Tyrosine ammonia lyase mutant and its use in production of p-coumaric acid

Abstract

The application provides a tyrosine deaminase mutant and application thereof in production of p-coumaric acid. The tyrosine deaminase mutant is obtained by mutating a wild-type tyrosine deaminase with an amino acid sequence shown in SEQ ID No. 1, and the mutation site is selected from at least one of Q108S, Y340Q and P375H. The application analyzes a catalytic active center of the wild-type tyrosine deaminase, and finds that different mutation sites, same mutation sites but different mutation targets can make the mutant show completely different enzyme catalytic activities, and the single mutation of Q108S, Y340Q and P375H can significantly improve the enzyme catalytic activity of the tyrosine deaminase mutant, and the tyrosine deaminase mutant with any two mutation sites, especially with three mutation sites also has excellent enzyme catalytic activity than the wild type, and has great application potential.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a L-tyrosine deaminase mutant and its application in the production of p-coumaric acid. BACKGROUND

[0002] P-coumaric acid (4-hydroxy cinnamic acid or trans-4-coumaric acid, p-CA) has the molecular formula C9H8O3 and the structural formula: ; It is an aromatic compound with broad application prospects, and has important applications in the fields of medicine, cosmetics, food and chemical industry, etc. It is the precursor or intermediate of many compounds with high application value. For example, in the field of medicine, p-coumaric acid can be used for the development of new antibacterial, antioxidant and other drugs, which have multiple biological activities such as antioxidant, anti-inflammatory, antibacterial and antitumor; in the field of cosmetics, p-coumaric acid can be added to skin care products as a natural antioxidant, which can delay skin aging, reduce the damage of ultraviolet light to the skin, reduce skin glycation, etc.; in the field of food, p-coumaric acid can be used as a spice and a nutritional fortifier, and also can be used as a natural preservative for food preservation, and also can be added to oil, meat and other foods to prevent oxidation and deterioration, prolong the shelf life of food. It can be seen that the market demand for p-coumaric acid is great.

[0003] The traditional production process of p-coumaric acid mainly relies on chemical synthesis, but there are problems such as high production cost, low production efficiency and serious environmental pollution. The use of biological synthesis technology to produce p-coumaric acid has the advantages of renewable raw materials, mild reaction conditions and environmental friendliness, which can solve the problems of high raw material cost, complex production process, low production efficiency, low product purity and serious environmental pollution in the existing p-coumaric acid production technology. Therefore, the biological transformation method will become an important development direction for the future production of p-coumaric acid, and will promote the large-scale production and application of p-coumaric acid.

[0004] The existing technology usually uses refined raw materials (such as p-hydroxyphenylpyruvic acid, L-tyrosine, etc.) as substrates when preparing p-coumaric acid by biological transformation method. For example, the Chinese patent application with the publication number CN113265431A discloses a method for synthesizing p-coumaric acid by using multi-enzyme (carbonyl reductase, glucose dehydrogenase and dehydrase) cascade catalysis with p-hydroxyphenylpyruvic acid as substrate, and the production efficiency reaches 50 g / L.h; and for example, the Chinese patent application with the publication number CN117965514A discloses a method for preparing p-coumaric acid by using phenylalanine deaminase mutant enzyme catalysis with L-tyrosine as substrate, and the yield of p-coumaric acid is 3.12 g / L, and the conversion rate is 95%; but the cost of refined p-hydroxyphenylpyruvic acid and L-tyrosine substrates is high, the production efficiency is not high, and the p-coumaric acid product obtained by production has no market competitive advantage.

[0005] At present, the demand for p-coumaric acid at home and abroad is huge, and the market size shows a steady growth trend, but the production scale of p-coumaric acid is far from meeting the growing market demand. Therefore, it is urgent to develop an efficient p-coumaric acid production process, so as to promote the large-scale industrial production of p-coumaric acid, reduce the production cost, improve the market competitiveness, and thus promote the large-scale market application of p-coumaric acid. SUMMARY

[0006] The purpose of the present application is to provide a tyrosine ammonia lyase mutant and its application in the production of p-coumaric acid, which has high enzyme catalytic activity and can efficiently convert L-tyrosine into p-coumaric acid.

[0007] To achieve the above-mentioned purpose of the application, the technical solutions of the present application are as follows:

[0008] The present application first provides a tyrosine ammonia lyase mutant, which is obtained by mutating the wild-type tyrosine ammonia lyase with the amino acid sequence shown in SEQ ID No. 1, and the mutation site is selected from at least one of Q108S, Y340Q and P375H.

[0009] The present application analyzes the catalytic activity center of the wild-type tyrosine ammonia lyase, and finds that after site-directed mutation of the five catalytic activity center sites (Y87, Q108, P239, Y340, P375), different mutation sites, the same mutation site but different mutation targets will make the mutant show completely different (increased or decreased) enzyme catalytic activity. Q108S (i.e. the 108th amino acid Q is mutated to S), Y340Q and P375H single mutation can significantly improve the enzyme catalytic activity of the tyrosine ammonia lyase mutant, and efficiently convert L-tyrosine into p-coumaric acid. Tyrosine ammonia lyase mutants with any two mutation sites (i.e. double mutation), especially three mutation sites, also have excellent enzyme catalytic activity than the wild type, and have great application potential.

[0010] As a preferred, the tyrosine ammonia lyase mutant has three mutation sites of Q108S, Y340Q and P375H.

[0011] The amino acid sequence of the tyrosine ammonia lyase mutant is shown in SEQ ID No. 3, i.e. the tyrosine ammonia lyase mutant with three mutations.

[0012] The present application also provides a polynucleotide encoding the above-mentioned tyrosine ammonia lyase mutant, a nucleic acid construct containing the polynucleotide, a recombinant expression vector containing the nucleic acid construct, and a recombinant engineering strain containing the recombinant expression vector.

[0013] The application also provides the use of the tyrosine ammonia-lyase mutant, polynucleotide, nucleic acid construct, recombinant expression vector or recombinant engineering strain in the production of p-coumaric acid. These substances can improve the production efficiency of p-coumaric acid, reduce the production cost, and improve the product quality; can realize the large-scale industrial production of p-coumaric acid, and can improve the market competitiveness of the product.

[0014] For example, the application of the recombinant engineering strain includes:

[0015] (1) inoculating the recombinant engineering strain into a fermentation medium for fermentation to obtain a catalyst;

[0016] (2) using L-tyrosine as a substrate, and using the catalyst obtained in step (1) to perform enzyme catalysis to obtain a p-coumaric acid conversion liquid, and after separation, extraction and purification of the p-coumaric acid conversion liquid, p-coumaric acid is obtained.

[0017] As a preferred, in step (1), the temperature of the fermentation system of the recombinant engineering strain is controlled at 32-42℃, the pH is controlled at 6.5-7.5, and the dissolved oxygen is controlled at more than 30%; during the fermentation process, glycerol is continuously supplemented at a rate of 100-150 mL / h to control the residual glycerol concentration in the fermentation system to be less than 1 g / L, and the bacterial growth rate is controlled at OD 600 =3-4 / h; when the bacterial concentration OD 600 reaches more than 120, the fermentation liquid temperature is reduced to 15℃ and the bacterial cells are collected. Using this high-density fermentation method can obtain a large amount of bacterial cells, which provides a solid foundation for enzyme catalysis.

[0018] In the application, the catalyst can be the original fermentation liquid of the recombinant engineering strain, or a tyrosine ammonia-lyase pure enzyme obtained by purifying the original fermentation liquid, or an immobilized enzyme obtained by immobilizing the tyrosine ammonia-lyase pure enzyme; or can be the fermentation bacterial cells of the recombinant engineering strain, or a liquid enzyme obtained by breaking the wall of the fermentation bacterial cells, or an immobilized enzyme obtained by immobilizing the liquid enzyme. Obviously, it is more convenient to directly use the original fermentation liquid or the fermentation bacterial cells of the recombinant engineering strain as the catalyst, which is suitable for industrial production.

[0019] In the catalytic reaction system of step (2), the substrate can be L-tyrosine, or the fermentation liquid of an L-tyrosine metabolic engineering strain (see Chinese patent No. 2019100195375); obviously, when the fermentation liquid containing L-tyrosine is directly used as the substrate, the production cost is lower, which is conducive to the large-scale industrial production of p-coumaric acid.

[0020] The content of L-tyrosine in the catalytic reaction system of step (2) is 40-100 g / L, the content of the fermentation bacteria of the recombinant engineering strain is 20-50 g / L, the pH of the catalytic reaction system is 8.0-11.0, and the temperature is 37-45℃;

[0021] As a further preference, in the catalytic reaction system of step (2), the content of L-tyrosine is 40-100 g / L, the content of the tyrosine deaminase engineering bacteria is 40-50 g / L, and the pH of the reaction system is 9.5-11.0, and the temperature is 37-40℃. Under this catalytic reaction system, the conversion rate of p-coumaric acid is higher.

[0022] As a preference, in step (2), the required time for enzyme catalytic synthesis is 6-12 h.

[0023] As a preference, a protective agent is added in the catalytic reaction system of step (2), and the protective agent is selected from at least one of sodium bisulfite, vitamin C, carbon dioxide and nitrogen; more preferably, it is composed of sodium bisulfite and nitrogen.

[0024] In the present application, there is no substrate residue in the p-coumaric acid conversion liquid (the molar conversion rate of the substrate is as high as 99.3%), so the p-coumaric acid conversion liquid can be directly treated as follows to obtain p-coumaric acid: first, add diatomite to the p-coumaric acid conversion liquid, stir, filter, and take the filtrate; then add activated carbon to the filtrate, stir, and suction filter, and take the suction filtrate; adjust the pH of the suction filtrate to 2-3, adjust the temperature to 15-20℃, and keep for 1-3 h to obtain a crystallization liquid; the crystallization liquid is suction filtered to obtain a p-coumaric acid crude product, which is washed with pure water, dried, and then p-coumaric acid is obtained.

[0025] The yield of p-coumaric acid extracted by the above separation and purification method can reach more than 95%; the content of the obtained p-coumaric acid product reaches more than 98.6%, the water content is ≤1.10%, the residue is ≤0.03%, the purity is more than 99.9%, no L-tyrosine substrate residue is detected, and the product quality is much higher than that of similar products on the market.

[0026] Compared with the prior art, the beneficial effects of the present application are reflected in:

[0027] (1) The application analyzes the catalytic active center of wild-type tyrosine deaminase, and finds that different mutation sites, same mutation sites but different mutation targets will make the mutant show completely different (increased or decreased) enzyme catalytic activity, and Q108S (i.e. the 108th amino acid Q is mutated to S), Y340Q and P375H single mutation will significantly improve the enzyme catalytic activity of tyrosine deaminase mutant, and efficiently convert L-tyrosine into p-coumaric acid; and the tyrosine deaminase mutant with any two mutation sites (i.e. double mutation), especially the tyrosine deaminase mutant with three mutation sites also has excellent enzyme catalytic activity than the wild type, and has great application potential.

[0028] (2) The tyrosine deaminase mutant of the application can directly use the metabolic engineering bacteria containing L-tyrosine fermentation broth as the substrate, without using L-tyrosine fine products, which is beneficial to reduce the substrate cost of p-coumaric acid production and facilitate large-scale industrialized production of p-coumaric acid.

[0029] (3) In the p-coumaric acid catalytic preparation reaction system of the application, the substrate concentration can reach 10%, the substrate molar conversion rate is as high as 99.3% after 8 h of conversion, no organic solvent is used in the separation and extraction process of p-coumaric acid, the extraction yield can reach 95.0%, the substrate dosage is high, the period is short, the conversion rate is high, the extraction yield is high, and the production efficiency is high.

[0030] (4) The p-coumaric acid product produced by the application has a p-coumaric acid content of 98.6% or more, a purity of 99.9% or more, and no L-tyrosine substrate residue is detected, and the product quality is much higher than that of similar products on the market. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 It is a structural schematic diagram of tyrosine deaminase recombinant expression vector pET24a- tal .

[0032] Figure 2 It is a protein electrophoresis diagram of tyrosine deaminase engineering bacteria correctly expressing tyrosine deaminase TAL;

[0033] Figure 3 It is a statistical diagram of the accumulation of bacteria at each time node in the fermentation process of tyrosine deaminase engineering bacteria;

[0034] Figure 4 It is a statistical diagram of the accumulation of L-tyrosine at each time node in the fermentation process of L-tyrosine metabolic engineering bacteria;

[0035] Figure 5 It is a statistical diagram of the substrate conversion rate at each time node in the enzyme catalytic preparation of p-coumaric acid with L-tyrosine-containing fermentation broth as the substrate.

[0036] Figure 6 Figure 2 is a liquid chromatogram of the p-coumaric acid conversion liquid;

[0037] Figure 7 Figure 3 is a liquid chromatogram of the p-coumaric acid product. DETAILED DESCRIPTION

[0038] The technical solutions of the present application are described in further detail below in combination with the drawings and examples.

[0039] Example 1: Construction of tyrosine ammonia-lyase mutant engineering bacteria

[0040] 1. Construction of wild-type tyrosine ammonia-lyase engineering bacteria

[0041] The construction process is as follows:

[0042] a. The wild-type tyrosine ammonia-lyase gene is optimized according to the codon bias of the microbial host and then subjected to whole gene synthesis;

[0043] In this example, a tyrosine ammonia-lyase TAL (amino acid sequence as shown in SEQ ID No. 1) with excellent enzymatic performance is screened from tyrosine ammonia-lyases from various microbial sources. In order to improve the protein expression amount of the recombinant tyrosine ammonia-lyase in the Escherichia coli host, the tyrosine ammonia-lyase TAL sequence is codon-optimized according to the codon bias of Escherichia coli. The optimized nucleic acid sequence is subjected to whole gene synthesis of the tyrosine ammonia-lyase TAL gene by GenScript Biotech Co., Ltd. (nucleotide sequence as shown in SEQ ID No. 2).

[0044] b. Construction of a protein expression vector and introduction of the recombinant expression vector into the target microbial host, i.e., to obtain the tyrosine ammonia-lyase engineering bacteria;

[0045] Specifically, the pET-24a(+) expression vector is first subjected to double digestion with restriction endonucleases H ind III and l ind III, the enzyme digestion products are subjected to nucleic acid electrophoresis separation, and then DNA gel recovery is performed to obtain a linearized pET-24a(+) vector fragment; the whole synthesis plasmid PUC-19 carrying the gene fragment is subjected to double digestion with restriction endonucleases H ind III and l ind III, the enzyme digestion products are subjected to nucleic acid electrophoresis separation, and then the gene fragment is subjected to DNA gel recovery to obtain the gene fragment; and finally, the gene fragment is ligated with the linearized pET-24a(+) vector fragment. Bam H Ⅰ and Xho l Ⅰ, the enzyme digestion products are subjected to nucleic acid electrophoresis separation, and then the gene fragment is subjected to DNA gel recovery to obtain the gene fragment; and finally, the gene fragment is ligated with the linearized pET-24a(+) vector fragment. TAL PUC-19 carrying the gene fragment is subjected to double digestion with restriction endonucleases H ind III and l ind III, the enzyme digestion products are subjected to nucleic acid electrophoresis separation, and then the gene fragment is subjected to DNA gel recovery to obtain the gene fragment; and finally, the gene fragment is ligated with the linearized pET-24a(+) vector fragment. tal H Ⅰ and Bam l Ⅰ, the enzyme digestion products are subjected to nucleic acid electrophoresis separation, and then the gene fragment is subjected to DNA gel recovery to obtain the gene fragment; and finally, the gene fragment is ligated with the linearized pET-24a(+) vector fragment. Xho H Ⅰ and TAL l Ⅰ, the enzyme digestion products are subjected to nucleic acid electrophoresis separation, and then the gene fragment is subjected to DNA gel recovery to obtain the gene fragment; and finally, the gene fragment is ligated with the linearized pET-24a(+) vector fragment. TA L H Ⅰ and Bam l Ⅰ, the enzyme digestion products are subjected to nucleic acid electrophoresis separation, and then the gene fragment is subjected to DNA gel recovery to obtain the gene fragment; and finally, the gene fragment is ligated with the linearized pET-24a(+) vector fragment. XhoThe linearized pET-24a(+) vector was recombined by one-step cloning reagent kit, and the recombination product was transformed into E. coli DH5a competent cells. The transformation liquid was spread on LB plates (with 50 μg / mL kanamycin). The transformants grown on the plates were picked into 5 mL LB test tubes (with kanamycin) and incubated at 37°C for 16 h. The plasmid of the transformants was extracted by a plasmid extraction kit and subjected to nucleic acid electrophoresis to screen the plasmid of the transformants, obtaining the recombinant expression vector pET24a- tal (see Figure 1 ).

[0046] Then, the recombinant expression vector pET24a- tal was added to E. coli BL21 (DE3) competent cells, which were subjected to ice bath for 30 min, 42°C heat shock for 90 s, ice bath for 2 min, addition of 1 mL LB medium for 37°C culture for 1 h, spreading of the transformation liquid on LB plates (with kanamycin) for 37°C culture for 24 h, and inoculation of the single colonies grown on the plates into 10 mL LB shake flasks (peptone 1%, yeast extract 0.5%, sodium chloride 1%, kanamycin 50 mg / L) for 37°C culture at 250 r / min for 12 h. The 50 mL LB shake flasks (with kanamycin 50 mg / L) were inoculated at a 10% inoculation amount and cultured at 37°C and 250 r / min for about 3 h until the OD 600 of the culture liquid reached 3-5. Then, 0.2 mM IPTG was added at a final concentration, and the culture was continued at 28°C overnight. After 12 h of induction culture, 2 mL of the culture liquid was centrifuged at 12000 r / min for 1 min to collect the bacterial cells. After removal of the supernatant, 1 mL of water was added to suspend the bacterial cells, which were broken by ultrasonic wave for 2 min. Then, 0.2 mL of the cell breaking liquid was added to 0.04 mL of 5X protein loading buffer, which was boiled in a water bath for 10 min. Then, 5 μL of the sample was subjected to SDS-PAGE protein electrophoresis, and the target protein band was about 70 kDa, indicating that the tyrosine deaminase gene tal has been successfully introduced into the E. coli host, and the TAL protein is correctly expressed in large amounts (see Figure 2 ). The wild-type tyrosine deaminase engineering bacteria TAL / BL21 (DE3) have the ability to biocatalyze the preparation of p-coumaric acid from L-tyrosine.

[0047] 2. Design of tyrosine deaminase mutants

[0048] The catalytic active center of wild-type tyrosine ammonia-lyase was analyzed by using the protein analysis website https: / / consurf.tau.ac.il, and a total of 5 catalytic active center sites (Y87, Q108, P239, Y340, P375) were predicted. Single-point site-directed mutation and multiple-site superposition mutation were performed on the 5 catalytic active sites to obtain the following single-mutant types: Y87D, Y87T, Y87A, Q108M, Q108S, Q108G, P239R, P239Y, P239V, Y340Q, Y340P, Y340M, P375T, P375K, P375H, and the following multiple-mutant types: Q108S / Y340Q, Y340Q / P375H, Q108S / P375H, Q108S / Y340Q / P375H.

[0049] 3. Construction of tyrosine ammonia-lyase mutant engineering bacteria

[0050] Similarly, according to the same steps as in “1. Construction of wild-type tyrosine ammonia-lyase engineering bacteria”, each tyrosine ammonia-lyase mutant engineering bacteria was constructed, and the bacterial cells were collected after induction and expression.

[0051] 4. Activity analysis of wild-type tyrosine ammonia-lyase and tyrosine ammonia-lyase mutants

[0052] The determination method of enzyme activity was as follows: 10 g of L-tyrosine substrate was weighed into a 500 mL shake flask, 80 g of pure water was added, the pH was adjusted to 10 with 40% NaOH, 2 g of bacterial enzyme was added, and the reaction was carried out at 40°C on a shaking bed at 220 r / min for 1 h. Then, the concentration of p-coumaric acid in the conversion solution was detected by liquid chromatography. According to the amount of generated p-coumaric acid, the specific activity of the bacterial enzyme was determined, and the relative activity value of the mutant enzyme was calculated and compared (with the enzyme activity of wild-type tyrosine ammonia-lyase as 100%). The results are shown in Table 1.

[0053] Enzyme activity definition (U / g): the amount of enzyme consumed per minute to generate 1 μmol of p-coumaric acid.

[0054] The detection method of p-coumaric acid was as follows: C18 chromatographic column, mobile phase 1‰ acetic acid / methanol = 55 / 45, flow rate 0.6 mL / min, column temperature 30°C, detection wavelength 280 nm, and the sample was diluted 1000 times with methanol before being injected into the liquid chromatograph, and the retention time was 25 minutes.

[0055] Table 1 Comparison of catalytic abilities of different tyrosine ammonia-lyases

[0056]

[0057] As can be seen from Table 1, the enzyme activities of the tyrosine ammonia lyase mutants obtained are quite different when the mutation sites are different, or the mutation sites are the same but the mutation targets are different. Among them, the Q108S, Y340Q and P375H single mutations increase the enzyme activity of the tyrosine ammonia lyase mutant by 56%, 37% and 49%, respectively. When two or more than two sites are combined in the single mutation sites with improved enzyme activity, it is found that the double mutations do not all lead to the superposition of enzyme activity improvement. Compared with the single mutation, only the Q108S / Y340Q double mutation and the Q108S / P375H double mutation produce synergistic effect. However, the Q108S / Y340Q / P375H triple mutation increases the enzyme activity of the tyrosine ammonia lyase mutant by 102%.

[0058] Example 2 Tyrosine ammonia lyase mutant engineering bacteria for producing p-coumaric acid

[0059] The method for producing p-coumaric acid in this embodiment comprises the following steps:

[0060] (1) The tyrosine ammonia lyase mutant (Q108S / Y340Q / P375H) engineering bacteria TAL19 / BL21(DE3) are inoculated into a fermentation medium for fermentation to obtain a catalyst;

[0061] Specifically, the tyrosine ammonia lyase engineering bacteria TAL19 / BL21(DE3) are inoculated from a frozen glycerol tube into a kanamycin-resistant LB solid medium (1% proteose peptone, 0.5% yeast extract, 1% sodium chloride, plus kanamycin 50 mg / L) for streak isolation, and cultured in a 37°C incubator for 24 h until single colonies are grown. The single colonies are picked into test tubes containing 5 mL of LB liquid medium (containing kanamycin 50 mg / L) and cultured at 220 r / min and 37°C for 16 h to obtain a primary seed liquid. 1 mL of the cultured primary seed liquid is inoculated into 500 mL of LB liquid medium (containing kanamycin 50 mg / L), and cultured at 220 r / min and 37°C for 12 h to obtain a secondary seed liquid;

[0062] The 500 mL secondary seed culture was inoculated into a 15 L fermenter containing 5 L sterilized fermentation medium at a 10% inoculation amount, and the fermentation medium had the following composition: 12 g / L tryptone, 24 g / L yeast extract, 9.4 g / L K2HPO4, 2.2 g / L KH2PO4, 5 g / L glycerol, sterilized at 121 ℃ for 30 min, and cooled to 37 ℃ before use, and the pH was adjusted to 7.0 with 20% ammonia water before inoculation. The culture was carried out at 37 ℃, with an initial rotation speed of 300 r / min and an initial air flow of 300 L / h. The pH was controlled at 7.0 during fermentation using 20% ammonia water, and the rotation speed (300-600 r / min) and air flow (300-600 L / h) were adjusted in sequence to maintain the dissolved oxygen above 30%. When the glycerol in the culture medium was depleted, the dissolved oxygen and pH would rise sharply, and 50% glycerol (sterilized at 121 ℃ for 30 min before use) was added. The initial feeding rate was controlled at 100 mL / h. When the cell concentration (OD 600 ) in the fermentation broth reached more than 30, the temperature was reduced to 28 ℃, and 1 mM IPTG inducer was added for protein induction expression. The cell concentration and glycerol concentration in the fermentation broth were measured every 3 h during fermentation, and the residual glycerol concentration was controlled below 1 g / L by adjusting the feeding rate to 100-150 mL / h, and the cell growth rate was OD 600 =3-4 / h, until the cell concentration continuously increased during fermentation, and the cell concentration OD 600 reached more than 120 at 30 h of fermentation, and the wet cell amount reached more than 15% (see Figure 3 ). At this time, the fermentation broth was cooled to 15 ℃, and the cells were collected by tubular centrifugation and stored in a -20 ℃ refrigerator for use.

[0063] (2) Using L-tyrosine as the substrate, the catalyst obtained in step (1) was used for enzyme catalysis to obtain a p-coumaric acid conversion liquid. After separation, extraction, and purification of the p-coumaric acid conversion liquid, p-coumaric acid was obtained.

[0064] Specifically, it includes:

[0065] 1) Preparation of a fermentation broth containing L-tyrosine;

[0066] First, the L-tyrosine metabolic engineering bacteria (see Chinese patent No. 2019100195375) were inoculated from the frozen glycerol tube to the kanamycin-resistant LB solid medium (1% proteose peptone, 0.5% yeast extract, 1% sodium chloride, plus kanamycin 50 mg / L) for streak isolation, and cultured in a 37°C incubator for 24 h until single colonies were grown. Then, single colonies were picked into a test tube containing 5 mL of LB liquid medium (containing kanamycin 50 mg / L) and cultured at 220 r / min and 37°C for 12 h to obtain a first-stage seed liquid. Subsequently, 1 mL of the cultured first-stage seed liquid was inoculated into 500 mL of LB liquid medium (containing kanamycin 50 mg / L) and cultured at 220 r / min and 37°C for 12 h to obtain a second-stage seed liquid; then, 500 mL of the cultured second-stage seed was inoculated into a 15 L fermenter containing 5 L of sterilized fermentation medium at a 10% inoculation amount, and the fermentation medium had the following composition: 5 g / L glucose, 10 g / L (NH4)2SO4, 5 g / L yeast extract powder, 10 g / L corn steep liquor, 1 g / L K2HPO4, 1 g / L KH2PO4, 0.5 g / L MgSO4.7H2O, 0.1 g / L FeSO4.7H2O, and 0.02 g / L MnSO4.H2O; after preparation, the medium was sterilized at 121°C for 30 min, cooled to 37°C, and then used, and the pH was adjusted to 7.0 with 20% ammonia water before inoculation. The culture was performed at 37°C, 300 r / min, and an air flow rate of 300 L / h, and the pH was controlled at 7.0 during the fermentation process using 20% ammonia water, and the rotation speed (300-600 r / min) and air flow rate (300-600 L / h) were adjusted in sequence to maintain the dissolved oxygen above 30%. When the glucose in the medium was consumed, the dissolved oxygen and pH would rise sharply, and the feeding of 70% glucose solution (sterilized at 118°C for 20 min and then used) was started. When the cell concentration (OD 600 ) in the fermentation broth reached more than 20, the temperature was increased to 42°C to induce the biosynthesis of L-tyrosine by the L-tyrosine metabolic engineering bacteria. The residual glucose in the fermentation broth was determined every 3 h after the start of the fermentation, and the feeding rate was adjusted to control the residual glucose between 10-15 g / L during 0-30 h of the fermentation, and the residual glucose was controlled between 5-10 g / L during 30-60 h of the fermentation until the end of the fermentation; the L-tyrosine content in the fermentation broth was detected every 4 h after 12 h of the start of the fermentation, and the cell concentration (OD 600 ) and the L-tyrosine content increased continuously, and the L-tyrosine content almost stopped increasing after 48 h of the fermentation, and the final L-tyrosine content in the fermentation broth reached 100.6 g / L ( Figure 4 ). After the L-tyrosine content stopped increasing, the fermentation broth was inactivated by heating at 80°C for 30 min to obtain the fermentation broth containing L-tyrosine;

[0067] 2) Using the fermentation broth containing L-tyrosine as the substrate of the enzyme catalytic reaction, and using the tyrosine ammonia lyase mutant (Q108S / Y340Q / P375H) engineering bacteria TAL19 / BL21(DE3) as the catalyst, the enzyme catalytic reaction was carried out to produce p-coumaric acid;

[0068] In order to improve the conversion efficiency of p-coumaric acid, the parameters of the enzyme catalytic reaction system were tested in this embodiment:

[0069] a. Effect of temperature on catalytic preparation of p-coumaric acid

[0070] In order to explore the effect of temperature on the catalytic preparation of p-coumaric acid, the following reaction system was carried out: 100 mL of fermentation broth containing L-tyrosine was added to a 500 mL flask, the pH was adjusted to 10.5 with 40% NaOH, 4 g of tyrosine ammonia lyase engineering bacteria was added, and the shaking bed at different temperatures (see Table 3) was reacted at 200 r / min for 8 h. The content of p-coumaric acid in the conversion liquid and the residual substrate were detected, and the substrate conversion rate was calculated (see Table 2).

[0071] Among them, the detection method of L-tyrosine: C18 chromatographic column, mobile phase 1‰ acetic acid / methanol=55 / 45, flow rate 0.8 mL / min, column temperature 30°C, detection wavelength 230 nm, sample was diluted 200 times with water and dilute hydrochloric acid before entering the liquid chromatograph.

[0072] Table 2 Effect of temperature on substrate conversion rate

[0073] Temperature / °C Substrate conversion % 37 91.1% 40 93.6% 45 89.3%

[0074] As can be seen from Table 2, the catalytic reaction is better at 37-40°C, and is best at 40°C.

[0075] b. Effect of pH on catalytic preparation of p-coumaric acid

[0076] In order to explore the effect of pH on the catalytic preparation of p-coumaric acid, the following reaction system was carried out: 100 mL of fermentation broth containing L-tyrosine was added to a 500 mL flask, the pH was adjusted to 8.0-11.0 with 40% NaOH, 4 g of tyrosine ammonia lyase engineering bacteria was added, and the shaking bed at 40°C was reacted at 200 r / min for 8 h. The content of p-coumaric acid and the residual substrate were detected, and the substrate conversion rate was calculated, and the results are shown in Table 3.

[0077] Table 3 Effect of pH on substrate conversion rate

[0078] pH Substrate conversion % 8.0 65.9% 8.5 70.4% 9.0 85.6% 9.5 90.3% 10.0 94.7% 10.5 93.1% 11.0 91.0%

[0079] As can be seen from Table 3, the catalytic reaction is better at pH 9.5-11.0, and is best at pH 10.0.

[0080] c、The influence of the amount of tyrosine deaminase engineering bacteria on the catalytic preparation of p-coumaric acid

[0081] In order to explore the influence of the amount of tyrosine deaminase engineering bacteria on the catalytic preparation of p-coumaric acid, the following reaction system was carried out: 100 mL of fermentation broth containing L-tyrosine was added to a 500 mL flask, the pH was adjusted to 10.0 with 40% NaOH, and different amounts of tyrosine deaminase engineering bacteria were added, and after 8 h of reaction at 40°C on a shaker at 200 r / min, the content of p-coumaric acid and the residual substrate were detected, and the substrate conversion rate was calculated. The results are shown in Table 4.

[0082] Table 4 Influence of the amount of tyrosine deaminase engineering bacteria on the substrate conversion rate

[0083] Bacterial amount (g / L) Substrate conversion % 20 60.5% 30 75.6% 40 94.3% 50 94.5%

[0084] As can be seen from Table 4, the substrate conversion rate is basically the same when the amount of bacteria is 40 g / L and the amount of bacteria is 50 g / L, but the amount of bacteria can be reduced to reduce the cost of bacteria, so the amount of bacteria is selected as 40 g / L for the catalytic reaction.

[0085] d、The influence of the amount of substrate on the preparation of p-coumaric acid

[0086] In order to explore the influence of the amount of substrate (i.e. fermentation broth containing L-tyrosine) on the catalytic preparation of p-coumaric acid, the following reaction system was carried out: L-tyrosine fermentation broth and appropriate amount of water were added to a 500 mL flask to prepare a substrate solution with L-tyrosine concentration of 40, 60, 80 or 100 g / L, the pH was adjusted to 10.0 with 40% NaOH, 4 g of tyrosine deaminase engineering bacteria was added, and after 8 h of reaction at 40°C on a shaker at 200 r / min, the content of p-coumaric acid and the residual substrate were detected, and the substrate conversion rate was calculated. The results are shown in Table 5.

[0087] Table 5 Influence of the amount of substrate on the catalytic preparation of p-coumaric acid

[0088] Substrate amount (g / L) Substrate conversion % 40 93.7% 60 94.1% 80 94.9% 100 94.6%

[0089] As can be seen from Table 5, the substrate conversion rate is above 93% when the amount of substrate is 40-100 g / L, but the yield of p-coumaric acid is the highest when the amount of substrate is 100 g / L, and the L-tyrosine concentration of the fermentation broth containing L-tyrosine is as high as 100.6 g / L, which can be directly used as the conversion substrate.

[0090] e、The influence of the amount of substrate on the catalytic preparation of p-coumaric acid

[0091] p-Coumaric acid is easily oxidized to other impurities, which can cause low substrate conversion rate, so it is necessary to explore the effects of different protective agents on the substrate conversion rate in the process of enzyme-catalyzed preparation of p-coumaric acid. The effects of sodium bisulfite, vitamin C, carbon dioxide and nitrogen on the substrate conversion rate were studied as follows:

[0092] ① 10L conversion tank was added with 5L fermentation broth containing L-tyrosine, stirred at 300r / min, and the temperature was raised to 40℃. The pH was adjusted to 10 with 40% NaOH, and 200g of tyrosine ammonia lyase bacteria was added to start the conversion. The pH was controlled at 10 with 40% NaOH during the conversion process. The content of p-coumaric acid and the residual substrate were detected until the substrate decreased slowly. The conversion was completed in 8h, and the substrate molar conversion rate was 94.0%.

[0093] ② 10L conversion tank was added with 5L fermentation broth containing L-tyrosine, stirred at 300r / min, and 5L fermentation broth containing L-tyrosine was added to the 10L conversion tank. The stirring speed was 300r / min, and the temperature was raised to 40℃. The pH was adjusted to 10 with 40% NaOH, and 10g of sodium bisulfite was added and stirred uniformly. 200g of tyrosine ammonia lyase bacteria was added to start the conversion. The pH was controlled at 10 with 40% NaOH during the conversion process. The content of p-coumaric acid and the residual substrate were detected until the substrate decreased slowly. The conversion was completed in 8h, and the substrate molar conversion rate was 97.1%.

[0094] ③ 10L conversion tank was added with 5L fermentation broth containing L-tyrosine, stirred at 300r / min, and the temperature was raised to 40℃. The pH was adjusted to 10 with 40% NaOH, and 10g of vitamin C was added and stirred uniformly. 200g of tyrosine ammonia lyase bacteria was added to start the conversion. The pH was controlled at 10 with 40% NaOH during the conversion process. The content of p-coumaric acid and the residual substrate were detected until the substrate decreased slowly. The conversion was completed in 8h, and the substrate molar conversion rate was 93.4%.

[0095] ④ 10L conversion tank was added with 5L fermentation broth containing L-tyrosine, stirred at 300r / min, and the temperature was raised to 40℃. The pH was adjusted to 10 with 40% NaOH, and 200g of tyrosine ammonia lyase bacteria was added. Carbon dioxide was introduced from the air line to maintain the tank pressure at 0.05MPa to start the conversion. The pH was controlled at 10 with 40% NaOH during the conversion process. The content of p-coumaric acid and the residual substrate were detected until the substrate decreased slowly. The conversion was completed in 8h, and the substrate molar conversion rate was 93.6%.

[0096] ⑤10L conversion tank is added 5L containing L-tyrosine broth, 300r / min stirring, heating to 40℃, pH 10 with 40% NaOH, adding 200 grams of tyrosine deaminase bacteria, from the air line into the nitrogen to maintain the tank pressure 0.05 MPa start conversion, conversion process with 40% NaOH control pH 10, the process detection p-coumaric acid content and substrate residual, until the substrate decreases slowly, 8 h conversion is completed, the substrate molar conversion rate 97.5%.

[0097] ⑥10L conversion tank is added 5L containing L-tyrosine broth, 300r / min stirring, heating to 40℃, pH 10 with 40% NaOH, adding 200 grams of tyrosine deaminase bacteria, from the air line into the nitrogen to maintain the tank pressure 0.05 MPa start conversion, conversion process with 40% NaOH control pH 10, the process detection p-coumaric acid content and substrate residual, until the substrate decreases slowly, 8 h conversion is completed, the substrate molar conversion rate 97.5%. Figure 5 )。

[0098] Table 6 The effect of different protective agents on substrate conversion rate

[0099] Protectant type Blank control Sodium bisulfite Vitamin C Carbon dioxide Nitrogen Sodium bisulfite + nitrogen Conversion % 94.0% 97.1% 94.7% 95.6% 97.5% 99.3%

[0100] From Table 6, compared with the blank control without adding any protective agent, the substrate conversion rate is improved to different degrees after adding the protective agent, among which the substrate conversion rate is the highest when adding sodium bisulfite + nitrogen as the protective agent, reaching 99.3%.

[0101] Based on the above experiments, the enzyme catalytic reaction system with the following parameters is summarized: temperature 40℃, pH 10.0, cell amount 40g / L, protective agent sodium bisulfite + nitrogen.

[0102] 3) Separation and extraction of p-coumaric acid;

[0103] Take 1L of p-coumaric acid conversion liquid obtained in step 2) (liquid chromatogram analysis chart see Figure 6 ), add 5% diatomite to the volume of the conversion liquid, stir at 40℃ for 0.5h, filter the solid-liquid separation with a Buchner funnel, add 0.5% activated carbon to the p-coumaric acid filtrate, stir at 40℃ for 1h, then filter, adjust the pH of the filtrate to 2.0 with 30% sulfuric acid, cool the obtained acidified liquid to 15-20℃ and keep for 2h, filter the obtained crystallization liquid to obtain p-coumaric acid wet crude product, wash the wet crude product with 1 volume of pure water for 3 times, and dry the wet product in an oven at 80℃ to constant weight, to obtain p-coumaric acid product.

[0104] The calculated extraction yield of p-coumaric acid was 95.0%, the content of the obtained p-coumaric acid product was 98.6%, the purity was more than 99.9% (see Figure 7 ), the moisture was 1.1%, the residue was 0.03%, no L-tyrosine substrate residue was detected in the product, and the product quality was qualified.

Claims

1. A mutant tyrosine deaminase enzyme, characterized in that, The mutant is obtained by mutating the wild-type tyrosine ammonia-lyase with the amino acid sequence shown in SEQ ID No. 1, and the mutation site is at least one selected from Q108S, Y340Q and P375H.

2. The tyrosine deaminase mutant of claim 1, wherein The mutant is obtained by mutating the wild-type tyrosine ammonia-lyase with the amino acid sequence shown in SEQ ID No. 1, and the mutation site is Q108S, Y340Q and P375H.

3. A polynucleotide encoding the mutant tyrosine ammonia-lyase according to claim 1 or 2.

4. A nucleic acid construct comprising the polynucleotide according to claim 3.

5. A recombinant expression vector comprising the nucleic acid construct according to claim 4.

6. A recombinant engineering strain comprising the recombinant expression vector according to claim 5.

7. Use of the mutant tyrosine ammonia-lyase according to claim 1 or 2 in the production of p-coumaric acid.

8. Use of the recombinant engineering strain according to claim 6 for the production of p-coumaric acid, characterized in that, The method comprises: (1) inoculating the recombinant engineering strain into a fermentation medium to perform fermentation, so as to obtain a catalyst; (2) using the catalyst obtained in step (1) to perform an enzyme catalysis reaction with L-tyrosine as a substrate, so as to obtain a p-coumaric acid conversion liquid, and after separation, extraction, purification of the p-coumaric acid conversion liquid, p-coumaric acid is obtained.

9. Use according to claim 8, wherein the compound is ###0002### In step (1), the fermentation system of the recombinant engineering strain is controlled at a temperature of 32-42°C, a pH of 6.5-7.5, and a dissolved oxygen of more than 30%; during the fermentation, glycerol is continuously supplemented at a rate of 100-150 mL / h to control the residual glycerol concentration in the fermentation system to be less than 1 g / L, and the growth rate of the bacteria is controlled at OD 600 =3-4 / h; when the bacterial concentration OD 600 reaches 120 or more, the fermentation broth temperature is reduced to 15°C and the bacteria are collected. The catalyst is the fermentation bacteria of the recombinant engineering strain, or the original fermentation liquid containing the fermentation bacteria, or a liquid enzyme obtained by breaking the wall of the fermentation bacteria, or a immobilized enzyme obtained by immobilization treatment of the liquid enzyme.

10. The use according to claim 8, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. In the catalysis reaction system of step (2), the content of L-tyrosine is 40-100 g / L, the content of the fermentation bacteria of the recombinant engineering strain is 20-50 g / L, and the pH of the catalysis reaction system is 8.0-11.0 and the temperature is 37-45℃; The required time for enzyme catalysis synthesis is 6-12 h; A protective agent is added to the catalysis reaction system, and the protective agent is at least one selected from sodium bisulfite, vitamin C, carbon dioxide and nitrogen; The p-coumaric acid conversion liquid is treated as follows to obtain p-coumaric acid: diatomite is added to the p-coumaric acid conversion liquid, stirred, filtered, and the filtrate is taken; then activated carbon is added to the filtrate, stirred, suction filtered, and the suction filtrate is taken; the pH of the suction filtrate is adjusted to 2-3, the temperature is adjusted to 15-20℃ and maintained for 1-3 h, so as to obtain a crystallization liquid; the crystallization liquid is suction filtered to obtain a p-coumaric acid crude product, and the p-coumaric acid crude product is washed with pure water, dried, so as to obtain p-coumaric acid.

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