Novel high-activity tyrosine ammonia lyase and application thereof

Abstract

The invention provides a novel high-activity tyrosine ammonia lyase and application thereof, the novel high-activity tyrosine ammonia lyase is derived from Trametes versicolor and is subjected to recombinant expression in Escherichia coli, a target gene is subjected to codon optimization, and an optimized DNA (deoxyribonucleic acid) sequence is shown as SEQ ID NO. 2. The enzyme can efficiently catalyze tyrosine to efficiently synthesize p-coumaric acid, and has great economic value in the field of p-coumaric acid production.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a novel high-activity tyrosine ammonia lyase and application thereof. Background Art

[0002] p-Coumaric acid (also known as p-hydroxycinnamic acid) is a natural phenolic compound. In the process of plant secondary metabolism, it is the precursor of many plant metabolites such as catechins and anthocyanins. It has anti-cancer, antioxidant, anti-inflammatory, antibacterial and antiviral pharmacological activities and is widely used in food, medical, health care products and cosmetics industries.

[0003] At present, the production methods of p-coumaric acid include plant extraction, chemical synthesis and biosynthesis. Among them, the plant growth cycle is long, the environmental impact is great, the p-coumaric acid content is low and the extraction cost is high, resulting in the high production cost of the plant extraction method and lack of market competitiveness. The traditional method of chemically synthesizing p-coumaric acid is mainly to prepare p-coumaric acid by Knoevenagel condensation reaction. The raw materials used in the condensation reaction are malonic acid and p-hydroxybenzaldehyde, and then morpholine, piperidine, etc. are used as catalysts for reaction, and the product in the reaction mixture is separated by sodium carbonate solution, and finally the precipitated product is obtained by acidification. This method has high requirements on conditions and power, is difficult to separate and purify, and is easy to pollute the environment.

[0004] There are currently two biosynthetic pathways for producing p-coumaric acid:

[0005] Pathway 1: Phenylalanine is used as the raw material, and phenylalanine ammonia lyase catalyzes deamination to generate cinnamic acid, and then cinnamic acid is catalyzed by cinnamic acid 4-hydroxylase (C4H) to generate p-coumaric acid. In this pathway, C4H can only function by binding to the endoplasmic reticulum membrane, and it is difficult to achieve active expression in prokaryotes such as Escherichia coli, so the synthesis efficiency of this route is low;

[0006] Pathway 2: Using tyrosine as raw material, tyrosine is catalyzed by tyrosine ammonia lyase to directly produce p-coumaric acid.

[0007]

[0008] Tyrosine ammonia-lyase (TAL, EC 4.3.1.23) is a member of the aromatic amino acid lyase family, which can catalyze the removal of amino groups from L-tyrosine to produce p-coumaric acid. Its catalytic efficiency directly determines the production efficiency and cost of p-coumaric acid. Therefore, the discovery and research of highly active tyrosine ammonia-lyases have been widely carried out at home and abroad, and applied to the production of p-coumaric acid. Trotman et al. have made an attempt to use TAL for production. They expressed Rg-TAL of Rhodotorula glutinis in Escherichia coli, stabilized TAL by fixing the whole cells in a calcium alginate matrix cross-linked with glutaraldehyde and polyethyleneimine (GA / PEI), and used the immobilized enzyme to convert 50 g / L of tyrosine into 39 g / L of p-coumaric acid in a 1L fermenter (Trotman RJ, et al. Biotechnol Prog. 2007, 23 (3): 638-44.). Huo Yanan et al. modified Rg-TAL from Rhodotorula glutinis through directed evolution and obtained the double mutant Rg-TALS9N / A11Y. Through shake flask fermentation, the strain produced 394.2 mg / L of p-coumaric acid at 48 h. The TAL enzyme activity of Rhodotorula glutinis was low and still could not meet the needs of industrial production (Huo Yanan, et al. Journal of Biotechnology. 2020, 36(11): 2367-2376.). Xue et al. found that TAL from Phanerochaete chrysosporium had good thermal stability and proved that the improvement of heat resistance could promote the improvement of enzyme activity. In 10 h, 50 g / L of tyrosine could be converted into 41.8 g / L of coumaric acid, which is also the highest yield of p-coumaric acid reported so far (Zhixiong Xue, et al. Enzyme and Microbial Technology. 2007, 42(1): 58-64.). Summary of the invention

[0009] The present invention aims to overcome the problem of low p-coumaric acid production efficiency caused by insufficient catalytic activity of tyrosine ammonia lyase in the prior art, discover a highly active tyrosine ammonia lyase, and optimize suitable reaction conditions for efficient production of p-coumaric acid.

[0010] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions:

[0011] The invention provides a new tyrosine ammonia lyase, which is derived from Trametes versicolor, and has an amino acid sequence of GenBank No. XP_008043251.1, and a specific sequence is shown in SEQ ID NO.1.

[0012] The present invention also provides a gene encoding the tyrosine ammonia lyase.

[0013] To facilitate expression in E. coli, the enzyme encoding gene (GenBank No: XP_008043251.1) was optimized according to the codon preference of E. coli. The optimized sequence is shown in SEQ ID NO.2.

[0014] The present invention also provides a recombinant vector for expressing the tyrosine ammonia lyase. Preferably, the recombinant vector is obtained by inserting the tyrosine ammonia lyase encoding gene SEQ ID NO.2 into an Escherichia coli expression vector to obtain a recombinant vector that can express a large amount of tyrosine ammonia lyase TvTAL. In the present invention, the pET-28a(+) vector is preferred.

[0015] The present invention also provides a recombinant bacterium containing the tyrosine ammonia lyase encoding gene. Preferably, the recombinant bacterium is obtained by introducing the tyrosine ammonia lyase encoding gene into Escherichia coli through the recombinant vector, and screening to obtain a recombinant bacterium expressing tyrosine ammonia lyase. In the present invention, the BL21 (DE3) strain of Escherichia coli is preferred.

[0016] The present invention also provides a detailed study on the enzymatic properties of the tyrosine ammonia lyase. Preferably, the method for studying the enzymatic properties of the tyrosine ammonia lyase is: fermenting and culturing the above-mentioned recombinant bacteria, inducing expression, breaking the cell wall of the cultured recombinant bacteria, and then separating and purifying to obtain the tyrosine ammonia lyase TvTAL.

[0017] On this basis, the effects of temperature, pH and other conditions on the catalytic activity and stability of the enzyme were studied respectively. The suitable reaction temperature of the enzyme is 45-65℃, and the suitable reaction pH is 9.0-11.0.

[0018] The present invention also provides application of the tyrosine ammonia lyase in producing p-coumaric acid.

[0019] Preferably, p-coumaric acid is produced by: the wet bacteria obtained by fermentation and the substrate L-tyrosine constitute a conversion system, and the appropriate reaction temperature and pH are controlled to produce p-coumaric acid by a whole-cell catalytic method.

[0020] Preferably, the initial concentration of the substrate L-tyrosine in the conversion system is 100 g / L, the amount of recombinant Escherichia coli added is 4%, the reaction temperature ranges from 45 to 55° C., and the reaction pH range is controlled to be 9.0 to 11.0 by adding sodium hydroxide solution.

[0021] Preferably, the initial concentration of the substrate L-tyrosine in the conversion system is 100 g / L, the amount of recombinant Escherichia coli added is 4%, the reaction temperature is 45°C, the reaction pH is controlled at about 10.5 by flowing sodium hydroxide solution, and after 12 hours of reaction, the final p-coumaric acid yield is 53.3 g / L.

[0022] The beneficial effects of the present invention are:

[0023] (1) The tyrosine ammonia lyase provided by the present invention is firstly discovered in the genus Trametes and confirmed by experiments. The amino acid sequence homology with TAL from Phanerochaete chrysosporium is 69.8%, and the homology with other experimentally confirmed tyrosine ammonia lyases is even lower. The TAL provided by the present invention is a new tyrosine ammonia lyase.

[0024] (2) The present invention provides a tyrosine ammonia lyase derived from Trametes versicolor. By constructing a high-expression recombinant engineered bacterium and measuring the enzymatic properties, it is found that the enzyme can efficiently catalyze the deamination of L-tyrosine to produce p-coumaric acid, thereby producing a higher concentration of p-coumaric acid.

[0025] (3) The enzyme was further applied to the preparation of p-coumaric acid and was found to have the advantages of rapid reaction and good stability, overcoming the defect of low p-coumaric acid yield and having important application value for the industrial preparation of p-coumaric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : SDS-PAGE gel electrophoresis detection of purified TvTAL.

[0027] Figure 2 : HPLC detection results of L-tyrosine and p-coumaric acid.

[0028] Figure 3 : The results of determination of enzymatic properties of TvTAL. DETAILED DESCRIPTION

[0029] The following examples are convenient for better understanding of the present invention, but are not limited to the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged. For the chromatograms of different reactions under the same condition parameters, the target peak retention time has a certain error range, which can be regarded as an error within 0.1min, and can be identified as the same target.

[0030] The present invention is described in more detail below with reference to specific embodiments.

[0031] Example 1

[0032] Construction, cultivation and inducible expression of genetically engineered Escherichia coli strains highly expressing tyrosine ammonia lyase TvTAL

[0033] (1) The protein sequence XP_008043251.1 of Trametes versicolor tyrosine ammonia lyase obtained from the NCBI database was codon-optimized to SEQ ID NO. 2, synthesized by Beijing Qingke Biotechnology Co., Ltd., and cloned into the pET28a(+) vector between the NdeI and BamHI restriction sites to obtain the pET28a(+)-TvTAL recombinant plasmid.

[0034] (2) The obtained recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells.

[0035] (3) Pick a single colony and inoculate it into a 5 mL LB test tube (kanamycin sulfate resistance) for overnight culture. Transfer it to TB medium containing 50 μg / mL kanamycin sulfate at a 2% inoculum, culture at 37°C for 3 h, add inducer IPTG to a final concentration of 0.2 mmol / L, and continue to culture at 18°C ​​for 20 hours to terminate the fermentation. Centrifuge at 7000 rpm and 4°C for 10 min, and collect the bacteria for later use.

[0036] Example 2

[0037] Purification of tyrosine ammonia lyase TvTAL

[0038] (1) The cells obtained in Example 1 were resuspended in a cell lysis buffer (50 mmol / L potassium hydrogen phosphate, 300 mmol / L sodium chloride, 5 mM imidazole, pH 8.0), centrifuged and the supernatant was discarded, the residual culture solution was washed away, the cells were resuspended in a cell lysis buffer, and then ultra-high pressure freezing was performed for cell disruption;

[0039] (2) After complete disruption, centrifuge at 4°C and 12,000 rpm for 30 min, filter the supernatant with a 0.45 μm filter membrane, and store on ice;

[0040] (3) Using a His Trap FF purification column, first equilibrate the column with a cell disruption buffer containing 5 mmol / L imidazole;

[0041] (4) After the sample is loaded, it is eluted with different concentrations of imidazole and the eluate is collected;

[0042] (5) Select only the target protein eluate and concentrate it, desalt it and retain the enzyme solution;

[0043] (6) Use SDS-PAGE gel electrophoresis to detect whether the target protein meets the requirements. The electrophoresis detection diagram is as follows Figure 1 As shown, a single band was obtained, and the protein size was 81.2 kDa, which was in line with expectations;

[0044] (7) The concentration of the target protein measured by the Brandford method was 100 mg / mL.

[0045] Example 3

[0046] Study on the Enzymatic Properties of Tyrosine Ammonia Lyase

[0047] (1) HPLC determination method of L-tyrosine and p-coumaric acid content:

[0048] HPLC system: Shimadzu LC-2030; Column: Welch Ultimate AQ-C18 (4.6×250 mm, 5 μm); mobile phase: 1% acetic acid aqueous solution: methanol = 55:45 (volume ratio).

[0049] Flow rate: 1.0 mL / min;

[0050] Column temperature: 30°C;

[0051] Detection wavelength: 280nm.

[0052] The detection pictures of L-tyrosine and p-coumaric acid are as follows Figure 2 As shown, the peak time of L-tyrosine is 2.84 min, and the peak time of p-coumaric acid is 4.56 min.

[0053] (2) Enzyme activity determination method:

[0054] The reaction system for determining enzyme activity was 1 mL, including 100 μL enzyme solution (enzyme concentration 6 μM), 900 μL L-tyrosine mother solution (mother solution concentration 10 g / L for suspension, prepared with 100 mmol / L, pH = 10.5 Gly-NaOH buffer solution), the reaction solution was reacted at 1500 rpm, 37°C for 10 min in an oscillating reactor, sampled, boiled in boiling water for 2 min to terminate the reaction, and centrifuged at 12000 rpm for 3 min to obtain the supernatant for subsequent HPLC detection.

[0055] The total amount of product was calculated based on the value and the standard curve, and the activity of the enzyme was calculated by dividing the total amount of product by the time (min) and the amount of enzyme added (mg). The amount of enzyme (mg) required to generate 1 μmol of p-coumaric acid per minute was defined as 1 enzyme activity unit.

[0056] (3) Effect of reaction temperature on TvTAL activity:

[0057] The reaction temperature selection range is 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, and 75°C, and the other conditions are consistent with the above enzyme activity determination protocol.

[0058] Test results such as Figure 3 As shown in a in the figure, the optimum temperature for tyrosine as substrate is 65°C.

[0059] (4) Study on the temperature stability of TvTAL:

[0060] The enzyme was incubated at 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C for 1 h, respectively, and the other conditions were consistent with the above enzyme activity assay protocol.

[0061] Test results such as Figure 3 As shown in b, the figure shows that when tyrosine substrate is used, more than 80% of the activity is retained between 45°C and 55°C.

[0062] (5) Effect of reaction pH on TvTAL activity:

[0063] The reaction pH selection range is pH=8, pH=8.5, pH=9.0, pH=9.5, 100mmol / L Tris-HCl and pH=9.5, pH=10.0, pH=10.5, pH=11, 100mmol / L Gly-NaOH, and the other conditions are consistent with the above enzyme activity determination scheme.

[0064] Test results such as Figure 3 As shown in c in the figure, when tyrosine is used as the substrate, the optimal pH is 10.5.

[0065] (6) pH stability study of TvTAL:

[0066] The enzyme was incubated for 1 h in buffers of pH=8, pH=8.5, pH=9.0, pH=9.5, 100 mmol / L Tris-HCl and pH=9.5, pH=10.0, pH=10.5, pH=11, 100 mmol / L Gly-NaOH. The other conditions were consistent with the above enzyme activity assay protocol.

[0067] Test results such as Figure 3 As shown in d in the figure, when tyrosine is used as the substrate, the activity can be maintained at more than 80% at pH 8-10.

[0068] (7) Tyrosine ammonia lyase enzymatic kinetic parameters:

[0069] The composition of each component in the reaction system: the concentration of L-tyrosine stock solution was prepared to be 12mmol / L, and it was diluted to: 0.5mmol / L, 1mmol / L, 2mmol / L, 3mmol / L, 5mmol / L, 6mmol / L, 8mmol / L, 12mmol / L respectively; purified enzyme: 2.4μM; the other conditions were consistent with the above enzyme activity determination protocol.

[0070] Test results such as Figure 3 As shown in e, the figure shows that when tyrosine is used as the substrate, the fitting result is y=8663.9198x+1423.8141. cat k is calculated cat 0.29s -1 , K m 6.08mM, k cat / K m 48.09M -1 ·s -1 .

[0071] Example 4

[0072] Application of Tyrosine Ammonia Lyase TvTAL in the Synthesis of p-Coumaric Acid

[0073] The composition of the reaction system: wet bacteria obtained by fermentation, substrate L-tyrosine;

[0074] The initial concentrations of the components in the reaction system are as follows: bacterial count is 4%; L-tyrosine concentration is 100 g / L. Reaction conditions: 45°C, water bath reaction.

[0075] During the reaction, 30% NaOH was added to control the pH of the reaction system to 10.5.

[0076] The p-coumaric acid production was detected by HPLC with interval sampling. At 45°C, the p-coumaric acid production was 53.3 g / L in 12 hours.

[0077] Example 5

[0078] Application of Tyrosine Ammonia Lyase TvTAL in the Synthesis of p-Coumaric Acid

[0079] The composition of the reaction system: wet bacteria obtained by fermentation, substrate L-tyrosine;

[0080] The initial concentrations of the components in the reaction system are as follows: bacterial count is 4%; L-tyrosine concentration is 100 g / L. Reaction conditions: 50° C., water bath reaction.

[0081] During the reaction, 30% NaOH was added to control the pH of the reaction system to 10.5.

[0082] The p-coumaric acid production was detected by HPLC with interval sampling. At 50°C, the p-coumaric acid production was 45.8 g / L in 12 hours.

[0083] Example 6

[0084] Application of Tyrosine Ammonia Lyase TvTAL in the Synthesis of p-Coumaric Acid

[0085] The composition of the reaction system: wet bacteria obtained by fermentation, substrate L-tyrosine;

[0086] The initial concentrations of the components in the reaction system are as follows: bacterial count is 4%; L-tyrosine concentration is 100 g / L. Reaction conditions: 55°C, water bath reaction.

[0087] During the reaction, 30% NaOH was added to control the pH of the reaction system to 10.5.

[0088] The p-coumaric acid production was detected by HPLC with interval sampling. At 55°C, the p-coumaric acid production was 38.9 g / L in 12 hours.

[0089] Although the present invention has been disclosed as above in the preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A tyrosine ammonia lyase, characterized in that It is derived from Trametes versicolor, and its amino acid sequence is shown in SEQ ID NO.

1. The target gene is codon optimized, and the optimized DNA sequence is shown in SEQ ID NO.

2.

2. A tyrosine ammonia lyase according to claim 1, characterized in that The enzyme has a very high catalytic efficiency, with an appropriate reaction temperature of 45-65°C and an appropriate reaction pH of 9.0-11.

0.

3. A use of the tyrosine ammonia lyase according to any one of claims 1 to 2, characterized in that: The application is that tyrosine ammonia lyase catalyzes the deamination reaction of substrate L-tyrosine to generate p-coumaric acid.

4. The use of a tyrosine ammonia lyase according to claim 3, characterized in that: The application method comprises the following steps: (1) Construction of recombinant Escherichia coli: The gene coding sequence shown in SEQ ID NO.2 was cloned into the expression vector pET28a(+), and then transformed into Escherichia coli BL21 for heterologous expression; (2) Fermentation culture: Pick a single colony and inoculate it into a 5 mL LB test tube for overnight culture. Transfer it to TB medium containing 50 μg / mL kanamycin sulfate at a 2% inoculum, culture at 37°C for 3 h, add the inducer IPTG to a final concentration of 0.2 mmol / L, and continue to culture at 18°C ​​for 20 h. End the fermentation, centrifuge at 7000 rpm and 4°C for 10 min, and collect the bacteria for later use; (3) Synthesis of p-coumaric acid: The wet cells obtained by fermentation are used to produce p-coumaric acid through the whole-cell catalytic method.

5. The use of a tyrosine ammonia lyase according to claim 4, characterized in that: The step (3): the whole-cell catalytic method for producing p-coumaric acid is as follows: the wet bacteria obtained by fermentation and the substrate L-tyrosine are used to form a conversion system, and p-coumaric acid is obtained by reaction.

6. The use of a tyrosine ammonia lyase according to claim 5, characterized in that: The initial concentration of the substrate L-tyrosine in the transformation system is 100 g / L, the amount of recombinant Escherichia coli added is 4%, the reaction temperature range is 45-55° C., and the reaction pH range is controlled to be 9.0-11.0 by adding sodium hydroxide solution.

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