An isocitrate dehydrogenase mutant with enhanced thermal stability, and its preparation method and application

Abstract

The present invention belongs to the technical field of enzyme genetic engineering, and specifically relates to an isocitrate dehydrogenase mutant with enhanced thermal stability, a preparation method thereof, and an application thereof. The mutant of the present invention is obtained by performing an I111Y mutation on isocitrate dehydrogenase with an amino acid sequence as shown in SEQ ID NO.1. Experiments show that its half-life is 1.3 times that of the wild-type enzyme, and the Tm value is increased by 6.7% compared with the wild-type enzyme. Therefore, the present invention expands the resources of isocitrate dehydrogenase genes and also provides an excellent isocitrate dehydrogenase for the conversion of isocitrate in the isocitrate metabolism process.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme genetic engineering, and specifically relates to an isocitrate dehydrogenase mutant with enhanced thermal stability, a preparation method thereof, and an application thereof. Background Art

[0002] Isocitrate dehydrogenase (ICDH) is one of the key enzymes in the tricarboxylic acid cycle. It not only provides energy for the metabolism of organisms, but also provides precursor substances and essential reducing power for the synthesis of biological macromolecules in cells. At the same time, it plays an important role in important physiological metabolisms such as defense against oxidative damage. In the field of scientific research, ICDH is often used as a biocatalyst in the laboratory for synthesizing specific compounds or studying metabolic pathways. Most of the isocitrate dehydrogenases on the market are derived from humans or animals, which have good activity and stability in the in vivo environment, but their stability in vitro is poor. During the use of related products, long-term exposure to room temperature will cause a decrease in the enzyme activity.

[0003] To solve this problem, it is expected to modify isocitrate dehydrogenase through genetic engineering technology, so as to extend the service life of the enzyme under normal temperature conditions and reduce the storage and transportation costs. Summary of the Invention

[0004] The purpose of the present invention is to provide an isocitrate dehydrogenase (ICDH) mutant with enhanced thermal stability, a preparation method thereof, and an application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention intends to introduce amino acid mutations related to thermal stability into ICDH by protein rational design and molecular biology techniques, and perform molecular modification on ICDH to improve its thermal stability. After the modification, the thermal stability of the ICDH mutant is significantly improved and the activity is also correspondingly improved, laying a good foundation for realizing lower-cost transportation and preservation in the future.

[0005] The present invention provides an isocitrate dehydrogenase mutant with enhanced thermal stability, which is obtained by mutating the I111 in the amino acid sequence shown in SEQ ID NO: 1.

[0006] Specifically, the isocitrate dehydrogenase mutant with improved thermal stability is obtained by the I111Y mutation in the amino acid sequence shown in SEQ ID NO: 1.

[0007] Preferably, the amino acid sequence of the isocitrate dehydrogenase mutant with improved thermal stability is as shown in SEQ ID NO: 2.

[0008] The present invention also provides a coding polynucleotide of the isocitrate dehydrogenase mutant with improved thermal stability.

[0009] Preferably, the nucleotide sequence of the isocitrate dehydrogenase mutant with improved thermal stability is as shown in SEQ ID NO: 4.

[0010] The present invention provides an expression vector containing the encoding polynucleotide.

[0011] The present invention further provides a recombinant host cell containing the encoding polynucleotide or the expression vector; preferably, the host cell is Escherichia ( Escherichia ).

[0012] The present invention also provides the application of the isocitrate dehydrogenase mutant with improved thermal stability or its encoding nucleotide in the preparation of the conversion of NADH, NADPH or isocitrate.

[0013] Preferably, its substrates are NADP oxidized coenzyme I and DL-isocitrate trisodium salt hydrate.

[0014] By performing the I111Y mutation on the ICDH sequence with the amino acid sequence as shown in SEQ ID NO.1, the mutant ICDH-Mutant2 was obtained. By calculating the half-lives of the wild-type enzyme and the mutant enzyme at 47 °C, the half-life of the mutant ICDH-Mutant2 at 47 °C was 12.96 h, which was increased by 237.5% compared with 3.84 h of the wild-type enzyme. That is, as the incubation time prolonged, the residual enzyme activity of the mutant enzyme of the present invention decreased more slowly compared with the wild-type enzyme. The Tm value of the mutant ICDH-Mutant2 was increased by 6.2 °C compared with that of the wild-type, that is, its stability was significantly improved. The present invention expands the resources of the isocitrate dehydrogenase gene and also provides an excellent isocitrate dehydrogenase for the conversion of isocitrate in the isocitrate metabolism process. Description of the Drawings

[0015] Figure 1 It is the map of the constructed pET28a-icd plasmid vector.

[0016] Figure 2 It is the map of the constructed pET28a-icd-Mutant2 plasmid vector.

[0017] Figure 3 It is the SDS-PAGE electrophoresis gel pattern of isocitrate dehydrogenase and its mutants.

[0018] Figure 4 It is the influence of the storage time at 47 °C on the specific enzyme activity of ICDH and its mutants.

[0019] Figure 5 It is the half-life of incubating ICDH and its mutants at 47 °C.

[0020] Figure 6 Tm values of wild-type ICDH and mutants. Specific implementation mode

[0021] The following further illustrates the present invention in conjunction with implementation examples. The following implementation examples are intended to enable those skilled in the art to better understand the present invention, but do not impose any limitations on the present invention.

[0022] The isocitrate dehydrogenase encoding gene selected by the present invention icd , with the Genebank accession number 945702. The isocitrate dehydrogenase encoded by it can catalyze isocitrate and nicotinamide adenine dinucleotide phosphate (NADP+) to generate α-ketoglutaric acid and reduced nicotinamide adenine dinucleotide phosphate (NADPH).

[0023] The original plasmid pET28a is from biovector (http: / / www.biovector.net / );

[0024] Original strain E. coli MG1655 is from laboratory storage;

[0025] E. coli The BL21(DE3) competent cells are from NEB (http: / / www.neb-china.com / );

[0026] The used NADPH standard product is purchased from sigma company (http: / / www.sigmaaldrich.com / sigma-aldrich);

[0027] The used restriction endonucleases, dephosphorylases, DNA ligases, etc., molecular biology reagents are purchased from thermo company (http: / / www.thermoscientificbio.com / fermentas);

[0028] The used other biochemical reagents (such as tryptone, yeast extract, NaCl, HEPES, TRIS, imidazole, isocitric acid, NADP + etc.) are purchased from Sangon Biotech (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0029] Example 1. Obtaining of isocitrate dehydrogenase mutants

[0030] First, search in the PDB database from E. coliThe structure of isocitrate dehydrogenase (ICDH, Genebank accession number 945702) of MG1655 is 4AJA. After splitting the ICDH dimer into monomers using PyMOL, analysis found that the inner side of the loop (101 - 113) where I111 is located is involved in the binding with the substrate, and the stability of this loop has a certain impact on the stability and activity of the enzyme. On the outer side of this loop, an interaction is formed with the loop (79 - 85). Increasing the interaction between the two loop regions can reduce the structural fluctuations of the target loop, thereby enhancing the stability of the enzyme and the stability of substrate binding. The I111Y mutation changes the single hydrophobic interaction residue I to the amphiphilic residue Y, which not only maintains the hydrophobic interaction with W83 but also increases the hydrogen bond interaction formed with D81. Compared with the wild-type residue I, it enhances the interaction strength between the two loops with insufficient stability, so it is beneficial to improve the stability of the enzyme and increase the enzyme activity.

[0031] The Gibson assembly method based on the principle of Overlap extension PCR is adopted. Gibson assembly requires introducing complementary overlapping sequences in the primers at both ends of the PCR, so that specific overlapping regions are formed between different fragments to be assembled. The 5'-exonuclease activity of T5 exonuclease is used to process different fragments. After PCR annealing, each overlapping region serves as a primer for each other, and then DNA polymerase extension and ligase are used to repair the nicks, and the assembly of multi-fragment DNA can be completed. If designed point mutations are introduced into the overlapping sequences, the corresponding point mutation plasmids can be constructed.

[0032] Then construct an overexpression vector of isocitrate dehydrogenase using the commercial protein expression vector pET28a icd , whose nucleotide sequence is SEQ ID NO.3. Using the genome of Escherichia coli E. coli MG1655 as a template, primers P0-1 and P0-2 are used to amplify the gene icd fragment (about 1.2 kb). icd The fragment and the pET28a plasmid are digested with Thermo Fast digest NdeI and EcoRI double digestion. After ligation and transformation, the expression vector pET28a icd of the gene is obtained -icd (see Figure 1 ), and the sequencing detection is correct. The plasmid with the correct sequencing result is transferred into the commercial competent Escherichia coli E. coli BL21(DE3) by the traditional calcium chloride method, and BL21-1 overexpressing isocitrate dehydrogenase ( icd ) is obtained.

[0033] Then, using the isocitrate enzyme geneicd The plasmid pET28a-icd where it is located (see Figure 1 ) was used as a template for PCR amplification. Using primers P1 and P2-Mutant2, amplifying with the pET28a-icd plasmid as a template, a target fragment F1-Mutant2 of 5.1 kb can be obtained. Using primers P3-Mutant2 and P4; amplifying with the pET28a-icd plasmid as a template, a target fragment F2-Mutant2 of 1.5 kb can be obtained. After treating the F1-Mutant2 and F2-Mutant2 fragments with T5 exonuclease, annealing, ligation and transformation were carried out to obtain icd The expression vector pET28a of the gene mutant Mutant2 (nucleotide sequence is SEQ ID NO.4) - icd-Mutant2 (see Figure 2 ), and the sequencing was detected without error. The plasmid with correct sequencing results was transferred into commercial competent Escherichia coli E. coli BL21(DE3) by the traditional calcium chloride method to obtain BL21-Mutant2 with overexpression of isocitrate dehydrogenase ( icd ).

[0034] Table 1. Primer sequences used for strain construction

[0035]

[0036] Example 2. Purification and concentration of isocitrate dehydrogenase and its mutants

[0037] The preparation method of isocitrate dehydrogenase is as follows:

[0038] 1) Inoculate Escherichia coli BL21 into 500 mL of LB medium, culture in a shaker at 37 °C and 220 rpm until the OD600 reaches 0.6, add the inducer IPTG to a final concentration of 0.5 mM, culture at 16 °C for 20 h, centrifuge at 4 °C and 4200 rpm for 20 min to collect the bacteria, and suspend them with 20 mL of lysis buffer.

[0039] 2) Collect the suspension of BL21 obtained in step 1), break the cells under the action of a high-pressure homogenizer, and treat them at 4 °C, 1200 bar, and an oil pressure of 18 Kg / cm 3 for 3 - 5 min. After breaking, centrifuge at 4 °C and 8000 rpm for 30 min, and collect the supernatant to obtain a crude enzyme solution.

[0040] 3) Purify the protein from the crude enzyme solution obtained in step 2) using gravity nickel column purification method. At 4°C, allow the crude enzyme solution to flow through a column packed with nickel filler, wash away the impurities with 1 L of washing solution to remove the contaminating proteins. Then elute with 20 mL of elution solution and collect the eluate to obtain a high-purity isocitrate dehydrogenase solution.

[0041] 4) Collect the target protein solution obtained in step 3) and concentrate the protein using an ultrafiltration tube with a pore size of 30 KDA. Centrifuge at 4800 rpm and 4°C. After the volume of the ultrafiltration tube reaches less than 5 mL, continue to add 15 mL of lysis buffer and centrifuge. Repeat this process 2 times, and continue to centrifuge until the remaining volume is 1 mL, then aliquot. Obtain a high-concentration isocitrate dehydrogenase solution. Then measure the concentration of isocitrate dehydrogenase using the BCA method.

[0042] 5) Perform SDS-PAGE electrophoresis on the isocitrate dehydrogenase solution obtained in step 4) to confirm that the size of isocitrate dehydrogenase is correct (see Figure 3 , lane 1 is wild-type isocitrate dehydrogenase (45 kDa), lane 2 is mutant enzyme (45 kDa), M is Marker, M: Marker; isocitrate dehydrogenase (45 kDa)).

[0043] Among them, the LB medium formula is 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, adjust the pH to 7.5. Sterilize at 0.1 Mpa for 20 min.

[0044] Lysis Buffer (1 L): 50 mM Tris-HCl, 500 mM NaCl, pH 8.5. 1 L formula: 29.22 g NaCl, 50 mL Tris-HCl, 950 mL ddH2O.

[0045] Washing solution: 50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.5. 1 L formula: 29.22 g NaCl, 50 mL 1 M Tris-HCl (pH 7.5), 1.36 g imidazole, make up to 1 L with ddH2O.

[0046] Elution solution: 50 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, pH 7.5. 1 L formula: 29.22 g NaCl, 50 mL 1 M Tris-HCl (pH 7.5), 17.025 g imidazole, make up to 1 L with ddH2O.

[0047] Example 3: Determination of the concentration and specific enzyme activity of isocitrate dehydrogenase

[0048] The specific steps for protein concentration quantification are as follows:

[0049] Dilute the sample to an appropriate dilution factor for color development. Take 25 μL and mix it thoroughly with 200 μL of the reaction solution in the kit, and incubate at 37 °C for 30 min. Detect using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 562 nm.

[0050] The specific steps for the determination of the specific enzyme activity of isocitrate dehydrogenase are as follows:

[0051] The reaction is carried out at 37 °C. By measuring the production amount of NADPH at 340 nm, the enzyme activity of isocitrate dehydrogenase is characterized. The reaction system contains: Tris-HCl (82.2 mmol / L, pH 8.0), MgCl2 (3 mmol / L), NADP oxidized coenzyme I (0.6 mmol / L), and DL-sodium isocitrate hydrate (5 mmol / L). Mix the above substances and incubate at 37 °C for 30 min. Start the reaction by adding the enzyme solution, and then monitor the production of NAD(P)H at 340 nm on an ELISA reader. The enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol of NAD(P)H per minute.

[0052] The specific enzyme activity results of the wild-type enzyme and the mutant enzyme are as Figure 4 shown. The initial specific enzyme activity of the mutant enzyme is 9.8% higher than that of the wild-type enzyme. However, after incubation at 47 °C for 8 hours, the specific enzyme activities of the wild-type enzyme and the mutant enzyme decreased by 76.4% and 67.1% respectively. At this time, the specific enzyme activity of the mutant enzyme is 53.1% higher than that of the wild-type enzyme, indicating that the stability of the mutant enzyme is significantly better than that of the wild-type enzyme and is more suitable for long-term storage.

[0053] Detection of the stability of the mutant enzyme in Example 4

[0054] The wild-type enzyme and the mutant enzyme are incubated at 47 °C. Samples are taken at 0 h, 2 h, 4 h, 6 h, and 8 h respectively and incubated at 37 °C for 30 min to detect the ICDH enzyme activity. The calculated half-life results are as Figure 5 shown. Under the same storage time at 47 °C, the half-life of the mutant enzyme is significantly higher than that of the wild-type enzyme, indicating that the stability of the mutant enzyme Mutant2 has also been significantly improved. By calculating the half-lives of the wild-type enzyme and the mutant enzyme under the condition of 47 °C, the half-life of the mutant enzyme Mutant2 at 47 °C is 4.81 h, which is 25.3% higher than that of the wild-type enzyme's 3.84 h.

[0055] Detection of the Tm value of the mutant enzyme in Example 5

[0056] Description of the detection method:

[0057] 1. Take 16 different 9 μL samples, with each sample repeated in 3 wells, for a total of 48 wells, which need to be added to 3 UNi tubes and placed in the instrument's sample chamber for detection simultaneously;

[0058] 2. This experiment can perform high-throughput detection of 48 samples at once, simultaneously detecting protein particle size and polydispersity, melting temperature (Tm), and onset aggregation temperature (Tagg). Protein intrinsic fluorescence (IF) is used to detect Tm; static light scattering (SLS) is used to detect Tagg; DLS is used to detect the current state of the protein in terms of particle size or aggregation;

[0059] 3. Set the detection parameters in the UNcle Client 5.03 software: For DLS parameter settings, each sample is detected 4 times, 5 s each time. The instrument will detect DLS data at the start and end of the temperature increase. For the temperature increase parameter settings, Start Temp is 20 °C, EndTemp is 95 °C, and the Rate of temperature increase is 0.25 °C / min. The Plate hold time is kept at 32 s without adjustment. Click "Apply";

[0060] 4. Place the sample-loaded UNi into the instrument's internal sample stage, and then click "Manual Configuration". Position the mouse at one of the sample wells (left click), and then click "Trigger" in the lower right corner. Observe the fluorescence spectrum curve area in the upper right corner. Usually, the excitation spectrum of protein intrinsic fluorescence will show a normal distribution peak between 300 - 400 nm. Click "Next"; Enter the "Please configure your DLS settings" interface, check "Auto", and keep other parameters at their default values. After the DLS Intensity stabilizes, click "Start" in the lower right corner to start the experiment;

[0061] 5. After the experiment, use the analysis software UNcle Analysis 5.03 to automatically calculate Tm&Tagg, and use the default BCM (Barycentric mean) method to analyze Tm.

[0062] The results are as Figure 6 shown. The Tm value of the mutant enzyme Mutant2 is 59.2 °C, which is 3.6 °C higher than that of the wild type (Tm value is 55.6 °C), indicating that the thermal stability of the mutant enzyme Mutant2 has been significantly improved.

Claims

1. A mutant isocitrate dehydrogenase with enhanced thermal stability, characterized in that, The heat stability-enhanced isocitrate dehydrogenase is obtained by performing the I111Y mutation on the amino acid sequence shown in SEQ ID NO:

1.

2. A polynucleotide encoding the heat stability-enhanced isocitrate dehydrogenase mutant as described in claim 1.

3. The encoded polynucleotide according to claim 2, wherein The nucleotide sequence of the heat stability-enhanced isocitrate dehydrogenase mutant is shown in SEQ ID NO:

4.

4. An expression vector containing the polynucleotide encoding as described in claim 2 or 3.

5. A recombinant host cell containing the polynucleotide encoding as described in claim 2 or 3, or the expression vector as described in claim 4.

6. The recombinant host cell according to claim 5, wherein, The host cell is Escherichia.

7. Use of the heat stability-enhanced isocitrate dehydrogenase mutant as described in claim 1 or its encoding nucleotide in the preparation of the conversion of NADH, NADPH or isocitrate.

8. The application according to claim 7, wherein Its substrates are NADP oxidized coenzyme I and DL-sodium isocitrate hydrate.

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