Polyphosphokinase mutant with improved thermal stability and application

Abstract

The invention discloses a polyphosphate kinase mutant with improved thermal stability and application. The polyphosphate kinase mutant is obtained by performing single mutation or multiple mutation on 59th, 68th and 190th amino acids of an amino acid sequence shown in SEQ ID NO.2. The invention further discloses a preparation method of the polyphosphate kinase mutant. The thermal stability of the polyphosphate kinase mutant is effectively improved, the longer catalytic activity can be kept, the half-life period is prolonged by 2-5 times compared with that of a wild type, and the half-life period at 35 DEG C can reach 53 h or above. When the polyphosphate kinase mutant disclosed by the invention is used for catalyzing ATP (adenosine triphosphate) synthesis, the ADP (adenosine diphosphate) conversion rate of the polyphosphate kinase mutant Q59R / D68H / T190Y can reach 80.57% after reaction for 2 hours. According to the invention, the thermal stability of the polyphosphate kinase is effectively improved, the industrial production cost of directly using ATP is reduced, and the method has a good application prospect in industrial production depending on an ATP circulating system.

Description

(I) Technical field

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

[0002] Adenosine triphosphate (ATP) is a high-energy phosphate compound and the most direct energy source in organisms. It participates in the synthesis of many important life substances as a precursor. Due to the complexity of ATP synthesis, poor stability and energy conversion efficiency, the exogenous addition of ATP as a raw material will increase production costs, thus 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 with other systems, polyphosphate kinases (PPKs)-mediated ATP synthesis and regeneration using polyphosphate as a phosphate donor has the advantages of cheap and stable substrates and efficient processes, which is more conducive to industrial applications.

[0003] Most of the 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 enzyme 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. (III) Summary of the invention

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

[0005] The technical solution adopted by 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 mutation or multiple mutation.

[0007] Furthermore, the polyphosphate kinase mutant is preferably one in which the amino acid sequence of SEQ ID NO.2 is mutated to one of the following: (1) the glutamine at position 59 is mutated to arginine or isoleucine, Q59R or Q59I; (2) the aspartic acid at position 68 is mutated to histidine, D68H, the amino acid sequence is as shown in SEQ ID NO.4, and the nucleotide sequence is as shown in SEQ ID NO.3; (3) the threonine at position 190 is mutated to tyrosine, T190Y; (4) the aspartic acid at position 68 is mutated to histidine, and the threonine at position 190 is mutated to tyrosine, D68H / T190Y, the amino acid sequence is as shown in SEQ ID NO.6, and the nucleotide sequence is as shown in SEQ ID NO.5; (5) the glutamine at position 59 is mutated to isoleucine, the aspartic acid at position 68 is mutated to histidine, and the threonine at position 190 is mutated to tyrosine, Q59I / D68H / T190Y, the amino acid sequence is as 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 coding gene of the polyphosphate kinase mutant, a recombinant vector comprising the coding gene, and a recombinant genetic engineering bacterium comprising the recombinant vector. Preferably, the original vector of the recombinant vector is pET-28a. The host cell for constructing the recombinant genetic engineering bacterium can be various conventional host cells in the art, and preferably, the host cell is Escherichia coli E.coli BL21.

[0009] The present invention also provides an application of the polyphosphate kinase mutant in the synthesis of adenosine triphosphate (ATP), and the application method is as follows: the application uses wet bacteria obtained by induction culture of recombinant genetic engineering bacteria expressing the polyphosphate kinase mutant or cell disruption liquid after ultrasonic disruption of wet bacteria as a catalyst, ADP as a substrate, MgCl2 and sodium hexametaphosphate are added, and a buffer solution with a pH of 6-8 is used as a reaction medium to form a reaction system, and the reaction is completed under the conditions of 30-60°C and 800-1000rpm (preferably 35°C and 1000rpm) to obtain ATP.

[0010] Furthermore, in the reaction system, the amount of catalyst is 1-5 g / L (preferably 1.25 g / L) based on the weight of wet bacteria, the volume concentration of cell disruption solution 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 substrate is added at a concentration of 1-10 mM (preferably 5 mM); the MgCl2 is added at a concentration of 5-15 mM (preferably 10 mM); and the sodium hexametaphosphate is added at a concentration of 1-10 mM (preferably 5 mM).

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

[0012] Further, the catalyst is prepared as follows: the recombinant genetic engineering bacteria expressing the polyphosphate kinase mutant are inoculated into a LB liquid culture medium test tube containing a final concentration of 0.1 mM kanamycin sulfate, and the seed solution is obtained after culturing at 37°C for 6-8 hours; the seed solution is transferred to an LB culture medium containing a final concentration of 0.1 mM kanamycin sulfate at an inoculum concentration of 1% by volume, and the culture is carried out 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) with 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;

[0013] The wet cells (preferably at 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 a working period of 1 s and an interval of 2 s to obtain a cell disruption solution.

[0014] The polyphosphate kinase mutant of the present invention can be used in the form of whole cells of engineered bacteria, in the form of crude enzymes without purification, or in the form of partially purified or completely purified enzymes. The nitrilase mutant of the present invention can also be made into a biocatalyst 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. The half-life is increased by 2-5 times compared with the wild type, and the half-life can reach more than 53 hours at 35°C.

[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 directly using ATP, and has good application prospects in industrial production that relies on the ATP circulation system. (IV) Description of the drawings

[0019] Figure 1 is the half-life measurement 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 in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0022] The molecular biology experiments in the embodiments, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., are mainly carried out with reference to "Molecular Cloning Experiment Guide" (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 with 20g / L agar powder added.

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

[0025] The gene fragment of polyphosphate kinase (GenBank accession number CP046566) PgPPK from Phnomibacter ginsenosidimutans 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. The gene fragment was inserted into the T7 promoter of pET-28a (+) to obtain the recombinant expression plasmid pET28a-PgPPK.

[0026] The recombinant plasmid pET28a-PgPPK was introduced into the host Escherichia coli BL21 (DE3) by heat shock transformation to obtain the recombinant Escherichia coli BL21 (DE3) / pET28a-PgPPK expressing the 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 with computer assistance, 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, the primers in Table 1 and the reaction system in Table 2 were used for PCR amplification.

[0032] Table 1 Saturation mutation 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 according to 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 was completed, the PCR product was analyzed as positive by 0.9% agarose gel electrophoresis, and 1 μL Dpn I was added to the PCR reaction solution, and the template plasmid DNA was removed by enzyme digestion at 37°C for 2 hours. Then, the cells were transformed into E. coli BL21 (DE3) competent cells by heat shock, and after recovery, they were spread on LB plates containing 0.1 mM kanamycin sulfate, cultured at 37°C overnight, and single colonies were picked and inoculated in LB liquid culture medium containing a final concentration of 0.1 mM kanamycin sulfate, cultured at 37°C and 200 rpm for 12 hours, and the plasmid was extracted for sequencing to obtain polyphosphate kinase mutants. The expression method of Example 3 was used, and the enzyme activity was tested by the method of Example 4. The results are shown in Table 3, and 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 pET28a-PgPPK-D68H plasmid (amino acid sequence as shown in SEQ ID NO.4, nucleotide sequence as shown in SEQ ID NO.3) as a template, the primers and reaction system of step 1 were used for mutation, and the method of step 2 was used for screening. The results are shown in Table 4, and the dominant mutants D68H / T190Y (amino acid sequence as shown in SEQ ID NO.6, nucleotide sequence as shown in SEQ ID NO.5), Q59I / D68H / T190Y (amino acid sequence as shown in SEQ ID NO.8, nucleotide sequence as shown in SEQ ID NO.7), and Q59R / D68H / T190Y (amino acid sequence as shown in SEQ ID NO.10, nucleotide sequence as shown in SEQ ID NO.9) were screened.

[0043] Table 4 Enzyme activities of combined mutants

[0044]

[0045] Example 3: Expression of polyphosphate kinase and mutants

[0046] The recombinant Escherichia coli pET28a-PgPPK / BL21 (DE3) expressing wild-type polyphosphate kinase constructed in Example 1 and the recombinant genetic engineering bacteria of the polyphosphate kinase mutant constructed in Example 2 were inoculated into LB liquid culture medium test tubes 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 was 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) with 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.

[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 a cell disruption solution.

[0048] Example 4: Enzyme activity assay of polyphosphate kinase and mutants

[0049] The wild-type polyphosphate kinase PgPPK expressed by the recombinant Escherichia coli obtained in Example 1 and the polyphosphate kinase mutant obtained in Example 2 were subjected to activity assay.

[0050] Enzyme activity assay reaction system: 50mM MgCl2 solution, 25mM sodium hexametaphosphate solution, and 25mM ADP solution were prepared with 50mM Tris-HCl buffer (pH 7.5). 200μL MgCl2 solution, 200μL sodium hexametaphosphate solution, and 200μL ADP solution were mixed, and then 400μL cell disruption solution was added. The reaction was shaken at 35°C and 1000rpm for 5min, and 200μL 2M HCl was added to terminate the reaction. The reaction was centrifuged at 10000r / min for 1min, and the supernatant was tested for ATP content by high performance liquid chromatography (HPLC).

[0051] Enzyme activity definition: The amount of enzyme required to generate 1 micromole of ATP per minute at 35°C is defined as 1 enzyme activity unit U.

[0052] ATP content HPLC detection conditions: C18 chromatographic 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 stability of the polyphosphate kinase mutant Q59R / D68H / T190Y was significantly improved compared with the wild-type polyphosphate kinase PgPPK, and its half-life was 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 kept warm at 60°C, and samples were taken at intervals of 6-12 hours. The residual enzyme activity was determined by the method of Example 4, and the half-life at 60°C was calculated. The half-life of the polyphosphate kinase mutant Q59R / D68H / T190Y at 60°C was 20.16 hours.

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

[0060] Weigh 4 mL of cell lysis solution of polyphosphate kinase WT and mutant D68H, mutant D68H / T190Y, and mutant Q59R / D68H / T190Y obtained in Example 3 (the enzyme activity of the cell lysis solution is 1514.29 U / g based on the amount of wet bacteria before lysis), add a final concentration of 10 mM MgCl2, 5 mM sodium hexametaphosphate, and 5 mM ADP, and make up to 20 mL with 50 mM Tris-HCl buffer (pH 7.5), react at 35°C and 1000 rpm for 2 h, determine the ADP content by the method of Example 4, and calculate the substrate conversion rate. The reaction progress of WT and mutant Q59R / D68H / T190Y is shown in Figure 2. Figure 2 .

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

[0062] The present invention is not limited by the above specific textual description. The present invention can make various changes within the scope summarized 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 obtained by subjecting the 59th, 68th and 190th amino acids of the amino acid sequence shown in SEQ ID NO.2 to single mutation or multiple mutation.

2. The polyphosphate kinase mutant according to claim 1, characterized in that The polyphosphate kinase mutant is SEQ ID The amino acid sequence shown in 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; (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; (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; (6) 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.

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

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

5. The use according to claim 4, characterized in that The application method is as follows: the application uses wet bacteria obtained by induction culture of recombinant genetic engineering bacteria expressing polyphosphate kinase mutants or cell fragmentation liquid after ultrasonic fragmentation of wet bacteria as a catalyst, ADP as a substrate, MgCl2 and sodium hexametaphosphate are added, a pH 6-8 buffer solution is used as a reaction medium to form a reaction system, and the reaction is completed under the conditions of 30-60°C and 800-1000rpm to obtain ATP.

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

7. The use according to claim 5, characterized in that The reaction medium is 50 mM Tris-HCl buffer, pH 7.

5.

8. The use according to claim 5, characterized in that The catalyst is prepared as follows: a recombinant genetic engineering bacterium expressing a polyphosphate kinase mutant is inoculated into a LB liquid culture medium test tube containing a final concentration of 0.1 mM kanamycin sulfate, and the seed solution is obtained after culturing at 37°C for 6-8 hours; the seed solution is transferred to an LB culture medium containing a final concentration of 0.1 mM kanamycin sulfate at an inoculum volume concentration of 1%, and the seed solution is 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 at 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 pH 7.5, 50 mM Tris-HCl buffer and disrupted by ultrasound at 200 W for 10 min, with a working period of 1 s and an interval of 2 s to obtain a cell disruption solution.

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