Mycobacterium tuberculosis tyrosine phenol lyase mutant and application thereof

Abstract

The invention discloses a mycobacterium tuberculosis tyrosine phenol lyase mutant and application thereof, and belongs to the field of molecular biology and the technical field of biological catalysis. The mutant is obtained by mutating 384 isoleucine of tyrosine phenol lyase from mycobacterium tuberculosis into valine, and is marked as a mutant I384V. The I384V and wild type tyrosine phenol lyase have similar optimum temperature and optimum pH, but the I384V shows higher specific enzyme activity and catalytic efficiency than the wild type tyrosine phenol lyase. Compared with a wild type, the tyrosine phenol lyase mutant I384V has the advantages that the synthesis efficiency of the tyrosine phenol lyase mutant I384V on the synthesis of levodopa is improved by 38.4%, and the tyrosine phenol lyase mutant I384V has wide prospects in large-scale industrial application.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology and biocatalysis technology, and specifically relates to a tyrosine phenol lyase mutant derived from Mycobacterium tuberculosis and an application thereof. Background Art

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

[0003] At present, the traditional preparation methods of levodopa mainly include plant extraction and organic chemical synthesis. However, plant extraction (such as extraction from cat beans or velvet beans) is limited by seasons and raw material supply, and the yield is low, which cannot meet market demand. Organic chemical synthesis usually uses vanillin and hydantoin as raw materials and produces levodopa through multiple steps of reaction, but this method requires a large amount of metal catalysts, has low reaction efficiency and product optical activity, and will cause serious environmental pollution, so it is gradually restricted in modern industry.

[0004] In contrast, the bio-enzyme catalysis method has become an important direction for the industrial production of L-DOPA due to its high catalytic efficiency and green environmental protection. Tyrosinase can catalyze L-tyrosine to produce L-DOPA, but it will further oxidize the product to dopaquinone, increasing the difficulty of subsequent separation; transaminase uses L-aspartate or L-glutamate and 3,4-dihydroxyphenylpyruvate as substrates to produce L-DOPA, but its catalytic activity is low and has not yet been industrialized; while tyrosine phenol lyase (TPL) catalyzes the synthesis of L-DOPA from catechol, pyruvate and ammonia, has higher catalytic activity and reaction rate, and requires the coenzyme pyridoxal phosphate. Based on its significant advantages, TPL shows great application potential in the industrial production of L-DOPA.

[0005] Currently known TPLs for the synthesis of levodopa are widely present in various microorganisms, especially intestinal microorganisms, including Erwinia herbicola ( Erwinia herbicola ), Citrobacter fluorescens ( Citrobacter frenudii )、Fusobacterium nucleatum( Fusobacterium nucleatum ), Kluyvera ( Kluyvera intermedia ), etc. However, in the current application scenarios, the 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 synthetic levodopa to improve its production efficiency. Summary of the invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a mutant of Mycobacterium tuberculosis tyrosine phenol lyase and its application. The mutant can improve the activity of synthesizing levodopa.

[0007] The present invention clones the tyrosine phenol lyase gene from Mycobacterium tuberculosis, rationally designs the tyrosine phenol lyase gene, and embeds the mutated gene into the two restriction sites of the expression plasmid pET-28a (+). N I and XOt I, transfer E. coli BL21 (DE3) expression strain E. coli BL21 (DE3) / pET28a-I384V, after purification and expression, obtained the tyrosine phenol lyase mutant I384V. I384V has similar optimum temperature and optimum pH as the wild-type tyrosine phenol lyase, but I384V shows higher specific enzyme activity and catalytic efficiency than the wild-type tyrosine phenol lyase. The tyrosine phenol lyase mutant I384V is about 40% higher than the wild-type in synthesizing L-DOPA.

[0008] The purpose of the present invention is achieved through the following technical solutions: A Mycobacterium tuberculosis tyrosine phenol lyase mutant, the tyrosine phenol lyase mutant is derived from Mycobacterium tuberculosis ( Mycobacterium tuberculosis ) tyrosine phenol lyase at position 384 isoleucine (Ile, I) is mutated to valine (Val, V), and its amino acid sequence is shown in SEQ ID NO: 3.

[0009] A gene encoding the Mycobacterium tuberculosis tyrosine phenol lyase mutant.

[0010] Preferably, the nucleotide sequence of the encoding gene is shown as SEQ ID NO: 2.

[0011] The mutant-related biological material is any one or more combinations of the following biological materials: (a) an expression cassette containing the above-mentioned coding gene; (b) a recombinant expression vector containing the above encoding gene; (c) a recombinant expression vector containing the expression cassette described in (a); (d) a recombinant microorganism containing the above encoding gene; (e) a recombinant microorganism containing the expression cassette described in (a); (f) A recombinant microorganism containing the recombinant expression vector described in (b) or (c).

[0012] Furthermore, the starting vector of the recombinant expression vector in (b) and (c) is a pET series vector, etc., preferably a pET-28a(+) vector.

[0013] Furthermore, the host microorganism corresponding to the recombinant microorganism in (d), (e), and (f) is selected from prokaryotes, etc.; the prokaryotes include Escherichia ( Escherichia ) and other bacteria. More specifically, the prokaryotic organism is Escherichia coli ( Escherichia coli , E. coli ), specifically, it can be Escherichia coli BL21 (DE3).

[0014] Application of the mutant, coding gene or mutant-related biological material in the preparation of a tyrosine phenol lyase mutant with improved levodopa synthesis activity.

[0015] The application of the mutant, encoding gene or mutant-related biomaterial in the synthesis of L-dopa is as follows: using the mutant, encoding gene or mutant-related biomaterial as a catalyst, catechol, pyruvic acid, and ammonium acetate as substrates, sodium sulfite and EDTA as auxiliary agents, and pyridoxal phosphate as a cofactor to react and obtain L-dopa.

[0016] A method for obtaining the above-mentioned Mycobacterium tuberculosis tyrosine phenol lyase mutant comprises the following steps: expressing a gene encoding the Mycobacterium tuberculosis tyrosine phenol lyase mutant to obtain the Mycobacterium tuberculosis tyrosine phenol lyase mutant.

[0017] Furthermore, the gene encoding the mutant of Mycobacterium tuberculosis tyrosine phenol lyase was transformed into Escherichia coli BL21 (DE3) for expression to obtain the mutant of Mycobacterium tuberculosis tyrosine phenol lyase.

[0018] Compared with the prior art, the present invention has the following advantages and effects: (1) The tyrosine phenol lyase mutant of the present invention retains the original optimum temperature and pH of the wild-type tyrosine phenol lyase, indicating that the mutant is still suitable for the synthesis of L-DOPA.

[0019] (2) The tyrosine phenol lyase mutant I384V of the present invention has higher specific enzyme activity and better catalytic efficiency, and the specific enzyme activity is increased by 40.5%.

[0020] (3) The tyrosine phenol lyase mutant of the present invention has a higher synthesis efficiency for the synthesis of L-DOPA and tyrosine. Compared with the wild type, the mutant has a 38.4% higher synthesis efficiency for L-DOPA, and has broad prospects for large-scale application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1The SDS-PAGE analysis of the tyrosine phenol lyase mutant; wherein, M: standard protein molecular weight; 1: precipitate after cell disruption; 2: purified flow cytometry collection fluid; 3: 10% Buffer B eluate; 4: purified TPL mutant I384V.

[0022] Figure 2 This is the optimal pH curve of the tyrosine phenol lyase mutant and the wild type.

[0023] Figure 3 This is the optimal temperature curve of tyrosine phenol lyase mutant and wild type.

[0024] Figure 4 This is a graph showing the production curve of L-DOPA produced by the mutant in fermentation tank scale-up culture. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0026] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0027] Example 1 Construction of recombinant plasmid pET28a-I384V (1) Design primers I384V-F / I384V-R to introduce mutant bases.

[0028] I384V-F: 5'-CGTGGTATCgtgTCTGCTGGTCGTAACAAAGA-3'; I384V-R: 5'-CCAGCAGAcacGATACCACGTTCCATAGAACG-3'; (2) Using I384V-F / I384V-R as primers and the recombinant plasmid pET28a-MtTPL as a template, PCR was used to amplify the upstream and downstream fragments containing the mutant base on the homology arm.

[0029] (3) The fragment of step (2) is recovered by PCR and transformed into a cloning host E. coli TOP10 was verified by colony PCR and sequencing, thus obtaining the successfully constructed recombinant plasmid pET28a-I384V; (4) Take the recombinant plasmid pET28a-I384V and add it to the competent E. coli E. coli Ice bath in BL21 (DE3); (5) Spread the ice bath product obtained in step (4) on an LB plate containing kanamycin and culture at 37°C to obtain a recombinant expression strain. E. coli BL21(DE3) / pET28a-I384V; (6) The cloned strain containing the correct recombinant plasmid pET28a-I384V was stored at -80°C with 15 wt% glycerol.

[0030] The recombinant plasmid pET28a-MtTPL is a plasmid in which the nucleotide sequence (SEQ ID NO.1) encoding the tyrosine phenol lyase MtTPL gene is inserted into the pET-28a(+) vector. N I and XOt I is constructed between.

[0031] Example 2 Expression and purification of mutant tyrosine phenol lyase I384V.

[0032] (1) Transform the successfully constructed recombinant plasmid pET28a-I384V into E. coli BL21 (DE3) competent cells to construct E. coli expression strains E. coli BL21 (DE3) / pET28a-I384V, specifically, the recombinant plasmid pET28a-I384V was added to the competent E. coli E. coli Incubate in BL21 (DE3) on ice for 5 min.

[0033] (2) Inoculate a single colony into 10 mL of LB medium (containing 50 μg / mL kanamycin) and culture at 37°C with shaking at 200 rpm for 12 h.

[0034] (3) Inoculate the seed solution 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.

[0035] (4) Add IPTG (isopropylthiogalactoside) at a final concentration of 0.5 mM and induce expression at 20°C and 200 rpm for 16 h.

[0036] (5) After induction of expression, the cells were collected by centrifugation and ultrasonically disrupted to extract the recombinant protein expressed intracellularly.

[0037] (6) Use Ni 2+ The purified tyrosine phenol lyase mutant was purified by SDS-PAGE analysis. Figure 1 As shown, where M is the molecular weight of the standard protein; 4 is the purified TPL mutant I384V. The results showed that the recombinant expression strain E. coliAn obvious protein band (55.35 kDa position) appeared in the protein expressed by BL21 (DE3) / pET28a-I384V.

[0038] Example 3 Determination of the enzymatic properties of wild-type tyrosine phenol lyase and mutant I384V (1) Reaction system for synthesis of levodopa and activity determination method The reaction system included 50mM Tris-HCL buffer (pH9.0), 10μg pure enzyme, 4.4g / L catechol, 4.4g / L sodium pyruvate, 27.5g / L ammonium acetate, 20μM PLP (pyridoxal phosphate), 1g / L sodium sulfite, 2g / L EDTA, 20mM KCl, and a total volume of 1mL. The reaction conditions were 30℃, pH=9.0. The reaction was terminated by adding an equal volume of 1M HCl. The sample was then centrifuged at 12000rpm for 2min and analyzed by HPLC. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1μmol of L-DOPA per minute. The specific enzyme activity is expressed in units per mg of protein.

[0039] (2) Optimum pH: Using the above reaction system at 40°C, the enzyme activities of the 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, respectively. The highest enzyme activity was defined as 100%. The results are as follows: Figure 2 The results showed that the optimum pH of tyrosine phenol lyase mutant I384V was similar to that of the wild type, both of which were 9.0.

[0040] (3) Optimum temperature: Using the above reaction system, at the optimum pH of 9.0, the enzyme activities of wild-type tyrosine phenol lyase and mutants were measured under different temperature conditions. The highest enzyme activity was defined as 100%. The results are as follows: Figure 3 The results showed that the optimum pH of tyrosine phenol lyase mutant I384V was similar to that of the wild type, both of which were 40℃.

[0041] (4) Analysis of reaction kinetics of mutant protein: The enzyme activity of tyrosine phenol lyase mutant at different catechol substrate concentrations was measured, and the Michaelis constant Km of tyrosine phenol lyase mutant I384V was obtained by Lineweaver-Burk double reciprocal plotting method, and the turnover number kcat value and enzyme catalytic efficiency kcat / Km value were calculated. Comparing the kinetic parameters of the wild type and the mutant, it can be seen from Table 1 that the Km of tyrosine phenol lyase mutant I384V is almost unchanged, while the specific 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 optimize the catalytic mechanism of the enzyme, improve the substrate conversion efficiency, and thus improve the catalytic efficiency of the enzyme.

[0042] Table 1 Kinetic parameters of wild type and mutants Specific 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 Example 4 5L fermentation tank to grow mutants to synthesize L-dopa Inoculation of wild type and mutants E. coli The BL21 (DE3) / pET28a-I384V strain was cultured in 500 mL of LB medium at 37 ° C 200 rpm overnight, and then transferred to a 5L fermenter containing a fermentation medium at a 5% inoculation rate. 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. IPTG was added for induction after 8 hours of culture, and the cells were collected in the tank after 14 hours of induction. React with the reaction solution.

[0043] The initial reaction solution included 8 g / L sodium pyruvate, 5 g / L catechol, 77 g / L ammonium acetate, 1 g / L Na2SO3, 2 g / LEDTA·2Na, and 1 mM PLP. Since high concentrations of catechol will have irreversible inhibitory effects on E. coli and enzymes, a batch feeding method was adopted. After the start of fermentation, catechol, sodium pyruvate and ammonium acetate were fed at a rate of 5.0 g / L, 5.0 g / L, and 3.5 g / L every 2 hours to ensure substrate consumption and enzyme activity. The results are shown in Figure 4 As shown, the final mutant I384V obtained 95.36 g / L of L-DOPA with a yield of 88%, which was 38.4% higher than the wild-type in terms of its ability to synthesize L-DOPA.

[0044] The above results indicate that the tyrosine phenol lyase mutant retains the optimal conditions of the wild-type tyrosine phenol lyase, and compared with the wild-type, the mutant has a significantly improved efficiency in synthesizing L-DOPA.

[0045] 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

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

3.

2. A gene encoding the Mycobacterium tuberculosis tyrosine phenol lyase mutant according to 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 biological material related to the Mycobacterium tuberculosis tyrosine phenol lyase mutant according to claim 1, characterized in that: Any one or more combinations of the following biological materials: (a) an expression cassette containing the gene according to claim 2 or 3; (b) a recombinant expression vector containing the gene according to claim 2 or 3; (c) a recombinant expression vector containing the expression cassette described in (a); (d) a recombinant microorganism containing the gene according to claim 2 or 3; (e) a recombinant microorganism containing the expression cassette described in (a); (f) A recombinant microorganism containing the recombinant expression vector described in (b) or (c).

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

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

7. Use of the Mycobacterium tuberculosis tyrosine phenol lyase mutant according to claim 1, 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 with improved activity in synthesizing levodopa.

8. Use of the Mycobacterium tuberculosis tyrosine phenol lyase mutant according to claim 1, 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 synthesis of levodopa.

9. The use according to claim 8, characterized in that: The method uses the Mycobacterium tuberculosis tyrosine phenol lyase mutant described in claim 1, the gene described in any one of claims 2 to 3, or the biological material described in any one of claims 4 to 6 as a catalyst, catechol, pyruvate, and ammonium acetate as substrates, sodium sulfite and EDTA as auxiliary agents, and pyridoxal phosphate as a cofactor to react and obtain levodopa.

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

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