Tryptophan hydroxylase mutant and application thereof

Abstract

The amino acid sequence of the mutant provided by the invention is as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and the gene sequence is as shown in SEQ ID NO: 3 or SEQ ID NO: 4. The mutant is obtained by carrying out multi-site site-specific mutagenesis on wild type tryptophan-5-hydroxylase of symbiotic bacteria of sponge animals. The relative enzyme activity of the mutant is 206% of that of a wild type at most. In a test of catalyzing tryptophan to produce 5-HTP, the 10min tryptophan conversion rate of the mutant can reach 49.82% and is 2.6 times that of a wild type. The tryptophan-5-hydroxylase mutant provided by the invention has the advantages that the enzyme activity is obviously improved, and the tryptophan-5-hydroxylase mutant can be completely used for efficiently synthesizing 5-HTP through industrial enzyme catalysis.

Description

Technical field:

[0001] The invention belongs to the technical field of gene engineering and enzyme engineering, and specifically relates to a tryptophan hydroxylase mutant with improved activity and application thereof. Background technology:

[0002] 5-Hydroxytryptophan (5-HTP) is a derivative of tryptophan, which is produced by replacing the 5′ hydrogen atom on the benzene ring of tryptophan with a hydroxyl group. 5-HTP is a non-protein amino acid and is a biosynthetic precursor of the neurotransmitter serotonin and the amine hormone melatonin in mammals. In the field of medical care, 5-HTP is widely used in clinical treatment as a mental regulator, including relieving headaches, promoting sleep, regulating appetite and treating depression. It has shown significant therapeutic ability, has extremely high medical care value, and has received widespread attention.

[0003] At present, natural product extraction is the main method for commercial production of 5-HTP. The raw material supply of this method is severely restricted by the origin and season, and the production capacity is limited, which makes it difficult to meet the growing market demand. The chemical synthesis of 5-HTP substrates is costly, the process route is complex, and involves many reactions and harsh conditions. At the same time, a large amount of organic solvents are used, which increases the environmental burden. Therefore, it is not suitable for large-scale production of 5-HTP. Microbial fermentation has the advantages of easy availability of raw materials, high efficiency, low cost and sustainable production, which is undoubtedly a more feasible option for the preparation of 5-HTP. However, when tryptophan hydroxylase (TPH) in the 5-HTP biosynthesis pathway is expressed in microorganisms, there are problems such as low activity, poor solubility, and insufficient supply of cofactor BH4. Although most current studies focus on the exploration and modification of highly active TPH to improve the activity and solubility of the enzyme, the enzymes in these studies are limited to the aromatic amino acid hydroxylase (AAAH) family. In the enzyme-catalyzed production of 5-HTP, oxygen is required as a co-substrate, tetrahydropterin (BH4) and Fe 2+ As a cofactor, the catalytic reaction involves the recycling and regeneration process of the cofactor BH4. During the reaction, TPH first catalyzes BH4, oxygen molecules and ferrous ions to form active hydroxylation intermediates, and then inserts one of the oxygen atoms into the aromatic ring of L-tryptophan to synthesize 5-HTP. However, the intermediate product quinone-type dihydropterin in the BH4 cycle has poor stability and can be converted into a BH4 structural analog that cannot participate in the tryptophan hydroxylation reaction under the action of non-enzymatic reactions, which reduces the catalytic activity of the enzyme and makes it impossible to efficiently synthesize the target product 5-HTP. Summary of the invention:

[0004] In view of the deficiencies of the above-mentioned prior art, the main purpose of the present invention is to obtain a tryptophan 5-hydroxylase (KERL) mutant by using site-directed mutagenesis technology and protein engineering technology, provide a recombinant plasmid or engineered strain expressing the above-mentioned tryptophan 5-hydroxylase mutant, and provide the application of the above-mentioned tryptophan 5-hydroxylase mutant.

[0005] The following definitions are used in the description of the technical solution of the present invention:

[0006] 1. Amino acid residues are represented by the three-letter abbreviations or single-letter symbols using the generally accepted IUPAC nomenclature. DNA sequences are represented by the generally accepted IUPAC nomenclature.

[0007] 2. Mutant identification: "amino acid replaced by the original amino acid position" is used to indicate the mutated amino acid in the tryptophan hydroxylase mutant. For example, I77A indicates that the amino acid at position 77 of the parent tryptophan-5-hydroxylase mutates from isoleucine Ile to alanine Ala; another example is I226A / P229C, indicating that the amino acid at position 226 of the parent mutates from isoleucine Ile to alanine Ala, and the amino acid at position 229 mutates from proline Pro to cysteine ​​Cys.

[0008] In the present invention, R275V / S282P and R275V / S282P / T379E represent two tryptophan 5-hydroxylase mutants obtained from wild-type tryptophan 5-hydroxylase (KERL), and lowercase italic kerl represents the coding gene of wild-type KERL.

[0009] The technical solution of the present invention is summarized as follows:

[0010] Through site-directed mutagenesis, with the goal of expanding the substrate pocket of tryptophan-5-hydroxylase and enhancing the activity of generating 5-HTP, the wild-type tryptophan-5-hydroxylase from the sponge symbiotic bacteria (Entotheonella factor) was used as the parent for semi-rational design and amino acid mutation using site-directed mutagenesis technology to obtain mutants. The details are as follows:

[0011] A tryptophan 5-hydroxylase mutant, characterized in that the amino acid sequence of the mutant is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] Among them, the mutant type corresponding to SEQ ID NO:1 is R275V / S282P, which is obtained by taking the wild-type KERL derived from the sponge symbiotic bacteria (Entotheonella factor) as the parent, mutating the arginine Arg at position 275 to valine Val, and mutating the serine Ser at position 282 to proline Pro in its amino acid sequence (as shown in SEQ ID NO:5).

[0013] The mutant type corresponding to SEQ ID NO:2 is R275V / S282P / T379E, which is also obtained by taking the above-mentioned wild-type KERL as the parent, and further introducing a mutation of the amino acid at position 379 (threonine Thr is mutated to glutamic acid Glu) on the basis of the R275V / S282P mutation shown in SEQ ID NO:1.

[0014] The present invention also provides the gene sequence of the tryptophan 5-hydroxylase mutant, as shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0015] The present invention also provides an expression vector carrying the gene sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0016] Preferably, the above expression vector is pQE-80L, pET-22b or pMA5.

[0017] The present invention also provides an engineering strain containing the above expression vector.

[0018] Preferably, the engineered strain is Escherichia coli, Bacillus subtilis or Pichia pastoris.

[0019] More preferably, the engineered strain is Escherichia coli K-12MG1655.

[0020] Application of the above tryptophan 5-hydroxylase mutant in producing 5-HTP.

[0021] The use of the above tryptophan-5-hydroxylase mutant in the reaction process of catalyzing tryptophan to produce 5-HTP.

[0022] Application of the above expression vector or engineered strain in the production of 5-HTP.

[0023] The experimental steps for obtaining the tryptophan 5-hydroxylase mutant of the present invention are summarized as follows:

[0024] 1. Using the wild-type tryptophan-5-hydroxylase gene ker1 from sponge symbiotic bacteria (Entotheonella factor) and the recombinant plasmid pQE-80L-ker1 constructed by plasmid pQE-80L as templates, design site-directed mutagenesis primers and perform site-directed mutagenesis PCR amplification reaction;

[0025] 2. After the site-directed mutagenesis PCR product is digested with Dpn I and self-circularized, it is transferred into the host bacteria and cultured. The obtained positive clones are screened with antibiotics and induced to express, and mutants with high activity are screened by converting tryptophan;

[0026] 3. The selected high-activity mutants were sequenced and identified, and the sequencing results were compared with the wild type to obtain two tryptophan 5-hydroxylase mutants with improved activity, R275V / S282P and R275V / S282P / T379E;

[0027] 4. Purify and determine the catalytic activity of the mutants;

[0028] 5. Use mutants to catalyze tryptophan to produce 5-HTP.

[0029] Beneficial effects of the present invention:

[0030] The present invention obtains mutants R275V / S282P and R275V / S282P / T379E of tryptophan-5-hydroxylase by site-directed mutagenesis, and the relative enzyme activities of mutants R275V / S282P and R275V / S282P / T379E are 185% and 206% of wild-type KERL, respectively. In the test of catalyzing tryptophan to produce 5-HTP, the 10-min tryptophan conversion rates of mutants R275V / S282P and R275V / S282P / T379E reach 35.76% and 49.82%, respectively, which are 1.8 times and 2.6 times of the wild-type, respectively. The tryptophan-5-hydroxylase mutant provided by the present invention significantly improves the enzyme activity and can be fully used for industrial enzyme catalysis and efficient synthesis of 5-HTP. Description of the drawings:

[0031] Figure 1 : HPLC identification results of the catalytic products. In the figure, “sample” corresponds to the product sample after the mutant R275V / S282P catalyzed the tryptophan reaction.

[0032] Figure 2 : Comparison of relative enzyme activities between wild type and mutant; in the figure, “WT” represents wild type KERL.

[0033] Figure 3 : Comparison of the conversion rates of tryptophan to 5-HTP catalyzed by the wild type and mutants; in the figure, "WT" represents the wild type KERL. Specific implementation method:

[0034] The present invention is further described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are all methods well known to those skilled in the art.

[0035] Example 1: Construction of recombinant plasmid

[0036] The kerl gene sequence (as shown in SEQ ID NO: 6) derived from the sponge animal symbiotic bacteria (Entotheonella factor) was optimized against the preference of the Escherichia coli host codon, and then the gene sequence was synthesized. The gene sequence was connected to the vector pQE-80L through two restriction sites, BamHI and HindIII. The connection product was transferred to the cloning host E.coli JM109, spread on LB solid medium for culture, and then the plasmid in the transformant was extracted for PCR and double enzyme verification, and the successful verification was sequenced. After the verification is correct, the pQE-80L-kerl recombinant plasmid is obtained.

[0037] Example 2: Construction of tryptophan 5-hydroxylase mutants

[0038] Using the pQE-80L-ker1 recombinant plasmid constructed in Example 1 as a template, mutation primers were designed, and site-directed mutation PCR amplification reactions were performed. Taking the introduction of mutations at the three amino acid sites of R275V / S282P / T379E as an example, the corresponding mutation primers included the following three pairs of primers: R275V-F / R, S282P-F / R, and T379E-F / R; wherein primer (5'-3') R275V-F and primer (5'-3') R275V-R are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; primer (5'-3') S282P-F and primer (5'-3') S282P-R are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; primer (5'-3') T379E-F and primer (5'-3') T379E-R are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. Shown in NO:12.

[0039] Add the reagents listed in Table 1 in order into a 0.2 mL EP tube to perform PCR amplification reaction.

[0040] Table 1 PCR amplification reaction system

[0041]

[0042]

[0043] The PCR reaction conditions and cycle process are shown in Table 2.

[0044] Table 2 PCR reaction conditions and cycles

[0045]

[0046] After the reaction, the product was demethylated, 0.35 μL Dpn I was added to the PCR product, and the reaction was carried out at 37°C for 1 h. The demethylated product was verified by agarose gel electrophoresis. The gene length was about 6000 bp. After successful verification, the next step of reaction was carried out.

[0047] For the self-cyclization of the PCR product, the reagents shown in Table 3 were added sequentially in a 0.2 mL EP tube to carry out the cyclization reaction.

[0048] Table 3 PCR product cyclization reaction system

[0049]

[0050] The ligation product after the cyclization reaction was added to the competent cells of E.coli K-12MG1655, and the following operations were performed in sequence: gently mixed, ice bath for 30 minutes; heat shock at 42℃ for 90 seconds; immediately ice bath for 5 minutes; add 600μL LB recovery solution, 37℃, 220r / min recovery culture for 40-60 minutes; centrifuge at 4000r / min for 5 minutes; suspend the precipitate obtained by centrifugation with 200μL LB culture medium, mix and spread on the resistance plate containing Amp, and after the bacterial solution is fully absorbed, invert and culture at 37℃ for 12-16 hours until a single colony appears; pick a single colony for activity detection. The above methods were used to obtain engineered strains containing mutant R275V / S282P and R275V / S282P / T379E genes. Among them, the amino acid sequence and gene sequence of the mutant R275V / S282P are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively; the amino acid sequence and gene sequence of the mutant R275V / S282P / T379E are shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively.

[0051] The detection peaks of samples and standards are as follows Figure 1 shown.

[0052] Example 3: Purification of mutant proteins

[0053] Protein purification: Resuspend the engineered bacterial cells containing the mutant gene in Lysis Buffer (50mM NaH2PO4, 10mM imidazole, 300mM NaCl, pH=8.0), add 200μL lysozyme and 120μL PMSF, mix well, pour into a beaker and place in an ice bath for 20min. The cell suspension is ultrasonically disrupted for 15min (ultrasound 2.5s, interval 3.0s). The broken liquid was placed at 4°C, 12,000r / min for 30min, the supernatant obtained by centrifugation was combined with Ni-NTA superflow equilibrated with Lysis Buffer for 1h, the combined liquid was passed through the purification column, and the impurities were eluted with Wash Buffer (50mM NaH2PO4, 50mMimidazole, 300mM NaCl, pH=8.0), and then the target protein was eluted with Elution Buffer (50mM NaH2PO4, 300mMimidazole, 300mM NaCl, pH=8.0), and then the Elution Buffer was dialyzed against 50mM HEPES (pH=8.0, containing 375mMNaCl), and the mutant pure enzyme solution was obtained by ultrafiltration concentration. The protein concentration was determined by Nanodrop2000. The activity of the mutant was determined by referring to the following catalytic reaction system: 3mM tryptophan, 3mM H2O2 and 20μmol enzyme solution were dissolved in 1mL 50mM HEPES (pH=8.0), reacted at 30°C for 10min, and the reaction was terminated by boiling for 10min. After centrifugation, the supernatant was diluted to an appropriate concentration and passed through a 0.22μm water filter into a chromatographic injection bottle. The product was identified and analyzed by HPLC. The HPLC detection conditions are as follows:

[0054] Chromatograph: Agilent 1260;

[0055] Detector: UV detector (Alltech Chrom, ELSD6000)

[0056] Injection: Agilent autosampler; injection volume 10 μL;

[0057] Chromatographic column: Diamonsil C18 (5 μm, 4.6 × 250 mm, DiKMA, Beijing); column temperature 33 °C;

[0058] Mobile phase: 10% acetonitrile aqueous solution; flow rate 1 mL / min.

[0059] The test results showed that both mutants could efficiently catalyze tryptophan to produce 5-HTP. Figure 1 shown.

[0060] Example 4: Determination of relative enzyme activity of mutants

[0061] The enzyme activity of the present invention is defined as follows: Under standard reaction conditions, 1 mL of 50 mM HEPES (pH = 8.0), 3 mM tryptophan, 3 mM H2O2 and 20 μM wild-type or mutant pure enzyme solution are uniformly mixed. The reaction mixture is incubated at 30°C for 10 min and boiled for 10 min to terminate the reaction. One unit (U) of enzyme is defined as the amount of 1 μM tryptophan consumed per minute under the above conditions. The relative enzyme activity of the mutant is based on the wild-type enzyme activity as 100%. The relative enzyme activities of the purified mutant and the wild-type were measured with reference to the enzyme reaction system of Example 3. The results are as follows: Figure 2 As shown, the relative enzyme activity of R275V / S282P is 185% of that of the wild type, and the relative enzyme activity of R275V / S282P / T379E is 206% of that of the wild type. Both mutants significantly increased their catalytic activity towards tryptophan compared to the wild type.

[0062] Example 5: Production of 5-HTP using mutants

[0063] In a 1L Erlenmeyer flask, add HEPES buffer (50mM, pH 8.0) containing 3mM tryptophan and 20μM wild-type or mutant pure enzyme solution, and react at 30℃ for 10min. After the reaction, boil for 10min and terminate the reaction by centrifugation at 12,000rpm for 5min. Detect the content of 5-HTP in the supernatant by HPLC, and compare the conversion rate of wild-type and mutant to produce 5-HTP at the same reaction time. The results are shown in Figure 3 As shown: After 10 minutes of reaction, the tryptophan conversion rate catalyzed by the wild-type KERL can reach 19.33%, the tryptophan conversion rate of the mutant R275V / S282P can reach 35.76%, which is 1.8 times that of the wild-type, and the tryptophan conversion rate of the mutant R275V / S282P / T379E can reach 49.82%, which is 2.6 times that of the wild-type.

Claims

1. A tryptophan 5-hydroxylase mutant, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. The tryptophan 5-hydroxylase mutant according to claim 1, characterized in that The gene sequence of the mutant is shown in SEQ ID NO:3 or SEQ ID NO:

4.

3. An expression vector, characterized in that: The expression vector carries the gene sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

4. The expression vector according to claim 3, characterized in that: The type of the expression vector is pQE-80L, pET-22b or pMA5.

5. An engineered strain, characterized in that: The engineered strain contains the expression vector according to claim 3.

6. The engineered strain according to claim 5, characterized in that: The engineering strain is Escherichia coli, Bacillus subtilis or Pichia pastoris.

7. The engineered strain according to claim 5, characterized in that: The engineering strain is Escherichia coli E.coliK-12MG1655, and the type of expression vector contained therein is pQE-80L.

8. Use of the tryptophan 5-hydroxylase mutant according to claim 1 or 2 in producing 5-hydroxytryptophan.

9. Use of the expression vector as claimed in claim 3 or 4 in producing 5-hydroxytryptophan.

10. Use of the engineered strain according to claims 5 to 7 in producing 5-hydroxytryptophan.

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