Tyrosine phenol lyase mutant and application thereof in synthesis of levodobar

Abstract

The invention discloses a tyrosine phenol lyase mutant and application of the tyrosine phenol lyase mutant in synthesis of levodobar. The tyrosine phenol lyase mutant is obtained by carrying out site-specific mutagenesis on the basis of wild type tyrosine phenol lyase. The mutant obtained through transformation can transform more substrates, the yield is also obviously improved, and the method is suitable for industrial production of levodopa.

Description

Technical Field

[0001] The invention belongs to the field of biocatalysis, and in particular relates to a tyrosine phenol lyase mutant and application thereof in the synthesis of levodopa. Background Art

[0002] Parkinson's disease is a motor dysfunction disease, and levodopa (L-DOPA, 3,4-Dihydroxy-L-phenylalanine) is a precursor of dopamine. It enters the brain and is converted into dopamine to play a role in treating Parkinson's disease. Levodopa, CAS: 59-92-7, C9H 11 NO4, molecular weight: 197 g / mol, density 1.5 g / cm 3 , boiling point 448 ℃, white or off-white crystalline powder, slightly soluble in water.

[0003] At present, there are three main methods for producing L-DOPA: extraction method, chemical method and bio-enzyme method. The enzymatic conversion method mainly uses aminoacylase, tyrosinase, tyrosine phenol lyase, and 4-hydroxyphenylacetic acid 3-hydroxylase as catalysts to synthesize L-DOPA. Among them, the tyrosine phenol lyase method for synthesizing L-DOPA has the advantages of high yield, high conversion rate, good enantioselectivity, simple reaction steps, and mild conditions. It has become increasingly popular in recent years. Tyrosine phenollyase (TPL, EC4.1.99.2), also known as β-tyrosinase, can catalyze catechol, sodium pyruvate and ammonium acetate to produce L-DOPA, using 5-pyridoxal phosphate (PLP) as a coenzyme and relying on K. + or NH 4+ , TPL is composed of 4 50 kDa subunits, and each subunit tetramer is bound to 4 PLP molecules. It should be noted that when the concentration of the reaction substrate catechol is higher than 10 g / L, it will inhibit the activity of tyrosine phenol lyase, and it is often necessary to adopt a substrate batch addition method to overcome the inhibitory effect of the high concentration of the initial substrate. In addition, pyruvate in the reaction system and the product L-dopa will generate by-products, resulting in waste of substrates and increased costs.

[0004] Tyrosine phenol-lyase TPL is derived from Erwinia herbicola and has excellent catalytic performance in enzymatic synthesis of L-DOPA (Forrest Foor, Nancy Morin and Keith A. Bostian. Production of L-dihydroxyphenylalanine in Escherichia coli with the tyrosine phenol-lyase gene cloned from Erwinia herbicola. Applied and Environmental Microbiology, 1993, 59(9): 3070-3075). In order to overcome the inhibitory effect of high concentration substrates and reduce the generation of by-products, it is necessary to further improve tyrosine phenol-lyase TPL. In order to obtain a tyrosine phenol-lyase mutant with higher activity, sequence analysis of tyrosine phenol-lyase TPL to determine the mutation site is the main technical means for those skilled in the art to improve the catalytic activity. However, not all mutations are beneficial mutations, and there is still a lack of technical solutions that can significantly improve the process of enzymatic synthesis of DOPA. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a tyrosine phenol-lyase mutant based on site-directed mutagenesis; another purpose of the present invention is to provide a gene fragment encoding the tyrosine phenol-lyase mutant; another purpose of the present invention is to provide a recombinant vector comprising the gene fragment encoding the tyrosine phenol-lyase, and a host cell comprising the recombinant vector; another purpose of the present invention is to provide the use of the aforementioned host cell in the catalytic synthesis of levodopa; another purpose of the present invention is to provide a method for preparing levodopa, a method for preparing levodopa by biocatalysis using a bacterial solution expressing a mutant tyrosine phenol-lyase as a catalyst and sodium pyruvate and catechol as substrates.

[0006] Technical solution: The tyrosine phenol lyase mutant provided by the present invention is derived from the wild-type Erwinia herbicola tyrosine phenol lyase (Erwinia herbicola) TPL, the length of the amino acid sequence is 456, as shown in SEQ ID NO: 11, and the protein molecular weight is about 50 kDa; the nucleotide sequence encoding the enzyme is shown in SEQ ID NO: 12. On the basis of the wild-type TPL, mutations are made at any one or more sites of position 142, position 157, and position 520, which can significantly improve the conversion efficiency of the expressed tyrosine phenol lyase to sodium pyruvate. Specifically, the site-directed mutation is selected from any one of R142C, N157D, and I420V, or a combination of any two of them. Among them, the mutant with the amino acid sequence shown in SEQ ID NO: 1-5 has a significant improvement effect, and its corresponding mutant type is shown as follows:

[0007]

[0008] As a preferred embodiment of the present invention, the mutant obtained by combining any two of the above-mentioned mutation sites has a better transformation efficiency, among which the two mutants TPL-I420V / R142C and TPL-I420V / N157D represented by the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5 are preferred.

[0009] For the tyrosine phenol lyase mutants with the amino acid sequences shown in SEQ ID NOs: 1-5, the nucleotide sequences of the encoding gene fragments are shown in SEQ ID NOs: 6-10.

[0010] The present invention further provides a recombinant vector comprising the aforementioned gene fragment, wherein the recombinant vector is derived from pET28a, and the vector comprises Nco I and Hind III sites for connecting the tyrosine phenol lyase encoding gene, thereby obtaining the recombinant plasmid pET28a-TPL.

[0011] The present invention further provides a host cell comprising the aforementioned recombinant vector, wherein the recombinant vector is transferred into a competent host cell E. coli BL21 (DE3) to obtain a recombinant strain E. coli BL21 (DE3) / pET28a-TPL. The genetically engineered strain can express tyrosine phenol lyase, which is used for biocatalysis of catechol and sodium pyruvate to prepare levodopa.

[0012] The host cell is used as a genetic engineering strain for catalytic synthesis of levodopa, which has higher production efficiency, safety and optical activity than the traditional extraction method and chemical method for producing L-DOPA. The reaction route shown in Formula I uses catechol and sodium pyruvate as substrates, adds tyrosine phenol lyase mutant enzyme solution, EDTA, ammonium acetate, sodium sulfite, and 5-pyridoxal phosphate, and performs a catalytic reaction in a reaction system of pH 7-9 and 20-30°C to obtain the tyrosine phenol lyase mutant enzyme solution; the tyrosine phenol lyase mutant enzyme solution is obtained by using the above-mentioned host cell after amplification culture, fermentation, and cell wall breaking.

[0013]

[0014] Furthermore, the amount of the tyrosine phenol lyase mutant enzyme solution added to the reaction system is 20-60 g / L in terms of wet bacterial body. The wet bacterial body is obtained by at least inoculating a genetically engineered strain carrying a tyrosine phenol lyase encoding gene into an LB liquid culture medium containing kanamycin, then adding IPTG for induction culture, then centrifuging and discarding the supernatant to collect and obtain the wet bacterial body.

[0015] Furthermore, 10-18 g / L of sodium pyruvate and 8-16 g / L of catechol are added to the reaction system; as a further optimization of the present invention, the mass ratio of sodium pyruvate and catechol added to the reaction system is 7:6. After the reaction starts, sodium pyruvate and catechol are added at regular intervals to control the final feed amount of catechol to be 75 g / L.

[0016] Furthermore, after the reaction is completed, the reaction solution is adjusted to be slightly acidic, and the crude crystals are collected by centrifugation or filtration, and then the acid is further adjusted to redissolve, activated carbon is added and the temperature is increased and stirred for decolorization, the clear liquid is filtered and the alkali is adjusted for crystallization, filtered and dried to obtain levodopa crystalline powder.

[0017] The present invention molecularly modified the gene encoding tyrosine phenol lyase TPL from Erwinia herbicola, and obtained 5 mutants with significantly improved enzyme activity, which all showed better catalytic efficiency in the enzymatic synthesis of levodopa. When the tyrosine phenol lyase mutant TPL-I420V / N157D was used as a biocatalyst, a catalytic system was constructed with catechol (75 g / L), sodium pyruvate, ammonium acetate, 5-pyridoxal phosphate (PLP), Na2SO3, and EDTA, and the reaction was carried out at 25°C and pH 8.3 for 8 h, and a maximum of 130.44 g / L of levodopa could be generated, while the levodopa concentration corresponding to the wild-type tyrosine phenol lyase TPL was only 71.91 g / L. Compared with before and after the modification of the tyrosine phenol lyase encoding gene, the production of L-DOPA increased by 81.4%, indicating that the tyrosine phenol lyase mutant TPL-I420V / N157D has better catalytic efficiency, which will help to further improve the economy of the enzymatic synthesis of L-DOPA in industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Agarose gel electrophoresis pattern of the construction of genetically engineered bacteria E. coli BL21 (DE3) / pET28a-TPL; Lane M is a marker; Lane 1 is the TPL gene fragment; Lane 2 is the pET28a linear vector;

[0019] Figure 2 It is the standard curve for the colorimetric detection of sodium pyruvate concentration;

[0020] Figure 3 is the HPLC chromatogram of the substrate catechol standard;

[0021] Figure 4 is the HPLC chromatogram of the product levodopa standard;

[0022] Figure 5 Agarose gel electrophoresis of PCR products obtained by mutation of key sites in recombinant plasmid pET28a-TPL; Lane M is a marker; Lanes 1-10 are: TPL-E30Q, TPL-T49I, TPL-T83K, TPL-Q115K, TPL-T126A, TPL-R142C, TPL-N157D, TPL-Y212F, TPL-S397R, TPL-I420V;

[0023] Figure 6SDS-PAGE detection of wet bacteria induced to express TPL and its mutants; Lane M, Marker; Lanes 1-10 are: TPL-E30Q, TPL-T49I, TPL-T83K, TPL-Q115K, TPL-T126A, TPL-R142C, TPL-N157D, TPL-Y212F, TPL-S397R, TPL-I420V. DETAILED DESCRIPTION

[0024] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to the specific embodiments of the accompanying drawings.

[0025] Example 1 Construction and cultivation of genetically engineered bacteria E. coli BL21 (DE3) / pET28a-TPL

[0026] The amino acid sequence of the tyrosine phenol lyase TPL from Erwinia herbicola is shown in SEQ ID NO: 11, and the nucleotide sequence of the codon-optimized TPL gene is shown in SEQ ID NO: 12, which was synthesized by Hangzhou Qingke Biotechnology Co., Ltd. The nucleotide sequence is 1368 bp long, the corresponding amino acid sequence is 456 aa long, and the protein molecular weight is about 50 kDa.

[0027] Then, PCR amplification was performed using primers TPL-F and TPL-R to obtain the gene encoding tyrosine phenol lyase TPL. PCR amplification was performed using primers pET28a-F and pET28a-R to obtain the pET28a linearized vector fragment. The agarose gel electrophoresis results are shown in Figure 1 As shown. Using the "one-step cloning" kit, the obtained tyrosine phenol lyase TPL encoding gene was inserted between the Nco I and Hind Ⅲ sites of the pET28a linearized vector to obtain the recombinant plasmid pET28a-TPL. The recombinant plasmid pET28a-TPL was transferred into the competent cell E. coli BL21 (DE3) to obtain the recombinant strain E. coli BL21 (DE3) / pET28a-TPL. The constructed genetic engineering bacteria were verified by plasmid sequencing, indicating that the tyrosine phenol lyase gene was correctly inserted.

[0028] TPL-F: 5'-GAAGGAGATATACATATGAACTATCCTGCCGAGCCTT-3';

[0029] TPL-R: 5'-TGCGGCCGCAAGCTTTTAAATAAAGTCAAAACGCGCAGTA-3';

[0030] pET28a-F: 5'-AAGCTTGCGGCCGCACT-3';

[0031] pET28a-R: 5'-ATGTATATCTCCTTCTTAAAGTTAAACAAAATTA-3'.

[0032] The recombinant strain was inoculated on LB solid medium containing 100 μg / mL kanamycin resistance and streaked, and cultured overnight at 37 °C to obtain a single colony. A single colony was picked and placed in 50 mL LB liquid medium containing 100 μg / mL kanamycin resistance, and cultured overnight at 37 °C and 150 rpm to obtain seed liquid. The fresh seed liquid was added with 40% glycerol at a volume ratio of 1:1 and stored in a -80 °C ultra-low temperature refrigerator.

[0033] Take the strain E. coli BL21(DE3) / pET28a-TPL out of the -80 ℃ freezer and place it on ice to melt. Take 5µL of the preserved bacterial solution and streak it on a LB solid medium plate containing 100 μg / mL kanamycin resistance, and incubate it in a constant temperature incubator at 37 ℃ for 12-16 h for activation. Pick a single colony to 50 mL of LB liquid medium containing 100 μg / mL kanamycin, and place it in a shaking incubator at 37 ℃ at 150 rpm for 12-16 h. The obtained seed solution is the genetic engineering bacterial seed solution of E. coli BL21(DE3) / pET28a-TPL. Extract the plasmid pET28a-TPL from the E. coli BL21(DE3) / pET28a-TPL bacterial solution.

[0034] LB liquid medium composition: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, solvent is distilled water, pH 7.0~7.5. LB solid medium is LB liquid medium with 20 g / L agar added.

[0035] Example 2 Inducible expression of tyrosine phenol lyase TPL and its mutants

[0036] Tyrosine phenol lyase E. coli BL21 (DE3) / pET28a-TPL and its mutants were streaked on LB solid plates containing 100 μg / mL kanamycin and inverted in a constant temperature incubator at 37 ° C for 12-16 h. Pick a single colony in 50 mL LB medium containing 100 μg / mL kanamycin and culture it at 37 ° C and 200 rpm for 12-16 h to obtain seed liquid. The seed liquid was transferred to 150 mL LB liquid medium containing the same kanamycin concentration at a volume ratio of 2% inoculation, and the bacterial concentration was cultured in a shaker at 37 ° C and 200 rpm. OD 600 to 0.6-0.8. Add the prepared IPTG solution to a final concentration of 0.2 mM, and induce expression at 24 °C and 150 rpm for 12-16 h. After the induction, transfer the bacterial solution to a centrifuge cup, centrifuge at 4 °C and 8000 rpm for 10 min, and discard the supernatant; resuspend the bacterial precipitate with 100 mM PBS buffer (pH 7.0), centrifuge at 4 °C and 8000 rpm for 10 min, and discard the supernatant. The obtained precipitate is the wet bacterial cell, which is stored in a -20 °C refrigerator for use.

[0037] Example 3 Construction of the catalytic reaction system of tyrosine phenol lyase TPL and its mutants

[0038] The wet cells of tyrosine phenol lyase TPL and its mutants were prepared according to the method described in Example 2, and a 15 mL reaction system was constructed. The reaction was carried out in a shaker at 30° C. and 200 rpm. The amount of each component added is shown in Table 1.

[0039] Table 1 Reaction system for the synthesis of L-DOPA catalyzed by tyrosine phenol lyase TPL

[0040]

[0041] Example 4 Rapid screening of mutation library by colorimetric detection of sodium pyruvate

[0042] Sodium pyruvate reacts with salicylaldehyde in a strong alkaline solution to generate a colored 1,5-bis(2-hydroxyphenyl)-1,4-pentadienone product with an absorption value at a wavelength of 465 nm. As the concentration of sodium pyruvate decreases, the color changes from orange-red to light yellow. The reaction detection system is 1 mL, and the reagents in Table 2 are added in sequence, mixed evenly, left at room temperature for 2 h, and detected with an enzyme reader at a wavelength of 465 nm. Aqueous solutions containing 2 mM, 5 mM, 8 mM, 10 mM, 20 mM, 50 mM and 80 mM sodium pyruvate are treated with alkali according to the above steps to obtain a standard curve of sodium pyruvate, OD 465 It is proportional to the concentration of sodium pyruvate. The standard curve is as follows: Figure 2As shown, its standard curve is y=0.0289x+0.2363, R 2 =0.9995.

[0043] When the enzymatic synthesis of L-DOPA was detected by colorimetry, the concentration of the substrate sodium pyruvate decreased as the reaction proceeded. Therefore, the faster the concentration of sodium pyruvate decreased within the same reaction time, the higher the activity of the tyrosine phenol lyase of the genetically engineered bacteria.

[0044] Table 2 Reaction system for colorimetric detection of sodium pyruvate

[0045]

[0046] Example 5 Liquid Chromatographic Analysis of the Synthesis of L-DOPA Catalyzed by Tyrosine Phenol Lyase

[0047] 1. Reaction solution treatment

[0048] A whole-cell catalytic reaction system was constructed according to the method of Example 3. After the reaction was completed, 500 μL of the reaction solution was taken, 500 μL of acetic acid and 250 μL of acetonitrile were added for extraction, and the mixture was centrifuged at 10,000 rpm for 2 min. The supernatant was taken and filtered through a membrane for loading.

[0049] 2. High performance liquid chromatography analysis method

[0050] The substrate catechol and the product levodopa can be detected by high performance liquid chromatography. The liquid column model used is ACE Excel 5 C18-PFP, the mobile phase is 10 mM potassium dihydrogen phosphate (adjusted to pH 3.0 with HCl): methanol = 9:1 (v / v), detection at 280 nm wavelength, column temperature 30 °C, injection volume 10 μL, holding time 20 min. The liquid phase spectrum of the substrate catechol standard is shown in Figure 3 As shown in Figure 1, the peak time is 4.635 min. The liquid phase spectrum of the product levodopa standard is as follows: Figure 4 As shown, the peak time is 18.652 min.

[0051] Example 6 Single-point mutation of tyrosine phenol lyase TPL

[0052] The tyrosine phenol lyase TPL gene was subjected to homology modeling and molecular docking analysis, and 10 amino acid sites were screened for single-point mutation. The amino acid sequences of the tyrosine phenol lyase TPL mutants are as follows:

[0053] The plasmid pET28a-TPL was used as the amplification template and the mutants were constructed by inverse PCR. The mutants and primers are shown in Table 3, and the PCR amplification system is shown in Table 4.

[0054] PCR reaction program: preliminary denaturation: 95 ℃, 5 min; complete denaturation: 95 ℃, 15 s; annealing: 58 ℃, 15 s; extension: 72 ℃, 90 s; 30 cycles; re-extension: 72 ℃, 5 min; reduce to 4 ℃ for insulation.

[0055] Table 3 Tyrosine phenol lyase TPL single point mutation primers

[0056]

[0057] Table 4 PCR system for full plasmid amplification

[0058]

[0059] Take an appropriate amount of PCR product and perform agarose gel electrophoresis verification. After electrophoresis is completed, observe under ultraviolet light and you will see a bright band between 5000-8000 bp, which is consistent with the theoretical value of the plasmid. The agarose gel electrophoresis results are as follows: Figure 5 As shown. Then add 1 μL of Dpn I restriction endonuclease to the PCR product and react at 37 ℃ for 1 h to remove the methylated template. Then use a DNA gel recovery and purification kit to recover and purify the PCR product. Store in a -20 ℃ low-temperature refrigerator for later use.

[0060] Transform the target fragment obtained by mutation directly into the host bacterium E. coli BL21 (DE3): take 50 μL E. coli BL21 (DE3) competent cells and put them on ice to melt, add 5 μL of mutant PCR product, and put them on ice for 30 min. After the ice bath, heat shock the competent cells at 42 ℃ for 90 s, and then immediately put them on ice for 3-5 min. Then add 1 mL of LB medium without antibiotics and incubate them in a shaker at 37 ℃ and 200 rpm for 1 h. After the culture is completed, centrifuge the culture at 4 ℃ and 4500 rpm for 5 min, discard 900 μL of the supernatant, fully resuspend the remaining bacteria, and take 100-150 μL of the bacterial solution to spread on the LB solid plate containing kanamycin resistance. The cells were incubated upside down in a 37°C constant temperature incubator for 12-16 h to obtain the genetically engineered Escherichia coli E. coli BL21(DE3) / pET28a-TPL-M, where M represents an amino acid replacement. For example, when the 30th glutamic acid in the amino acid sequence is replaced by glutamine, the genetically engineered bacteria is named E. coli BL21(DE3) / pET28a-TPL-E30Q.

[0061] The method of Example 2 was used to induce the expression of tyrosine phenol lyase and its mutants to obtain wet cells of the engineered bacteria, and the expression effect was verified by SDS-PAGE electrophoresis experiment. The results of the electrophoresis experiment are as follows: Figure 6As shown, the protein molecular weight of TPL and its mutants is about 50 kDa, and the protein bands expressed by each mutant strain are obviously thickened and the band position is around 50 kDa, which is the correct size.

[0062] To verify the activity of different mutants, wet cells of TPL and its mutants were prepared by the method of Example 2, and TPL and its mutants were subjected to catalytic reaction by the method of Example 3. The reaction solutions of 0 min, 20 min, 40 min, and 1 h were taken respectively, and the colorimetric method was used for verification by the method described in Example 4. The results are shown in Table 5. Compared with TPL, amino acid substitutions such as TPL-R142C, TPL-N157D, and TPL-I420V resulted in a faster decrease rate of the concentration of the substrate sodium pyruvate, which were confirmed to be beneficial mutations. The amino acid sequences corresponding to these three mutants are SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; the corresponding nucleotide sequences are SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0063] Table 5 Colorimetric verification of the catalytic results of TPL and its mutants

[0064]

[0065] In order to further verify the situation that each beneficial mutation improves the catalytic activity of the engineered bacteria, the catalytic results of tyrosine phenol lyase and its mutant engineered bacteria were analyzed by liquid phase detection using the method of Example 5. The catalytic effect was compared by comparing the consumption of substrate catechol and the production of product levodopa in the 1 h catalytic reaction liquid. The results are shown in Table 6. Compared with tyrosine phenol lyase TPL, multiple mutations led to a significant increase in catalytic activity, namely TPL-R142C, TPL-N157D and TPL-I420V. The liquid phase detection results were basically consistent with the results obtained by the colorimetric method and could be mutually verified. Among these mutations, TPL-I420V performed best. After 1 h of reaction, the substrate consumption was increased by 87% compared with TPL, and the product production was increased by 71% compared with TPL.

[0066] Table 6 Liquid phase detection of catalytic results of TPL and its mutants

[0067]

[0068] Example 7 Combination mutation of tyrosine phenol lyase TPL

[0069] Using plasmid pET28a-TPL-I420V as an amplification template and inverse PCR technology, a combined mutant strain was constructed according to the method described in Example 6, wherein the following amino acids were replaced:

[0070] (1) The isoleucine at position 420 of the amino acid sequence shown in SEQ ID No: 11 was replaced with valine, and the arginine at position 142 was replaced with cysteine ​​to obtain a mutant strain E. coli BL21(DE3) / pET28a-TPL-I420V / R142C; the amino acid sequence is shown in SEQ ID NO: 4, and the encoded amino acid sequence is shown in SEQ ID NO: 9;

[0071] (2) The isoleucine at position 420 of the amino acid sequence shown in SEQ ID NO: 1 was replaced with valine, and the asparagine at position 157 was replaced with aspartic acid to obtain a mutant strain E. coli BL21(DE3) / pET28a-TPL-I420V / N157D; the amino acid sequence of which is shown in SEQ ID NO: 5, and the encoded amino acid sequence is shown in SEQ ID NO: 10.

[0072] The wet cells of TPL and its mutants were prepared by the method of Example 2, the wet cells were used for catalytic reaction by the method of Example 3, the catalytic results of the cells were analyzed by liquid phase detection by the method of Example 5, and the catalytic effects of the mutants were compared by comparing the consumption of the substrate catechol and the production of the product levodopa in the 1 h catalytic reaction liquid. The results are shown in Table 7. Compared with tyrosine phenol lyase TPL, these combined mutations significantly improved the catalytic activity, among which TPL-I420V / N157D performed the best, with a substrate consumption of 146% and a product production of 128% in a reaction of 1 h.

[0073] Table 7 Liquid phase detection of catalytic results of TPL and its mutant engineering bacteria

[0074]

[0075] Example 8 High-density fermentation of tyrosine phenol lyase TPL and mutant TPL-I420V / N157D and enzyme solution preparation

[0076] 1. Seed liquid culture

[0077] Take out strains E. coli BL21(DE3) / pET28a-TPL and strains E. coli BL21(DE3) / pET28a-TPL-I420V / N157D from the -80 ℃ freezer, and use a glycerol tube to streak on the slant of a test tube containing 100 μg / mL kanamycin. Incubate at 37 ℃ for 13-15 h. Add 6 mL of sterile water to the incubated slant test tube, and use an inoculation loop to scrape the bacteria on the slant into the sterile water. Inoculate 1% of the inoculation volume into LB medium, and incubate at 37 ℃, 220 rpm, for 10-11 h. At this time, OD 600Around 5.0.

[0078] LB liquid medium composition: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, solvent is distilled water, pH 7.0-7.5. LB solid medium is LB liquid medium with 20 g / L agar added.

[0079] 2. High-density fermentation

[0080] Prepare 5 L of fermentation medium, the formula is shown in Table 8. Prepare 1 L of feed medium, the formula is shown in Table 9. The fermentation medium and feed medium were sterilized at 121 °C for 30 min. After sterilization, when the temperature dropped to 60 °C, kanamycin was added to the fermentation medium at 100 μg / L. When the temperature dropped to 37 °C, the seed liquid was inoculated into the fermentation medium at a 2.0% inoculation amount, i.e. 100 mL / 5 L, and cultured at 37 °C. The ventilation volume was 1 vvm at the beginning and increased to 1.2 vvm after 3 h. The tank pressure was maintained at 0.05-0.06 Mpa. The stirring speed was 500-700 rpm. The dissolved oxygen was controlled to be no less than 20%. The dissolved oxygen dropped rapidly after 2 h of fermentation and rose rapidly after about 5 h. At this time, the feed medium was added to control the dissolved oxygen at about 30%. The pH may rise slowly in the early stage of fermentation, and drop after feeding. Thereafter, the pH can be adjusted with ammonia water and controlled at 6.7-6.9. 1-2 h after the start of feeding, slowly cool down to 30-33 ℃. 600 When the temperature reaches 20-25, cool it down to 25°C. Add 0.3 mM IPTG to induce for 14-16 hours and then release the tank.

[0081] Table 8 Fermentation medium formula (5 L)

[0082]

[0083] Table 9 Feed medium formula (1 L)

[0084]

[0085] 3. Preparation of cell wall breaking enzyme solution

[0086] Before the fermentation liquid is put into the tank, the fermentation liquid temperature is reduced to 10-15 ℃ in advance by using the fermentation tank cooling system. The fermentation liquid passes through the ceramic membrane and is top washed twice with tap water to ensure that the fermentation liquid is washed away as much as possible and concentrated to a bacterial content of 200 g / L. The membrane process will generate heat and needs to be cooled to below 20 ℃. The cooled bacterial suspension is broken by a high-pressure homogenizer with a pressure of 60-80 kg. The wall breaking process will heat up. During the period, ensure that the temperature does not exceed 30 ℃, and the wall breaking cycle is repeated twice. The pH of the enzyme solution needs to be controlled during the whole wall breaking process, and the pH is adjusted to about 7.0-7.2 with sodium hydroxide aqueous solution. After the wall breaking is completed, the temperature is lowered to 0 ℃, the enzyme solution is divided into packages, and placed in a cold storage for freezing.

[0087] Example 9 Scale-up of the synthesis of L-DOPA catalyzed by tyrosine phenol lyase

[0088] In order to further confirm the difference in activity between strain E. coli BL21(DE3) / pET28a-TPL and strain E. coli BL21(DE3) / pET28a-TPL-I420V / N157D, the two strains were subjected to high-density fermentation production and the corresponding enzyme solution was prepared by the method of Example 8. Subsequently, a 200 mL catalytic reaction system was constructed, 2.8 g of sodium pyruvate, 3.0 g of catechol, 0.2 g of EDTA, 8 g of ammonium acetate, 0.4 g of sodium sulfite, 0.02 g of pyridoxal 5-phosphate (PLP), 60 mL of the enzyme solution prepared in Example 9 (60 g / L based on the wet bacterial cell measurement) was added, and the catalytic reaction system was made up to 200 mL with pure water, and the pH was set to 8.3 and the temperature was set to 25 °C. After the reaction started, 2.4 g of sodium pyruvate and 2.0 g of catechol were added every 1 h for a total of 6 times (sampling was completed before addition). After the addition was completed, the reaction was continued for 2 h. The final amount of catechol was 75 g / L.

[0089] The reaction solution of 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, and 8 h was taken, and the catalytic results of strain E. coli BL21 (DE3) / pET28a-TPL and strain E. coli BL21 (DE3) / pET28a-TPL-T4C / A2T were analyzed by liquid phase detection using the method of Example 5 to monitor the residual amount of catechol and the amount of levodopa produced during the reaction process. The residual amount of catechol and the amount of levodopa produced during the reaction process are shown in Table 10. The tyrosine phenol lyase TPL enzyme solution prepared by strain E. coli BL21(DE3) / pET28a-TPL catalyzed the reaction for 8 h, and the yield of L-DOPA was 71.91 g / L; the tyrosine phenol lyase TPL enzyme solution prepared by strain E. coli BL21(DE3) / pET28a-TPL-I420V / N157D catalyzed the reaction for 8 h, and the yield of L-DOPA was 125.44 g / L, which increased the yield of L-DOPA by 74.4%. In addition, compared with the enzyme solution prepared by strain E. coli BL21(DE3) / pET28a-TPL, the residual catechol in the enzyme solution prepared by strain E. coli BL21(DE3) / pET28a-TPL-I420V / N157D was lower, only 1.23 g / L. The pH of the reaction solution is adjusted to slightly acidic with acid solution, and crude crystals are collected by centrifugation or filtration. Then, the acid is further adjusted to redissolve, activated carbon is added, and the temperature is increased and stirred for decolorization, the clear liquid is filtered, and then alkali is adjusted for crystallization, which is filtered and dried to obtain levodopa crystalline powder.

[0090] Table 10 The residual amount of catechol and the amount of L-DOPA produced during the catalytic reaction of different strains

[0091]

[0092] The tyrosine phenol lyase mutant provided by the present invention is catalyzed in a reaction system containing 10-18 g / L sodium pyruvate and 8-16 g / L catechol, and 12 g / L sodium pyruvate and 10 g / L catechol are added every 1 h after the reaction, so that the residual amount of catechol does not exceed 1 g / L in 4 hours before the reaction, and the residual amount of catechol does not exceed 2.5 g / L in 8 hours, which reflects the excellent enzyme conversion efficiency of the mutant and avoids the inhibition of the substrate concentration on the enzymatic activity of the tyrosine phenol lyase.

[0093] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A tyrosine phenol lyase mutant, characterized in that: The amino acid sequences of the mutants are shown in SEQ ID NOs: 1-5.

2. A tyrosine phenol lyase mutant according to claim 1, characterized in that: The amino acid sequence of the mutant is SEQ ID NO: 4 or SEQ ID NO:

5.

3. A gene fragment encoding the tyrosine phenol lyase mutant according to claim 1 or 2, characterized in that: The nucleotide sequences of the gene fragments are shown in SEQ ID NOs: 6-10.

4. A recombinant vector, characterized in that: Comprising the gene fragment as described in claim 3.

5. A host cell, characterized in that: Comprising the recombinant vector as described in claim 4.

6. Use of the host cell as claimed in claim 5 in catalyzing the synthesis of levodopa.

7. A method for preparing levodopa, characterized in that: The tyrosine phenol lyase mutant enzyme solution is obtained by taking catechol and sodium pyruvate as substrates, adding tyrosine phenol lyase mutant enzyme solution, EDTA, ammonium acetate, sodium sulfite and 5-pyridoxal phosphate, and carrying out catalytic reaction in a reaction system of pH 7-9 and 20-30° C. The tyrosine phenol lyase mutant enzyme solution is obtained by using the host cell according to claim 5, after amplification culture, fermentation and cell wall breaking.

8. The method for preparing levodopa according to claim 7, characterized in that: The amount of tyrosine phenol lyase mutant enzyme solution added to the reaction system is 20-60 g / L based on the wet bacterial cell measurement.

9. The method for preparing levodopa according to claim 8, characterized in that: 10-18 g / L of sodium pyruvate and 8-16 g / L of catechol are added to the reaction system; sodium pyruvate and catechol are added at regular intervals after the reaction starts, and the final feeding amount of catechol is controlled to be 75 g / L.

10. The method for preparing levodopa according to claim 9, characterized in that: After the reaction is completed, the reaction solution is adjusted to be slightly acidic, and the crude crystals are collected by centrifugation or filtration. The acid is then adjusted to redissolve, activated carbon is added, and the temperature is increased and stirred for decolorization. The clear liquid is filtered, and the alkali is adjusted for crystallization, filtered, and dried to obtain levodopa crystalline powder.

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