A polyphosphorylase with improved thermostability

Abstract

The application provides a polyphosphate kinase (PPK) mutant and a preparation method and application thereof, and belongs to the technical field of enzyme engineering. The polyphosphate kinase mutant is mutated on the amino acid sequence shown in SEQ ID NO. 1. The application also provides an engineering bacterium for expressing the polyphosphate kinase mutant and a construction method of the polyphosphate kinase mutant. The polyphosphate kinase mutant provided by the application has improved thermal stability compared with the wild type, and the enzyme activity can be maintained for more than 1 h under the condition of 37 DEG C, and under the same heat treatment condition, the enzyme activity is 1.5 times that of the wild type, and is more suitable for industrialization.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, and particularly relates to a mutant of polyphosphate kinase, its preparation method and application. Background Technology

[0002] Adenosine triphosphate (ATP) is a high-energy phosphate compound that serves as a phosphate group donor in the industrial synthesis of many important phosphate-containing compounds. However, in biosynthesis, using ATP in stoichiometric amounts (or even several times greater) than the substrate as a raw material would significantly increase production costs, which is the main bottleneck limiting the industrial application of ATP as a phosphate group donor.

[0003] Polyphosphate kinase (PPK) can use polyphosphate (Poly P) as a phosphate donor to carry out ATP synthesis and regeneration reactions. Compared with other ATP regeneration systems, such as acetate kinase-acetyl phosphate, pyruvate kinase-phosphoenolpyruvate, and creatine kinase-phosphocreatine systems, the required substrates are inexpensive and stable, making them more suitable for industrial scale-up. Choosing to use a PPK-mediated ATP regeneration system in a cascade with the main product production enzyme to jointly carry out the biocatalytic reaction can significantly reduce raw material costs (Cheng et al., 2023).

[0004] The industrialization process of PPK needs to consider not only the cost of biocatalysts and products, but also other limiting factors affecting its catalytic process (such as mass transfer limitations). Currently, the industrialization of PPK needs to adapt to more conditions, such as improving enzyme activity; enhancing enzyme thermostability; improving acceptance of Poly P with different chain lengths; and optimizing the optimal reaction conditions after the enzyme is coupled with PPK. Summary of the Invention

[0005] To address the aforementioned issues, this invention first employs enzyme engineering techniques to directionally enhance the thermal stability and enzyme activity of PauPPK2.

[0006] To date, PPK has been isolated and purified from hundreds of bacterial species, but research on PPK in eukaryotes is limited. Studies have found that the PPK of *D. discoideum* has 1050 amino acid residues, while that of *E. coli* has only 688. Structural analysis of the *E. coli* PPK shows that it is a dimer with four domains, and its active site is located within an ATP-binding pocket (ABP) channel, which regulates the migration of poly-P (Brown and Kornberg, 2008). Kumble et al. showed that during poly-P formation in *E. coli*, the terminal phosphate group of ATP is first transferred to histidine residues H441 and H460 of PPK to form a phosphatase mediator. Subsequently, under the catalysis of PPK, the chain length of poly-P increases. Site-directed mutagenesis of H441 and H460 revealed that the mutant protein lacked the ability to synthesize poly-P. Analysis of histidine tag proteins in *Streptomyces lividans* revealed that its PPK has similar enzymatic properties to that of *Escherichia coli*. The difference is that the phosphatase mediator in *S. lividans* may be formed by the transfer of the terminal phosphate group of ATP to conserved histidine residues H517 and H536 (Yuan et al., 2015).

[0007] PPK2 subtypes are divided into three types: type I, type II, and type III. Type I and type II tend to catalyze the phosphorylation of ADP to ATP and AMP to ADP, respectively. Type III is active for both ADP and AMP substrates and can simultaneously achieve phosphorylation from AMP to ADP and from ADP to ATP.

[0008] Several bacterial PPK2 crystal structures from different sources have been published, including representative ones: Class I: *Sinorhizobium meliloti* and *Franciscellella tularensis*; Class II: *Pseudomonas aeruginosa*; Class III: *Arthrobacter aureus*, *Meiothermus ruber*, *Cytophagahutchinsonii*, and *Deinococcus radiodurans*. PPK2 sequences from different species show low sequence similarity, but all possess an α / β / α structure, and distinct Walker A (GXXXXGK) and Walker B (DR) modules can be observed (Cheng et al., 2023).

[0009] In this invention, PPK2 derived from Paenarthrobacter aurescens is used as the modification target. It belongs to class III PPK2, and the thermal stability of the enzyme is improved through enzyme engineering technology.

[0010] Based on this, the first objective of the present invention is to provide a polyphosphate kinase (PPK2) mutant, which is mutated on the amino acid sequence shown in SEQ ID NO.1, wherein the mutation includes the substitution of amino acid residues at positions 39, 263, 22, 211, 191 and / or 195 on the corresponding sequence.

[0011] Preferably, the substitution of the amino acid residue at position 39 is a mutation of valine at position 39 to aspartic acid, abbreviated as V39D.

[0012] Preferably, the substitution of the amino acid residue at position 263 is a mutation of glycine at position 263 to glutamic acid, abbreviated as G263E.

[0013] Preferably, the substitution of the amino acid residue at position 22 is a mutation of glycine at position 22 into lysine or arginine, abbreviated as G22K or G22R.

[0014] Preferably, the substitution of the amino acid residue at position 211 is a mutation of aspartic acid at position 211 to lysine, abbreviated as D211K.

[0015] Preferably, the substitution of the amino acid residue at position 191 is a mutation of lysine at position 191 to glutamic acid, abbreviated as K191E.

[0016] Preferably, the substitution of the amino acid residue at position 195 is a mutation of isoleucine at position 195 to leucine, abbreviated as I195L.

[0017] Preferably, the amino acid residues are substituted at any two or more sites corresponding to the sequence V39D, G263E, G22K or G22R, D211K, K191E, I195L.

[0018] Preferably, the mutant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the polypeptide of SEQ ID NO:2, and wherein the mutant has at least improved properties compared with the wild-type polyphosphate kinase.

[0019] Regarding amino acid sequence homology, considering that when one or more amino acids are replaced by structurally related amino acids, the performance of the mutant does not significantly improve or enhance compared to the wild type, such as when the amino acid residue is replaced by an amino acid residue with a similar side chain, such mutants fall within the scope of protection of this invention.

[0020] Regarding the improved properties relative to the wild type, the improved properties of the enzyme mutant of the present invention include one or more of the following: increased expression efficiency, increased catalytic efficiency, increased catalytic rate, increased chemical stability, increased thermal stability, increased pH stability, increased specific activity, increased stability under storage conditions, increased substrate binding activity, increased substrate catalytic activity, broadened substrate spectrum, increased substrate stability, and increased surface properties.

[0021] Furthermore, the improved properties include an increase in the thermal stability of the mutant by at least 1°C compared to the wild-type polyphosphokinase; more preferably, an increase of 2°C; and even more preferably, an increase of 6°C.

[0022] Furthermore, the improved properties include the ability of the mutant enzyme to maintain its activity at 37°C for at least 1 hour.

[0023] A second objective of this invention is to provide a nucleic acid molecule comprising a nucleotide sequence encoding the aforementioned polyphosphate kinase mutant or its complementary sequence.

[0024] Regarding nucleotide sequence homology, on the one hand, due to the degeneracy of codons, the nucleotide sequence encoding a protein is not unique. Therefore, any nucleotide sequence that can encode the amino acid sequence of the mutant is within the scope of protection of this invention.

[0025] The encoding includes proteins produced by transcription of DNA molecules to form an RNA product, followed by translation; or proteins produced by transcription of DNA molecules to provide an RNA product, processing to provide a processed RNA product, followed by translation.

[0026] A third objective of this invention is to provide a recombinant expression vector containing the aforementioned nucleic acid molecules.

[0027] Vectors include any nucleic acid molecule derived from any source and capable of genome integration or autonomous replication (e.g., plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecules), comprising one or more nucleic acid molecules that are operatively linked. Vectors may include, for example, one or more selectable markers, one or more origins of replication (e.g., prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the host cell genome.

[0028] Preferably, the recombinant expression vector is also linked to a NusA tag, which can increase the expression level of the target protein in the supernatant.

[0029] Preferably, the recombinant expression vector is also linked to a 6His tag to purify the target protein.

[0030] A fourth objective of this invention is to provide a recombinant cell containing the aforementioned expression vector, or having an exogenous nucleic acid molecule integrated into its chromosome, or having an exogenous nucleic acid molecule integrated into its chromosome and the nucleotide sequence of the aforementioned lysis-promoting tag. In this invention, *E. coli* is selected for recombinant expression of the enzyme mutant.

[0031] The fourth objective of this invention is to provide a method for preparing the aforementioned polyphosphate kinase mutant, comprising the following preparation steps:

[0032] i) The aforementioned nucleic acid molecules are cloned into plasmids to construct a recombinant expression vector;

[0033] ii) The recombinant expression vector obtained in i) is then transformed into recombinant cells for induced expression. The cell pellet is collected, the cells are resuspended and then broken to obtain a lysed bacterial solution containing the polyphosphate kinase mutant, or the lysed bacterial solution is further purified to obtain a polyphosphate kinase mutant enzyme solution.

[0034] A fifth objective of this invention is to provide an enzyme preparation comprising the aforementioned polyphosphate kinase mutant.

[0035] The enzyme preparations are products prepared from the polyphosphate kinase mutants or complexes, and can be used to enhance or accelerate biochemical reactions, especially dehydrogenation reactions. The dosage forms include solid enzyme preparations and liquid enzyme preparations.

[0036] In one embodiment, the enzyme preparation may contain only the glycosidase described in this invention. In another embodiment, it may also contain enzymes from other sources, selectively enzymatically hydrolyzing the polyphosphate by using an enzyme combination that includes, in addition to polyphosphate kinase, one or more enzymes that jointly regulate the synthesis and catabolism of poly-P, such as polyphosphate esterase (PPX), polyphosphate endonuclease (PPN), polyphosphate glucokinase (PPGK), polyP-AMP-phosphotransferase (PAP), polyphosphate fructose kinase, and polyphosphate mannokinase.

[0037] The sixth objective of this invention is to provide the aforementioned polyphosphokinase mutant, or the aforementioned nucleic acid molecule, or the aforementioned recombinant expression vector, or the aforementioned recombinant cell, or the aforementioned lysed bacterial solution containing the polyphosphokinase mutant, or the polyphosphokinase mutant enzyme solution obtained after further purification of the lysed bacterial solution, or the aforementioned enzyme preparation, or the aforementioned enzyme preparation in catalyzing substrate phosphorylation.

[0038] More preferably, it is used in the catalytic phosphorylation of AMP or ADP.

[0039] In addition, the polyphosphate kinase mutant of the present invention can be used to obtain highly active immobilized enzymes through various immobilization methods, and continuously carry out a large number of hydrolysis reactions. The immobilization methods utilize known methods such as carrier binding, cross-linking, gel encapsulation, microencapsulation, etc. to prepare immobilized enzymes.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The present invention provides several mutants with improved thermal stability. The preferred mutant has a TM value of 52-54°C, which is 6°C higher than that of wild-type PPK2. After heat treatment at 37°C for 1 hour, the relative enzyme activity of wild-type PPK2 decreases by 30%, while the enzyme activity of the mutant is not significantly lost. That is, under the same heat treatment conditions, the efficiency of catalytic substrate phosphorylation is improved compared with wild-type PPK2. The preferred mutant enzyme activity is more than 1.5 times that of wild-type, which shows that the enzyme mutant of the present invention is more suitable for industrialization. Attached Figure Description

[0042] Figure 1 The effect of different promotion tags on the soluble expression of PauPPK2 in Escherichia coli was analyzed by SDS-PAGE.

[0043] Figure 2 Protein purification for SDS-PAGE analysis of different polyphosphate kinase mutant PM1.

[0044] Figure 3 Protein purification for SDS-PAGE analysis of different polyphosphate kinase mutants PM2.

[0045] Figure 4 Protein purification for SDS-PAGE analysis of different polyphosphate kinase mutant PM8.

[0046] Figure 5 Protein purification for SDS-PAGE analysis of different polyphosphate kinase mutant PM9.

[0047] Figure 6 Protein purification for SDS-PAGE analysis of different polyphosphate kinase mutants PM10.

[0048] Figure 7 Protein purification for SDS-PAGE analysis of different polyphosphate kinase mutant PM13.

[0049] Figure 8 Protein purification for SDS-PAGE analysis of different polyphosphate kinase mutant PM14.

[0050] Figure 9 The Tm value of polyphosphate kinase mutant PM1 was detected by qPCR.

[0051] Figure 10 The Tm value of polyphosphate kinase mutant PM2 was detected by qPCR.

[0052] Figure 11 The Tm value of polyphosphate kinase mutant PM8 was detected by qPCR.

[0053] Figure 12 The Tm value of polyphosphate kinase mutant PM9 was detected by qPCR.

[0054] Figure 13 The Tm value of polyphosphate kinase mutant PM10 was detected by qPCR.

[0055] Figure 14 The Tm value of polyphosphate kinase mutant PM13 was detected by qPCR.

[0056] Figure 15 The Tm value of polyphosphate kinase mutant PM14 was detected by qPCR. Detailed Implementation

[0057] This invention does not specifically limit the preparation method of the recombinant vector; any conventional recombinant vector preparation method in the art can be used. In the specific implementation of this invention, it is preferred to recombine the gene into the initial vector via homologous recombination. In this invention, the gene can be synthesized by a biotechnology company. This invention does not specifically limit the separation and purification method; any conventional protein separation and purification method in the art can be used; preferred technical solutions are described in the examples.

[0058] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1 Construction of Recombinant Plasmid

[0060] The synthesized pUC-GW-PPK2 was used as a template for PCR amplification to obtain the PPK2 target gene fragment. The target fragment was then ligated into expression vectors fused with different solubilization tags via enzyme digestion and ligation.

[0061] Add 10 μL of the constructed plasmid to DH5α, incubate on ice for 30 min, heat shock at 42°C for 90 s, incubate on ice for another 5 min, add 500 μL of antibiotic-free LB medium, and incubate on a shaker at 37°C for 45-60 min. After incubation, centrifuge at 6000 rpm for 3 min, resuspend the bacterial cells, and plate them on a plate containing KANA resistance, and incubate overnight.

[0062] Two single colonies were picked from each plate for colony PCR identification. The PCR system and procedure are shown in Tables 1 and 2.

[0063] Table 1. PCR reaction system for colony identification

[0064]

[0065] Table 2. PCR Procedure for Colony Identification

[0066]

[0067] The correctly identified colonies were inoculated into 4 mL of LB liquid medium containing kanamycin and cultured overnight at 37°C and 220 rpm. Plasmids were extracted using the Tiangen Rapid Plasmid Mini-Prep Kit, and plasmid concentration was measured using NanoDrop One. The plasmids were then transformed into the expression strain BL21. Three single colonies of BL21 were picked and cultured until OD (Organic Demand) was reached. 600 The concentration was 0.6-0.8. 0.1 mM IPTG was added and the mixture was induced overnight at 16 °C and 220 rpm in a shaker.

[0068] Centrifuge the overnight induced strain at 12,000 rpm for 5 min, discard the liquid culture medium, resuspend the cells in 1 ml Lysis Buffer, transfer the resuspended cells to a 2 mL centrifuge tube, and disrupt the cells using an ultrasonic cell disruptor.

[0069] SDS-PAGE analysis showed that fusing the NusA tag increased the expression level of the target protein. Figure 1 ).

[0070] Example 2 Construction of PPK2 mutant

[0071] The inventors studied the interaction between enzymes and substrates and mutated at specific sites to enhance enzyme stability. The mutants constructed are shown in Table 5.

[0072] Table 3 PPK2 mutants

[0073]

[0074] The mutant was amplified by PCR on the constructed 6His tag plasmid. The PCR system and procedure are shown in Tables 4 and 5. After PCR, the residual template plasmid in the PCR product was digested with DpnI in a 37°C water bath for 30 minutes. The digestion system is shown in Table 6.

[0075] Table 4 Amino acid mutation PCR system

[0076]

[0077] Table 5. Amino acid mutation PCR program

[0078]

[0079] Table 6. Dpn I enzyme digestion system

[0080]

[0081] Following Example 1, the PCR product was transformed into the cloning strain DH5α. Two single colonies were randomly selected from the plate and inoculated into 400 uL LB liquid medium. The culture was then incubated in a shaker at 37°C and 220 rpm for 6 h. The bacterial culture was then sent to Shenzhen Qingke Biotechnology Co., Ltd. for sequencing verification.

[0082] Single colonies with correct sequencing results were inoculated into 4 mL of LB liquid medium (Kanamic final concentration 50 μg / mL) and incubated overnight at 37°C and 220 rpm. Plasmids were extracted using the Tiangen Rapid Plasmid Mini-Prep Kit, and plasmid concentration was measured using NanoDrop One. Following Example 1, the plasmids were transformed into expression strain BL21 to induce expression. The cells were then collected by centrifugation at 12000 rpm for 10 min at 4°C. The cells were resuspended in Lysis Buffer. The cells were homogenized using an autoclave. After homogenization, the cells were centrifuged at 12000 rpm for 30 min, and the supernatant was collected. Protein was purified by nickel column chromatography and then dialyzed. SDS-PAGE analysis was performed on the protein expression levels in the whole cells and supernatant. Figures 2-8 ).

[0083] Example 3: Establishment of the enzyme activity system

[0084] The purified enzyme solution after dialysis was subjected to enzyme activity testing. The enzyme activity reaction system is shown in Table 7, and the reaction was carried out in a 40℃ water bath for 30 min. After the reaction was completed, the ATP production content was detected using the Beyotime ATP assay kit (S0026). Since the ATP production content detectable by this assay kit needs to be within a certain standard curve range, wild-type PPK2 was diluted to a concentration of 0.1 mg / ml. It was found that at an enzyme concentration of 0.1 mg / ml, the production of ATP showed a linear relationship within a certain range when different amounts of enzyme were added. Furthermore, 0.89 mg of enzyme was within the range of this linear relationship. Based on this, the same amount of enzyme was added to both wild-type and mutant strains for enzyme activity testing. The experimental results showed that ATP was produced, and the enzyme activity of most mutant strains was higher than that of the wild-type.

[0085] Table 7 Enzyme activity reaction system of wild-type PPK2 and its mutants

[0086]

[0087] Example 4: Tm value test of wild-type PPK2 and mutant

[0088] The dialyzed enzyme solution was concentrated to a concentration of 1 mg / ml, and the Tm value was detected by qPCR. The PCR reaction system and procedure are shown in Tables 8 and 9.

[0089] Table 8 TM Value Detection System

[0090]

[0091] Table 9 qPCR reaction procedure

[0092]

[0093] TM value detection experiment results ( Figures 8-14 As shown in Table 10, the Tm values ​​of mutants PM1, PM2, PM8, PM9, PM10, PM13, and PM14 were higher than those of wild-type PPK2.

[0094] Table 10 Results of Tm value test for mutants

[0095]

[0096] Example 5: Thermal stability test of wild-type PPK2 and its mutants

[0097] Wild-type PPK2 pure enzyme solution was placed in a 37°C water bath. Samples of protein were collected at 0, 30, 45, 60, 72, and 90 minutes. The heat-treated protein was added to the enzyme activity test reaction system and reacted at 37°C for 30 minutes. After the reaction, the ATP content was measured using an ATP assay kit. The residual enzyme activity of wild-type PPK2 gradually decreased with increasing water bath time at 37°C. Wild-type PPK2 and mutant pure enzyme solutions were heat-treated at 37°C for 1 hour, and their enzyme activities were measured simultaneously with untreated pure enzyme solutions. The results (Table 11) showed that the enzyme activity of wild-type PPK2 decreased by approximately 30% after 1 hour of treatment, while the enzyme activity of the mutant of this invention showed no significant loss. This indicates that the mutant of this invention can adapt to a wider range of reaction conditions and is suitable for industrial applications.

[0098] Table 11 Residual enzyme activity of wild-type PPK2 and mutants after heat treatment

[0099]

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polyphosphate kinase mutant, characterized in that it is Mutations are made on the amino acid sequence shown in SEQ ID NO.1, wherein the mutations include substitution of amino acid residues at any one or more of the following positions: position 39, position 263, position 22, position 211, position 191, and position 195.

2. The polyphosphate kinase mutant according to claim 1, characterized in that it is Mutations are made on the amino acid sequence shown in SEQ ID NO.1, including substitution of amino acid residues at any one or more sites corresponding to the sequence, such as V39D, G263E, G22K or G22R, D211K, K191E, I195L.

3. The polyphosphate kinase mutant according to claim 1, characterized in that, Furthermore, the mutant exhibits at least 1°C greater thermal stability compared to the wild-type polyphosphokinase with the amino acid sequence shown in SEQ ID NO.

1.

4. The polyphosphate kinase mutant according to claim 1, characterized in that, Furthermore, the enzyme activity of the mutant can be maintained at 37°C for at least 1 hour.

5. The polyphosphate kinase mutant according to claim 1, characterized in that, The mutant has at least 99% but less than 100% sequence identity with any amino acid sequence in SEQ ID NO:

1.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleotide sequence or its complementary sequence encoding the polyphosphokinase mutant as described in any one of claims 1-5.

7. A recombinant expression vector, characterized in that, It contains the nucleic acid molecule as described in claim 6.

8. A recombinant cell, characterized in that, The recombinant cells contain the recombinant expression vector of claim 7, or have an exogenous nucleic acid molecule of claim 6 integrated into their chromosomes.

9. The method for preparing the polyphosphokinase mutant according to any one of claims 1-5, characterized in that, The preparation steps include the following: i) Cloning the nucleic acid molecule described in claim 6 into a plasmid to construct a recombinant expression vector, wherein the recombinant expression vector is linked with a purification tag and / or a lysis-promoting tag; ii) The recombinant expression vector obtained in i) is then transformed into recombinant cells for induced expression. The cell pellet is collected, the cells are resuspended and then broken to obtain a lysed bacterial solution containing the polyphosphate kinase mutant, or the lysed bacterial solution is further purified to obtain a polyphosphate kinase mutant enzyme solution.

10. An enzyme preparation, characterized in that, It includes the mutant as described in any one of claims 1-5.

11. The polyphosphate kinase mutant of any one of claims 1-5, or the nucleic acid molecule of claim 6, or the recombinant expression vector of claim 7, or the recombinant cell of claim 8, or the lysate containing the polyphosphate kinase mutant of claim 9, or the polyphosphate kinase mutant enzyme solution obtained by further purifying the lysate, or the enzyme preparation of claim 10 in catalyzing AMP or ADP phosphorylation.

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