A mutant of Mycobacterium tuberculosis tyrosine phenol lyase and its application

Abstract

This invention discloses a mutant of Mycobacterium tuberculosis tyrosine lyase and its applications, belonging to the fields of molecular biology and biocatalysis. This mutant, denoted as mutant I384V, is obtained by mutating isoleucine at position 384 of the Mycobacterium tuberculosis tyrosine lyase to valine. I384V has similar optimal temperature and pH to the wild-type tyrosine lyase, but exhibits higher specific enzyme activity and catalytic efficiency. Compared to the wild-type, the mutant I384V improves the synthesis efficiency of levodopa by 38.4%, showing broad prospects for large-scale industrial application.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and biocatalysis, specifically relating to a tyrosine phenol lyase mutant derived from Mycobacterium tuberculosis and its applications. Background Technology

[0002] Levodopa (L-dopa, full name 3,4-dihydroxyphenylalanine) is a dopamine prodrug with no direct pharmacological activity. It can cross the blood-brain barrier into the central nervous system, where it is converted into dopamine by dopa decarboxylase, thereby exerting a therapeutic effect. Therefore, it is widely used in the drug treatment of Parkinson's disease.

[0003] Currently, traditional methods for preparing levodopa mainly include plant extraction and organic chemical synthesis. However, plant extraction (such as from kohlrabi or quinoa) is limited by seasonality and raw material supply, resulting in low yields that cannot meet market demand. Organic chemical synthesis typically uses vanillin and hydantoin as raw materials, producing levodopa through multiple steps. However, this method requires large amounts of metal catalysts, has low reaction efficiency and low product optical activity, and causes serious environmental pollution, thus gradually being restricted in modern industry.

[0004] In contrast, bio-enzymatic catalysis, due to its high catalytic efficiency and environmental friendliness, has become an important direction for the industrial production of levodopa. Tyrosinase can catalyze the conversion of L-tyrosine to levodopa, but it further oxidizes the product to dopaquinone, increasing the difficulty of subsequent separation. Transaminase produces levodopa using L-aspartic acid or L-glutamic acid and 3,4-dihydroxyphenylpyruvic acid as substrates, but its catalytic activity is low and it has not yet been industrialized. Tyrosine phenol lyase (TPL), on the other hand, catalyzes the synthesis of levodopa from catechol, pyruvic acid, and ammonia, exhibiting higher catalytic activity and reaction rate, but requiring the coenzyme pyridoxal phosphate. Based on its significant advantages, TPL shows great application potential in the industrial production of levodopa.

[0005] Currently known synthetic levodopa, TPL, is widely found in various microorganisms, especially gut microbiota, specifically including Erwinia herbivora (…). Erwinia herbicola ), Citrobacter fluocinolone ( Citrobacter frenudii ), Fusobacterium nucleatum ( Fusobacterium nucleatum Kluwer ( Kluyvera intermedia However, in current applications, existing TPL is still limited by insufficient enzyme activity. Therefore, there is still a need for TPL mutants that can effectively improve the activity of synthesizing levodopa in order to improve its production efficiency. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide a Mycobacterium tuberculosis tyrosine phenol lyase mutant and its applications. This mutant can enhance the activity of levodopa synthesis.

[0007] This invention clones a tyrosine phenol lyase gene derived from Mycobacterium tuberculosis, rationally designs the tyrosine phenol lyase gene, and inserts the mutated gene into the expression plasmid pET-28a(+) at two restriction enzyme sites. Nde I and Xho Between I, transition to E. coli BL21(DE3) expression strain E. coli BL21(DE3) / pET28a-I384V was purified and expressed to obtain the tyrosine phenol lyase mutant I384V. I384V has similar optimal temperature and pH to the wild-type tyrosine phenol lyase, but I384V exhibits higher specific activity and catalytic efficiency than the wild-type tyrosine phenol lyase. The tyrosine phenol lyase mutant I384V synthesizes approximately 40% more levodopa than the wild-type.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A mutant of Mycobacterium tuberculosis tyrosine lyase, wherein the tyrosine lyase mutant is derived from Mycobacterium tuberculosis (Mycobacterium tuberculosis) Mycobacterium tuberculosis The isoleucine (Ile, I) at position 384 of the tyrosine phenol lyase is mutated to valine (Val, V), and its amino acid sequence is shown in SEQ ID NO: 3.

[0010] The encoding gene of a mutant Mycobacterium tuberculosis tyrosine phenol lyase.

[0011] Preferably, the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 2.

[0012] The aforementioned mutant-related biological materials are any one or more combinations of the following biological materials:

[0013] (a) An expression cassette containing the above-mentioned encoded genes;

[0014] (b) Recombinant expression vectors containing the above-mentioned coding genes;

[0015] (c) A recombinant expression vector containing the expression cassette described in (a);

[0016] (d) Recombinant microorganisms containing the above-mentioned encoding genes;

[0017] (e) Recombinant microorganisms containing the expression cassette described in (a);

[0018] (f) Recombinant microorganisms containing the recombinant expression vector described in (b) or (c).

[0019] Furthermore, the starting vector for the recombinant expression vector described in (b) and (c) is a pET series vector, etc.; preferably, it is a pET-28a(+) vector.

[0020] Furthermore, the host microorganisms corresponding to the recombinant microorganisms mentioned in (d), (e), and (f) are selected from prokaryotes, etc.; the prokaryotes include Escherichia coli (…). Escherichia Bacteria such as Escherichia coli. More specifically, the prokaryotes are Escherichia coli (E. coli). Escherichia coli , E. coli Specifically, it could be Escherichia coli BL21 (DE3).

[0021] The application of the above-mentioned mutants, encoding genes, or mutant-related biological materials in the preparation of tyrosine phenol lyase mutants that enhance the activity of synthetic levodopa.

[0022] The application of the aforementioned mutants, encoding genes, or mutant-related biological materials in the synthesis of levodopa. Specifically, levodopa is obtained by using the aforementioned mutants, encoding genes, or mutant-related biological materials as catalysts, using catechol, pyruvate, and ammonium acetate as substrates, using sodium sulfite and EDTA as auxiliaries, and using pyridoxal phosphate as a cofactor.

[0023] A method for obtaining the above-mentioned Mycobacterium tuberculosis tyrosine phenol lyase mutant includes the following steps: expressing the gene encoding the Mycobacterium tuberculosis tyrosine phenol lyase mutant to obtain the Mycobacterium tuberculosis tyrosine phenol lyase mutant.

[0024] Furthermore, the gene encoding the Mycobacterium tuberculosis tyrosine phenol lyase mutant was transformed into Escherichia coli BL21(DE3) for expression, thus obtaining the Mycobacterium tuberculosis tyrosine phenol lyase mutant.

[0025] The present invention has the following advantages and effects compared with the prior art:

[0026] (1) The tyrosine phenol lyase mutant of the present invention retains the original optimal temperature and pH of wild-type tyrosine phenol lyase, indicating that it is still suitable for the synthesis of levodopa after mutation.

[0027] (2) The tyrosine phenol lyase mutant I384V of the present invention has higher specific enzyme activity, better catalytic efficiency, and the specific enzyme activity is increased by 40.5%.

[0028] (3) The tyrosine phenol lyase mutant of the present invention has higher synthesis efficiency for the synthesis of levodopa and tyrosine. Compared with the wild type, the synthesis efficiency of levodopa by this mutant is increased by 38.4%, which has broad prospects for large-scale application. Attached Figure Description

[0029] Figure 1 SDS-PAGE analysis of the tyrosine phenol lyase mutant; where M: standard protein molecular weight; 1: precipitate after cell disruption; 2: purified flow-through collection solution; 3: 10% Buffer B elution solution; 4: purified TPL mutant I384V.

[0030] Figure 2 The optimal pH curves for the tyrosine phenol lyase mutant and the wild type are shown.

[0031] Figure 3 The optimal temperature curves for the tyrosine phenol lyase mutant and the wild type are shown.

[0032] Figure 4 The yield curve of levodopa synthesis from mutants cultured in a fermenter at scale-up. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0034] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0035] Example 1: Construction of recombinant plasmid pET28a-I384V

[0036] (1) Design primers I384V-F / I384V-R to introduce mutant bases.

[0037] I384V-F: 5'-CGTGGTATCgtgTCTGCTGGTCGTAACAAAGA-3';

[0038] I384V-R: 5'-CCAGCAGAcacGATACCACGTTCCATAGAACG-3';

[0039] (2) Using I384V-F / I384V-R as primers and recombinant plasmid pET28a-MtTPL as template, upstream and downstream fragments containing mutant bases on homologous arms were amplified by PCR.

[0040] (3) The fragment from step (2) is recovered as a PCR product and then transformed into a cloning host. E. coli The TOP10 were verified by colony PCR and sequencing, thus obtaining the successfully constructed recombinant plasmid pET28a-I384V;

[0041] (4) Take the recombinant plasmid pET28a-I384V and add it to competent cells of Escherichia coli. E. coli Ice bath in BL21 (DE3);

[0042] (5) Spread the ice bath product obtained in step (4) onto an LB agar plate containing kanamycin and incubate at 37°C to obtain the recombinant expression strain. E. coli BL21(DE3) / pET28a-I384V;

[0043] (6) The cloned strain containing the correct recombinant plasmid pET28a-I384V was stored at -80℃ with 15wt% glycerol.

[0044] The recombinant plasmid pET28a-MtTPL is formed by inserting the nucleotide sequence (SEQ ID NO.1) of the gene encoding the tyrosine phenol lyase MtTPL, optimized using E. coli codons, into the pET-28a(+) vector. Nde I and Xho It is constructed from I.

[0045] Example 2 Expression and purification of mutant tyrosine phenol lyase I384V.

[0046] (1) Transform the successfully constructed recombinant plasmid pET28a-I384V into E. coli Escherichia coli expression strain was constructed using BL21(DE3) competent cells. E. coli BL21(DE3) / pET28a-I384V, specifically, involves adding the recombinant plasmid pET28a-I384V into competent E. coli cells. E. coli Ice bath in BL21 (DE3) for 5 min.

[0047] (2) Inoculate a single colony in 10 mL of LB medium (containing 50 μg / mL kanamycin) and culture at 37°C with shaking at 200 rpm for 12 h.

[0048] (3) Inoculate the seed culture into 500 mL of LB medium (containing 50 μg / mL kanamycin) at a volume ratio of 1:100, and culture at 37°C with shaking at 200 rpm until the OD reaches 0.8.

[0049] (4) Add IPTG (isopropyl thiogalactoside) to a final concentration of 0.5 mM and induce expression at 20°C and 200 rpm for 16 h.

[0050] (5) After the induction expression is completed, the bacterial cells are collected by centrifugation and the recombinant protein expressed in the cells is extracted by ultrasonic disruption.

[0051] (6) Use Ni 2+ The column was used for purification, and the SDS-PAGE analysis of the purified tyrosine phenol lyase mutant is shown in the figure below. Figure 1 As shown, M represents the molecular weight of the standard protein; 4 represents the purified TPL mutant I384V. The results show that the recombinant expression strain... E. coli A distinct protein band (at 55.35 kDa) was observed in the BL21(DE3) / pET28a-I384V expressed protein.

[0052] Example 3 Determination of the enzymatic properties of wild-type tyrosine phenol lyase and mutant I384V

[0053] (1) Reaction system for the synthesis of levodopa and method for activity determination

[0054] The reaction system consisted of 50 mM Tris-HCl buffer (pH 9.0), 10 μg purified enzyme, 4.4 g / L catechol, 4.4 g / L sodium pyruvate, 27.5 g / L ammonium acetate, 20 μM PLP (pyridoxal phosphate), 1 g / L sodium sulfite, 2 g / L EDTA, and 20 mM KCl, with a total volume of 1 mL. The reaction conditions were 30 °C and pH 9.0. The reaction was terminated by adding an equal volume of 1 M HCl. The sample was then centrifuged at 12,000 rpm for 2 min and analyzed by HPLC. One unit of enzyme activity was defined as the amount of enzyme catalyzing the formation of 1 μmol of levodopa per minute. Specific enzyme activity was expressed in units per mg of protein.

[0055] (2) Optimal pH: Using the above reaction system, the enzyme activities of wild-type tyrosine phenol lyase and mutants were measured at pH 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 at 40℃. The highest enzyme activity was defined as 100%. The results are as follows: Figure 2 The results showed that the optimal pH for the tyrosine phenol lyase mutant I384V was similar to that of the wild type, both being 9.0.

[0056] (3) Optimal temperature: Using the above reaction system, the enzyme activities of wild-type tyrosine lyase and mutant were measured under different temperature conditions at the optimal pH of 9.0. The highest enzyme activity was defined as 100%. The results are as follows: Figure 3 The results showed that the optimal pH for the tyrosine phenol lyase mutant I384V was similar to that of the wild type, both being 40°C.

[0057] (4) Kinetic analysis of mutant proteins: The enzyme activity of tyrosine phenol lyase mutants with different catechol substrate concentrations was measured. The Michaelis constant Km of tyrosine phenol lyase mutant I384V was obtained by Lineweaver-Burk double reciprocal plotting method, and the turnover number kcat and enzyme catalytic efficiency kcat / Km were calculated. Comparing the kinetic parameters of wild type and mutant, it can be seen from Table 1 that the Km of tyrosine phenol lyase mutant I384V is almost unchanged, while the enzyme activity is increased by 40.5% and Kcat is increased by 26.5%. Analysis of its substrate binding constant and turnover number shows that mutant I384V may have optimized the enzyme catalytic mechanism, improved substrate conversion efficiency, and thus improved enzyme catalytic efficiency.

[0058] Table 1 Kinetic parameters of wild type and mutant

[0059] Enzyme activity (U / mg) Km (mM) <![CDATA[k cat (s -1 )]]> <![CDATA[k cat / K m (mM -1 S -1 )]]> Wt 2.57 12.55 4.91 0.391 I384V 3.610 11.81 6.21 0.526

[0060] Example 4: Scale-up culture of mutants in a 5L fermenter to synthesize levodopa

[0061] Inoculation with wild-type and mutant E. coli BL21(DE3) / pET28a-I384V strains were cultured overnight in 500 mL LB medium at 37°C and 200 rpm. They were then transferred at a 5% inoculum to 5 L fermenters containing fermentation medium. The fermentation medium contained 12 g / L peptone, 24 g / L yeast extract, 30 g / L glycerol, 2.31 g / L KH₂PO₄, and 12.54 g / L K₂HPO₄. After 8 h of culture, IPTG was added for induction. The cells were collected after 14 h of induction and then reacted with the reaction solution.

[0062] The initial reaction solution consisted of 8 g / L sodium pyruvate, 5 g / L catechol, 77 g / L ammonium acetate, 1 g / L Na₂SO₃, 2 g / L EDTA·2Na, and 1 mM PLP. Because high concentrations of catechol can have an irreversible inhibitory effect on *E. coli* and the enzyme, a fed-batch method was adopted. After fermentation began, catechol, sodium pyruvate, and ammonium acetate were added every 2 hours at rates of 5.0 g / L, 5.0 g / L, and 3.5 g / L to ensure substrate consumption and enzyme activity. Results are as follows... Figure 4 As shown, the final mutant I384V yielded 95.36 g / L of levodopa with a yield of 88%, which is 38.4% higher than that of the wild type.

[0063] The above results indicate that the tyrosine phenol lyase mutant retains the optimal conditions for wild-type tyrosine phenol lyase, and compared with the wild type, the mutant has a significantly improved efficiency in synthesizing levodopa.

[0064] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A mutant of Mycobacterium tuberculosis tyrosine phenol lyase, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO:

3.

2. The gene encoding the Mycobacterium tuberculosis tyrosine phenol lyase mutant of claim 1.

3. The gene according to claim 2, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

4. The biomaterial related to the Mycobacterium tuberculosis tyrosine phenol lyase mutant as described in claim 1, characterized in that, It can be any one or more combinations of the following biological materials: (a) An expression cassette containing the gene of claim 2 or 3; (b) A recombinant expression vector containing the gene of claim 2 or 3; (c) A recombinant expression vector containing the expression cassette described in (a); (d) A recombinant microorganism containing the gene described in claim 2 or 3; (e) Recombinant microorganisms containing the expression cassette described in (a); (f) Recombinant microorganisms containing the recombinant expression vector described in (b) or (c).

5. The biomaterial according to claim 4, characterized in that: The starting vector for the recombinant expression vectors described in (b) and (c) is a pET series vector; The host microorganisms corresponding to the recombinant microorganisms described in (d), (e), and (f) are selected from prokaryotes.

6. The biomaterial according to claim 5, characterized in that: The starting vector for the recombinant expression vectors described in (b) and (c) is the pET-28a(+) vector; The host microorganisms corresponding to the recombinant microorganisms described in (d), (e), and (f) are selected from Escherichia coli.

7. The use of the gene according to any one of claims 2 to 3 or the biomaterial according to any one of claims 4 to 6 in the preparation of a tyrosine phenol lyase mutant that enhances the activity of synthetic levodopa.

8. The use of the Mycobacterium tuberculosis tyrosine lyase mutant of claim 1, the gene of any one of claims 2-3, or the biomaterial of any one of claims 4-6 in the synthesis of levodopa.

9. The application according to claim 8, characterized in that: Using the Mycobacterium tuberculosis tyrosine phenol lyase mutant of claim 1, the gene of any one of claims 2-3, or the biological material of any one of claims 4-6 as a catalyst, catechol, pyruvic acid, and ammonium acetate as substrates, sodium sulfite and EDTA as auxiliaries, and pyridoxal phosphate as a cofactor, levodopa is obtained by reaction.

10. A method for obtaining the Mycobacterium tuberculosis tyrosine phenol lyase mutant of claim 1, characterized in that, The method includes the following steps: expressing the gene encoding the Mycobacterium tuberculosis tyrosine phenol lyase mutant with the amino acid sequence shown in SEQ ID NO: 3 to obtain the Mycobacterium tuberculosis tyrosine phenol lyase mutant as described in claim 1.

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