Isocitrate dehydrogenase mutant with enhanced thermal stability and application thereof

Abstract

The invention belongs to the technical field of enzyme gene engineering, and particularly relates to an isocitrate dehydrogenase mutant with enhanced thermal stability and application thereof. According to the invention, a mutant is obtained by carrying out A44V mutation on isocitrate dehydrogenase with an amino acid sequence as shown in SEQ ID NO. 1. Experiments show that the half-life period is 3.4 times that of a wild type enzyme, and the Tm value is increased by 11% compared with that of the wild type enzyme. Therefore, the resource of the isocitrate dehydrogenase gene is expanded, and excellent isocitrate dehydrogenase is provided for the conversion of isocitric acid in the metabolic process of isocitric acid.

Description

Technical Field

[0001] The invention belongs to the field of enzyme gene engineering, and particularly relates to an isocitrate dehydrogenase mutant with enhanced thermal stability and 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 precursors and essential reducing power for the synthesis of intracellular biomacromolecules. It also plays an important role in important physiological metabolism such as defense against oxidative damage. In the field of scientific research, ICDH is often used as a biocatalyst in laboratories for the synthesis of specific compounds or for metabolic pathway research. Most isocitrate dehydrogenases on the market are derived from humans or animals. They have good activity and stability in the in vivo environment, but poor stability in vitro. Long-term exposure of related products to room temperature during use will lead to reduced enzyme activity.

[0003] To solve this problem, it is hoped that isocitrate dehydrogenase can be modified through genetic engineering technology to extend the service life of the enzyme at room temperature and reduce storage and transportation costs. Summary of the invention

[0004] The purpose of the present invention is to provide an isocitrate esterase (ICDH) mutant with enhanced thermal stability and a preparation method and 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 using protein rational design and molecular biology technology, and to perform molecular transformation of ICDH to improve the thermal stability. After the transformation, the thermal stability of the ICDH mutant is significantly improved and the activity is also correspondingly improved, which lays a good foundation for realizing low-cost transportation and preservation in the future.

[0005] The present invention provides an isocitrate dehydrogenase mutant with enhanced thermal stability, which is obtained by mutation of the amino acid sequence A44 as shown in SEQ ID NO:1.

[0006] Specifically, the isocitrate dehydrogenase mutant with improved thermal stability is obtained by the presence of A44V mutation in the amino acid sequence as shown in SEQ ID NO:1.

[0007] Preferably, the amino acid sequence of the isocitrate dehydrogenase mutant with improved thermal stability is 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 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 a member of the genus Escherichia ( Escherichia ).

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

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

[0014] The present invention obtains a mutant ICDH-Mutant by performing A44V mutation on the ICDH sequence shown in SEQ ID NO.1. By calculating the half-life of the wild-type enzyme and the mutant enzyme at 47°C, the half-life of the mutant ICDH-Mutant at 47°C is 12.96h, which is 237.5% higher than that of the wild-type enzyme at 3.84h. That is, as the incubation time increases, the residual enzyme activity of the mutant enzyme of the present invention decreases more slowly than that of the wild-type enzyme. The Tm value of the mutant ICDH-Mutant is increased by 6.2°C compared to that of the wild-type, that is, its stability is significantly improved. The present invention expands the resources of isocitrate dehydrogenase genes and also provides excellent isocitrate dehydrogenase for the conversion of isocitrate in the process of isocitrate metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0016] Figure 2 This is a map of the constructed pET28a-icd-Mutant plasmid vector.

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

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

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

[0020] Figure 6 are the Tm values ​​of wild-type ICDH and mutants. DETAILED DESCRIPTION

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

[0022] The isocitrate dehydrogenase encoding gene selected by the present invention icd , Genebank accession number is 945702.

[0023] The isocitrate dehydrogenase encoded by it can catalyze isocitrate and nicotinamide adenine dinucleotide phosphate (NADP+) to produce α-ketoglutarate and reduced nicotinamide adenine dinucleotide phosphate (NADPH).

[0024] The original plasmid pET28a was from biovector ( http: / / www.biovector.net / ); Original strain E. coli The source of MG1655 was laboratory storage; E. coli BL21 (DE3) competent cells were sourced from NEB (http: / / www.neb-china.com / ); The NADPH standard used was purchased from Sigma ( http: / / www.sigmaaldrich.com / sigma-aldrich ); The restriction enzymes, dephosphorylase, DNA ligase, and molecular biology reagents used were purchased from Thermo Corporation ( http: / / www.thermoscientificbio.com / fermentas ); Other biochemical reagents used (such as tryptone, yeast extract, NaCl, HEPES, TRIS, imidazole, isocitrate, NADP + etc.) were purchased from Sangon Biotechnology (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0025] Example 1. Obtaining isocitrate dehydrogenase mutants First search the PDB database for E. coliThe structure 4AJA of isocitrate dehydrogenase (ICDH, Genebank accession number 945702) of MG1655 was analyzed, and the ICDH dimer was divided into monomers using PyMOL. The analysis found that the 44th residue was at the interface between the inside and outside of the protein structure, and in the helix structure, the relatively short side chain was different from the D40 of the previous helix and the V48 of the next helix, and the corresponding residue of the helix to which it was bound was G409, a residue with a missing side chain. This allows a channel to be formed at this position, allowing water molecules to penetrate from here to the inside 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 length of the side chains of D and V at the corresponding positions of the upper and lower helices is formed, thereby forming a tight bond with the adjacent helix structure, blocking this channel and preventing water molecules from penetrating into the enzyme structure.

[0026] The Gibson assembly method based on the principle of Overlap extension PCR is used. Gibson assembly requires the introduction of overlapping sequences that can be complementary to each other in the primers at both ends of PCR, so that specific overlapping regions are formed between the different fragments to be assembled, and the 5' exonuclease activity of T5 exonuclease is used to treat different fragments. After PCR annealing, each overlapping region is used as a primer for each other, and then the DNA polymerase is extended and the ligase is used to repair the incision to complete the assembly of multi-fragment DNA. If a designed point mutation is introduced into the overlapping sequence, the corresponding point mutation plasmid can be constructed.

[0027] Then, a commercial protein expression vector pET28a was constructed to overexpress isocitrate dehydrogenase ( icd , whose nucleotide sequence is SEQ ID NO.3). E. coli The MG1655 genome was used as a template and primers P0-1 and P0-2 were used to amplify the gene icd Fragment (about 1.2kb). icd The fragment and pET28a plasmid were digested using Thermo Fast digest N i and EcoRI Double enzyme digestion, ligation and transformation to obtain icd Gene expression vector pET28a -icd (See Figure 1 The plasmid with the correct sequencing result was transferred into commercial competent Escherichia coli by traditional calcium chloride method. E. coli BL21 (DE3), obtained isocitrate dehydrogenase ( icd ) overexpressed BL21-1.

[0028] Isocitrate gene icd The plasmid pET28a-icd (see Figure 1 ) as a template for PCR amplification. Using primers P1 and P2-Mutant, a 5.1 kb target fragment F1-Mutant can be obtained by amplification with pET28a-icd plasmid as a template. Using primers P3-Mutant and P4; a 1.5 kb target fragment F2-Mutant can be obtained by amplification with pET28a-icd plasmid as a template. The F1-Mutant and F2-Mutant fragments were treated with T5 exonuclease, annealed, connected and transformed to obtain icd Expression vector pET28a of gene mutant Mutant (nucleotide sequence is SEQ ID NO.4) - icd-Mutant (see Figure 2 The plasmid with the correct sequencing result was transferred into commercial competent Escherichia coli by traditional calcium chloride method. E. coli BL21 (DE3), obtained isocitrate dehydrogenase ( icd ) BL21-Mutant overexpressing the mutant Mutant.

[0029] Table 1. Primer sequences used for strain construction

[0030] Example 2: Purification and concentration of isocitrate dehydrogenase and its mutants The preparation method of isocitrate dehydrogenase is as follows: 1) Inoculate E. coli BL21 into 500 mL LB medium, culture at 37°C, 220 rpm in a shaker until OD600 reaches 0.6, add inducer IPTG to a final concentration of 0.5 mM, culture at 16°C for 20 h, collect the cells by centrifugation at 4°C, 4200 rpm for 20 min, and suspend in 20 mL lysis buffer.

[0031] 2) Collect the suspension of BL21 obtained in step 1) and break the cells in a high-pressure homogenizer at 4°C, 1200 bar, and oil pressure 18 kg / cm 3 The mixture was treated under the above conditions for 3-5 min, and after crushing, it was centrifuged at 8000 rpm and 4°C for 30 min, and the supernatant was collected to obtain the crude enzyme solution.

[0032] 3) The crude enzyme solution obtained in step 2) is purified by gravity nickel column purification method. At 4°C, the crude enzyme solution is completely passed through a column filled with nickel filler, and washed with 1L of washing solution to remove impurities. Then, 20mL of eluent is used for elution, and the effluent is collected to obtain a high-purity isocitrate dehydrogenase solution.

[0033] 4) Collect the target protein solution obtained in step 3) and concentrate the protein using an ultrafiltration tube with a pore size of 30KDA. Centrifuge at 4800rpm and 4℃. When the volume of the ultrafiltration tube reaches 5mL, continue to add 15mL of lysis buffer and continue centrifugation. Repeat this twice, continue centrifugation until the remaining volume is 1mL, and divide it into packages. A high-concentration isocitrate dehydrogenase solution is obtained. The concentration of isocitrate dehydrogenase is then determined using the BCA method.

[0034] 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 the wild-type isocitrate dehydrogenase (45 kDa), lane 2 is the mutant enzyme (45 kDa), M is a marker; isocitrate dehydrogenase (45 kDa)).

[0035] The LB medium formula is 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and the pH is adjusted to 7.5. Sterilize at 0.1 Mpa pressure for 20 min.

[0036] Lysis Buffer (1L): 50mM Tris-HCl, 500mM NaCl, pH 8.5. 1L formula: 29.22g NaCl, 50ml Tris-HCl, 950ml ddH2O.

[0037] Washing solution: 50mM Tris-HCl, 500mM NaCl, 20mM imidazole, pH 7.5. 1L formula: 29.22g NaCl, 50mL 1MTris-HCl (pH 7.5), 1.36g imidazole, ddH2O to make up to 1L.

[0038] Elution buffer: 50mM Tris-HCl, 500mM NaCl, 250mM imidazole, pH 7.5. 1L formula: 29.22g NaCl, 50ml 1MTris-HCl (pH 7.5), 17.025g imidazole, ddH2O to make up to 1L.

[0039] Example 3, determination of isocitrate dehydrogenase concentration and specific enzyme activity The specific steps for protein concentration quantification are as follows: Dilute the sample to a suitable color development multiple. Take 25 μL and 200 μL of the reagent kit reaction solution and mix thoroughly, incubate at 37°C for 30 minutes. Use an ELISA reader to detect at a wavelength of 562 nm.

[0040] The specific enzyme activity of isocitrate dehydrogenase is determined by the following steps: The reaction was carried out at 37°C, and the activity of isocitrate dehydrogenase was characterized by measuring the amount of NADPH generated at 340nm. The reaction system contained: Tris-HCl (82.2mmol / L, pH8.0), MgCl2 (3mmol / L), NADP oxidized coenzyme I (0.6mmol / L) and DL-isocitrate trisodium salt hydrate (5mmol / L). The above substances were mixed and incubated at 37°C for 30min, and the reaction was started by adding enzyme solution, and then the generation of NAD(P)H was monitored at 340nm on a microplate reader. The enzyme activity unit was defined as the amount of enzyme required to generate 1μmol of NAD(P)H per minute.

[0041] The results of the enzyme activity comparison between the wild-type enzyme and the mutant enzyme are shown in Figure 4 As shown in the figure, the initial specific enzymatic activity of the wild-type enzyme was 39% higher than that of the mutant enzyme, but after incubation at 47°C for 8 hours, the specific enzymatic activities of the wild-type enzyme and the mutant enzyme decreased by 76.4% and 36.4%, respectively. At this time, the specific enzymatic activity of the mutant enzyme exceeded that of the wild-type enzyme by 93.6%, indicating that the stability of the mutant enzyme was significantly better than that of the wild-type enzyme and was more suitable for long-term storage.

[0042] Example 4 Stability test of mutant enzyme The wild-type enzyme and mutant enzyme were stored at 47 °C for incubation. Samples were taken at 0 h, 2 h, 4 h, 6 h, and 8 h and incubated at 37 °C for 30 min to detect ICDH enzyme activity. The half-life results were calculated as follows: Figure 5 As shown in the figure, the half-life of the mutant enzyme is significantly higher than that of the wild-type enzyme when stored at 47°C for the same time, indicating that the stability of the mutant enzyme has also been significantly improved. By calculating the half-life of the wild-type enzyme and the mutant enzyme at 47°C, the half-life of the mutant enzyme at 47°C is 12.96h, which is 237.5% higher than that of the wild-type enzyme at 3.84h.

[0043] Example 5 Detection of Tm value of mutant enzyme Description of the test method: 1. Take 16 different 9 μL samples, repeat 3 wells for each sample, a total of 48 wells, add them to 3 UNi tubes, and place them in the instrument sample compartment for testing; 2. This experiment can detect 48 samples at a time with high throughput, and simultaneously detect protein particle size and polydispersity, melting temperature (Tm) and starting aggregation temperature (Tagg). Protein endogenous 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 protein particle size or aggregation; 3. Set the detection parameters in UNcle Client 5.03 software: DLS parameter setting, test each sample 4 times, 5 seconds each time, the instrument will detect DLS data at the beginning and end of the heating. Set the heating parameters to Start Temp at 20 °C, End Temp at 95 °C, and Rate at 0.25 °C / min. Keep the Plate hold time at 32 s without adjustment, and click "Apply"; 4. Place the sample-loaded UNi on the sample stage in the instrument, and then click "Manual Configuration". Position the mouse to one of the sample wells (left click), then click "Trigger" in the lower right corner, and observe the fluorescence spectrum curve area in the upper right corner. Usually, the protein endogenous fluorescence excitation spectrum will show a normal distribution peak between 300-400 nm, and click "Next"; enter the "Please configure your DLS settings" interface, check "Auto", and keep the other parameters at the default values. After the DLS Intensity is stable, click "Start" in the lower right corner to start the experiment; 5. After the experiment, the Tm&Tagg were automatically calculated using the analysis software UNcle Analysis 5.03, and the BCM (Barycentric mean) method was used by default to analyze Tm.

[0044] The results are as follows Figure 6 It showed that the Tm value of the mutant enzyme Mutant was 61.8℃, which was 6.2℃ higher than that of the wild type (Tm value was 55.6℃), indicating that the thermal stability of the mutant enzyme Mutant was significantly improved.

Claims

1. A mutant of isocitrate dehydrogenase with enhanced thermal stability, characterized in that: The isocitrate dehydrogenase with enhanced thermal stability is obtained by performing substitution mutation on the amino acid sequence A44 as shown in SEQ ID NO:

1.

2. The isocitrate dehydrogenase mutant with enhanced thermal stability according to claim 1, characterized in that The isocitrate dehydrogenase with enhanced thermal stability is obtained by subjecting the amino acid sequence shown in SEQ ID NO: 1 to A44V mutation.

3. The isocitrate dehydrogenase mutant with enhanced thermal stability according to claim 2, characterized in that The amino acid sequence of the isocitrate dehydrogenase with enhanced thermal stability is shown in SEQ ID NO:

2.

4. A polynucleotide encoding the isocitrate dehydrogenase mutant with enhanced thermal stability according to any one of claims 1 to 3.

5. The coding polynucleotide according to claim 4, characterized in that The nucleotide sequence of the isocitrate dehydrogenase mutant with enhanced thermal stability is shown in SEQ ID NO:

4.

6. An expression vector containing the encoding polynucleotide according to claim 4 or 5.

7. A recombinant host cell containing the encoding polynucleotide according to claim 4 or 5, or the expression vector according to claim 6.

8. The recombinant host cell according to claim 7, characterized in that The host cell is Escherichia Escherichia ).

9. Use of the isocitrate dehydrogenase mutant with enhanced thermal stability or the encoding nucleotide thereof according to any one of claims 1 to 3 in the conversion of preparing NADH, NADPH or isocitrate.

10. The use according to claim 9, characterized in that Its substrates are NADP oxidized coenzyme I and DL-isocitrate trisodium salt hydrate.

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