Phosphotransferase mutant with improved catalytic activity

Abstract

The invention discloses a phosphotransferase mutant with improved catalytic activity, and belongs to the field of biology and food. On the basis of a phosphotransferase female parent sequence, the 72nd site, the 104th site and the 136th site are substituted. The enzyme activity of the constructed mutant is improved from 46.2 U / mg to 213.2 U / mg in a whole-cell catalytic enzyme activity test of inosine compared with that of a female parent mature enzyme. The phosphotransferase variant disclosed by the invention benefits from the improved catalytic activity, is more beneficial to stable application in a harsh industrial environment, can be used for producing food flavoring agents such as disodium inosinate and the like, and is widely applied to the fields of food and biology.

Description

Technical Field

[0001] The invention relates to a phosphotransferase mutant with improved catalytic activity, belonging to the fields of biology and food. Background Art

[0002] Phosphotransferases from Klebsiella pneumonia are enzymes that catalyze the hydrolysis of phosphate esters and the transfer of phosphate groups. They have important applications in the food industry and biocatalysis, and are widely used in the industrial production of nucleotides (such as IMP and GMP). Their advantages include high substrate specificity and conversion efficiency. However, these enzymes face several challenges in practical applications, such as a significant decrease in catalytic efficiency at high substrate concentrations, low phosphate transfer efficiency, and poor thermal stability. These issues severely limit their use in industrial-scale reactions.

[0003] To address these issues, studies have attempted to optimize and modify phosphotransferases using various methods. First, through directed mutagenesis, some studies have optimized the structure of the catalytic pocket of phosphatases, for example, optimizing the substrate binding site to reduce steric hindrance and improve substrate binding efficiency. Second, molecular dynamics simulations (MD) and quantum mechanics / molecular mechanics (QM / MM) analysis have been used to identify key sites in the enzyme and design site-directed mutagenesis to improve the enzyme's catalytic performance. Finally, some studies have attempted to enhance the electron transfer capacity of the catalytic center by adjusting the charge distribution of the enzyme molecule to improve the efficiency of phosphate group transfer. These methods have achieved certain results in improving the activity and stability of phosphotransferases.

[0004] However, existing research still has many shortcomings. On the one hand, the optimized mutants show good catalytic activity under laboratory conditions, but their catalytic efficiency and stability under high substrate concentration conditions still fail to meet the needs of industrial production. On the other hand, most studies focus on the modification of a single mutation site, ignoring the impact of synergistic effects between different sites on the overall performance of the enzyme. In addition, the structural stability of the modified mutants in long-term reactions is also insufficient, limiting their continued use in industrial production. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a phosphotransferase mutant with high catalytic activity, which has a mutation at position 115 and one or more amino acid mutations at positions 72, 104, and 136 based on the sequence shown in SEQ ID NO.1.

[0006] In one embodiment, the phosphotransferase mutant is based on the starting sequence, wherein lysine at position 115 is mutated to leucine, and has at least one of the following mutations (a) to (c):

[0007] (a) Alanine at position 72 was mutated to glutamine;

[0008] (b) mutating glutamic acid at position 104 to lysine;

[0009] (c) Mutate glutamic acid at position 136 to glycine.

[0010] In one embodiment, the phosphotransferase mutant is based on the sequence shown in SEQ ID NO. 1, with lysine at position 115 mutated to leucine, and alanine at position 72 mutated to glutamine.

[0011] In one embodiment, the phosphotransferase mutant is based on the sequence shown in SEQ ID NO. 1, with the 115th lysine mutated to leucine and the 104th glutamic acid mutated to glycine.

[0012] In one embodiment, the phosphotransferase mutant is based on the sequence shown in SEQ ID NO.1, with lysine at position 115 mutated to leucine, alanine at position 72 mutated to glutamine, and glutamate at position 136 mutated to glycine.

[0013] In one embodiment, the phosphotransferase mutant is based on the sequence shown in SEQ ID NO.1, with lysine at position 115 mutated to leucine, alanine at position 72 mutated to glutamine, and glutamate at position 104 mutated to glycine.

[0014] The present invention also provides a gene encoding the mutant.

[0015] The invention also provides a plasmid containing the gene.

[0016] The present invention also provides a recombinant microbial cell expressing the mutant.

[0017] In one embodiment, the cells include Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, etc.

[0018] The present invention also provides recombinant Escherichia coli, which uses pET-22b as a vector to express the phosphotransferase mutant.

[0019] In one embodiment, the E. coli is E. coli BL21.

[0020] A cell catalyst containing the recombinant microbial cell.

[0021] The present invention also relates to methods for producing the phosphotransferase variants of the present invention, comprising:

[0022] a) cultivating a host cell of the invention under conditions suitable for expression of the variant; and

[0023] b) optionally recovering the variant.

[0024] The present invention also provides a method for preparing disodium inosinate using the phosphotransferase mutant. The method uses inosine, sodium pyrophosphate and sodium acetate as substrates, the recombinant microbial cells as catalysts, and the reaction is carried out at 30-35° C. for at least 8 hours.

[0025] In one embodiment, the method comprises adding 20 g / L to 200 g / L of microbial cells expressing phosphotransferase in a reaction system to a solution containing 0.05 M to 0.5 M inosine, 0.1 M anhydrous sodium pyrophosphate, and 0.17 M to 1.7 M sodium acetate, and carrying out whole-cell catalysis to prepare disodium inosinate at 32° C. and pH 5.0.

[0026] The present invention also relates to applications in the development of drug targets, biomarkers and detection, synthesis of energy molecules and energy storage, regulation of signal transduction, regulation of protein activity, production of food additives, and production of other organophosphate compounds or phosphorylated bioactive molecules.

[0027] In one embodiment, the use comprises using the phosphotransferase variant of the present invention in any of the above-described product production processes.

[0028] In one embodiment, the food flavor enhancer includes but is not limited to disodium inosinate.

[0029] In one embodiment, the use is for producing ascorbic acid phosphate derivatives, ascorbic acid phosphate esters, phosphorylated vitamin B6 derivatives or phosphatase-related inhibitors.

[0030] Beneficial effects:

[0031] The phosphotransferase variants provided herein are based on the parent enzyme, i.e., substitutions at positions 72, 104, 115, and 136 of the sequence shown in SEQ ID NO. 1. In a whole-cell catalytic activity assay of inosine, the constructed variant mature enzyme increased its activity from 46.2 U / mg to 213.2 U / mg, a 361.47% increase compared to the parent mature enzyme. This activity was also 22.38% higher than the previously reported highest-activity mutant, A72Q-E104G-E136G.

[0032] The phosphotransferase variants of the present invention have significantly enhanced catalytic activity and can be used in the production of food additives and organophosphate compounds. The enhanced catalytic activity of the phosphotransferase variants facilitates their stable application in harsh industrial environments, such as the production of the food flavor enhancer "I+G." BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is SDS-PAGE analysis of samples with lower enzyme activity to confirm their expression; M is a protein marker (kDa); lanes 1 to 11 are A72D, A72G, E104K, E104Q, E104D, E136K, E136Q, K139Q, K139A, K139D, and K139G, respectively.

[0034] Figure 2 The enzyme activities of wild-type and mutant phosphotransferases in cells. DETAILED DESCRIPTION

[0035] (1) Reagents and materials

[0036] Escherichia coli BL21(DE3) was purchased from Takara Biotech (Beijing) Co., Ltd., and the pET-22b(+) plasmid was purchased from Novagen. Disodium inosinate was purchased from Glpbio (Tianjin, China). NaCl, K2HPO4, KH2PO4, tryptone, glucose, yeast extract, and glycerol were all purchased from Sangon Biotech (Shanghai, China).

[0037] (2) Detection method

[0038] Enzyme activity detection:

[0039] Substrate solution A: 0.05 M inosine, 0.1 M anhydrous sodium pyrophosphate, 0.17 M sodium acetate, and glacial acetic acid to adjust the pH to 4.9-5.0.

[0040] Stop solution B: saturated NaOH solution.

[0041] Enzyme activity definition: One activity unit (U) is the amount of enzyme required to catalyze the production of 1 μmol of disodium inosinate in 1 minute in a basic reaction system comprising a 50 g / L wet weight microbial cell suspension at 32°C and pH 5.0.

[0042] The enzyme activity was determined by measuring the activity of the phosphotransferase in 1 ml of a standard reaction mixture containing 100 μM sodium acetate buffer (pH 5.0), 40 μM inosine, 100 μM tetrasodium pyrophosphate, and enzyme solution. 2 ml of OD600 A bacterial resuspension of 80 (wet bacterial weight at this OD is 100 g / L) and 2 ml of substrate solution A were placed in a shaker at 32°C for reaction for 20 minutes, taken out, and 0.5 ml of stop solution B was added to terminate the reaction. The reaction solution was adjusted to pH 10.0 with HCl, passed through a membrane, and detected by HPLC.

[0043] (3) Culture medium

[0044] LB liquid medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, ampicillin 100 μg / L.

[0045] LB solid medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L, ampicillin 100 μg / L.

[0046] TB medium: yeast extract 24 g / L, tryptone 12 g / L, dipotassium hydrogen phosphate trihydrate 12.84 g / L, potassium dihydrogen phosphate 2.31 g / L, glycerol 4 mL / L, ampicillin 100 μg / L.

[0047] Example 1 Prediction of key mutation sites of phosphotransferase

[0048] This example optimizes the catalytic efficiency of Klebsiella pneumonia phosphotransferase using a three-step progressive strategy. First, a three-dimensional model of the enzyme was constructed based on AlphaFold2, and molecular docking was used to determine the binding conformation of the substrate p-NPP in the catalytic pocket. Molecular dynamics simulations revealed that the E104 side chain carboxylic acid group forms a stable hydrogen bond network with the substrate phosphate group, resulting in the substrate maintaining a stable bond with the catalytic residue H189. Ineffective distance. Alanine scanning mutagenesis binding free energy calculations confirmed that the E104 mutation can reduce the binding energy by 6.8kcal / mol. Based on this, a saturation mutation library of E104 site was constructed, and the E104G mutant obtained by experimental screening had a 2.7-fold increase in activity. Crystal structure analysis (PDB:9JQ0) showed that this mutation increased the substrate channel diameter from Expand to QM / MM calculations confirmed that the energy barrier for phosphate group transfer was reduced by 21.7%.

[0049] Based on the E104G modification, trajectory clustering analysis (RMSD cutoff = 0.25 nm) found that the conformation of the phosphate acceptor inosine in the catalytic pocket fluctuated significantly, suggesting that the receptor binding stability was insufficient. To address this issue, a three-level optimization was implemented. First, the key residues were identified, and the contact frequency of the substrate binding trajectory was extracted to determine that sites such as A72, E136, and R183 were receptor binding hotspots. Second, virtual saturation mutagenesis was used to screen potential mutants, and a large number of single-point mutants were obtained through simulation. Based on the evaluation of mutant binding energy and stability, 17 candidate mutants such as A72Q, K115L, and E136G were selected.

[0050] Example 2 Construction of recombinant E. coli expressing mutants

[0051] The parent gene (nucleotide sequence as shown in SEQ ID NO: 2, amino acid sequence as shown in SEQ ID NO: 1, insertion sequence and enzyme cutting site as shown in SEQ ID NO: 3) was synthesized by Suzhou Jinyu Zhi Company, and inserted into the plasmid pET-22b(+) through restriction enzyme sites NdeI and BlpI to obtain pET-22b / KpAP. The primers used for polymerase chain reaction (PCR) were synthesized by Jinyu Zhi Company, and when preparing single-point mutants, pET-22b / KpAP was used as the template, and primers (as shown in Table 1) were designed for the mutation sites predicted in Example 1. Linear DNA with point mutations was obtained through PCR, and was circularized by one-step cloning enzyme, and was transformed into E. coli BL21. The PCR process was referred to the instructions of Baorai Biotechnology (Beijing) Co., Ltd. The linear DNA circularization method was referred to the instructions of Novizen (Nanjing) Co., Ltd. ClonExpress MultiS One Step Cloning Kit.

[0052] Table 1 Primer gene sequence

[0053]

[0054]

[0055] Example 3 Preparation of mutants

[0056] The transformed product of the recombinant E. coli E. coli BL21 carrying the mutant coding sequence constructed in Example 2 was spread on LB solid medium, incubated at 37°C for 10 h, and the transformants were picked for sequence determination. The recombinant plasmid with correct sequencing was obtained, and was expressed by E. coli E. coli BL21(DE3).

[0057] The obtained recombinant E. coli was spread on LB solid medium and cultured at 37°C for 10 h. The transformants were picked and inoculated into LB liquid medium (containing 100 μg / mL ampicillin). The cells were cultured at 37°C for 10 h and then transferred to TB liquid medium (containing 100 μg / mL ampicillin) at a 1% transfer volume. E. coli BL21 (DE3) was cultured at 37°C until the OD 600 When the pH value was between 1.0 and 1.5, IPTG was added to a final concentration of 0.01 mM and the recombinant E. coli cells expressing the mutant were obtained by continuous culture at 25°C for 30 h. All liquid cultures were cultured on a shaker at 220 rpm.

[0058] Example 4 Preparation of whole-cell catalyst

[0059] The mutant strain expressing the above phosphotransferase constructed in Example 2 was cultured overnight in a 3 ml shake tube in LB, and then transferred to a 250 ml conical shake flask containing 50 ml fermentation medium at a 2% inoculum size and cultured at 37°C until the strain OD 600 When the OD value was between 1.0 and 1.5, 0.01 mM IPTG was added for induction, and the fermentation temperature was lowered to 25 °C and cultured for 30 h at a speed of 220 rpm. 600 When the temperature reaches 17-20 °C, the fermentation broth is collected and centrifuged at 5000 rpm / min for 10 minutes to collect the cells. The cells are resuspended in a certain volume of phosphate buffer (1 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, pH 7.0) until the cell wet weight reaches 100 g / L, thereby preparing a whole-cell catalyst containing phosphotransferase.

[0060] Example 5 Determination of enzyme activity of cell catalyst expressing mutant

[0061] Prepare solution A (calculated by final concentration): 0.05 M inosine, 0.1 M anhydrous sodium pyrophosphate, 0.17 M sodium acetate, and adjust the pH to 4.9-5.0 by adding glacial acetic acid.

[0062] To each 10 mL reactor, 1 mL of the whole-cell catalyst containing the phosphotransferase prepared in Example 4 and 4 mL of Solution A were added, and the mixture was allowed to react in a constant-temperature shaking incubator at 32°C for 8 hours. After the reaction, the pH was adjusted to approximately 10.0. The treated samples were filtered through a 0.22 μm microporous membrane, and the products were accurately quantitatively analyzed using high-performance liquid chromatography (HPLC).

[0063] As shown in Table 2, the enzyme activity of the phosphotransferase mutants AP-A72Q, AP-E104G, AP-K115L, AP-E136G, AP-A72Q-E104G, AP-A72Q-E136G, AP-E104G-K115L, AP-E104G-E136G, AP-A72Q-E104G-K115L, and AP-A72Q-E104G-E136G mature enzymes was significantly improved compared to that of the parent phosphotransferase mature enzyme.

[0064] Table 2 Specific enzyme activity and inosine disodium production of mutants

[0065]

[0066]

[0067] Example 5 Whole cell catalyzed preparation of inosine disodium

[0068] Preparation of solution reaction liquid (in final concentration): 0.4 M inosine, 0.5 M anhydrous base pyrophosphate sodium, 1 M sodium acetate, and add glacial acetic acid to adjust pH to 4.9-5.0.

[0069] Into a 5 L reactor, 1 L of whole cell catalyst containing phosphotransferase E104G-K115G-E136G prepared according to the method of Reference Example 4 and 4 L of solution reaction liquid were sequentially added, and the reaction was carried out in a constant temperature shaking incubator at 30°C for 20 hours. After the reaction was completed, the pH was adjusted to 10.0. The treated sample was filtered through a 0.22 μm microporous filter, and finally the product was accurately quantitatively analyzed by high performance liquid chromatography (HPLC) method. The results showed that the yield of inosine disodium was 116.7 g / L.

[0070] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the scope of protection of the present application should be defined by the claims.

Claims

1. A phosphotransferase mutant, characterized in that Based on the sequence shown in SEQ ID NO.1, it has a mutation at position 115, and one or more amino acid mutations at positions 72, 104, and 136.

2. The phosphotransferase mutant according to claim 1, characterized in that The phosphotransferase mutant is based on the starting sequence, with lysine at position 115 mutated to leucine, and has at least one of the following mutations (a) to (c): (a) Alanine at position 72 was mutated to glutamine; (b) mutating glutamic acid at position 104 to lysine; (c) Mutate glutamic acid at position 136 to glycine.

3. The phosphotransferase mutant according to claim 1, characterized in that The mutant is any one of (a) to (d): (a) Based on the sequence shown in SEQ ID NO. 1, lysine at position 115 is mutated to leucine, and alanine at position 72 is mutated to glutamine; (b) Based on the sequence shown in SEQ ID NO. 1, the lysine at position 115 is mutated to leucine, and the glutamic acid at position 104 is mutated to glycine; (c) Based on the sequence shown in SEQ ID NO. 1, lysine at position 115 is mutated to leucine, alanine at position 72 is mutated to glutamine, and glutamic acid at position 136 is mutated to glycine; (d) Based on the sequence shown in SEQ ID NO. 1, the lysine at position 115 is mutated to leucine, the alanine at position 72 is mutated to glutamine, and the glutamic acid at position 104 is mutated to glycine.

4. A gene encoding the phosphotransferase mutant according to any one of claims 1 to 3.

5. A plasmid containing the gene according to claim 4.

6. A recombinant microbial cell expressing the phosphotransferase mutant according to any one of claims 1 to 3.

7. Recombinant Escherichia coli, characterized in that The pET-22b vector is used to express the phosphotransferase mutant according to any one of claims 1 to 3.

8. A method for producing the phosphotransferase mutant according to any one of claims 1 to 3, characterized in that: include: a) cultivating the host cell of the present invention under conditions suitable for expressing the variant; as well as b) optionally recovering the variant.

9. A method for preparing disodium inosinate, characterized in that: Inosine, anhydrous sodium pyrophosphate and sodium acetate are used as substrates, and the recombinant microbial cells according to claim 6 are used as catalysts, and the reaction is carried out at 30-35°C.

10. Use of the phosphotransferase mutant according to any one of claims 1 to 3, or the recombinant Escherichia coli according to claim 7, or the method according to claim 8 in the development of drug targets, biomarkers and detection, synthesis of energy molecules and energy storage, regulation of signal transduction, regulation of protein activity, production of food additives, and production of other organophosphate compounds or phosphorylated bioactive molecules.

Images