A halotolerant adenylate kinase mutant and application thereof

Abstract

The application discloses a salt-tolerant adenylate kinase mutant and application thereof. Through modification on an amino acid sequence of a wild-type adenylate kinase (adenylate kinase, EC 2.7.4.3) from Bacillus subtilis (strain 168), three salt-tolerant adenylate kinase mutants are obtained; the mutant adenylate kinase has higher salt tolerance compared with the wild-type adenylate kinase, and can tolerate a salt concentration of 300-2000 mmol / L in an adenosine diphosphate reaction system.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a salt-loving adenosine monophosphate kinase mutant and its applications. Background Technology

[0002] In the preparation of adenosine diphosphate (ADP), the substrate acetyl phosphate is continuously added during the batch feeding process to produce acetic acid, followed by the addition of alkali to maintain pH stability. As the acid and alkali are continuously neutralized, the salt concentration in the reaction solution gradually increases, and the inhibitory effect on adenylate kinase (ADK) activity (EC: 2.7.4.3) also increases with increasing salt concentration. When the salt concentration exceeds 300 mM, ADK activity is significantly inhibited; when the salt concentration reaches 1200 mM, ADK activity decreases by more than 50%. To adapt to the salt environment of the reaction, it is necessary to find a highly efficient, salt-tolerant adenylate kinase. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a salt-loving adenosine kinase mutant in response to the shortcomings of the prior art.

[0004] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned salt-loving adenosine monophosphate kinase mutant in the catalytic reaction to prepare adenosine diphosphate.

[0005] To address the aforementioned technical problems, this invention provides a saline adenosine kinase mutant.

[0006] The amino acid sequence of the salami-adenosine kinase mutant is obtained by mutating any one or more of the following positions from the wild-type adenosine kinase: lysine at position 40, lysine at position 74, arginine at position 79, serine at position 131, lysine at position 152, glutamine at position 159, asparagine at position 164, arginine at position 19, serine at position 51, lysine at position 117, lysine at position 177, asparagine at position 189, glutamine at position 198, lysine at position 55, lysine at position 69, serine at position 169, and lysine at position 209.

[0007] Preferably, the amino acid sequence of the salami-adenosine kinase mutant is obtained by mutating the following positions of wild-type adenosine kinase: lysine at position 40, lysine at position 74, arginine at position 79, serine at position 131, lysine at position 152, glutamine at position 159, asparagine at position 164, arginine at position 19, serine at position 51, lysine at position 117, lysine at position 177, asparagine at position 189, and glutamine at position 198.

[0008] Specifically, the following mutations occur: lysine at position 40 mutates to aspartic acid; lysine at position 74 mutates to glutamic acid; arginine at position 79 mutates to aspartic acid; serine at position 131 mutates to aspartic acid; lysine at position 152 mutates to glutamic acid; glutamine at position 159 mutates to aspartic acid; asparagine at position 164 mutates to aspartic acid; arginine at position 19 mutates to aspartic acid; serine at position 51 mutates to glutamic acid; lysine at position 117 mutates to glutamic acid; lysine at position 177 mutates to glutamic acid; lysine at position 189 mutates to glutamic acid; and glutamine at position 198 mutates to aspartic acid.

[0009] The amino acid sequence of the salamicin kinase mutant is shown in any one of SEQ ID NO. 2-4, and the nucleotide sequence of the corresponding encoding gene is shown in any one of SEQ ID NO. 6-8.

[0010] Preferably, the amino acid sequence of the salamidone kinase mutant is shown in SEQ ID NO.3, and the nucleotide sequence of the corresponding encoding gene is shown in SEQ ID NO.7, namely, adenosine kinase mutant M2.

[0011] The wild-type adenosine kinase is derived from Bacillus subtilis, and its amino acid sequence is shown in SEQ ID NO.1. The corresponding nucleotide sequence encoding the wild-type adenosine kinase gene is shown in SEQ ID NO.5.

[0012] A recombinant expression vector containing the nucleotide sequence of the above-mentioned salt-loving adenosine kinase mutant encoding gene is also within the scope of protection of this invention.

[0013] A recombinant strain containing the nucleotide sequence of the above-mentioned salt-adenosine kinase mutant encoding gene or containing the above-mentioned recombinant expression vector is also within the scope of protection of this invention.

[0014] The application of the above-mentioned saline adenosine monophosphate kinase mutant in the catalytic reaction to prepare adenosine diphosphate is also within the scope of protection of this invention.

[0015] The reaction system of the catalytic reaction uses adenosine monophosphate, adenosine triphosphate, and acetyl phosphate as substrates, and adds a purified enzyme of a protein-purified halophilic adenosine kinase mutant to catalyze the synthesis of adenosine diphosphate.

[0016] Specifically, in the salt tolerance test of the salinity kinase mutant, the salinity kinase mutant can tolerate acetate concentrations of 300-2000 mmol / L.

[0017] The acetate salt includes any one of Kac, NaAc, LiAc, and NH4Ac, with Kac being the preferred acetate salt.

[0018] Among them, the enzyme activities of the salicylate kinase mutants M2 and M3 are not significantly different under different concentrations of KAc. By screening the enzyme kinetic parameters of the two, the enzyme activity kinetic parameters of M2 are higher than those of M3. Therefore, the salicylate kinase mutant M2 is the most preferred salicylate kinase.

[0019] Beneficial effects: Compared with the prior art, the salt-loving adenosine monophosphate kinase mutants M1-M3 of the present invention have higher salt tolerance than wild-type salt-loving adenosine monophosphate kinase. The best-performing salt-loving adenosine monophosphate kinase mutant M2 can tolerate a salt solution concentration of 2000mM compared to wild-type adenosine monophosphate kinase. At the same time, KAc was selected as the acetate with the lowest inhibitory effect on enzyme activity. Therefore, potassium hydroxide was chosen as an alkaline reagent to neutralize the acetic acid produced in the system to maintain a constant pH value because it has the least negative impact on enzyme activity and can be better applied to the preparation of adenosine diphosphate (ADP). Attached Figure Description

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0021] Figure 1 It is a recombinant expression vector containing the adenosine kinase encoding gene (ADK).

[0022] Figure 2 It is a recombinant expression vector that displays the gene encoding the adenosine kinase mutant M1.

[0023] Figure 3 It is a recombinant expression vector that displays the gene encoding the adenosine monophosphate kinase mutant M2.

[0024] Figure 4 It is a recombinant expression vector that displays the gene encoding the adenosine monophosphate kinase mutant M3. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments. The embodiments will help to understand the present invention, but the scope of protection of the present invention is not limited to the following embodiments.

[0026] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0027] Example 1: Modification of the salt-loving adenosine monophosphate kinase mutants M1-M3

[0028] First, the crystal structure of wild-type adenosine monophosphate kinase protein from Bacillus subtilis (strain 168) (PDB ID: 1P3J) was found using the PDB database. Then, the substrate adenosine monophosphate was molecularly docked with adenosine monophosphate using Autodock vina software to determine its active pocket location and prevent the selected mutated residue sites from affecting its active pocket. Homology sequence alignment was performed using the Consurf database to predict the conserved and non-conserved regions of adenosine monophosphate kinase. Then, a batch of specific sites with uniform distribution and positive or no charge on the surface and structural space of the enzyme protein were anchored in the non-conserved sequence mutation region. These sites were gradually mutated into negatively charged amino acids to maximize the distribution of negative charge on the protein surface. Using the structure-based open-source computational software Rosetta, the folding free energy change (ΔG) of the mutant and wild type was estimated using empirical force field formulas. Then, the difference between the two was used to predict the difference in free energy change (ΔGG) of the corresponding mutation. Mutations with smaller ΔGG were selected. At the same time, the increase of surface salt bridges and hydrogen bonds and the reduction of solvent-accessible surface area caused by the mutation were considered to maintain the stability of the structure in a high-salt environment. Finally, the determined mutation sites were screened out.

[0029] Wild-type adenosine monokinase (WT) was synthesized from the genome of Bacillus subtilis (strain 168) through whole-genome synthesis (by GENEWIZ). Its amino acid sequence is shown in SEQ ID NO.1, and the corresponding nucleotide sequence is shown in SEQ ID NO.5.

[0030] Based on the selected sites, the above-mentioned wild-type adenosine kinase WT was subjected to site-directed mutagenesis. The modification sites and modification methods are as follows:

[0031] (1) The wild-type adenosine kinase WT was mutated by changing the 40th lysine to aspartic acid; the 74th lysine to glutamic acid; the 79th arginine to aspartic acid; the 131st serine to aspartic acid; the 152nd lysine to glutamic acid; the 159th glutamine to aspartic acid; and the 164th asparagine to aspartic acid, i.e., K40D, K74E, R79D, S131D, K152E, Q159D, and N164D. This yielded the adenosine kinase mutant M1.

[0032] Specifically, the amino acid sequence of the adenosine kinase mutant M1 is shown in SEQ ID NO.2, and the nucleotide sequence of the corresponding artificially synthesized coding gene is shown in SEQ ID NO.6 (synthesized by GENEWIZ).

[0033] (2) Mutate lysine at position 40 of wild-type adenosine kinase (WT) to aspartic acid; lysine at position 74 to glutamic acid; arginine at position 79 to aspartic acid; serine at position 131 to aspartic acid; lysine at position 152 to glutamic acid; glutamine at position 159 to aspartic acid; asparagine at position 164 to aspartic acid; arginine at position 19 to aspartic acid; and lysine at position 51 to aspartic acid. Serine was mutated to glutamic acid; lysine at position 117 was mutated to glutamic acid; lysine at position 177 was mutated to glutamic acid; aspartic acid at position 189 was mutated to glutamic acid; and glutamine at position 198 was mutated to aspartic acid, namely K40D, K74E, R79D, S131D, K152E, Q159D, N164D, R19D, S51E, K117E, K177E, K189E, and Q198D. This yielded the adenosine kinase mutant M2.

[0034] Specifically, the amino acid sequence of the adenosine kinase mutant M2 is shown in SEQ ID NO.3, and the nucleotide sequence of the corresponding artificially synthesized coding gene is shown in SEQ ID NO.7 (synthesized by GENEWIZ).

[0035] (3) Mutate the 40th lysine of wild-type adenosine kinase (WT) to aspartic acid; mutate the 74th lysine to glutamic acid; mutate the 79th arginine to aspartic acid; mutate the 131st serine to aspartic acid; mutate the 152nd lysine to glutamic acid; mutate the 159th glutamine to aspartic acid; mutate the 164th asparagine to aspartic acid; mutate the 19th arginine to aspartic acid; mutate the 51st serine to glutamic acid; mutate the 117th lysine to glutamic acid; mutate the 177th lysine to glutamic acid; mutate the 189th lysine to glutamic acid; mutate the 198th glutamine to aspartic acid; mutate the 55th lysine to glutamic acid; mutate the 69th lysine to aspartic acid; mutate the 169th serine to glutamic acid; and mutate the 209th lysine to glutamic acid. Specifically, K40D, K74E, R79D, S131D, K152E, Q159D, N164D, R19D, S51E, K117E, K177E, K189E, Q198D, K55E, K69D, S169E, and K209E were used. This yielded the adenosine kinase mutant M3.

[0036] Specifically, the amino acid sequence of the adenosine kinase mutant M3 is shown in SEQ ID NO.4, and the nucleotide sequence of the corresponding artificially synthesized coding gene is shown in SEQ ID NO.8 (synthesized by GENEWIZ).

[0037] Example 2: Construction of salamiphilic adenosine kinase WT and its mutant M1-M3 recombinant strains

[0038] According to Example 1, the corresponding nucleotide sequences of the amino acid sequences of the adenosine kinase mutants M1-M3 were artificially synthesized. The upstream primer F (5'-CGCCATATGATGAACTTAGTCTTAATGGGGC-3') and the downstream primer R (5'-CCGCTCGAGTCATTTTTTTAATCCTCCAAGAAG-3') were designed and synthesized to obtain the wild-type adenosine kinase WT and its mutant M1-M3 genes, and they were used as templates for PCR amplification.

[0039] The PCR reaction system included: 25 μL 2×PCR Buffer, 10 μL 2 mM dNTPs, 1.5 μL each of 10 pmol / μL primer F and primer R, 1 μL template DNA, 1 μL high-fidelity polymerase, and finally, sterile double-distilled water to a final volume of 50 μL. The PCR reaction parameters were: 94℃ melting for 2 minutes, 98℃ denaturation for 10 seconds, 60℃ annealing for 30 seconds, 68℃ extension for 1.5 minutes, 30 cycles, with a final 5 minutes at 68℃, followed by storage at 4℃.

[0040] The wild-type adenosine kinase WT and its mutant M1-M3 genes, as well as the pET-28a expression vector (purchased from Novagen), were digested with NdeI and XhoI restriction endonucleases (purchased from TaKaRa). The digested products of the wild-type adenosine kinase WT and its mutant M1-M3 genes were purified by 1% agarose gel electrophoresis using a DNA fragment purification kit (purchased from TaKaRa). The purified target fragments were then ligated into the digested pET28a expression vector using the following ligation system: 4 μL of purified DNA fragment product, 6 μL of linearized pET-28a plasmid, and 10 μL of Solution I. The mixture was incubated at 16°C for 1.5 h to obtain the recombinant expression vector. Figure 1-4 As shown, the recombinant expression vector was then transformed into Escherichia coli BL21(DE3) competent cells using the calcium chloride method to obtain recombinant Escherichia coli. The obtained recombinant Escherichia coli were cultured in LB medium at 37°C and 200 rpm (final concentration 50 mg / L Kan) for 12 hours, and single colonies were picked for verification and sequencing to determine the sequence.

[0041] Example 3: Enzyme activity detection of wild-type adenosine monophosphate kinase WT and its mutants M1-M3

[0042] Recombinant Escherichia coli containing wild-type adenosine kinase WT and its mutants M1-M3 were cultured in 1 L TB medium (final concentration 50 mg / L Kan, 1% v / v inoculum). When OD... 600 When the OD value was 0.6-0.8, 0.2 mM isopropyl β-d-1-mercaptogalactoside (IPTG) was added for induction, and the mixture was incubated at 30℃ and 150 rpm for 16 h. The OD value was then measured. 600 If the bacterial count is 5 or higher, the cells are collected by centrifugation for subsequent protein purification.

[0043] Remove the bacterial cells collected from the induced expression container from the -80℃ freezer. Weigh 2g into a 50mL centrifuge tube, wash twice with 0.1mol / L PBS (pH 7.5), centrifuge to collect the wet cells, and resuspend in 20mL of 0.1mol / L PBS (pH 7.5) to obtain a 100g / L bacterial suspension. Transfer 20mL of the bacterial suspension into a 50mL centrifuge tube and sonicate the cells using an ultrasonic cell disruptor under ice-water bath conditions. Set the instrument parameters to 200W power, sonicate for 3 seconds, pause for 7 seconds, and continue for approximately 40 minutes until the bacterial suspension becomes clear. After completion, centrifuge at 8000rpm for 10 minutes at 4℃. The supernatant in the centrifuge tube is the crude enzyme solution. Add 20 mL of crude enzyme solution to the nickel column in two portions to ensure complete chelation between the enzyme and the packing material (Ni-NTA). Rinse the nickel column three times with 10 mL of 5 mmol / L phosphate-imidazole (pH 7.5) to remove impurities and proteins. Then add 10 mL of 20 mmol / L phosphate-imidazole (pH 7.5) and let stand for 15 min to release the pure enzyme. Add an equal volume of glycerol. At this point, the cell concentration of the pure enzyme is 100 g / L. Refrigerate the pure enzyme at -80°C.

[0044] Preparation of a standard reaction mixture (5 mL): The reaction system consisted of 100 mmol / L Tris-HCl (pH 7.5), 10 mmol / L adenosine monophosphate (AMP), 2 mmol / L adenosine triphosphate (ATP), 15 mmol / L acetyl phosphate, and 4 mmol / L MgCl2. 100 μL of 100 g / L purified adenosine kinase was added at 37 °C. After standing for 5 min, the reaction mixture was shaken. Samples were taken every 10 min and quenched by boiling in a water bath. Enzyme activity was defined as the amount of enzyme required to produce 1 μmol of ADP per minute.

[0045] Table 1. Relative enzyme activities of wild-type adenosine kinase WT and its mutants M1-M3

[0046]

[0047] As shown in Table 1, the results obtained by comparing the relative enzyme activity of mutants M1, M2, and M3 with those of wild-type strains show that as the surface negative charge of adenosine kinase gradually increases, its relative enzyme activity gradually decreases in a salt-free environment. Considering that the relative enzyme activity of M3 decreased too much, further mutation was not carried out on M3. Finally, the three mutant strains M1, M2, and M3 were set up for subsequent experimental verification.

[0048] Example 4: Salt tolerance test of salt-loving adenosine monophosphate kinase mutants M1-M3

[0049] To test the effect of high-salt solutions on enzymes, four acetate solutions (Kac, NaAc, LiAc, and NH4Ac) with salt concentrations controlled between 300 and 2000 mmol / L were prepared. After mixing all acetate solutions into the reaction system (as in Example 3), pure enzymes of adenosine kinase WT and mutant M1-M3 were added to start the reaction to simulate the actual catalytic environment in the catalytic process and test the effect of high-salt environment on enzymes. The results are shown in Table 2.

[0050] Table 2. Differences in the activity of wild-type adenosine kinase WT and mutant M1-M3 at different concentrations of acetate solution.

[0051]

[0052]

[0053] The experimental results are shown in Table 2. With increasing acetate concentration, the enzyme activity of wild-type WT decreased compared to mutants M1, M2, and M3. Among the four different acetate solutions, KAc showed the lowest inhibitory effect on adenosine kinase; therefore, KOH was the preferred alkaline reagent for neutralizing acetic acid in the reaction system. Based on the differences in activity between wild-type adenosine kinase WT and mutants M1-M3 at different KAc concentrations in the table above, the enzyme activities of M2 and M3 were not significantly different under different KAc concentrations. Therefore, suitable halophilic adenosine kinase mutants need to be screened based on their enzymatic kinetic parameters.

[0054] Example 5: Kinetic parameters of saline adenosine monophosphate kinase mutants M2 and M3

[0055] Following the standard reaction of Example 3, the pH of the reaction was maintained at 7.0-7.5 by supplementing KOH, the substrate AMP concentration ranged from 1 mM to 30 mM, and the reaction was carried out at room temperature for 5-15 minutes, with kinetic parameters calculated at 30-second intervals. The kinetic parameter K was obtained using nonlinear regression analysis of the Michaelis-Menten equation. cat and K m The kinetic parameters of the wild-type strain and the adenosine kinase mutants M2 and M3 are shown in Table 3.

[0056] Table 3. Enzymatic activity mechanics parameters of adenosine kinase

[0057]

[0058] Table 3 shows that in mutants M2 and M3, Kt against the substrate AMP... cat Compared to WT, this is an increase, and M2 and M3 have different K values ​​for the substrate AMP. cat / K m Compared to the wild-type strain, the activity was increased by approximately 1.6 times and 1.5 times, respectively. This indicates that the enzyme activity kinetics of M2 are higher than those of M3, therefore M2 was selected as the most suitable natriuretic adenosine kinase.

[0059] This invention provides a concept and method for a saline adenosine monophosphate kinase mutant and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A mutant halophilic adenosine kinase, characterized in that, The amino acid sequence of the halophilic adenosine kinase mutant is shown in SEQ ID NO. 2, or SEQ ID NO. 3, or SEQ ID NO.

4.

2. The halophilic adenosine kinase mutant of claim 1, wherein, The nucleotide sequence of the coding gene corresponding to the amino acid sequence of the halophilic adenosine kinase mutant shown in SEQ ID NO. 2, or SEQ ID NO. 3, or SEQ ID NO. 4 is shown in SEQ ID NO. 6-8.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleotide sequence of the coding gene corresponding to the amino acid sequence of the halophilic adenosine kinase mutant shown in claim 2.

4. A recombinant bacterial strain, characterized in that, The nucleotide sequence of the coding gene corresponding to the amino acid sequence of the halophilic adenosine kinase mutant shown in claim 2 or the recombinant expression vector shown in claim 3.

5. The use of the halophilic adenosine kinase mutant shown in claim 1 in catalytic reaction for preparing adenosine diphosphate.

6. Use according to claim 5, characterized in that, The reaction system of the catalytic reaction is to use adenosine monophosphate, adenosine triphosphate and acetyl phosphate as substrates, and to add the pure enzyme of the halophilic adenosine kinase mutant to catalyze the synthesis of adenosine diphosphate.

7. Use according to claim 5, characterized in that, The halophilic adenosine kinase mutant is resistant to the concentration of 300-2000 mmol / L of acetate in the catalytic reaction.

8. Use according to claim 7, characterized in that, The acetate includes any one of KAc, NaAc, LiAc and NH4Ac. The amino acid sequence of the halophilic adenosine kinase mutant is shown in SEQ ID NO. 2, or SEQ ID NO. 3, or SEQ ID NO.

4. The nucleotide sequence of the coding gene corresponding to the amino acid sequence of the halophilic adenosine kinase mutant shown in SEQ ID NO. 2, or SEQ ID NO. 3, or SEQ ID NO. 4 is shown in SEQ ID NO. 6-8. The recombinant expression vector contains the nucleotide sequence of the coding gene corresponding to the amino acid sequence of the halophilic adenosine kinase mutant shown in claim 2. The nucleotide sequence of the coding gene corresponding to the amino acid sequence of the halophilic adenosine kinase mutant shown in claim 2 or the recombinant expression vector shown in claim 3.

5. The use of the halophilic adenosine kinase mutant shown in claim 1 in catalytic reaction for preparing adenosine diphosphate. The reaction system of the catalytic reaction is to use adenosine monophosphate, adenosine triphosphate and acetyl phosphate as substrates, and to add the pure enzyme of the halophilic adenosine kinase mutant to catalyze the synthesis of adenosine diphosphate. The halophilic adenosine kinase mutant is resistant to the concentration of 300-2000 mmol / L of acetate in the catalytic reaction. The acetate includes any one of KAc, NaAc, LiAc and NH4Ac.

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