A polyphosphate kinase mutant with improved thermal stability and its application

Abstract

The present invention discloses a polyphosphate kinase mutant and application with improved thermal stability, wherein the polyphosphate kinase mutant is obtained by single mutation or multiple mutation of the 59th, 68th, and 190th amino acids of the amino acid sequence shown in SEQ ID NO.2. The thermal stability of the polyphosphate kinase mutant of the present invention is effectively improved, and the catalytic activity can be maintained for a longer time. The half-life is improved by 2-5 times compared to the wild type, and the half-life can reach more than 53h at 35°C. The polyphosphate kinase mutant of the present invention is in catalysis of ATP synthesis, and the ADP conversion rate of the polyphosphate kinase mutant Q59R / D68H / T190Y can reach 80.57% after 2h of reaction. The present invention effectively improves the thermal stability of polyphosphate kinase, helps to reduce the industrial production cost of directly using ATP, and has good application prospects in the industrial production that relies on the ATP circulation system.

Description

(1) Technical field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to a polyphosphate kinase mutant with improved thermal stability and its application in adenosine triphosphate (ATP) synthesis. (2) Background technology

[0002] Adenosine triphosphate (ATP), a high-energy phosphate compound, is the most direct source of energy in organisms. It serves as a precursor for the synthesis of numerous essential biomolecules. Due to the complexity of ATP synthesis, poor stability, and energy conversion efficiency, exogenous addition of ATP as a raw material increases production costs, limiting its large-scale industrial application. Commonly used ATP synthesis and regeneration systems include acetate kinase-acetyl phosphate, polyphosphate kinase-polyphosphate, pyruvate kinase-phosphoenolpyruvate, and creatine kinase-phosphokinase systems. Compared to these other systems, polyphosphate kinases (PPKs)-mediated ATP synthesis and regeneration using polyphosphate as a phosphate donor offer advantages such as inexpensive and stable substrates and efficient processes, making them more suitable for industrial applications.

[0003] Most research focuses on obtaining polyphosphate kinases with high catalytic activity through screening and modification. Rao et al. molecularly modified the polyphosphate kinase from Cytophaga hutchinsonii, increasing its enzymatic activity by 4.3 times and effectively improving the ATP regeneration efficiency (Biotechnol. Biofuels, 2023, 16, 2-13), but the mutant had poor thermal stability. The stability of polyphosphate kinases reported so far is generally not high. Li et al. obtained a polyphosphate kinase from Sulfurovum lithotrophicum with a half-life of 14.1h at 60°C (Appl. Environ. Microb., 2024, 90: e01574-23), but its stability still cannot meet industrial needs, limiting the efficient regeneration of ATP and its application in biosynthesis systems. (3) Summary of the invention

[0004] The present invention aims to provide a polyphosphate kinase mutant with improved thermal stability and its application in adenosine triphosphate synthesis. The present invention transforms the polyphosphate kinase derived from Phnomibacter ginsenosidimutans through protein engineering technology to obtain a polyphosphate kinase with improved thermal stability. The polyphosphate kinase is used to efficiently catalyze the synthesis of ATP from adenosine diphosphate (ADP), thereby constructing an efficient ATP regeneration system, which is of great significance for the industrial application of ATP regeneration systems.

[0005] The technical solution adopted in the present invention is:

[0006] The present invention provides a polyphosphate kinase mutant with improved thermal stability. The polyphosphate kinase mutant is obtained by subjecting the 59th, 68th and 190th amino acids of the amino acid sequence shown in SEQ ID NO.2 to single or multiple mutations.

[0007] Furthermore, it is preferred that the polyphosphate kinase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.2 is mutated into one of the following: (1) glutamine at position 59 is mutated into arginine or isoleucine, Q59R or Q59I; (2) aspartic acid at position 68 is mutated into histidine, D68H, the amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.3; (3) threonine at position 190 is mutated into tyrosine, T190Y; (4) aspartic acid at position 68 is mutated into histidine, and threonine at position 190 is mutated into tyrosine, D68H / T190Y, the amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO.5; (5) glutamine at position 59 is mutated into isoleucine, aspartic acid at position 68 is mutated into histidine, and threonine at position 190 is mutated into tyrosine, Q59I / D68H / T190Y, the amino acid sequence is shown in SEQ ID NO.8, and the nucleotide sequence is shown in SEQ ID NO.7; (6) the glutamine at position 59 is mutated to arginine, the aspartic acid at position 68 is mutated to histidine, and the threonine at position 190 is mutated to tyrosine, Q59R / D68H / T190Y, the amino acid sequence is shown in SEQ ID NO.10, and the nucleotide sequence is shown in SEQ ID NO.9.

[0008] The present invention also provides a gene encoding the polyphosphate kinase mutant, a recombinant vector comprising the gene encoding the gene, and a recombinant genetically engineered bacterium comprising the recombinant vector. Preferably, the original vector of the recombinant vector is pET-28a. The host cell for constructing the recombinant genetically engineered bacterium can be any conventional host cell in the art, preferably, Escherichia coli BL21.

[0009] The present invention also provides an application of the polyphosphate kinase mutant in adenosine triphosphate (ATP) synthesis. The application method comprises the following steps: using wet bacteria obtained by induction culture of recombinant genetically engineered bacteria expressing the polyphosphate kinase mutant or cell fragmentation liquid after ultrasonic disruption of the wet bacteria as a catalyst, using ADP as a substrate, adding MgCl2 and sodium hexametaphosphate, and using a pH 6-8 buffer as a reaction medium to form a reaction system, and carrying out the reaction at 30-60°C and 800-1000 rpm (preferably 35°C and 1000 rpm) to complete the reaction and obtain ATP.

[0010] Furthermore, in the reaction system, the amount of catalyst used is 1-5 g / L (preferably 1.25 g / L) based on the weight of wet bacteria, the volume concentration of cell disruption solution added is 10-30% (preferably 20%), and the enzyme activity of the cell disruption solution is 1415.05-2531.28 U / g based on the weight of wet bacteria before disruption; the concentration of substrate added is 1-10 mM (preferably 5 mM); the concentration of MgCl2 added is 5-15 mM (preferably 10 mM); and the concentration of sodium hexametaphosphate added is 1-10 mM (preferably 5 mM).

[0011] Furthermore, preferably, the reaction medium is 50 mM Tris-HCl buffer (pH 7.5).

[0012] Furthermore, the catalyst was prepared as follows: a recombinant genetically engineered bacterium expressing a polyphosphate kinase mutant was inoculated into a LB liquid culture medium test tube containing a final concentration of 0.1 mM kanamycin sulfate, and cultured at 37°C for 6-8 hours to obtain a seed solution; the seed solution was transferred to an LB medium containing a final concentration of 0.1 mM kanamycin sulfate at an inoculum concentration of 1% by volume, and cultured at 37°C and 200 rpm until the OD 600 When the pH value is 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM and culture at 28°C and 200 rpm for 12-16 h; then centrifuge at 8000 rpm for 10 min at 4°C, discard the supernatant, and collect the wet cells;

[0013] The wet cells (preferably 0.5 g / L) were resuspended in 50 mM Tris-HCl buffer (pH 7.5) and ultrasonically disrupted at 200 W for 10 min, with an operating speed of 1 s and an interval of 2 s to obtain a cell disruption solution.

[0014] The polyphosphate kinase mutants of the present invention can be used in the form of whole engineered bacterial cells, unpurified crude enzymes, partially purified enzymes, or completely purified enzymes. The polyphosphate kinase mutants of the present invention can also be prepared as biocatalysts in the form of immobilized enzymes or immobilized cells using immobilization techniques known in the art.

[0015] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0016] (1) The thermal stability of the polyphosphate kinase mutant of the present invention is effectively improved, and the catalytic activity can be maintained longer. Compared with the wild type, the half-life is increased by 2-5 times, and the half-life at 35°C can reach more than 53 hours.

[0017] (2) When the polyphosphate kinase mutant of the present invention catalyzes ATP synthesis, the ADP conversion rate of the polyphosphate kinase mutant Q59R / D68H / T190Y can reach 80.57% after a reaction of 2 h.

[0018] (3) The present invention effectively improves the thermal stability of polyphosphate kinase, helps to reduce the cost of industrial production that directly uses ATP, and has good application prospects in industrial production that relies on the ATP circulation system. (IV) Description of the accompanying drawings

[0019] Figure 1 is the half-life determination result of the polyphosphate kinase mutant in Example 5 of the present invention;

[0020] Figure 2 This is the reaction process in which the wild-type polyphosphate kinase and the polyphosphate kinase mutant Q59R / D68H / T190Y are used for ATP synthesis in Example 7 of the present invention. (V) Specific implementation methods

[0021] The present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0022] The molecular biology experiments in the examples, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., were mainly carried out with reference to Molecular Cloning Laboratory Manual (3rd edition, edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002).

[0023] LB liquid medium: 10g / L sodium chloride, 10g / L peptone, 5g / L yeast extract, solvent is water, pH is natural. LB solid medium is LB liquid medium supplemented with 20g / L agar powder.

[0024] Example 1: Construction of recombinant Escherichia coli expressing wild-type polyphosphate kinase

[0025] A gene fragment of polyphosphate kinase (PgPPK) from Phnomibacter ginsenosidimutans (GenBank accession number CP046566) was artificially synthesized. The nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2. This gene fragment was inserted into the T7 promoter of pET-28a(+) to generate the recombinant expression plasmid pET28a-PgPPK.

[0026] The recombinant plasmid pET28a-PgPPK was transformed into the host Escherichia coli BL21 (DE3) by heat shock to obtain the recombinant E. coli BL21 (DE3) / pET28a-PgPPK expressing wild-type polyphosphate kinase.

[0027] Example 2: Screening of polyphosphate kinase mutants

[0028] 1. Selection of mutation sites

[0029] Homology modeling of polyphosphate kinase was performed, saturation mutagenesis was performed using computer-assisted methods, and amino acids 59, 68, and 190 were selected for site-directed mutagenesis.

[0030] 2. Screening of single mutants

[0031] According to the gene sequence of PgPPK in Example 1, primers were designed to mutate sites 59, 68, and 190, as shown in Table 1. Using the plasmid pET28a-PgPPK in Example 1 as a template, PCR amplification was performed using the primers in Table 1 and the reaction system in Table 2.

[0032] Table 1 Saturation mutagenesis primers for amino acid sequences 59, 68, and 190 of polyphosphate kinase

[0033]

[0034] N stands for A / C / G / T, K stands for G / T;

[0035] Table 2 Polyphosphate kinase mutant PCR system

[0036]

[0037] PCR conditions: pre-denaturation at 94°C for 5 min; 30 cycles with the following parameters: denaturation at 94°C for 1 min, annealing at 62°C for 30 s, extension at 72°C for 5 min; final extension for 10 min.

[0038] After the PCR reaction, the PCR product was analyzed as positive by 0.9% agarose gel electrophoresis. 1 μL of Dpn I was added to the PCR reaction mixture and digested at 37°C for 2 hours to remove the template plasmid DNA. The cells were then heat-shocked and transformed into E. coli BL21(DE3) competent cells. After recovery, the cells were plated on LB plates containing 0.1 mM kanamycin sulfate and cultured overnight at 37°C. A single colony was picked and inoculated into LB liquid medium containing a final concentration of 0.1 mM kanamycin sulfate. The cells were cultured at 37°C and 200 rpm for 12 hours. The plasmid was extracted and sequenced to obtain the polyphosphate kinase mutants. Expression was performed using the method in Example 3, and enzyme activity was tested using the method in Example 4. The results are shown in Table 3. The dominant single mutants Q59R, Q59I, D68H, and T190Y were screened.

[0039] Table 3 Enzyme activities of mutants

[0040]

[0041] 3. Combination Mutation

[0042] Using the pET28a-PgPPK-D68H plasmid (amino acid sequence shown in SEQ ID NO. 4, nucleotide sequence shown in SEQ ID NO. 3) as a template, mutation was performed using the primers and reaction system of step 1, and screening was performed using the method of step 2. The results are shown in Table 4, and the dominant mutants D68H / T190Y (amino acid sequence shown in SEQ ID NO. 6, nucleotide sequence shown in SEQ ID NO. 5), Q59I / D68H / T190Y (amino acid sequence shown in SEQ ID NO. 8, nucleotide sequence shown in SEQ ID NO. 7), and Q59R / D68H / T190Y (amino acid sequence shown in SEQ ID NO. 10, nucleotide sequence shown in SEQ ID NO. 9) were screened.

[0043] Table 4 Enzyme activities of combined mutants

[0044]

[0045] Example 3: Expression of polyphosphate kinase mutants

[0046] The recombinant Escherichia coli pET28a-PgPPK / BL21 (DE3) expressing wild-type polyphosphate kinase constructed in Example 1 and the recombinant genetically engineered bacteria of the polyphosphate kinase mutant constructed in Example 2 were inoculated into LB liquid culture medium containing a final concentration of 0.1 mM kanamycin sulfate, and cultured at 37°C for 6-8 hours to obtain seed liquid. The seed liquid was transferred to LB medium containing a final concentration of 0.1 mM kanamycin sulfate at an inoculum concentration of 1% by volume, and cultured at 37°C and 200 rpm until the OD 600When the pH value is 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM and culture at 28°C and 200 rpm for 12-16 hours. Then, centrifuge at 8000 rpm for 10 minutes at 4°C, discard the supernatant, and collect the wet cells.

[0047] The collected wet cells were resuspended in 50 mM Tris-HCl buffer (pH 7.5) at 0.5 g / L and ultrasonically disrupted at 200 W for 10 min, with a working period of 1 s and an interval of 2 s to obtain cell disruption solution.

[0048] Example 4: Determination of enzyme activity of polyphosphate kinase and mutants

[0049] The activity of the wild-type polyphosphate kinase PgPPK expressed by the recombinant E. coli obtained in Example 1 and the polyphosphate kinase mutant obtained in Example 2 were determined.

[0050] Enzyme activity assay reaction system: Prepare 50 mM MgCl2 solution, 25 mM sodium hexametaphosphate solution, and 25 mM ADP solution in 50 mM Tris-HCl buffer (pH 7.5). Mix 200 μL of MgCl2 solution, 200 μL of sodium hexametaphosphate solution, and 200 μL of ADP solution, then add 400 μL of cell disruption buffer. Incubate at 35°C and 1000 rpm for 5 minutes. Terminate the reaction by adding 200 μL of 2 M HCl. Centrifuge at 10,000 rpm for 1 minute. The supernatant is analyzed for ATP content by high-performance liquid chromatography (HPLC).

[0051] Enzyme activity definition: 1 enzyme activity unit (U) is the amount of enzyme required to generate 1 μmole of ATP per minute at 35°C.

[0052] HPLC detection conditions for ATP content: C18 column (4.6×150 mm), mobile phase: 50 mM dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (adjusted to pH 7.0), flow rate: 1 mL / min, injection volume: 10 μL, column temperature: 40°C, detection at a wavelength of 254 nm.

[0053] Example 5: Half-life of polyphosphate kinase at 35°C

[0054] The cell lysate obtained by the method of Example 3 for the enzymes in Table 5 was kept at 35°C, and samples were taken at intervals of 1-12 hours. The residual enzyme activity was determined by the method of Example 4, and the half-life at 35°C was calculated. Figure 1As shown in Table 5, the polyphosphate kinase mutant Q59R / D68H / T190Y has significantly improved stability compared to the wild-type polyphosphate kinase PgPPK, and its half-life is 4.64 times that of the wild-type.

[0055] Table 5 Half-life of polyphosphate kinase

[0056]

[0057] Example 6: Half-life of polyphosphate kinase mutant Q59R / D68H / T190Y at 60°C

[0058] The cell lysate of the polyphosphate kinase mutant Q59R / D68H / T190Y obtained in Example 3 was incubated at 60°C, and samples were taken at intervals of 6-12 h. The residual enzyme activity was determined using the method of Example 4, and its half-life at 60°C was calculated. The half-life of the polyphosphate kinase mutant Q59R / D68H / T190Y at 60°C was 20.16 h.

[0059] Example 7: Application of wild-type polyphosphate kinase and its mutant Q59R / D68H / T190Y in catalyzing ATP synthesis

[0060] 4 mL of cell lysate of the polyphosphate kinase WT and mutant D68H, mutant D68H / T190Y, and mutant Q59R / D68H / T190Y obtained in Example 3 was weighed respectively (the enzyme activity of the cell lysate was 1514.29 U / g based on the amount of wet cells before lysis). A final concentration of 10 mM MgCl2, 5 mM sodium hexametaphosphate, and 5 mM ADP was added, and the volume was made up to 20 mL with 50 mM Tris-HCl buffer (pH 7.5). The mixture was reacted at 35°C and 1000 rpm for 2 h. The ADP content was determined by the method of Example 4, and the substrate conversion rate was calculated. The reaction progress of WT and mutant Q59R / D68H / T190Y is shown in FIG. Figure 2 .

[0061] The results showed that after 2 h of reaction, the ADP conversion rate of wild-type polyphosphate kinase PgPPK was 37.09%, while the ADP conversion rate of mutant Q59R / D68H / T190Y reached 80.57%.

[0062] The present invention is not limited by the above specific description. Various changes can be made to the present invention within the scope outlined by the claims, and these changes are all within the scope of the present invention.

Claims

1. A polyphosphate kinase mutant with improved thermal stability, characterized in that The polyphosphate kinase mutant is a polyphosphate kinase mutant in which the amino acid sequence shown in SEQ ID NO. 2 is mutated into one of the following: (1) aspartic acid at position 68 is mutated into histidine, D68H; (2) aspartic acid at position 68 is mutated into histidine, and threonine at position 190 is mutated into tyrosine, D68H / T190Y; (3) glutamine at position 59 is mutated into isoleucine, aspartic acid at position 68 is mutated into histidine, and threonine at position 190 is mutated into tyrosine, Q59I / D68H / T190Y; (4) glutamine at position 59 is mutated into arginine, aspartic acid at position 68 is mutated into histidine, and threonine at position 190 is mutated into tyrosine, Q59R / D68H / T190Y.

2. A recombinant genetically engineered bacterium containing the gene encoding the polyphosphate kinase mutant according to claim 1.

3. Use of the polyphosphate kinase mutant according to claim 1 in ATP synthesis.

4. The use according to claim 3, characterized in that The application method comprises the following steps: using wet bacteria obtained by induction culture of recombinant genetically engineered bacteria expressing a polyphosphate kinase mutant or cell disruption liquid after ultrasonic disruption of wet bacteria as a catalyst, using ADP as a substrate, adding MgCl2 and sodium hexametaphosphate, and using a pH 6-8 buffer as a reaction medium to form a reaction system, and carrying out a complete reaction at 30-60°C and 800-1000 rpm to obtain ATP.

5. The use according to claim 4, characterized in that In the reaction system, the catalyst dosage is 1-5 g / L based on the weight of wet bacteria, the volume concentration of the cell disruption solution is 10-30%, the enzyme activity of the cell disruption solution is 1415.05-2531.28 U / g based on the weight of wet bacteria before disruption; the substrate is added at a concentration of 1-10 mM; the MgCl2 is added at a concentration of 5-15 mM; and the sodium hexametaphosphate is added at a concentration of 1-10 mM.

6. The use according to claim 4, characterized in that The reaction medium is 50 mM Tris-HCl buffer, pH 7.

5.

7. The use according to claim 4, characterized in that The catalyst was prepared as follows: a recombinant genetically engineered bacterium expressing a polyphosphate kinase mutant was inoculated into a LB liquid culture medium containing a final concentration of 0.1 mM kanamycin sulfate, and cultured at 37°C for 6-8 h to obtain a seed solution; the seed solution was transferred to an LB medium containing a final concentration of 0.1 mM kanamycin sulfate at an inoculum concentration of 1% by volume, and cultured at 37°C and 200 rpm until the OD 600 When the RI is 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.1 mM and culture at 28°C and 200 rpm for 12-16 h; then centrifuge at 8000 r / min for 10 min at 4°C, discard the supernatant, and collect the wet cells; The wet cells were resuspended in 50 mM Tris-HCl buffer (pH 7.5) and ultrasonically disrupted at 200 W for 10 min, with a 1 s interval and a 2 s interval, to obtain a cell disruption solution.

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