A polyphosphate kinase mutant and its application in catalyzing synthesis of glutathione

Abstract

The application discloses a polyphosphate kinase mutant and application thereof in catalyzing synthesis of glutathione, wherein the mutant is obtained by mutating the 22th amino acid in the amino acid sequence shown in SEQ ID NO:2.The application provides a polyphosphate kinase mutant from Cytophaga hutchinsonii, which has 2 times of catalytic activity compared to that before modification, and an ATP regeneration system formed by the mutant can be used in a glutathione synthesis reaction, and more than 19g of glutathione is generated in 2h, and the product generation rate is close to 90%.

Description

(I) TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and enzyme catalysis, and particularly relates to a polyphosphate kinase mutant and application thereof in glutathione synthesis by converting ADP to ATP with polyphosphate as substrate. (II) BACKGROUND

[0002] Glutathione (GSH) is an important tripeptide compound composed of L-cysteine, L-glutamic acid and glycine. As one of the most abundant thiol compounds in cells, GSH has multiple important physiological functions such as antioxidant, immune regulation and regulation of neurotransmitter receptor activity, and is widely used in the fields of medicine, food, cosmetics and health products.

[0003] At present, the one-step enzymatic synthesis of glutathione bifunctional enzyme is an ideal way for industrial synthesis of GSH, which has the advantages of easy access to raw materials, high specificity, high atom economy and environmental friendliness. This process realizes the synthesis of GSH by glutathione bifunctional enzyme catalysis of L-cysteine, L-glutamic acid and glycine, which highly depends on the continuous supply of energy by adenosine triphosphate (ATP). However, the high cost of ATP and the difficulty in effective separation of by-products adenosine diphosphate (ADP) and adenosine monophosphate (AMP) have become the limiting factors for its application in industry. Therefore, it is of great significance to develop an economic and efficient ATP synthesis and recycling system.

[0004] Polyphosphate kinase (PPK) is a kind of enzyme that catalyzes the generation of ATP from ADP or AMP with polyphosphate as the phosphate donor, and is an important enzyme catalyst for ATP regeneration. At present, there are many reports on the improvement of polyphosphate kinase catalytic activity based on protein engineering. For example, Gao et al. used error-prone PCR technology to modify the polyphosphate kinase derived from Cytophaga hutchinsonii, and obtained a double mutant with 4.3-fold higher enzyme activity; Gao et al. used computer-aided technology to rationally modify the polyphosphate kinase (ChPPK) derived from C. hutchinsonii, and obtained a mutant with 3.3-fold higher enzyme activity after remodeling its double substrate channel. Cao et al. rationally designed the polyphosphate kinase derived from Sinorhizobium meliloti, which greatly improved its catalytic activity for synthesizing ATP from short-chain polyphosphate. It can be seen that rational / non-rational design is an important means to improve the catalytic activity of polyphosphate kinase.

[0005] Previously, the inventors modified polyphosphokinases derived from Cytophaga hutchinsonii using protein engineering, obtaining a PPK mutant with significantly enhanced activity, and utilized its ATP regeneration system to assist glutathione synthesis (CN202310871912.5). However, the catalytic activity of existing polyphosphokinases remains low, making them unsuitable for industrial applications. This invention, through a semi-rational design method, constructs a highly efficient polyphosphokinase mutant suitable for glutathione synthesis, laying the foundation for enzymatic glutathione synthesis. (III) Summary of the Invention

[0006] The purpose of this invention is to provide a polyphosphate kinase mutant and its application in the catalytic synthesis of glutathione. This is achieved through protein engineering techniques, employing random and site-directed mutagenesis to modify the Cytophaga hutchinsonii-derived polyphosphate kinase mutant ChPPK. M0 Further modifications will be made to enhance its catalytic activity in synthesizing ATP, meeting the requirements for industrial production.

[0007] The technical solution adopted in this invention is:

[0008] The present invention provides a polyphosphate kinase mutant, which is obtained by mutating the 22nd position of the amino acid sequence shown in SEQ ID NO:2.

[0009] Furthermore, preferably, the polyphosphate kinase mutant is obtained by replacing serine at position 22 of the amino acid sequence shown in SEQ ID NO:2 with alanine, and is denoted as polyphosphate kinase mutant ChPPK. M1 The amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO:3.

[0010] This invention also relates to the coding gene of the polyphosphate kinase mutant, a recombinant vector constructed from the coding gene, and a recombinant genetically engineered bacterium constructed by transforming the recombinant vector into a host bacterium, wherein the recombinant vector is based on pET-28a. The host cell can be any conventional host cell in the art; preferably, it is *Escherichia coli* BL21(DE3).

[0011] The present invention also provides an application of the polyphosphate kinase mutant in the construction of an ATP regeneration system. The application involves using the polyphosphate kinase mutant and polyphosphate (polyP) to replace part of the ATP (adenine nucleoside triphosphate) to assist glutathione synthesis. The polyphosphate mutant is used in the form of crude enzyme solution or pure enzyme prepared from wet cells obtained by fermentation culture of polyphosphate kinase mutant genetically engineered bacteria.

[0012] This invention also provides the application of the polyphosphate kinase mutant or its expressing microorganism in the synthesis of glutathione. The method of application is as follows: using wet bacterial cells obtained after induced expression of the polyphosphate kinase mutant recombinant genetically engineered bacteria, or cell lysate after ultrasonic disruption, as a catalyst, and sodium hexametaphosphate as a substrate, ATP and Mg are added. 2+ The reaction system consisted of glutathione bifunctional synthase (GshF), cysteine, glycine, glutamic acid, and a buffer solution at pH 7.0. The reaction was carried out completely at 35°C to obtain the product glutathione. Mg... 2+ It is an activator of GshF, sodium hexametaphosphate is a substrate of polyphosphate kinase mutants, ATP provides energy for glutathione synthesis, and Mg... 2+ It is MgSO4 or MgCl2.

[0013] In the reaction system, the wet bacterial cells are 1-5 g / L (preferably 2.5 g / L) based on whole cells, and the cell lysis solution is 1-5 g / L (preferably 2.5 g / L) based on whole cells before lysis; the amount of glutathione bifunctional synthase added is 5-20 g / L (preferably 10 g / L), the amount of sodium hexametaphosphate added is 20-70 mM (preferably 30-50 mM), the amount of ATP added is 1-20 mM (preferably 2 mM), and Mg... 2+ The amount added is 20-70mM (preferably 60mM), cysteine ​​30-90mM (preferably 60mM), glycine 60-100mM (preferably 80mM), and glutamic acid 60-100mM (preferably 80mM).

[0014] The catalyst was prepared as follows: Recombinant genetically engineered bacteria containing a polyphosphate kinase mutant were inoculated into liquid LB medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 180 rpm. Subsequently, the inoculum was transferred at a volume concentration of 2% to fresh liquid LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 180 rpm until the bacterial cell concentration reached OD0.05. 600 The concentration was 0.6–0.8. IPTG was then added to the culture medium to a final concentration of 0.1 mM, and the culture was induced at 28°C and 180 rpm for 12 h. The fermentation broth was centrifuged at 4°C and 8000 rpm for 10 min to collect the wet cells. The wet cells were resuspended at 10 g / L in 50 mM Tris-HCl buffer (pH = 7.5), and sonicated at a power of 200 W for 1 second of operation followed by a 2-second rest, for a total working time of 10 min. The resulting cell lysate was obtained. The LB liquid culture medium consisted of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and water as the solvent, pH 7.0.

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

[0016] This invention provides a polyphosphate kinase mutant derived from Cytophaga hutchinsonii, which has a 2-fold increase in catalytic activity compared to the original mutant. The ATP regeneration system formed by the mutant can be used for glutathione synthesis, producing more than 19g of glutathione within 2 hours, with a product yield of nearly 90%. (iv) Description of the attached drawings

[0017] Figure 1 It is the polyphosphate kinase mutant ChPPK in Example 4. M1 The results of enzyme activity assay.

[0018] Figure 2 It is the polyphosphate kinase mutant ChPPK in Example 5. M1 and the originating strain polyphosphate kinase ChPPK M0 Comparison of reaction processes in the catalytic synthesis of ATP.

[0019] Figure 3 It is the polyphosphate kinase mutant ChPPK in Example 6. M1 and the originating strain polyphosphate kinase ChPPK M0 A comparison chart of the reaction process for the catalytic synthesis of GSH. (V) Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0021] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, solvent: water, pH: natural. LB solid medium is LB liquid medium with 20 g / L agar powder added.

[0022] Example 1: Induction of polyphosphate kinase expression in engineered bacteria

[0023] The T25S mutant of polyphosphate kinase (GenBank accession number ABG57400.1) from Cytophaga hutchinsonii, disclosed in patent application CN202310871912.5, is designated as ChPPK. M0 The engineered bacteria (nucleotide sequence as shown in SEQ ID NO:1, amino acid sequence as shown in SEQ ID NO:2) were inoculated into 10 μL of glycerol culture stored at -80°C in 10 mL of liquid LB medium (containing 50 μg / mL kanamycin) and cultured overnight at 37°C and 200 rpm. The culture was then transferred to 100 mL of fresh LB medium (containing 50 μg / mL kanamycin) at a 2% (v / v) inoculation concentration and cultured until OD200. 600The pH was 0.4–0.8, and IPTG was added to a final concentration of 0.1 mM. The cells were then induced and cultured at 28°C for 12 h. After the culture was completed, the cells were collected by centrifugation at 8000 rpm for 10 min at 4°C. The cells were washed twice with 0.9% physiological saline and the wet cells were collected.

[0024] SEQ ID NO:2

[0025] MATDFSKLSKYVETLRVKPKQSIDLKKDFDTDYDHKMLTKEEGEELLNLGISKLSEIQEKLYASGTKSVLIVFQAMDAAGKDGTVKHIMTGLNPQGVKVTSFKVPSKIELSHDYLWRHYVALPASGEIGIFNRSHYENVLVTRVHPEYLLSEQTSG VTAIEQVNQKFWDKRFQQINNFEQHISENGTIVLKFFLHVSKKEQKKRFIERIELDTKNWKFSTGDLKERAHWKDYRNAYEDMLANTSTKQAPWFVIPADDKWFTRLLIAEIICTELEKLNLTFPTVSLEQKAELEKAKAELVAEKSSDHHHHHH.

[0026] Example 2: Polyphosphate kinase saturation mutation

[0027] Predicting ChPPK using HotSpot Wizard 3.1 M0 The hotspot for modification, according to ChPPK in Example 1 M0 The predicted S22 site was subjected to saturation mutation based on the gene sequence (SEQ ID NO:1). Mutation primers were designed as shown in Table 1, with pET28a-ChPPK as the primary primer. M0 The plasmid was used as a template for full plasmid amplification. The PCR system consisted of: 25 μL of 2×phanta Max buffer, 1 μL of dNTP mixture (10 mM), 1 μL of each of the 10 μM mutant primers shown in Table 1, 1 μL of plasmid, 1 μL of Phanta Max DNA polymerase, and ddH2O to a final volume of 50 μL. The PCR conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 65℃ annealing for 30 s, and 72℃ extension for 6.5 min, for 30 cycles; and a final extension at 72℃ for 10 min. The PCR products were analyzed by 0.9% agarose gel electrophoresis. 20 μL of the PCR product was mixed with 1 μL of DpnI, digested at 37℃ for 3 h to remove the template plasmid DNA, and then inactivated at 65℃ for 10 min.

[0028] Table 1. Primers for site-directed mutagenesis of polyphosphokinase site 22

[0029]

[0030] Note: N=A / G / C / T, K=G / T, M=A / C

[0031] After the PCR reaction, the PCR products were analyzed by 0.9% agarose gel electrophoresis and found to be positive. Then, 1 μl of Dpn1 was added to the PCR reaction solution, and the template plasmid DNA was removed by enzyme digestion at 37℃ for 2 h, yielding 19 mutant vectors. These mutant vectors were then heat-transformed into E. coli BL21(DE3) competent cells. After recovery, the cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37℃. Single colonies were picked and inoculated into LB liquid medium containing a final concentration of 0.1 mmol / L kanamycin sulfate. After incubation, the cells were collected by centrifugation, plasmids were extracted, and sequencing was performed, yielding 19 polyphosphate kinase mutant engineered strains.

[0032] Example 3: Expression of polyphosphate kinase mutant and preparation of crude enzyme solution

[0033] The recombinant E. coli strain containing the polyphosphokinase constructed in Example 1 and the mutant polyphosphokinase strain constructed in Example 2 were inoculated into LB medium containing 0.1 mmol / L kanamycin in test tubes. After culturing at 37°C and 200 rpm for 6-8 hours, seed culture was obtained. The seed culture was then transferred at a volume concentration of 2% to 50 mL of fresh LB medium containing 0.1 mmol / L kanamycin and cultured at 37°C and 200 rpm until OD200. 600 When the pH is 0.6-0.8, add isopropyl-BD-thiogalactopyranoside (IPTG) to the above culture medium to a final concentration of 0.1 mmol / L, and incubate at 28°C for 12-16 h. Collect the fermentation broth and centrifuge at 8000 r / min for 10 min at 4°C, discard the supernatant, and obtain wet cells. Freeze at -20°C for 1 h.

[0034] The collected wet bacterial cells were resuspended in 50mM Tris-HCl buffer (pH=7.5) at a concentration of 10g / L, and then sonicated at a power of 200W for 1 second of operation followed by a 2-second rest for a total working time of 10 minutes. The resulting cell lysate was the crude enzyme solution.

[0035] Example 4: Polyphosphokinase activity assay

[0036] The activity of the crude enzyme solution obtained in Example 3 was determined.

[0037] The enzyme reaction system (10 mL) consisted of 15 mM sodium hexametaphosphate, 20 mM MgCl2, 5 mM ADP (adenosine diphosphate), and 50 mM Tris-HCl buffer (pH = 7.5). Crude enzyme solution at a concentration of 10 g / L (based on the pre-lysis cell concentration) was added. The reaction was incubated at 35°C with shaking for 5 min. The reaction was then terminated with 50 μL of 2 M HCl, followed by neutralization with 50 μL of NaOH aqueous solution. The mixture was centrifuged at 10000 r / min for 1 min. The supernatant was diluted 5-fold with ultrapure water and filtered through a 0.22 μm aqueous filter. ATP content was determined by HPLC. Relative enzyme activity was calculated, and the results are shown below. Figure 1 The results showed that the mutant with S replaced by A at position 22 of the amino acid sequence shown in SEQ ID NO:2 was denoted as ChPPK. M1 The nucleotide sequence is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:4. The activity is increased by 2 times, and the corresponding engineered bacterium is E. coli BL21(DE3) / pET28-ChPPK. M1 .

[0038] Enzyme activity is defined as the amount of enzyme required to generate 1 micromole of ATP per minute at 35°C, which is defined as 1 enzyme activity unit (U).

[0039] HPLC detection conditions for ATP content: C18 column (4.6×mmol / L, 5μm), 50mmol / L dipotassium hydrogen phosphate as mobile phase (pH adjusted to 7.0 with potassium dihydrogen phosphate, freshly prepared), 10μL injection volume, 1mL / min flow rate, 40℃, 10min retention time, detection at 254nm, ATP, ADP, and AMP peaked sequentially.

[0040] Example 5: Application of polyphosphokinase in whole-cell catalytic synthesis of ATP

[0041] E. coli BL21(DE3) / pET28-ChPPK were respectively M0 , E.coli BL21(DE3) / pET28-ChPPK M1 The crude enzyme solution was prepared according to the method in Example 3. The enzyme activity was assessed according to Example 4 to determine the reaction progress of catalyzing the synthesis of ATP from ADP. The reaction was carried out at 35°C for 4 hours, with samples taken every hour to detect the conversion rate. The reaction progress is shown in [Figure 4]. Figure 2 The results showed that, after 3 hours of reaction, based on a cell dosage of 10 g / L, the starting strain of polyphosphate kinase ChPPK... M0 The ADP conversion rate reached 69.57%, and the polyphosphate kinase mutant ChPPK... M1 The ADP conversion rate reached 72.33%.

[0042] Example 6: Application of whole-cell polyphosphoric acid kinase in glutathione synthesis

[0043] The initial reaction system (50 mL) contained the following raw materials at the following concentrations: 60 mmol / L cysteine, 80 mmol / L glycine, 80 mmol / L glutamic acid, and the crude enzyme solution prepared in Example 3 (using ChPPK). M0 or ChPPK M1 A 50 mL reaction system was prepared using whole-cell GSH (2.5 g / L), glutathione bifunctional synthase (10 g / L), 60 mmol / L MgSO4, 35 mM sodium hexametaphosphate, 2 mmol / L ATP, and 50 mM Tris-HCl buffer (pH 7.0). The reaction temperature was 35 °C, and the reaction time was 2 h. Intermittently, 1 mL of the reaction system was taken, and 100 μL of 2 mol / L HCl was added to terminate the reaction, followed by neutralization with 100 μL of 2 mol / L NaOH aqueous solution. The mixture was centrifuged at 10000 r / min for 1 min, the precipitate was discarded, and the supernatant was diluted 10-fold with ultrapure water and filtered through a 0.22 μm aqueous filter. The GSH content was detected by HPLC, and the concentration of GSH in the product was calculated using a GSH standard curve. The results are shown in [Figure 1]. Figure 3 The results showed that the starting strain contained polyphosphate kinase ChPPK. M0 Used in the glutathione synthesis system, the GSH yield reached 16.8 g / L after 2 hours of reaction; the mutant ChPPK M1 After 2 hours of reaction, the GSH yield reached 19.0 g / L, with a product formation rate of 87%.

[0044] Chromatographic conditions: C18 column (4.6×mmol / L, 5μm), mobile phase (dipotassium hydrogen phosphate and sodium heptanesulfonate added to 50mM Tris-HCl buffer (pH=7.0) to make final concentrations of 6.8g / L and 2.02g / L respectively, then adjusted to pH=2.8 with phosphoric acid to obtain a mixed solution; 950mL of the mixed solution was added to 50mL of methanol, freshly prepared for use), 10μL injection volume, 1mL / min flow rate, 30℃, 20min retention time, detected at 210nm.

Claims

1. A mutant of polyphosphate kinase, characterized in that, The polyphosphate kinase mutant is obtained by replacing the serine at position 22 of the amino acid sequence shown in SEQ ID NO: 2 with alanine, and the amino acid sequence is shown in SEQ ID NO.

4.

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

3. Use of the polyphosphate kinase mutant of claim 1 in constructing an ATP regeneration system.

4. Use of the polyphosphate kinase mutant of claim 1 or the recombinant genetically engineered bacterium of claim 2 in synthesizing glutathione.

5. The use according to claim 4, wherein the compound is ###0002### The application method is: using the wet bacteria body obtained after the induced expression of the polyphosphokinase mutant recombinant genetically engineered bacteria or the cell broken liquid after ultrasonic breaking as a catalyst, using sodium hexametaphosphate as a substrate, adding ATP, Mg 2+ , glutathione bifunctional synthetic enzyme, cysteine, glycine, glutamic acid and buffer solution with pH 7.0 to constitute a reaction system, and reacting completely at 35 DEG C to obtain product glutathione.

6. The use according to claim 5, wherein the compound is ###0002### The reaction system contains 1-5 g / L of wet bacteria in terms of whole cells, 1-5 g / L of cell broken solution in terms of whole cells before breaking, 5-20 g / L of glutathione bifunctional synthesis enzyme, 20-70 mM of sodium hexametaphosphate, 1-20 mM of ATP, 1-20 mM of Mg 2+ 20-70 mM of sodium hexametaphosphate, 30-90 mM of cysteine, 60-100 mM of glycine, and 60-100 mM of glutamic acid.

7. The use according to claim 5, wherein the compound is ###0002### The catalyst is prepared as follows: the polynucleotide kinase mutant recombinant genetically engineered bacteria are inoculated into liquid LB medium containing 50 μg / mL kanamycin, cultured at 37 °C, 180 rpm overnight, then transferred into fresh liquid LB medium containing 50 μg / mL kanamycin at a volume concentration of 2%, cultured at 37 °C, 180 rpm until the bacterial concentration OD 600 is 0.6-0.8, then IPTG is added to the culture medium at a final concentration of 0.1 mM, and the culture is induced at 28 °C, 180 rpm for 12 h; the fermentation broth is centrifuged at 8000 rpm for 10 min at 4 °C, and the wet bacterial cells are collected; the wet bacterial cells are resuspended with 50 mM Tris-HCl buffer at pH=7.5 at a concentration of 10 g / L, ultrasonically broken, the breaking power is 200 W, 1 s of work, 2 s of rest, the total working time is 10 min, and after breaking, the cell broken liquid is obtained.

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