A mutant of L-threonine dehydrogenase and its application
By mutation of L-threonine dehydrogenase V115H and N152R sites, the enzyme activity was improved, the problem of low catalytic activity of TDH enzyme was solved, and the efficient synthesis of 2,5-dimethylpyrazine was achieved and the cost reduction of 2,5-dimethylpyrazine was achieved.
Patent Information
- Application Number
- CN202510474414.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-16
- Publication Date
- 2025-07-25
- Estimated Expiration
- 2045-04-16
AI Technical Summary
In the prior art, the TDH enzyme derived from wild strains has low catalytic activity and the conversion rate of L-threonine is low, limiting the large-scale industrial production of 2,5-dimethylpyrazine.
By performing site-directed mutations of L-threonine dehydrogenase, especially the modification of V115H and N152R sites, L-threonine dehydrogenase mutants were constructed and expressed in E. coli to improve enzyme activity.
The mutant enzyme activity was increased by 1.76 times, significantly reducing the enzyme dosage, improving the synthesis capacity of 2,5-dimethylpyrazine, reducing production costs, and stable expression.
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Figure CN119979493B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biocatalysis and enzyme engineering, and relates to a mutant of L-threonine dehydrogenase and its application. Background Art
[0002] 2,5-Dimethylpyrazine is a high-value-added alkyl pyrazine compound, which is widely used in the food and pharmaceutical industries. In the pharmaceutical field, it can be used as an intermediate for the synthesis of anti-hypertensive and hypoglycemic drugs such as glipizide and acipimox; in the food field, due to its strong baking smell and extremely low odor detection threshold, it has become a food additive permitted to be added in China and is an important food flavor.
[0003] At present, the main synthesis route of 2,5-dimethylpyrazine is chemical synthesis. However, the chemical synthesis method has many problems such as many by-products, difficult to obtain raw materials, large equipment requirements, difficult separation and purification, and high production costs. In addition, the chemical synthesis process often requires harsh conditions such as high temperature, high pressure and catalysts, which are likely to cause the emission of toxic gases and explosion risks, resulting in serious environmental pollution. In contrast, the biotransformation method has the advantages of good selectivity and mild reaction conditions, and has a fast synthesis speed, strong reaction specificity, no generation of toxic by-products, environmental friendliness, simple product purification process, and cheap and easily available substrates. Therefore, the biosynthesis method of 2,5-dimethylpyrazine has become a research hotspot.
[0004] Threonine dehydrogenase (abbreviated as TDH enzyme) is the only key enzyme in the biosynthetic pathway of 2,5-dimethylpyrazine. By specifically catalyzing the dehydrogenation reaction of L-threonine, it generates key intermediates and then participates in the biosynthetic pathway of 2,5-dimethylpyrazine. The by-products generated by this reaction are few, which is beneficial to reducing production costs and making the subsequent separation of 2,5-dimethylpyrazine more convenient. It is currently the most ideal method for large-scale industrial production of 2,5-dimethylpyrazine.
[0005] However, the TDH enzyme derived from wild strains without modification has low enzyme activity and catalytic activity. When used for the production of 2,5-dimethylpyrazine, the conversion rate of L-threonine is low, and there are certain limitations in industrial applications, which restricts the large-scale industrial production of 2,5-dimethylpyrazine. Summary of the Invention
[0006] Aiming at the deficiencies of the existing technology, the present invention provides a mutant of L-threonine dehydrogenase and its application. Site-directed mutagenesis is carried out on L-threonine dehydrogenase to obtain a mutant of L-threonine dehydrogenase, and the enzyme activity and catalytic activity of L-threonine dehydrogenase are improved.
[0007] The technical solution of the present invention is as follows:
[0008] To solve the above technical problems, the present invention adopts the following technical solutions
[0009] A mutant of L-threonine dehydrogenase (TDH), wherein the mutant is a point mutation at a specific amino acid site of L-threonine dehydrogenase; the nucleotide sequence of the coding gene of the TDH is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.
[0010] The mutant is the L-threonine dehydrogenase mutant V115H, and the nucleotide sequence and amino acid sequence of its coding gene are SEQ ID NO.3 and SEQ ID NO.4 respectively; it is to mutate valine (V) at the 115th position of the L-threonine amino acid sequence into histidine (H).
[0011] The mutant is the L-threonine dehydrogenase mutant N152R, and the nucleotide sequence and amino acid sequence of its coding gene are SEQ ID NO.5 and SEQ ID NO.6 respectively; it is to mutate asparagine (N) at the 152nd position of the L-threonine dehydrogenase amino acid sequence into arginine (R).
[0012] The mutant is the TDH mutant V115H-N152R, and the nucleotide sequence and amino acid sequence of its coding gene are SEQ ID NO.7 and SEQ ID NO.8 respectively; it is to mutate valine (V) at the 115th position of the L-threonine dehydrogenase amino acid sequence into histidine (H), and asparagine (N) at the 152nd position into arginine (R).
[0013] A recombinant vector is obtained by inserting the coding gene of the above TDH mutant into a plasmid vector. Preferably according to the present invention, the plasmid vector is pET-28a.
[0014] The strain expressing the mutant is recombinant Escherichia coli.
[0015] The present invention also provides the application of the above mutant of L-threonine dehydrogenase in the synthesis of 2,5-dimethylpyrazine.
[0016] Compared with the prior art, the present invention has the following beneficial effects:
[0017] The mutant of L-threonine dehydrogenase described in the present invention contains V115H and N152R mutation sites, and the enzyme activity is significantly improved compared with the wild-type L-threonine dehydrogenase. The enzyme activity of the mutant of L-threonine dehydrogenase described in the present invention is 1.76 times that of the wild-type L-threonine dehydrogenase under the same conditions.
[0018] When the mutant of L-threonine dehydrogenase of the present invention is used to catalyze the dehydrogenation reaction of L-threonine to produce 2,5-dimethylpyrazine, the synthesis ability of 2,5-dimethylpyrazine is improved, the amount of enzyme used is reduced, and it has important industrial application value.
[0019] The nucleotide sequence of the L-threonine dehydrogenase mutant of the present invention has a high expression level and stable expression in Escherichia coli, and can significantly reduce the cost of preparing L-threonine dehydrogenase. Brief Description of the Drawings
[0020] Figure 1 It is the SDS-PAGE analysis map of the L-threonine dehydrogenase mutant;
[0021] Figure 2 It is the gas chromatogram of 2,5-dimethylpyrazine catalyzed by the wild-type engineering bacteria;
[0022] Figure 3 It is the gas chromatogram of 2,5-dimethylpyrazine catalyzed by the engineering bacteria with double-site mutation (V115H / N152R). Detailed Embodiments
[0023] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, the embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but such modifications and replacements all fall within the protection scope of the present invention.
[0024] LB liquid medium: Tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, the solvent is ultrapure water, the pH value is natural pH, and it is autoclaved at 121 °C for 20 min.
[0025] LB liquid medium containing kanamycin: 50 μg / mL (final concentration) of kanamycin is added to the LB liquid medium before use.
[0026] LB solid medium: 2% agar is added to the LB liquid medium and autoclaved at 121 °C for 20 min.
[0027] LB solid medium containing kanamycin: 50 μg / mL (final concentration) of kanamycin is added to the LB solid medium before use.
[0028] 100 mM L-threonine solution: Weigh 120 mg of L-threonine, dissolve it in 10 mL of ultrapure water, filter it with a 0.22 μm filter membrane, and dispense it into 1.5 mL centrifuge tubes.
[0029] 40 mM NAD ＋ Solution: Weigh 133 mg of NAD ＋ and dissolve it in 5 mL of ultrapure water, then filter it through a 0.22 μm filter membrane and dispense it into 1.5 mL centrifuge tubes.
[0030] Example 1: Construction of Recombinant Plasmids Containing L-Threonine Dehydrogenase Mutants
[0031] 1. Perform bioinformatics analysis on the wild-type TDH enzyme (shown as SEQ ID NO.1 in the sequence listing) derived from Escherichia coli to determine the key amino acid sites. Analyze the model structure of the enzyme through computer simulation of homology modeling of this amino acid sequence, predict the active sites of the enzyme, and use computer simulation to perform alanine scanning on the key amino acid residues to screen for mutants that may improve enzyme activity. The following 12 potential key amino acid sites are obtained: Gly39, Val42, Thr40, His43, Trp50, HIs63, His90, Gly112, Val115, Asn152, Ile175, Arg289. Perform virtual saturation mutagenesis on these 12 amino acid residues, use the computer to perform protein modeling on the mutants and predict the protein stability and free energy changes of each mutant, and finally determine that the two amino acid sites Val115 and Asn152 may affect the catalytic activity and stability of the TDH enzyme.
[0032] 2. Construction of mutant plasmids: Insert the nucleotide sequence of the wild-type TDH enzyme into the pET-28a plasmid, and add a nucleotide sequence capable of expressing a 6×His protein tag after the nucleotide sequence of the wild-type TDH enzyme for subsequent purification of the crude enzyme solution to obtain the recombinant plasmid pET-28a-TDH (commercially synthesized); use the whole plasmid PCR technique to perform site-directed mutagenesis with the recombinant plasmid pET-28a-TDH as the template to obtain recombinant plasmids pET-28a-TDH V115H, pET-28a-TDH N152R, and pET-28a-TDH V115H / N152R containing the TDH enzyme mutant genes.
[0033] The specific mutation process is as follows:
[0034] (1) Mutation primers
[0035] V115H-F: Its nucleotide sequence is shown as SEQ ID NO.9 in the sequence listing;
[0036] V115H-R: Its nucleotide sequence is shown as SEQ ID NO.10 in the sequence listing;
[0037] N152R-F: Its nucleotide sequence is as shown in SEQ ID NO.11 in the sequence listing;
[0038] N152R-R: Its nucleotide sequence is as shown in SEQ ID NO.12 in the sequence listing;
[0039] (2) The PCR system is shown in Table 1.
[0040] Table 1 PCR system
[0041]
[0042] (3) PCR amplification
[0043] Conditions: Pre-denaturation at 95°C for 3 min; then enter the following cycle: denaturation at 95°C for 15 s, annealing at 65°C for 15 s, extension at 72°C for 3 min 30 s, 30 cycles; extension at 72°C for another 5 min; incubation at 4°C.
[0044] (4) Verification
[0045] After the PCR amplification is completed, the amplification product is detected by agarose gel electrophoresis. An obvious bright band appears at the target band, and the target band is purified and recovered using a DNA recovery and purification kit.
[0046] The recovered PCR product is digested with DNPI enzyme to remove the DNA template. The volume of the digestion system is 50 μl, and the digestion system is shown in Table 2.
[0047] Table 2 Digestion system
[0048]
[0049] Digestion conditions:
[0050] 37°C: 30 min; 80°C: 5 min; incubation at 4°C.
[0051] The digested PCR amplification product is transformed into Escherichia coli DH5a competent cells, and the transformants are selected for sequencing. The sequencing verification results show that no random mutations occur except for the required mutation sites, indicating that the recombinant plasmids pET-28a-TDH V115H, pET-28a-TDH N152R, and pET-28a-TDH V115H / N152R are successfully constructed, and each recombinant plasmid is extracted. pET-28a-TDH V115H / N152R is obtained by mutating the two sites sequentially.
[0052] Example 2: Construction of recombinant Escherichia coli containing L-threonine dehydrogenase mutants
[0053] Take E.coilThe BL21(DE3) competent cells were taken out from -80 °C and quickly melted on ice. 5 μl of the recombinant plasmid obtained in Example 1 was added to the competent cell suspension respectively. The tube wall was flicked gently to mix evenly, and it was left standing on ice for 30 min, heat-shocked at 42 °C for 90 s, quickly cooled on ice for 2 min, 900 μL of sterile LB liquid medium was added, and it was cultured on a shaker at 37 °C and 200 rpm for 1 h. Then it was centrifuged at 5,000 rpm for 3 min, 900 μl of the supernatant was discarded, the cell pellet was resuspended with the remaining medium, and then evenly spread on the LB solid medium plate containing kanamycin. After the bacterial liquid was completely absorbed by the medium, the plate was inverted and cultured in a 37 °C constant temperature incubator for 16 h.
[0054] Clones were randomly picked from the colonies grown on the plate and the plasmids were extracted and identified by agarose gel electrophoresis to obtain the genetically engineered bacteria containing the TDH enzyme mutant gene: E.coil BL21(DE3)-pET-28a-TDH V115H, E.coil BL21(DE3)-pET-28a-TDH N152R, E.coil BL21(DE3)-pET-28a-TDH V115H / N152R. Similarly, the recombinant plasmid pET-28a-TDH was constructed into Escherichia coli according to the above method to obtain the wild-type genetically engineered bacteria E.coil BL21(DE3)-pET-28a-TDH.
[0055] Example 3: Induced expression of recombinant Escherichia coli containing L-threonine dehydrogenase mutant
[0056] Each of the genetically engineered bacteria prepared in Example 2 was streaked on the LB solid medium containing 50 μg / mL kanamycin and inverted and cultured overnight in a 37 °C constant temperature incubator to activate the strains. The next day, single colonies were picked from the plate and inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured on a shaker at 37 °C and 200 rpm for 12 h. The bacterial liquid was inoculated into 100 mL of LB liquid medium containing 50 μg / mL kanamycin at an inoculation amount of 1%, and cultured on a shaker at 37 °C and 200 rpm until the OD 600 reached 0.6 - 0.8 (about 2 - 3 h), then IPTG with a final concentration of 1 mmol / L was added, and it was cultured on a shaker at 16 °C and 180 rpm for 16 h.
[0057] Obtaining of crude enzyme solution
[0058] The induced bacterial solution was centrifuged at 4°C and 6000 rpm using a high-speed refrigerated centrifuge to collect the bacterial cells. The collected bacterial cells were washed twice with PBS buffer (pH = 7.5). The washed bacterial cells were resuspended in Lysis Buffer at a ratio of bacterial cells: Lysis Buffer = 1:10 (W / V). The resuspended and mixed bacterial cells were placed on ice, and the bacterial cells were lysed using an ultrasonic cell disruptor on ice until the bacterial solution was basically clear. The clear lysate was transferred to a centrifuge tube and centrifuged at 4°C and 6000 rpm for 20 min. The obtained supernatant was the crude enzyme solution, and the crude enzyme solution was detected by polyacrylamide gel electrophoresis. The results were as Figure 1 shown. Among them, lane M represents the protein Marker, lane 1 represents V115H, lane 2 represents N152R, and lane 3 represents V115H-N152R. The band size is about 37 kDa, which is consistent with the expected value.
[0059] Purification of the crude enzyme solution
[0060] As described in Example 1, for the convenience of purifying L-threonine dehydrogenase, a 6×His protein tag was fused after the target gene when constructing the expression plasmid of the TDH enzyme. The target protein was purified using a NiNTABeads gravity column based on affinity chromatography. The basic principle of purification was loading with low imidazole and eluting with high imidazole.
[0061] The preparation method of the protein purification buffer is shown in Table 3.
[0062] Table 3 Preparation method of the protein purification buffer
[0063]
[0064] The specific purification method is as follows:
[0065] Add 5 column volumes of Lysis Buffer to a 1 mL NiNTABeads gravity column to equilibrate the gravity column. Add the sample to the equilibrated gravity column and keep it for 2 - 5 min to allow the target protein to fully contact with the medium. Wash with 10 - 15 column volumes of Wash Buffer to remove non-specifically adsorbed impurity proteins. Use 10 column volumes of Elution Buffer to elute the target protein, collect in fractions, collect one tube for each column volume, and the obtained eluate is the purified enzyme solution of TDH.
[0066] Using bovine serum albumin as a standard, the enzyme concentration was determined by the Bradford method.
[0067] Example 4: Enzyme activity assay of L-threonine dehydrogenase mutants
[0068] The reaction system for measuring TDH enzyme activity (total volume is 1 mL) is shown in Table 4.
[0069] Table 4 Reaction system for measuring TDH enzyme activity
[0070]
[0071] After incubating 800 μl of Tris-Hcl buffer, 100 μl of L-threonine solution, and 50 μl of NAD ＋ solution in a water bath at 37 °C for 10 min, add 50 μl of the purified TDH enzyme solution. After mixing, use a UV spectrophotometer to detect the change in absorbance of the system at 340 nm. Record the absorbance every 5 s, with a total duration of 60 s. Independently repeat the parallel experiment three times. Define one enzyme activity unit (U) as the amount of enzyme required to catalyze the reduction of 1 μmol of coenzyme NAD+ per minute.
[0072] The specific enzyme activities of the purified enzyme solutions obtained from the fermentation of each genetically engineered bacterium are shown in Table 5.
[0073] Table 5 Specific enzyme activities of the purified enzyme solutions
[0074]
[0075] As can be seen from the above table, the purified enzyme solution obtained from the fermentation of the genetically engineered bacterium containing the TDH enzyme mutant gene is significantly improved compared to the wild-type genetically engineered bacterium; compared to the genetically engineered bacterium with a single-site mutation, the genetically engineered bacterium with two-site mutations has significantly higher enzyme activity of the TDH enzyme in the purified enzyme solution obtained from its fermentation. At the same time, computer-aided design for enzyme modification can preliminarily screen out possible positive mutants from a large number of mutations, greatly saving the time cost of enzyme modification and achieving technical effects significantly superior to the prior art.
[0076] Example 5: Whole-cell catalysis of recombinant strains containing TDH enzyme mutants for the synthesis of 2,5-dimethylpyrazine
[0077] Streak-activate the recombinant Escherichia coli strain containing the TDH enzyme mutant obtained in Example 2 on an LB plate containing 50 μg / mL kanamycin resistance. Invert the plate and culture it in a constant temperature incubator at 37 °C for 12 h. Pick a single colony and inoculate it into 10 mL of liquid LB medium containing 50 μg / mL kanamycin. Incubate it on a shaker at 37 °C and 200 rpm for 10 - 12 h. Then transfer the seed solution to 100 mL of liquid LB medium containing 50 μg / mL kanamycin at an inoculation amount of 1%. Make the L-threonine dehydrogenase of the recombinant strain highly expressed according to the method of Example 3. Centrifuge and collect the cells at 4 °C and 6000 rpm, and wash the collected cells twice with PBS buffer (pH 7.5).
[0078] The washed cells were resuspended in Tris-Hcl buffer (pH 9.0) to a cell concentration of 30 g / L. Subsequently, the whole-cell catalysis conditions were adjusted as follows: the substrate L-threonine was 10 g / L, the initial reaction pH was 9.0, the reaction temperature was 40 °C, and the stirring speed was 200 rpm. After 24 h of whole-cell catalysis, the ability of each recombinant strain to synthesize 2,5-dimethylpyrazine was measured. The gas chromatogram of the whole-cell catalysis synthesis of 2,5-dimethylpyrazine by the wild-type genetically engineered bacterium E.coil BL21(DE3)-pET-28a-TDH is as Figure 2 shown, and the gas chromatogram of the whole-cell catalysis synthesis of 2,5-dimethylpyrazine by E.coil BL21(DE3)-pET-28a-TDH V115H / N152R is as Figure 3 shown.
[0079] The analysis method of 2,5-dimethylpyrazine is as follows: The fermentation broth was centrifuged at 12000 rpm for 10 minutes, and the supernatant was treated by liquid-liquid extraction. The extractant was ethyl acetate, and the extraction volume ratio was 1:1. The obtained extract was filtered through a 0.22 μm filter membrane and loaded into a sample bottle, and gas chromatography analysis was immediately carried out. The instrument model was Shimadzu GC-2030, and the detection conditions were: the column temperature was 80 °C, the injector and detector temperatures were 240 °C, the injection volume was 0.2 μL, and the chromatographic column specifications were: the column length was 30 m, the inner diameter was 0.32 mm, and the liquid film thickness was 0.5 μm.
[0080] The ability of each recombinant strain to synthesize 2,5-dimethylpyrazine by whole-cell catalysis is shown in Table 6.
[0081] Table 6 Ability to synthesize 2,5-dimethylpyrazine
[0082]
[0083] The results showed that: the ability of the recombinant strain E.coil BL21(DE3)-pET-28a-TDH V115H / N152R to synthesize 2,5-dimethylpyrazine by whole-cell catalysis was significantly improved, and its synthesis ability was increased by 28% compared with the wild-type engineered bacterium. Thus, it can be seen that in the reaction of catalyzing L-threonine to produce 2,5-dimethylpyrazine, the catalytic activity of the TDH enzyme with two-site mutations is significantly higher than that of the wild-type TDH enzyme, which can significantly improve the synthesis ability of 2,5-dimethylpyrazine, and the co-mutation is better than the single-point mutation.
Claims
1. A mutant of L-threonine dehydrogenase, characterized in that, The mutant is L-threonine dehydrogenase mutant V115H, and the nucleotide sequence and amino acid sequence of its encoding gene are SEQ ID NO.3 and SEQ ID NO.4 respectively; it is the mutation of valine (V) at position 115 of the L-threonine amino acid sequence to histidine (H).
2. A mutant of L-threonine dehydrogenase, characterized in that, The mutant is L-threonine dehydrogenase mutant N152R, and the nucleotide sequence and amino acid sequence of its encoding gene are SEQ ID NO.5 and SEQ ID NO.6 respectively; it is the mutation of asparagine (N) at position 152 of the L-threonine dehydrogenase amino acid sequence to arginine (R).
3. A mutant of L-threonine dehydrogenase, characterized in that, The mutant is L-threonine dehydrogenase mutant V115H-N152R, and the nucleotide sequence and amino acid sequence of its encoding gene are SEQ ID NO.7 and SEQ ID NO.8 respectively; it is the mutation of valine (V) at position 115 of the L-threonine dehydrogenase amino acid sequence to histidine (H), and the mutation of asparagine (N) at position 152 to arginine (R).
4. A recombinant vector, characterized in that, The recombinant vector inserts the encoding gene of the mutant of L-threonine dehydrogenase described in any one of claims 1-3 into a plasmid vector, and the plasmid vector is pET-28a.
5. A recombinant Escherichia coli containing the mutant of L-threonine dehydrogenase described in any one of claims 1-3.
6. Use of the mutant of L-threonine dehydrogenase described in any one of claims 1-3 in the synthesis of 2,5-dimethylpyrazine.
7. Use of the recombinant vector according to claim 4 in the synthesis of 2,5-dimethylpyrazine.
8. Use of the recombinant Escherichia coli according to claim 5 in the synthesis of 2,5-dimethylpyrazine.