An isocitrate dehydrogenase mutant with enhanced thermal stability and its applications

Abstract

The present invention belongs to the technical field of enzyme genetic engineering, and particularly relates to an isocitrate dehydrogenase mutant with enhanced thermal stability and its application. The mutant of the present invention is obtained by performing the A44V mutation on isocitrate dehydrogenase with the amino acid sequence shown in SEQ ID NO.1. Experiments show that its half-life is 3.4 times that of the wild-type enzyme, and the Tm value is increased by 11% 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 during 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 and its application. 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 biological catalyst in the laboratory for synthesizing specific compounds or conducting research on 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 the enzyme activity to decrease.

[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 enzyme (ICDH) mutant with enhanced thermal stability, its preparation method and application, 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 conduct molecular modification to improve the thermal stability of ICDH. 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 44th amino acid A in the amino acid sequence shown in SEQ ID NO: 1.

[0006] Specifically, the isocitrate dehydrogenase mutant with improved thermal stability is obtained by mutating A44V 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 polynucleotide encoding 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-sodium isocitrate hydrate.

[0014] In the present invention, by performing an A44V mutation on the ICDH sequence with the amino acid sequence as shown in SEQ ID NO.1, the mutant ICDH-Mutant 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-Mutant at 47 °C is 12.96 h, which is increased by 237.5% compared with 3.84 h of the wild-type enzyme. That is, as the incubation time prolongs, the residual enzyme activity of the mutant enzyme of the present invention decreases more slowly compared with the wild-type enzyme. The Tm value of the mutant ICDH-Mutant is increased by 6.2 °C compared with that of the wild-type, that is, its stability has been 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. BRIEF 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-Mutant plasmid vector.

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

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

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

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

[0021] The present invention will be further described below 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 limit the present invention in any way.

[0022] The isocitrate dehydrogenase encoding gene selected by the present invention icd has a Genebank accession number of 945702.

[0023] The isocitrate dehydrogenase encoded by it can catalyze the formation of α-ketoglutaric acid and reduced nicotinamide adenine dinucleotide phosphate (NADPH) from isocitric acid and nicotinamide adenine dinucleotide phosphate (NADP+).

[0024] The original plasmid pET28a is sourced from biovector (http: / / www.biovector.net / );

[0025] The original strain E. coli MG1655 is sourced from laboratory storage;

[0026] E. coli The BL21(DE3) competent cells are sourced from NEB (http: / / www.neb-china.com / );

[0027] The used NADPH standard product is purchased from sigma company (http: / / www.sigmaaldrich.com / sigma-aldrich);

[0028] The used restriction endonucleases, dephosphorylases, DNA ligases, etc., and molecular biology reagents are purchased from thermo company (http: / / www.thermoscientificbio.com / fermentas);

[0029] 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 / ).

[0030] Example 1. Obtaining of isocitrate dehydrogenase mutants

[0031] First, search in the PDB database for those from E. coliThe structure of isocitrate dehydrogenase (ICDH, Genebank accession number 945702) of MG1655 is 4AJA. After using PyMOL to split the ICDH dimer into monomers, analysis found that the residue at position 44 is at the interface between the inside and outside of the protein structure and is on the helix structure. The relatively short side chain forms a difference with D40 of the previous helix and V48 of the next helix, and the helix it binds to has G409 at the corresponding residue, a residue lacking a side chain. This forms a channel at this position that allows water molecules to penetrate into the inner side of the enzyme structure, which is not conducive to the stability of the enzyme. Therefore, by mutating A to V, a structure with the same side chain length as D and V at the corresponding positions of the upper and lower helices is formed, thus forming a tight binding with the adjacent helix structure, blocking this channel, and preventing water molecules from penetrating into the inner side of the enzyme structure.

[0032] 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 repair of the nicks are carried out to complete the assembly of multi-fragment DNA. If designed point mutations are introduced into the overlapping sequences, the corresponding point mutation plasmids can be constructed.

[0033] Then construct an overexpression 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 double digested with Thermo Fast digest NdeI and EcoRI . 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 to obtain BL21-1 overexpressing isocitrate dehydrogenase ( icd ).

[0034] Again, using the plasmid pET28a-icd where the isocitrate enzyme gene icd is located (seeFigure 1 ) Perform PCR amplification using it as a template. Using primers P1 and P2-Mutant, amplify with the pET28a-icd plasmid as the template to obtain a 5.1 kb target fragment F1-Mutant. Using primers P3-Mutant and P4, amplify with the pET28a-icd plasmid as the template to obtain a 1.5 kb target fragment F2-Mutant. After treating the F1-Mutant and F2-Mutant fragments with T5 exonuclease, perform annealing, ligation, and transformation to obtain icd The expression vector pET28a of the gene mutant Mutant (nucleotide sequence is SEQ ID NO.4) - icd-Mutant (see Figure 2 ), and the sequencing is correct. Transfer the plasmid with correct sequencing results into commercial competent Escherichia coli E. coli BL21(DE3) by the traditional calcium chloride method to obtain BL21-Mutant overexpressing the isocitrate dehydrogenase ( icd ) mutant Mutant.

[0035] Table 1. Primer sequences used for strain construction

[0036]

[0037] Example 2. Purification and concentration of isocitrate dehydrogenase and its mutants

[0038] The preparation method of isocitrate dehydrogenase is as follows:

[0039] 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 cells, and suspend them with 20 mL of lysis buffer.

[0040] 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.

[0041] 3) Use the gravity nickel column purification method to purify the protein from the crude enzyme solution obtained in step 2). At 4 °C, allow the crude enzyme solution to flow through a column filled with nickel packing, wash away the miscellaneous proteins with 1 L of washing solution. Then elute with 20 mL of elution solution, collect the effluent, and obtain a high-purity isocitrate dehydrogenase solution.

[0042] 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 operation 2 times, and continue to centrifuge until the remaining volume is 1 mL, then aliquot. A high-concentration isocitrate dehydrogenase solution is obtained. Then, use the BCA method to determine the concentration of isocitrate dehydrogenase.

[0043] 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; isocitrate dehydrogenase (45 kDa)).

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] Example 3: Determination of the Concentration and Specific Enzyme Activity of Isocitrate Dehydrogenase

[0049] The specific steps for protein concentration quantification are as follows:

[0050] Dilute the sample to an appropriate multiple for color development. Take 25 μL and mix it well with 200 μL of the kit reaction solution, and incubate at 37 °C for 30 min. Detect using an enzyme-labeled instrument at a wavelength of 562 nm.

[0051] The specific steps for measuring the specific enzyme activity of isocitrate dehydrogenase are as follows:

[0052] 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 them 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 a microplate reader. The enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol of NAD(P)H per minute.

[0053] 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 wild-type enzyme is 39% higher than that of the mutant 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 36.4% respectively. At this time, the specific enzyme activity of the mutant enzyme exceeded that of the wild-type enzyme by 93.6%, 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.

[0054] Example 4 Detection of the stability of the mutant enzyme

[0055] 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 the ICDH enzyme activity is detected after incubating at 37 °C for 30 min. The half-life results are calculated as Figure 5 shown. When the mutant enzyme is stored at 47 °C for the same time, the half-life is significantly higher than that of the wild-type enzyme, indicating that the stability of the mutant enzyme Mutant 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 Mutant at 47 °C is 12.96 h, which is 237.5% higher than that of the wild-type enzyme's 3.84 h.

[0056] Example 5 Detection of the Tm value of the mutant enzyme

[0057] Description of the detection method:

[0058] 1. Take 16 different 9 μL samples, repeat each sample in 3 wells, a total of 48 wells, and add them to 3 UNi tubes. At the same time, place them in the instrument sample chamber for detection;

[0059] 2. This experiment can detect 48 samples in a high-throughput manner at one time, and simultaneously detect protein particle size and polydispersity, melting temperature (Tm), and onset aggregation temperature (Tagg). Intrinsic protein fluorescence (IF) is used to detect Tm; static light scattering (SLS) is used to detect Tagg; DLS is used to detect the particle size or aggregation of the current protein state;

[0060] 3. Set the detection parameters in the UNcle Client 5.03 software: For DLS parameter settings, each sample is detected 4 times for 5 s each. The instrument will detect DLS data at the beginning and end of the temperature increase. Set the temperature increase parameters: 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 32 s and does not need to be adjusted. Click "Apply".

[0061] 4. Place the sample-loaded UNi on the sample stage in the instrument, 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 spectral curve area in the upper right corner. Usually, the excitation spectrum of intrinsic protein 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 as default values. After the DLS Intensity is stable, click "Start" in the lower right corner to start the experiment;

[0062] 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.

[0063] The results are as Figure 6 shown. The Tm value of the mutant enzyme Mutant is 61.8 °C, which is 6.2 °C higher than that of the wild type (Tm value is 55.6 °C), indicating that the thermal stability of the mutant enzyme Mutant has been significantly improved.

Claims

1. An isocitrate dehydrogenase mutant with enhanced thermal stability, characterized in that, The thermostability-enhanced isocitrate dehydrogenase is obtained by A44V mutation of the amino acid sequence shown in SEQ ID NO:

1.

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

3. The encoded polynucleotide according to claim 2, wherein, The nucleotide sequence of the thermostability-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 described is Escherichia ( Escherichia ).

7. Use of the thermostability-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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