Mutant of L-threonine dehydrogenase and application thereof

Abstract

The invention belongs to the technical field of biological catalysis and enzyme engineering, and relates to a mutant of L-threonine dehydrogenase and application thereof. The invention discloses a mutant of L-threonine dehydrogenase (TDH). The mutant is characterized in that a specific amino acid site of the L-threonine dehydrogenase is subjected to point mutation; the nucleotide sequence of the coding gene of the TDH is as shown in SEQ ID NO. 1, and the amino acid sequence of the coding gene of the TDH is as shown in SEQ ID NO. 2. The L-threonine dehydrogenase mutant disclosed by the invention contains V115H and N152R mutation sites, the enzyme activity of the L-threonine dehydrogenase mutant is obviously improved compared with that of wild type L-threonine dehydrogenase, the L-threonine dehydrogenase mutant can be used for catalytically synthesizing 2, 5-dimethylpyrazine, the dosage of the enzyme is reduced, and the L-threonine dehydrogenase mutant has important industrial application value. The L-threonine dehydrogenase mutant disclosed by the invention is high in expression quantity and stable in expression in escherichia coli, and the cost of preparing the L-threonine dehydrogenase can be remarkably reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis and enzyme engineering, and relates to a mutant of L-threonine dehydrogenase and application thereof. Background Art

[0002] 2,5-Dimethylpyrazine is a high value-added alkylpyrazine 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 antihypertensive 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 allowed in my country and is an important food flavoring.

[0003] At present, the main synthetic route of 2,5-dimethylpyrazine is chemical synthesis. However, chemical synthesis methods have many problems such as many by-products, difficult raw materials to obtain, large equipment requirements, difficult separation and purification, and high production costs. In addition, harsh conditions such as high temperature, high pressure and catalysts are often required in the chemical synthesis process, which easily lead to the dispersion and explosion risk of toxic gases, causing serious environmental pollution. In contrast, the biotransformation method has the advantages of good selectivity and mild reaction conditions, and fast synthesis speed, strong reaction specificity, no toxic by-product generation, environmentally friendly, simple product purification process, and cheap and easy-to-obtain substrates. Therefore, the biosynthesis of 2,5-dimethylpyrazine has become a hot topic in research.

[0004] Threonine dehydrogenase (abbreviated as TDH enzyme) is the only key enzyme in the biosynthetic pathway of 2,5-dimethylpyrazine. It specifically catalyzes the dehydrogenation reaction of L-threonine to generate key intermediates, and then participates in the biosynthetic pathway of 2,5-dimethylpyrazine. This reaction generates few by-products, which is conducive 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 unmodified wild strains has low enzyme activity and low catalytic activity. When used to produce 2,5-dimethylpyrazine, the conversion rate of L-threonine is low. There are certain limitations in its industrial application, which restricts the large-scale industrial production of 2,5-dimethylpyrazine. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a mutant of L-threonine dehydrogenase and application thereof, performs site-directed mutagenesis on L-threonine dehydrogenase to obtain a mutant of L-threonine dehydrogenase, and improves the enzymatic activity and catalytic activity of L-threonine dehydrogenase.

[0007] The technical solution of the present invention is as follows: In order to solve the above technical problems, the present invention adopts the following technical solutions A mutant of L-threonine dehydrogenase (TDH), wherein a point mutation occurs at a specific amino acid position of the L-threonine dehydrogenase; the nucleotide sequence of the gene encoding the TDH is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0008] The mutant is an L-threonine dehydrogenase mutant V115H, the nucleotide sequence and amino acid sequence of the encoding gene are SEQ ID NO.3 and SEQ ID NO.4 respectively; the valine (V) at position 115 of the L-threonine amino acid sequence is mutated into histidine (H).

[0009] The mutant is L-threonine dehydrogenase mutant N152R, the nucleotide sequence and amino acid sequence of the encoding gene are SEQ ID NO.5 and SEQ ID NO.6 respectively; the asparagine (N) at position 152 of the amino acid sequence of L-threonine dehydrogenase is mutated into arginine (R).

[0010] The mutant is TDH mutant V115H-N152R, the nucleotide sequence and amino acid sequence of the encoding gene are SEQ ID NO.7 and SEQ ID NO.8 respectively; the valine (V) at position 115 of the amino acid sequence of L-threonine dehydrogenase is mutated into histidine (H), and the asparagine (N) at position 152 is mutated into arginine (R).

[0011] A recombinant vector is a plasmid vector into which the coding gene of the TDH mutant is inserted. Preferably, according to the present invention, the plasmid vector is pET-28a.

[0012] The strain expressing the mutant is a recombinant Escherichia coli.

[0013] The present invention also provides the use of the mutant of the L-threonine dehydrogenase in synthesizing 2,5-dimethylpyrazine.

[0014] Compared with the prior art, the present invention achieves the following beneficial effects: The mutant of L-threonine dehydrogenase of 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 of the present invention is 1.76 times that of the wild-type L-threonine dehydrogenase under the same conditions.

[0015] The mutant of L-threonine dehydrogenase of the present invention improves the synthesis ability of 2,5-dimethylpyrazine and reduces the amount of enzyme when used for catalyzing the dehydrogenation reaction of L-threonine to generate 2,5-dimethylpyrazine, and has important industrial application value.

[0016] The nucleotide sequence of the L-threonine dehydrogenase mutant of the invention has a high expression level in Escherichia coli and a stable expression, and can significantly reduce the cost of preparing the L-threonine dehydrogenase. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the SDS-PAGE analysis pattern of L-threonine dehydrogenase mutant; Figure 2 The gas chromatogram of 2,5-dimethylpyrazine catalyzed by wild-type engineered bacteria; Figure 3 This is the gas chromatogram of 2,5-dimethylpyrazine produced by double-site mutation (V115H / N152R) engineered bacteria. DETAILED DESCRIPTION

[0018] 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 proceeds. However, the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0019] LB liquid culture medium: tryptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, solvent is ultrapure water, pH value is natural pH, high pressure sterilization at 121 °C for 20 min.

[0020] LB liquid medium containing kanamycin: LB liquid medium was supplemented with 50 μg / mL (final concentration) of kanamycin before use.

[0021] LB solid medium: Add 2% agar to LB liquid medium and sterilize at 121℃ for 20 minutes.

[0022] LB solid medium containing kanamycin: 50 μg / mL (final concentration) of kanamycin was added to LB solid medium before use. 100 mM L-threonine solution: Weigh 120 mg of L-threonine and 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.

[0023] 40mM NAD ＋ Solution: weigh 133 mg of NAD ＋ Dissolve in 5 mL of ultrapure water, filter with a 0.22 μm filter membrane, and dispense into 1.5 mL centrifuge tubes.

[0024] Example 1: Construction of a recombinant plasmid containing an L-threonine dehydrogenase mutant 1. From Escherichia coli The wild-type TDH enzyme (as shown in SEQ ID NO.1 in the sequence list) was subjected to bioinformatics analysis to determine the key amino acid sites. The model structure of the enzyme was analyzed by computer simulation of homology modeling of the amino acid sequence, the active site of the enzyme was predicted, and the mutants that may improve the enzyme activity were screened by alanine scanning of the key amino acid residues by computer simulation, and the following 12 potential key amino acid sites were screened: Gly39, Val42, Thr40, His43, Trp50, HIs63, His90, Gly112, Val115, Asn152, Ile175, Arg289. Virtual saturation mutations were performed on these 12 amino acid residues, and protein modeling of the mutants was performed by computer to predict the protein stability and free energy changes of each mutant, and it was finally determined that the two amino acid sites of Val115 and Asn152 may affect the catalytic activity and stability of the TDH enzyme.

[0025] 2. Construction of mutant plasmids: The nucleotide sequence of the wild-type TDH enzyme was inserted into the pET-28a plasmid, and a nucleotide sequence capable of expressing a 6×His protein tag was added 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); using the whole plasmid PCR technology, the recombinant plasmid pET-28a-TDH was used as a template for site-directed mutagenesis to obtain the recombinant plasmids pET-28a-TDH V115H, pET-28a-TDH N152R, and pET-28a-TDH V115H / N152R containing the TDH enzyme mutant gene.

[0026] The specific mutation process is as follows: (1) Mutation primers V115H-F: Its nucleotide sequence is shown in SEQ ID NO.9 in the sequence listing; V115H-R: Its nucleotide sequence is shown in SEQ ID NO.10 in the sequence listing; N152R-F: Its nucleotide sequence is shown in SEQ ID NO.11 in the sequence listing; N152R-R: Its nucleotide sequence is shown in SEQ ID NO.12 in the sequence listing; (2) The PCR system is shown in Table 1.

[0027] Table 1 PCR system

[0028] (3) PCR amplification The conditions were as follows: pre-deformation at 95°C for 3 min; then entering 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; and insulation at 4°C.

[0029] (4) Verification After PCR amplification, the amplified product was detected by agarose gel electrophoresis, and an obvious bright band appeared at the target band. The target band was purified and recovered using a DNA recovery and purification kit.

[0030] The recovered PCR product was digested with DNPI enzyme to remove the DNA template. The volume of the digestion system was 50 μl. The digestion system is shown in Table 2.

[0031] Table 2 Digestion system

[0032] Digestion conditions: 37℃: 30min; 80℃: 5min; 4℃: keep warm.

[0033] The digested PCR amplification products were transduced into E. coli DH5a competent cells, and transformants were selected for sequencing. The sequencing verification results showed that no random mutations occurred except for the desired mutation sites, indicating that the recombinant plasmids pET-28a-TDH V115H, pET-28a-TDH N152R, and pET-28a-TDH V115H / N152R were successfully constructed, and each recombinant plasmid was extracted. pET-28a-TDH V115H / N152R was obtained by sequentially mutating two sites.

[0034] Example 2: Construction of recombinant Escherichia coli containing L-threonine dehydrogenase mutant Will E.coil The BL21 (DE3) competent cells were taken out from -80 ° C, quickly placed on ice to melt, and 5 μl of the recombinant plasmid obtained in Example 1 was added to the competent cell suspension, the tube wall was gently tapped to mix, and the mixture was allowed to stand on ice for 30 min. The mixture was heat-shocked at 42 ° C for 90 s, and quickly placed on ice to cool for 2 min. 900 μL of sterile LB liquid culture medium was added, and the mixture was placed on a shaker at 37 ° C and 200 rpm for 1 h. The mixture was centrifuged at 5,000 rpm for 3 min, and 900 μl of supernatant was discarded. After the bacteria were resuspended with the remaining culture medium, they were evenly spread on an LB solid culture medium plate containing kanamycin. After the bacterial solution was completely absorbed by the culture medium, the plate was inverted and cultured in a 37 ° C constant temperature incubator for 16 h.

[0035] Randomly pick clones from the colonies grown on the plate and extract plasmids for agarose gel electrophoresis identification to obtain 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 wild-type genetically engineered bacteria. E.coil BL21(DE3)-pET-28a-TDH.

[0036] Example 3: Inducible expression of recombinant Escherichia coli containing L-threonine dehydrogenase mutant Each genetically engineered bacterium prepared in Example 2 was streaked on a LB solid medium containing 50 μg / mL kanamycin, and inverted in a 37°C constant temperature incubator for overnight culture to activate the strain. The next day, a single colony was picked from the plate and inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin, and placed in a shaker at 37°C and 200 rpm for 12 h. The bacterial solution was inoculated with 1% inoculum into 100 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured in a shaker at 37°C and 200 rpm until OD 600 When the concentration reaches 0.6-0.8 (approximately 2-3 h), add IPTG with a final concentration of 1 mmol / L, and culture in a shaker at 16°C and 180 rpm for 16 h.

[0037] Obtaining crude enzyme solution The induced bacterial liquid was centrifuged and collected using a high-speed refrigerated centrifuge at 4°C and 6000rpm. The collected bacteria were washed twice with PBS buffer (PH=7.5). The washed bacteria were added to Lysis Buffer at the ratio of bacteria: Lysis Buffer=1:10 (W / V) to resuspend the precipitated bacteria. The resuspended and mixed bacteria were placed on ice and disrupted with an ultrasonic cell disruptor on ice until the bacterial liquid remained basically clear. The clarified disrupted liquid was transferred to a centrifuge tube and centrifuged at 4°C and 6000rpm for 20 minutes. The obtained supernatant was the crude enzyme solution, which was detected by polyacrylamide gel electrophoresis. The results are as shown below. Figure 1 As shown, lane M represents the protein marker, lane 1 represents V115H, lane 2 represents N152R, and lane 3 represents V115H-N152R. The band size is around 37 kDa, which is consistent with the expected value.

[0038] Purification of crude enzyme solution As described in Example 1, in order to facilitate the purification of L-threonine dehydrogenase, a 6×His protein tag was fused after the target gene when constructing the expression plasmid of TDH enzyme. The target protein was purified by affinity chromatography using NiNTABeads gravity column, and the basic principle of purification was low imidazole loading and high imidazole elution.

[0039] The preparation method of protein purification buffer is shown in Table 3.

[0040] Table 3 Preparation method of protein purification buffer

[0041] The specific purification method is as follows: Add 5 column volumes of Lysis Buffer to a 1 mL NiNTABeads gravity column to balance the gravity column. Add the sample to the balanced gravity column and keep for 2-5 minutes to allow the target protein to fully contact the medium. Wash with 10-15 column volumes of Wash Buffer to remove non-specifically adsorbed impurities. Use 10 column volumes of Elution Buffer to elute the target protein, collect in sections, and collect one tube for each column volume. The resulting eluate is the purified enzyme solution of TDH.

[0042] The enzyme concentration was determined by Bradford method using bovine serum albumin as standard.

[0043] Example 4: Enzyme activity assay of L-threonine dehydrogenase mutants The reaction system for determining TDH enzyme activity (total system is 1 mL) is shown in Table 4.

[0044] Table 4 Reaction system for determining TDH enzyme activity

[0045] 800 μl of Tris-HCl buffer, 100 μl of L-threonine solution, 50 μl of NAD ＋ After the solution was kept in a water bath at 37°C for 10 minutes, 50 μl of TDH purified enzyme solution was added and mixed. The absorbance change of the system at 340 nm was detected by UV spectrophotometer. The absorbance was recorded every 5 seconds for a total of 60 seconds. The parallel experiment was repeated three times independently. One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the reduction of 1 μmol of coenzyme NAD+ per minute.

[0046] The specific enzyme activities of the pure enzyme solutions obtained by fermentation of various genetically engineered bacteria are shown in Table 5.

[0047] Table 5 Specific enzyme activity of pure enzyme solution

[0048] As can be seen from the above table, the purified enzyme liquid obtained by fermentation of genetically engineered bacteria containing TDH enzyme mutant genes is significantly improved compared with wild-type genetically engineered bacteria; compared with genetically engineered bacteria with single-site mutations, the enzyme activity of TDH enzyme in the purified enzyme liquid obtained by fermentation of genetically engineered bacteria with two-site mutations at the same time is significantly improved. At the same time, computer-aided design of 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 that are significantly better than the existing technology.

[0049] Example 5: Synthesis of 2,5-dimethylpyrazine by whole-cell catalysis of a recombinant strain containing a TDH enzyme mutant The recombinant E. coli strain containing the TDH enzyme mutant obtained in Example 2 was streaked on an LB plate containing 50 μg / mL kanamycin resistance to activate the strain, the plate was inverted in a 37°C constant temperature incubator for 12 hours, a single colony was picked and inoculated in 10 mL of liquid LB medium containing 50 μg / mL kanamycin, and cultured in a shaking table at 37°C and 200 rpm for 10-12 hours, and then the seed liquid was transferred to 100 mL LB liquid medium containing 50 μg / mL kanamycin at an inoculum of 1%, and the L-threonine dehydrogenase of the recombinant strain was efficiently expressed according to the method of Example 3. The bacterial cells were collected by centrifugation at 4°C and 6000 rpm, and the collected bacterial cells were washed twice with PBS buffer (pH 7.5).

[0050] The washed cells were resuspended in Tris-HCl buffer (pH 9.0) to a cell concentration of 30 g / L, and then the whole cell catalytic conditions were adjusted to: substrate L-threonine 10 g / L, initial reaction pH 9.0, reaction temperature 40 ° C, stirring speed 200 rpm. After 24 hours of whole cell catalysis, the synthesis ability of 2,5-dimethylpyrazine of each recombinant strain was determined. The gas chromatogram of the wild-type genetically engineered bacteria E.coil BL21 (DE3)-pET-28a-TDH whole cell catalytic synthesis of 2,5-dimethylpyrazine is shown in Figure 2. Figure 2 As shown, the gas chromatogram of the synthesis of 2,5-dimethylpyrazine catalyzed by E.coil BL21 (DE3)-pET-28a-TDH V115H / N152R whole cells is shown in Figure 3 shown.

[0051] The specific analysis method of 2,5-dimethylpyrazine is as follows: the fermentation broth is centrifuged at 12000 rpm for 10 minutes, the supernatant is treated by liquid-liquid extraction, the extractant is ethyl acetate, the extraction volume ratio is 1:1, the obtained extract is filtered through a 0.22μm filter membrane and filled into a sample bottle, and gas chromatography analysis is performed immediately. The instrument model is Shimadzu GC-2030, the detection conditions are: column temperature 80 ℃, injector and detector temperature 240 ℃, injection volume 0.2μL, chromatographic column specifications: column length 30m, inner diameter 0.32 mm, liquid film thickness 0.5μm.

[0052] The ability of the whole cells of each recombinant strain to catalyze the synthesis of 2,5-dimethylpyrazine is shown in Table 6.

[0053] Table 6 Ability to synthesize 2,5-dimethylpyrazine

[0054] The results showed that the ability of the recombinant strain E.coil BL21 (DE3)-pET-28a-TDH V115H / N152R whole cell to catalyze the synthesis of 2,5-dimethylpyrazine was significantly improved, and its synthesis ability was increased by 28% compared with the wild-type engineered bacteria. This shows 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 synergistic mutation is better than the single-point mutation.

Claims

1. A mutant of L-threonine dehydrogenase, characterized in that: The mutant is a point mutation at a specific amino acid site of L-threonine dehydrogenase; the nucleotide sequence of the gene encoding the L-threonine dehydrogenase is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. The mutant according to claim 1, characterized in that The mutant is an L-threonine dehydrogenase mutant V115H, the nucleotide sequence and amino acid sequence of the encoding gene are SEQ ID NO.3 and SEQ ID NO.4 respectively; the valine (V) at position 115 of the L-threonine amino acid sequence is mutated into histidine (H).

3. The mutant according to claim 1, characterized in that The mutant is L-threonine dehydrogenase mutant N152R, the nucleotide sequence and amino acid sequence of the encoding gene are SEQ ID NO.5 and SEQ ID NO.6 respectively; the asparagine (N) at position 152 of the amino acid sequence of L-threonine dehydrogenase is mutated into arginine (R).

4. The mutant according to claim 1, characterized in that The mutant is an L-threonine dehydrogenase mutant V115H-N152R, and the nucleotide sequence and amino acid sequence of the encoding gene are SEQ ID NO.7 and SEQ ID NO.8 respectively; the valine (V) at position 115 of the L-threonine dehydrogenase amino acid sequence is mutated into histidine (H), and the asparagine (N) at position 152 is mutated into arginine (R).

5. A recombinant vector, characterized in that: The recombinant vector is a plasmid vector in which a gene encoding a mutant of L-threonine dehydrogenase according to any one of claims 1 to 4 is inserted. The plasmid vector is pET-28a.

6. A recombinant Escherichia coli comprising the mutant of L-threonine dehydrogenase according to any one of claims 1 to 4.

7. Use of the mutant of L-threonine dehydrogenase according to any one of claims 1 to 4 in the synthesis of 2,5-dimethylpyrazine.

8. Use of the recombinant vector as claimed in claim 5 in the synthesis of 2,5-dimethylpyrazine.

9. Use of the recombinant Escherichia coli as claimed in claim 6 in the synthesis of 2,5-dimethylpyrazine.

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