP or the phosphorylation of ADP to generate ATP (Ishige K, Zhang H, Kornberg A. Polyphosphate kinase (PPK2), apotent, polyphosphate-driven generator of GTP [J]. Proceedings of the National Academy of Sciences, 2002, 99 (26): 16684-16688.). Most importantly, the forward reaction rate of PPK2 catalyzing the production of ATP / GTP from Poly P is 75 times that of its reverse reaction (Poly P synthesis), far exceeding the catalytic efficiency of PPK1, providing an enzymatic basis for efficient ATP regeneration. In terms of substrate preference, PPK1 primarily utilizes long-chain Poly P, while PPK2 prefers short-chain Poly P as a phosphorus donor. Furthermore, when the Poly P concentration in the reaction system reaches saturation, oligomerization can occur between Poly P molecules, increasing the apparent catalytic activity of PPK2 by 10 times. This characteristic makes PPK2 an ideal enzyme for constructing Poly P-dependent ATP regeneration systems, widely adaptable to various in vitro enzymatic reactions requiring ATP energy.

[0004] Based on sequence functional characteristics and phylogenetic analysis, the PPK2 family is further divided into three subtypes: I, II, and III. The substrate specificity of each subtype is significantly different (Nocek BP, Khusnutdinova AN, Ruszkowski M, et al. Structural insights into substrate selectivity and activity of bacterial polyphosphate kinases[J]. ACS Catalysis, 2018, 8(11): 10746-10760.).PPK2-I type primarily catalyzes the phosphorylation of ADP to ATP, exhibiting a narrow substrate range but high catalytic efficiency (LINDNER SN, VIDAURRED, WILLBOLD S, SCHOBERTH SM, WENDISCH VF. NCgl2620 encodes a class II polyphosphate kinase in Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2007, 73(15): 5026-5033.); PPK2-II type prefers AMP as a substrate, catalyzing its phosphorylation to ADP, but cannot directly generate ATP, requiring co-action with adenosine kinase to complete ATP regeneration (Bonting CF, Kortstee GJ, Zehnder A J. Properties of polyphosphate: AMPphosphotransferase of Acinetobacter strain 210A[J]. Journal of bacteriology, 1991, 173(20): 6484-6488.); while PPK2-III has bifunctional catalytic activity, which can catalyze the phosphorylation of AMP to ADP, and further catalyze the ADP to ATP. It can achieve the complete regeneration from AMP to ATP without the need for additional enzymes, and has high flexibility in multi-enzyme cascade reactions (MOTOMURA K, HIROTA R, OKADA M, IKEDA T, ISHIDA T, KURODA A. A new subfamily of polyphosphate kinase 2 (class IIIPPK2) catalyzes both nucleoside monophosphate phosphorylation and nucleoside diphosphate phosphorylation[J]. Applied and Environmental Microbiology, 2014,80(8): 2602-2608.). The molecular mechanism of PPK2 catalyzing ATP regeneration has been reported so far. PPK2 first reacts with Poly P (in the form of Poly P). n (represented), ADP via Mg 2+ Bridging reaction forms a ternary enzyme-substrate complex, Mg 2+It not only stabilizes the spatial conformation of substrate molecules but also mediates the directional transfer of phosphate groups through electrostatic interactions; subsequently, the oxygen atom of β-phosphate in the ADP molecule pairs with Poly P n The terminal phosphorus atom initiates a nucleophilic attack, forming a five-coordinate phosphorus transition state intermediate; ultimately, the transition state decomposes to generate ATP and Poly P. n-1 After the enzyme-product complex dissociates, it releases ATP and completes the catalytic cycle (Nocek BP, Khusnutdinova AN, Ruszkowski M, et al. Structural insights into substrate selectivity and activity of bacterial polyphosphate kinases[J]. ACSCatalysis, 2018, 8(11): 10746-10760.).

[0005] In industrial applications, the thermal stability, catalytic efficiency, and substrate tolerance of enzymes are key indicators determining the feasibility of the process. However, existing PPK2 enzymes for ATP regeneration generally suffer from insufficient thermal stability: when the reaction temperature exceeds 50°C, the enzyme's spatial conformation is prone to irreversible denaturation, resulting in a decrease in catalytic activity of more than 80% within one hour, making it impossible to maintain stable catalysis for a long time (Gao H, Li M, Wang Q, et al. A high-throughput dual system to screen polyphosphate kinase mutants for efficient ATP regenerationin L-theanine biocatalysis[J]. Biotechnology for biofuels and bioproducts,2023, 16(1): 122.)(Nocek B, Kochinyan S, Proudfoot M, et al. Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases inbacteria[J]. Proceedings of the National Academy of Sciences, 2008, 105(46):17730-17735.) According to the Arrhenius equation, higher reaction temperatures have a positive promoting effect on increasing the rate of enzyme-catalyzed reactions. At the same time, higher reaction temperatures can also inhibit contamination by miscellaneous bacteria and reduce the consumption of substrates or products by microorganisms in the fermentation system. However, the thermal instability of existing PPK2 is fundamentally contradictory to this requirement. Although existing studies have reported that a few PPK2s derived from thermophilic microorganisms (such as Thermosynechococcus elongatus BP-1) have certain heat resistance and can maintain some activity above 50℃, their catalytic efficiency is only 30%-50% of that of mesophilic PPK2, which cannot meet the requirements of efficient ATP regeneration. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a polyphosphokinase mutant with both high catalytic activity and thermal stability, its encoding gene, and its application in ATP regeneration. Based on a semi-rational design strategy and combined with the three-dimensional structure analysis of PPK2, this invention precisely selects key amino acid sites for mutation, thereby retaining the high catalytic efficiency of PPK2 while significantly improving its thermal stability. This meets the requirements of in vitro multi-enzyme cascade reactions for ATP regeneration under high-temperature conditions, making it suitable for industrial-grade high-temperature reaction conditions and breaking through the bottleneck of existing ATP regeneration technology.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a polyphosphokinase mutant, which is derived from *Sphingomyelinus siyangensis* with an amino acid sequence as shown in SEQ ID NO.1. S. siyangensis 1.6855 The PPK2 source is obtained by amino acid mutation, wherein the mutation site is at least one of position 125, position 192, and position 228, and isoleucine at position 125 is mutated to phenylalanine, isoleucine at position 192 is mutated to proline, and cysteine ​​at position 228 is mutated to alanine.

[0008] Based on the above technical solution, further, the amino acid sequence of the mutant PPK2-I125F, in which isoleucine at position 125 is mutated to phenylalanine, is shown in SEQ ID NO.2; The amino acid sequence of the mutant PPK2-I192P, in which isoleucine at position 192 is mutated to proline, is shown in SEQ ID NO. 3; The amino acid sequence of the mutant PPK2-S228A, in which cysteine ​​at position 228 is mutated to alanine, is shown in SEQ ID NO. 4. The amino acid sequence of the mutant PPK2-I125F-I192P, in which isoleucine at position 125 is mutated to phenylalanine and isoleucine at position 192 is mutated to proline, is shown in SEQ ID NO.5. The amino acid sequence of the mutant PPK2-I192P-S228A, in which isoleucine at position 192 is mutated to proline and cysteine ​​at position 228 is mutated to alanine, is shown in SEQ ID NO.6. The amino acid sequence of the mutant PPK2-I125F-S228A, in which isoleucine at position 125 is mutated to phenylalanine and cysteine ​​at position 228 is mutated to alanine, is shown in SEQ ID NO.7. The amino acid sequence of the mutant PPK2-I125F-I192P-S228A, in which isoleucine at position 125 is mutated to phenylalanine, isoleucine at position 192 is mutated to proline, and cysteine ​​at position 228 is mutated to alanine, is shown in SEQ ID NO. 8.

[0009] Based on the above technical solution, the thermal stability of the above polyphosphokinase mutant is significantly improved compared to wild-type PPK2.

[0010] Secondly, the present invention provides the encoding gene of the above-mentioned polyphosphokinase mutant.

[0011] Thirdly, the present invention provides a recombinant expression vector comprising the coding gene of the above-mentioned polyphosphoric acid kinase mutant.

[0012] Based on the above technical solution, the recombinant expression vector further includes the pET-29a plasmid.

[0013] Fourthly, the present invention provides a recombinant bacterium carrying the above-described recombinant expression vector.

[0014] Based on the above technical solution, the recombinant bacteria further include Escherichia coli. BL21 (DE3).

[0015] Fourthly, the present invention provides a method for preparing the above-mentioned polyphosphoric acid kinase mutant, comprising the following steps: inoculating the above-mentioned recombinant bacteria into a culture medium and culturing to OD200. 600 The bacterial cells were added to a concentration of 0.4-0.8, and IPTG was added to induce protein expression for 10-20 hours. The cells were then collected, lysed, and purified by nickel affinity chromatography to obtain the polyphosphoric acid kinase mutant.

[0016] Based on the above technical solution, the culture medium further includes LB liquid medium, with culture conditions of 35~38℃ and 150~300 rpm; the culture conditions for inducing protein expression are 15~20℃ and 150~250 rpm, and the final concentration of IPTG is 30~80 mM.

[0017] Fifthly, the present invention provides the application of the above-mentioned polyphosphokinase mutant in ATP regeneration.

[0018] Based on the above technical solution, the application specifically involves using a polyphosphoric acid kinase mutant as the enzyme, ADP and polyphosphates as substrates, and Mg... 2+ As a cofactor, ATP was obtained by reacting in a buffer solution with a pH of 7-12 at 25-65°C.

[0019] Based on the above technical solution, the amount of polyphosphokinase mutant added to the reaction system is 200~300 μg / mL, preferably 250 μg / mL.

[0020] Based on the above technical solution, the concentration of the substrate ADP in the reaction system is further 140~200 mM, preferably 170 mM.

[0021] Based on the above technical solution, the pH range of the buffer solution is 8 to 11, preferably 9.5.

[0022] Based on the above technical solution, further, Mg in the reaction system 2+ The concentration range is 60-90 mM, preferably 75 mM.

[0023] Based on the above technical solution, the polyphosphate further includes sodium tripolyphosphate, sodium tetrapolyphosphate and sodium hexametaphosphate, with a concentration of 100~1000mM, more preferably 200 mM.

[0024] Based on the above technical solution, the reaction temperature is further 45-65℃, preferably 52℃.

[0025] Compared with the prior art, the present invention has the following beneficial effects: (1) Precise design of a PPK2 enzyme mutant with both excellent thermal stability and catalytic efficiency: Based on the three-dimensional structure of the enzyme, the conformation of the active site and substrate binding ability of the enzyme are analyzed. The PPK2 enzyme mutant obtained by site-directed mutagenesis in this invention, when catalyzing the generation of ATP with ADP as substrate, has the following characteristics: on the one hand, the catalytic activity is 1.98 times higher than that of wild-type PPK2 under the same reaction conditions, and no by-products are generated throughout the process. This characteristic not only greatly improves the ATP synthesis efficiency and reduces substrate waste, but also avoids the increased process complexity and cost due to the presence of by-products in the subsequent product separation and purification process, providing a new and efficient enzyme source for increasing production capacity and controlling costs in industrial production; on the other hand, after high-temperature heat treatment, its residual catalytic activity is 20.2 times higher than that of wild-type. This breakthrough effectively resolves the contradiction between "high temperature to accelerate reaction rate and inhibit microbial contamination" and "enzyme thermal inactivation" in industrial scenarios, enabling enzymatic reactions to proceed stably within a more optimal temperature range. This not only ensures the continuity of catalytic efficiency but also reduces the risk of substrate loss and product impurities caused by microbial contamination, providing a key guarantee for process stability.

[0026] (2) Promoting the industrialization of high-temperature adapted ATP regeneration systems and expanding industrial applications: This invention innovatively constructs an ATP regeneration system adaptable to high-temperature industrial conditions, using the PPK2 enzyme mutant pure enzyme, which has excellent thermal stability and catalytic efficiency, as the core component. This system not only fills the application gap of existing technologies in high-temperature scenarios, but also has the industrialization advantage of "no need for additional temperature control equipment and simplified process flow"—compared to traditional ATP regeneration systems that rely on low-temperature temperature control, it can significantly reduce equipment investment and energy consumption costs. At the same time, this system can be widely integrated into various in vitro multi-enzyme cascade reactions that rely on ATP for energy, solving the bottleneck of existing systems that "cannot stably supply energy at high temperatures," and providing a brand-new solution for the development of biocatalysis processes towards high temperature, high efficiency, and low cost, possessing extremely strong industrial promotion value and scenario expansion capabilities. Attached Figure Description

[0027] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0028] Figure 1 This is a map of the wild-type plasmid for polyphosphokinase (PPK2).

[0029] Figure 2 This is a molecular docking model diagram of the PPK2 wild-type enzyme and its substrate ADP. Figure 3 This is a liquid chromatogram of a mixture of standards ATP and ADP.

[0030] Figure 4 The results show the catalytic activity of PPK2 wild-type enzymes when using polyphosphates with different degrees of polymerization as phosphate donors. In this study, A represents the screening experiment for polyphosphates with different degrees of polymerization, and B represents the screening experiment for PolyP6 concentration.

[0031] Figure 5 The liquid chromatogram shows the product obtained by reacting the PPK2 mutant I125F with the substrate ADP.

[0032] Figure 6 This is a comparison of the initial relative activities of polyphosphokinases expressed by recombinant strains constructed using different mutation sites. Figure 7 This is a comparison of the residual relative activities of polyphosphokinases expressed by recombinant strains constructed using different mutation sites after heat treatment. Detailed Implementation The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0033] Unless otherwise specified, all raw materials used in this invention are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art. Nucleic acids are written from left to right in a 5' to 3' direction, and amino acid sequences are written from left to right in a direction from the amino terminus to the carboxyl terminus. Example 1: Design and Construction of PPK2 Mutants The PPK2 enzyme described in this embodiment is derived from *Sphingosine monocytogenes*. S. siyangensis 1.6855 The amino acid sequence of PPK2 is shown in SEQ ID NO.1.

[0034] 1. Design of PPK2 mutation sites 125, 192, and 228 The selection of PPK2 enzyme mutation sites (such as I125F, I192P, S228A, etc.) in this invention is based on an integrative semi-rational design strategy, aiming to improve the enzyme's thermostability while ensuring that its catalytic activity is not compromised. The specific process is as follows: First, by using SWISS-MODEL homology modeling and Autodock Vina molecular docking, the binding site of the substrate ADP was clarified, thus avoiding the active site residues directly involved in catalysis and preventing functional loss at the source. Second, a systematic site screening was conducted using five complementary computational tools: FoldX energy assessment: calculating the change in free energy (ΔΔG) of each residue mutation to screen for potential stability-enhancing sites with ΔΔG < 0 kcal / mol; HotSpot Wizard structural flexibility analysis: identifying key sites that regulate conformational dynamics and thermal stability based on flexible regions and conserved characteristics; FireProt distal effect prediction: predicting distal mutation sites that can stabilize the structure through long-range interactions through evolutionary coupling and energy calculation; PROSS evolutionary information mining: screening for evolutionarily high-frequency substitution sites that may enhance stability through multiple sequence alignment; and EVcouplings co-evolutionary analysis: selecting high-resolution residue pairs that can synergistically optimize the local interaction network based on the co-evolutionary relationship between residues.

[0035] Based on the above analysis, sites I125F, I192P, and S228A, recommended by at least two methods, were selected. Further molecular dynamics simulations confirmed that mutations at these sites have a stabilizing effect on conformational dynamics.

[0036] 2. Construction of PPK2 unit site mutants at points 125, 192, and 228 Using the wild-type plasmid pET-29a-PPK2 as a template (the nucleotide sequence of PPK2 is shown in SEQ ID NO.9), mutant plasmids were constructed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, United States), resulting in pET-29a-PPK2-I125F, pET-29a-PPK2-I192P, and pET-29a-PPK2-S228A. Single point mutations were performed on isoleucine (I) at position 125, isoleucine (I) at position 192, and cysteine ​​(S) at position 228 of the wild-type amino acid sequence. Corresponding primers were designed, as shown in Table 1.

[0037] Table 1. Primers used for mutations at each site

[0038] The constructed mutant plasmid was added to E. coli. E. coli BL21 (DE3) competent cells, mixed well, placed on ice for 30 min, and then E. coli were added. E. coli BL21 (DE3) competent cells were heat-shocked at 42℃ for 60s and then placed on ice for 5 min to thaw. Then, 1 mL of antibiotic-free LB medium was added and cultured at 37℃ for 45 min. After the thaw, the cells were centrifuged at 6000 rpm for 5 min, and then resuspended in 1 mL of LB medium. 100 μL of the resuspended cells were spread on LB plates containing 50 μg / mL kanamycin and incubated at 37℃ for 16 h. Pick a single colony from the plate and place it into an activation vial containing 3 mL of LB (kanamycin-resistant) medium. After incubating for 8 hours, take 1 mL of the vial for sequencing. For colonies with correct sequencing results, add an equal volume of 40% glycerol solution to the remaining bacterial culture and store it in a -80℃ freezer for later use. PPK2 mutant engineered bacteria were obtained respectively E. coli BL21 (DE3) / pET-29a-PPK2-I125F、 E. coli BL21 (DE3) / pET-29a-PPK2-I192P E. coli BL21Sequencing results for (DE3) / pET-29a-PPK2-S228A showed that the codon ATT encoding isoleucine at position 125 (I) was mutated to the codon TTT encoding phenylalanine (F); the codon ATC encoding isoleucine at position 192 (I) was mutated to the codon CCG encoding proline (P); and the codon TCG encoding cysteine ​​at position 228 (S) was mutated to the codon GCG encoding alanine (A). The amino acid sequences of the PPK2 mutants I125F, I192P, and S228A are shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively.

[0039] 3. Construction of PPK2 combinatorial mutants Using pET-29a-PPK2-I125F, pET-29a-PPK2-I192P, and pET-29a-PPK2-S228A constructed in step 2 as templates, the mutant plasmids and recombinant bacteria were constructed using a point mutation kit, following the same method as in step 2. Sequencing verification confirmed the results were correct, and PPK2 mutant engineered bacteria were obtained. E. coli BL21 (DE3) / pET-29a-PPK2-I125F-I192P, E. coli BL21 (DE3) / pET-29a-PPK2-I192P-S228A, E. coli BL21 The amino acid sequences of the mutants (DE3) / pET-29a-PPK2-I125F-S228A are shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively.

[0040] Using the aforementioned plasmid as a template, the mutant plasmid and recombinant bacteria were constructed using a point mutation kit, following the same method as above. Sequencing results correctly yielded the PPK2 mutant engineered bacteria. E. coli BL21 (DE3) / pET-29a-PPK2-I125F-I192P-S228A, the corresponding amino acid sequence is shown in SEQ ID NO.8.

[0041] Example 2: Purification of various mutant enzymes Based on the mutant strains constructed in Example 1, the strains were purified using the corresponding protein purification methods to obtain purified PPK2 enzyme solution. Each mutant recombinant strain was activated on LB agar plates containing 50 μg / mL kanamycin resistance and cultured at 37°C for 12 h. Single colonies were picked and placed in 5 mL LB tubes containing 50 μg / mL kanamycin resistance and cultured at 37°C and 220 rpm for approximately 12 h. Then, 1% inoculum (v / v) was added to 200 mL LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm until the bacterial concentration reached OD500. 600 When the concentration reaches approximately 0.6, add IPTG to a final concentration of 60 mM, and induce culture at 18℃ and 180 rpm for 16 h. Collect the bacterial cells by centrifugation at 4℃ and 8000 rpm for 10 min to obtain the wet bacterial cells of the engineered bacteria expressing the mutant enzyme. Resuspend the cells in PBS buffer at a volume ratio of 10:1 in the wet bacterial cells, and sonicate them using an ultrasonic homogenizer at 300 W for 30 min with a 2 s interval between sonication cycles. After homogenization, centrifuge at 8000 rpm for 30 min, and retain the supernatant for subsequent protein purification.

[0042] Using Ni 2+ Protein purification was performed using affinity chromatography, with the following steps: (1) Wash the nickel column stored in 20% ethanol with 6 column volumes of 20 mM imidazole solution; (2) Filter the obtained supernatant through a 0.22 μm filter membrane and load the sample; (3) Wash 6 column volumes with PBS buffer containing 20 mM imidazole to remove non-specifically bound proteins; (4) Elute the target protein with PBS buffer containing 300 mM imidazole and collect the eluted sample; (5) Wash the chromatographic column with 500 mM imidazole solution and sterile water sequentially for 6 column volumes, and finally wash with 20% ethanol and store at 4°C. Next, the collected recombinant protein was concentrated by ultrafiltration using an ultrafiltration tube (10 kDa) (centrifuged at 5000 rpm and 4°C), retaining a certain final ultrafiltration volume. Then, Tris-HCl buffer was added, and ultrafiltration was performed 5 times to remove salt and imidazole and replace the buffer. Glycerol with a final concentration of 20% was added, aliquoted, and stored at -80°C, or used for subsequent enzyme activity verification.

[0043] Example 3: ATP preparation from various mutants 1. Optimization of pure enzyme reaction conditions This experiment mainly determined the catalytic activity of wild-type polyphosphokinase (PPK2-WT) with polyphosphates of different degrees of polymerization as phosphate donors, and clarified its substrate specificity.

[0044] Experimental materials: Enzyme sample: purified wild-type polyphosphoric acid kinase (PPK2-WT), protein concentration 1.2 mg / mL; Substrate: Adenosine diphosphate (ADP); Phosphate donors: sodium tripolyphosphate (PolyP3), sodium tetrapolyphosphate (PolyP4), sodium hexametaphosphate (PolyP6). Cofactor: Mg 2+ ; Reaction system buffer: 50 mM Tris-HCl buffer (pH 9.5) Detection: The concentrations of ADP and ATP were detected using high-performance liquid chromatography (HPLC), with C0.05... 18 The column was used with a mobile phase of 20 mmol / L potassium dihydrogen phosphate in 5% methanol at a flow rate of 0.8 mL / min, a column temperature of 25℃, and a UV detector at a detection wavelength of 254 nm. The HPLC chromatograms of ATP and ADP standards are shown below. Figure 3 As shown.

[0045] The reaction system consists of the following components: The total volume of the reaction system was 1 mL, and the solvent was 50 mM Tris-HCl buffer (pH 9.5). The main components of the system are shown in Table 2. Table 2. Components of the reaction system

[0046] The experimental procedure is as follows: (1) Premix all components except enzymes and incubate them in a constant temperature water bath at the set temperature (52℃) for 5 minutes; (2) Add PPK2-WT enzyme solution to start the reaction and immediately vortex to mix; (3) React precisely at the set temperature for 5 min, 30 min, and 60 min; (4) Move the reaction tube to a 99°C metal bath and heat for 10 minutes to terminate the reaction; (5) After the reaction solution is cooled to room temperature, it is centrifuged at 12,000 rpm for 5 minutes, and the supernatant is collected for testing; (6) Take 200 μL of supernatant and use high performance liquid chromatography to detect ATP production.

[0047] The effect of PolyP6 concentration on the catalytic activity of PPK2-WT was investigated according to the above experimental procedure. The difference was that the PolyP6 concentrations were 100 mM, 200 mM, 300 mM, 500 mM and 1000 mM, and the reaction time was 60 min. The highest conversion rate in the experimental results was defined as 100%.

[0048] Experimental results are as follows Figure 4As shown, the results indicate that wild-type polyphosphokinase (PPK2-WT) can catalyze the conversion of ADP to ATP using polyphosphates with different degrees of polymerization as phosphate donors, but their catalytic activities vary significantly. Sodium hexametaphosphate (PolyP6) is the optimal phosphate donor. Under the same reaction conditions for 60 min, when PolyP6 is used as the substrate, the average enzyme activity of PPK2-WT, calculated based on the cumulative product amount, is approximately 12.7 U / mg, significantly higher than other tested substrates. Compared with PolyP6, when sodium tetrapolyphosphate (PolyP4) is used as the substrate, the enzyme activity of PPK2-WT decreases by approximately 51% (relative activity 39.0%); when sodium tripolyphosphate (PolyP3) is used as the substrate, the enzyme activity decreases by approximately 70% (relative activity 30.5%). The degree of polymerization of the phosphate donor is a key factor affecting the catalytic activity of PPK2-WT. Within the tested range, the catalytic activity of the enzyme was positively correlated with the degree of polymerization of polyphosphates; that is, the higher the degree of polymerization, the higher the catalytic efficiency. An optimal working window existed for PolyP6 concentration. When the concentration was below or above 200 mM, the catalytic activity of PPK2-WT decreased significantly, indicating that insufficient substrate concentration limited the reaction rate, while excessively high concentrations may inhibit enzyme activity through ionic strength effects or non-specific binding. In the ATP regeneration reaction system, a PolyP6 concentration of 200 mM achieved the best ATP regeneration efficiency and system compatibility. In conclusion, PolyP6 is the most suitable phosphate donor for PPK-WT to achieve efficient ATP regeneration, with an optimal working concentration of 200 mM. This conclusion provides a crucial basis for selecting phosphate donors for constructing efficient ATP regeneration systems.

[0049] 2. Verification of mutant thermostability and detection of absolute enzyme activity This experiment verified the thermostability of each mutant (using PPK2-WT enzyme as a control) under the corresponding reaction conditions. The specific procedure is as follows: The function of PPK2 is to synthesize ATP using ADP and polyphosphate as substrates. The reaction system is 1 mL, and the main components of the system are shown in Table 3. The concentration of the mutant protein PPK2 is 250 μg / mL, and the solvent of the reaction system is Tris-HCl buffer with a pH of 9.5.

[0050] Table 3. Components of the reaction system

[0051] Untreated enzyme solution was added to the reaction system, and the temperature was controlled at 52℃ for 10 min. The reaction was then terminated by heating at 99℃ for 10 min, and enzyme activity was measured. The same batch of enzyme solution was then incubated at 60℃ for 1 h. Following the same procedure, the heat-treated enzyme solution was added to the prepared reaction system, and the temperature was controlled at 52℃ for 10 min. The reaction was then terminated by heating at 99℃ for 10 min. After the reaction, ATP production was analyzed using high-performance liquid chromatography (HPLC), and the absolute enzyme activity of each mutant was calculated. See Table 4 for details.

[0052] Table 4. Absolute enzyme activity of each mutant

[0053] The initial enzyme activity percentage of each mutant before heat treatment and the residual enzyme activity percentage of each mutant after heat treatment were calculated respectively. The test results are as follows: Figure 6 and 7 As shown.

[0054] This invention, through site-directed mutagenesis, yielded a PPK2 enzyme mutant that, when catalyzing the production of ATP using ADP and polyphosphate as substrates, exhibited a 1.98-fold increase in catalytic activity compared to the wild-type PPK2 under the same reaction conditions. Furthermore, after high-temperature heat treatment, its residual catalytic activity was 20.2 times higher than the wild-type. This breakthrough effectively resolves the contradiction between "high-temperature acceleration of reaction rate and inhibition of microbial contamination" and "enzyme thermal inactivation" in industrial settings. It allows the enzymatic reaction to proceed stably within a more optimal temperature range, ensuring the continuity of catalytic efficiency while reducing substrate loss and product impurity risks caused by microbial contamination, thus providing crucial assurance for process stability.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polyphosphokinase mutant, characterized in that, It is obtained by mutating the amino acid sequence PPK2 as shown in SEQ ID NO.1, wherein the mutated amino acid sites are at least one of positions 125, 192, and 228, and isoleucine at position 125 is mutated to phenylalanine, isoleucine at position 192 is mutated to proline, and cysteine ​​at position 228 is mutated to alanine.

2. The polyphosphokinase mutant according to claim 1, characterized in that, The amino acid sequences of the polyphosphokinase mutants are shown in SEQ ID NO.2~8.

3. The encoding gene of the polyphosphokinase mutant according to claim 1 or 2.

4. A recombinant expression vector comprising the encoding gene of the polyphosphokinase mutant of claim 3.

5. A recombinant bacterium carrying the recombinant expression vector of claim 4.

6. The method for preparing the polyphosphokinase mutant according to claim 1 or 2, characterized in that, The process includes the following steps: inoculating the recombinant bacteria of claim 5 into a culture medium and culturing it until the OD value reaches 50%. 600 The bacterial cells were added to a concentration of 0.4-0.8, and IPTG was added to induce protein expression for 10-20 hours. The cells were then collected, lysed, and purified by nickel affinity chromatography to obtain the polyphosphoric acid kinase mutant.

7. The use of the polyphosphokinase mutant according to claim 1 or 2 in ATP regeneration.

8. The application according to claim 7, characterized in that, The specific application involves using a polyphosphoric acid kinase mutant as the enzyme, ADP and polyphosphates as substrates, and Mg... 2+ As a cofactor, ATP was obtained by reacting in a buffer solution with a pH of 7-12 at 25-65°C.

9. The application according to claim 8, characterized in that, The amount of polyphosphokinase mutant added to the reaction system is 200-300 μg / mL, preferably 250 μg / mL; the concentration of substrate ADP is 140-200 mM, preferably 170 mM; the polyphosphate includes sodium tripolyphosphate, sodium tetrapolyphosphate, and sodium hexametaphosphate, with a concentration of 100-1000 mM, preferably 200 mM; Mg in the reaction system 2+ The concentration range is 60-90 mM, preferably 75 mM.

10. The application according to claim 8, characterized in that, The pH range of the buffer solution is 8-11, preferably 9.5; the reaction temperature is 45-65℃, preferably 52℃.

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