A recombinant vector of tyrosine phenol lyase and its application in the synthesis of levodopa

Abstract

The present invention discloses a recombinant vector of tyrosine phenol lyase and its application in the synthesis of levodopa. Any one or more of the first base T, the second base A, the fourth base T, and the fifth base A of the T7 promoter in the vector pET28a are replaced with other bases to increase the strength and activity of the promoter, thereby obtaining an engineered bacterium with high tyrosine phenol lyase activity in the enzymatic synthesis of levodopa. This engineered bacterium can efficiently convert catechol in the catalytic reaction system and increase the yield of levodopa, which is much higher than that of the engineered strain corresponding to the unmodified T7 promoter. Based on this, the amount of biocatalyst can be reduced, and the economy of the enzymatic synthesis process of levodopa can be improved.

Description

Technical Field

[0001] The present invention belongs to the field of biocatalysis, and particularly relates to a recombinant vector of tyrosine phenol lyase and its application in the synthesis of L-DOPA. Background Art

[0002] Parkinson's disease is a movement disorder disease. The incidence of Parkinson's disease increases with age. The incidence rate in people over 65 years old is 2%-3%. The incidence rate is relatively low before the age of 50, but currently this trend is changing, and the number of patients with early-onset Parkinson's disease (under 50 years old) is increasing year by year. Levodopa (L-DOPA, 3,4-dihydroxy-L-phenylalanine) is the main drug for the treatment of Parkinson's disease. L-DOPA is a precursor of dopamine and has no pharmacological activity itself. After crossing the blood-brain barrier, it is decarboxylated to form dopamine, solving the problem that dopamine cannot directly reach the brain. In 1967, levodopa was finally approved by the US FDA for the treatment of Parkinson's disease. Levodopa is a white or almost white crystalline powder, slightly soluble in water, its CAS number is 59-92-7, the molecular formula is C9H 11 NO4, the molecular weight is 197 g / mol, and the density is 1.5 g / cm 3 , and the boiling point is 448 °C.

[0003] Currently, there are mainly three methods for producing L-DOPA: 1. Extraction method: extracting from plants such as Mucuna pruriens and Stizolobium hassjoo. The raw material source is scarce, the yield is small, and the production cost is high; 2. Chemical method: the process is complex, and both the conversion rate and the optical rotation are relatively low. It is easy to produce a mixture of D-LOPA and L-LOPA, and D-LOPA can cause toxic reactions in the human body; 3. Biocatalytic method: Enzyme-catalyzed synthesis mainly includes tyrosinase synthesis method, tyrosine phenol lyase synthesis method, transaminase synthesis method, etc. Among the three, the biocatalytic method has specificity and high efficiency, and can greatly simplify the manufacturing process. In the biocatalytic method, tyrosine phenol lyase is the most important biocatalyst at present. Tyrosine phenol lyase (TPL, E.C.4.1.99.2), also known as β-tyrosinase, can catalyze catechol, sodium pyruvate and ammonium acetate to generate levodopa, with pyridoxal 5'-phosphate (PLP) as a coenzyme, dependent on K + or NH 4+ , TPL is composed of 4 subunits of 50 kDa, and each subunit tetramer binds with 4 molecules of PLP.

[0004] Improving the catalytic activity or expression activity of tyrosine phenol lyase is very important for further improving the economy of the enzymatic synthesis process of levodopa. Existing technologies often focus on the tyrosine phenol lyase coding sequence, performing directed evolution or rational design on it to improve enzyme activity, which involves a large amount of protein engineering work and often yields unsatisfactory results. In contrast, promoter engineering is a key means to achieve efficient and precise gene expression regulation at the transcriptional level. Currently, the strategies of promoter engineering mainly involve the construction of promoter libraries, promoter replacement, promoter modification, and the rational design of hybrid promoters. Mutating and modifying the promoter can significantly increase the promoter strength and improve promoter activity, thereby enhancing the expression activity of tyrosine phenol lyase and further improving the enzymatic synthesis process of levodopa. Summary of the Invention

[0005] Objectives of the Invention: The objectives of the present invention are to provide a recombinant vector of tyrosine phenol lyase; another objective of the present invention is to provide a host cell containing the aforementioned recombinant vector of tyrosine phenol lyase; another objective of the present invention is to provide the application of the aforementioned recombinant vector of tyrosine phenol lyase in the catalytic synthesis of levodopa; another objective of the present invention is to provide the application of a recombinant pET T7 promoter obtained by site-directed mutagenesis in the preparation of tyrosine phenol lyase with high conversion rate; another objective of the present invention is to provide a method for preparing levodopa, which is a method for biocatalytically preparing levodopa using the bacterial liquid cultured from the aforementioned host cell containing the recombinant vector of tyrosine phenol lyase as a catalyst and using sodium pyruvate and catechol as substrates.

[0006] Technical Solution: A recombinant vector of tyrosine phenol lyase provided by the present invention is derived from a pET vector and includes a site-directed mutagenized T7 promoter and a tyrosine phenol lyase coding gene, and the site-directed mutagenesis is selected from any one or a combination of multiple of T1A, A2T, T4C, T4G, A5C; the nucleotide sequence of the tyrosine phenol lyase coding gene has a homology of not less than 95% compared with SEQ ID NO: 2.

[0007] The tyrosine phenol lyase TPL of the present invention is derived from Erwinia herbicola, and its amino acid sequence is as shown in SEQ ID NO: 1, and the nucleotide sequence of its coding gene is as shown in SEQ ID NO: 2. Those skilled in the art can appropriately modify the aforementioned sequences on the basis of not departing from the inventive principle of the present application. As a preferred embodiment, the nucleotide sequence of the tyrosine phenol lyase coding gene has a homology of not less than 98% compared with SEQ ID NO: 2. As an alternative embodiment of the present invention, the nucleotide sequence of the tyrosine phenol lyase coding gene is as shown in SEQ ID NO: 2.

[0008] Furthermore, any site-directed mutation of the pET T7 promoter provided by the present invention has been demonstrated by experiments to be beneficial to improving the conversion efficiency of the substrate. The initial T7 promoter sequence of pET is 5'-TAATACGACTCACTATAG-3', which is the site recognized and bound by T7 RNA polymerase. Among them, the preferred site-directed mutation scheme is any one of the three mutation combinations of T4C / T1A, T4C / A2T, and T4C / A5C. As the optimal scheme of the present invention, the mutation site is T4C / A2T, and the mutated T7 promoter sequence is 5'-TTACACGACTCACTATAG-3'.

[0009] Furthermore, the recombinant vector described in the present invention is derived from pET28a, and this vector includes Nco I and Hind III sites for ligating the tyrosine phenol lyase encoding gene, thereby obtaining the recombinant plasmid pET28a-TPL.

[0010] Furthermore, the recombinant plasmid pET28a-TPL is transferred into the competent host cell E. coli BL21(DE3) to obtain the recombinant strain E. coli BL21(DE3) / pET28a-TPL. This genetically engineered strain can express tyrosine phenol lyase and is used for biocatalytically preparing levodopa from catechol and sodium pyruvate. Among them, the site-directed mutation of the pET T7 promoter makes an important contribution to the improvement of the substrate catalytic efficiency and the yield of levodopa.

[0011] A method for preparing levodopa provided by the present invention uses catechol and sodium pyruvate as substrates, adds tyrosine phenol lyase enzyme solution, EDTA, ammonium acetate, sodium sulfite, and pyridoxal 5'-phosphate, and conducts a catalytic reaction in a reaction system at pH 7-9 and 20-30 °C; the tyrosine phenol lyase enzyme solution is obtained by using the aforementioned host cells through scale-up culture, fermentation, and cell disruption.

[0012] Furthermore, the addition amount of the tyrosine phenol lyase enzyme solution in the reaction system is 20-60 g / L in terms of wet cells. The acquisition of the wet cells at least includes: inoculating the genetically engineered strain carrying the tyrosine phenol lyase encoding gene into an LB liquid medium containing kanamycin, adding IPTG for induced culture, then centrifuging to discard the supernatant, and collecting the wet cells.

[0013] 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 to catechol added to the reaction system is 7:6. After the reaction starts, sodium pyruvate and catechol are replenished at regular intervals, and the final feeding amount of catechol is controlled to be 75 g / L.

[0014] 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. Then, the solution is adjusted to be acidic again for redissolution, activated carbon is added, and the mixture is heated and stirred for decolorization. The filtrate is adjusted to be alkaline for crystallization, filtered, and dried to obtain the crystalline powder of levodopa.

[0015] The T7 promoter of the vector pET28a was modified in the present invention, and the corresponding engineered bacteria obtained had improved catalytic activity in the enzymatic synthesis of levodopa. In particular, when the engineered strain E. coli BL21(DE3) / pET28a-TPL-T4C / A2T was used as the biocatalyst, in a catalytic reaction system containing catechol and sodium pyruvate as substrates, reacting at 25 °C and pH 8.3 for 8 h, 99.52 g / L of levodopa could be produced, while the concentration of levodopa converted by the engineered strain corresponding to the unmodified T7 promoter was only 71.91 g / L. It can be seen that the yield of levodopa increased by 38.4% after the promoter modification, which helps to reduce the amount of catalyst used in the enzymatic synthesis of levodopa and improve the economic efficiency of the enzymatic synthesis process of levodopa. Description of the Drawings

[0016] Figure 1 Schematic diagram of the catalytic synthesis of levodopa by tyrosine phenol lyase;

[0017] Figure 2 Agarose gel electrophoresis pattern of constructing the genetically engineered bacterium E. coli BL21(DE3) / pET28a-TPL; Lane M is the marker; Lane 1 is the TPL gene fragment; Lane 2 is the linear pET28a vector;

[0018] Figure 3 Standard curve for detecting the concentration of sodium pyruvate by colorimetry;

[0019] Figure 4 High-performance liquid chromatography (HPLC) chromatogram of the substrate catechol standard;

[0020] Figure 5 High-performance liquid chromatography (HPLC) chromatogram of the product levodopa standard;

[0021] Figure 6 Agarose gel electrophoresis pattern of the PCR product obtained by modifying the T7 promoter in the recombinant plasmid pET28a-TPL; Lane M is the marker; Lanes 1 - 18 are in sequence: base substitutions T1A, T1C, T1G, A2T, A3T, T4A, T4C, T4G, -A5C, A5T, C6G, C6T, G7A, A8T, A12T, C13G, T16A, A17T;

[0022] Figure 7SDS-PAGE detection of induced expression of wet bacteria by TPL under different promoters; Lane M, Marker; Lanes 1-19 are in sequence: T7 and its base substitutions T1A, T1C, T1G, A2T, A3T, T4A, T4C, T4G, A5C, A5T, C6G, C6T, G7A, A8T, A12T, C13G, T16A, A17T;

[0023] Figure 8 It is the plasmid map of the optimized recombinant vector pET28a-TPL-T4C / A2T. Detailed implementation manners

[0024] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] Example 1 Construction and culture of genetic engineering bacterium E. coli BL21(DE3) / pET28a-TPL

[0026] Tyrosine phenol lyase TPL is derived from Erwinia herbicola and has excellent catalytic performance in the enzymatic synthesis of L-dopa (Forrest Foor, Nancy Morin and Keith A. Bostian. Production ofL-dihydroxyphenylalanine in Escherichia coli with the tyrosine phenol-lyasegene cloned from Erwinia herbicola. Applied and Environmental Microbiology,1993, 59(9):3070-3075). The synthesis route of this method is as Figure 1 shown. The amino acid sequence of tyrosine phenol lyase TPL is as shown in SEQ ID NO.1, and the nucleotide sequence of the codon-optimized TPL gene is as shown in SEQ ID NO.2, which was synthesized by Hangzhou Qingke Biotechnology Co., Ltd. Its nucleotide sequence is 1368 bp in length, corresponding to an amino acid sequence of 456 aa, and the protein molecular weight is about 50 kDa.

[0027] Subsequently, PCR amplification was carried out using primers TPL-F and TPL-R to obtain the tyrosine phenol lyase TPL coding gene. PCR amplification was carried out using primers pET28a-F and pET28a-R to obtain the linearized pET28a vector fragment. The results of agarose gel electrophoresis are as Figure 2As shown in the figure. 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 linearized pET28a 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. After the constructed genetically engineered bacteria were verified to be correct by plasmid extraction and sequencing, it indicated that the tyrosine phenol lyase gene was inserted correctly.

[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 streaked and isolated on an LB solid medium containing 100 μg / mL kanamycin resistance and cultured overnight at 37 °C to obtain single colonies. Single colonies were picked and inoculated into 50 mL of LB liquid medium containing 100 μg / mL kanamycin resistance and cultured overnight at 37 °C and 150 rpm to obtain a seed solution. Fresh seed solution was added with 40% glycerol according to a volume ratio of 1:1 and stored in a -80 °C ultra-low temperature refrigerator.

[0033] The strain E. coli BL21(DE3) / pET28a-TPL was taken out from the -80 °C refrigerator and melted on ice. 5 μL of the preserved bacterial solution was streaked on an LB solid medium plate containing 100 μg / mL kanamycin resistance and cultured at 37 °C in a constant temperature incubator for 12 - 16 h for activation. Single colonies were picked into 50 mL of LB liquid medium containing 100 μg / mL kanamycin and placed in a shaking incubator at 150 rpm at 37 °C for 12 - 16 h. The obtained seed solution was the seed solution of the genetically engineered bacteria of E. coli BL21(DE3) / pET28a-TPL, and the plasmid pET28a-TPL was extracted from the E. coli BL21(DE3) / pET28a-TPL bacterial solution.

[0034] Composition of LB liquid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, with distilled water as the solvent, pH 7.0 - 7.5. LB solid medium is prepared by adding 20 g / L agar to LB liquid medium.

[0035] Example 2 Induced expression of tyrosine phenol lyase TPL under different promoters

[0036] Tyrosine phenol lyase E. coli BL21(DE3) / pET28a-TPL was streaked on an LB solid plate containing 100 μg / mL kanamycin and incubated inverted in a constant temperature incubator at 37 °C for 12 - 16 h. A single colony was picked and cultured in 50 mL of LB medium containing 100 μg / mL kanamycin at 37 °C and 200 rpm for 12 - 16 h to obtain a seed solution. The seed solution was transferred to 150 mL of LB liquid medium containing the same kanamycin concentration at an inoculation amount of 2% (v / v) and cultured in a shaker at 37 °C and 200 rpm until the cell concentration OD600 reached 0.6 - 0.8. IPTG solution prepared was added to a final concentration of 0.3 mM, and induced expression was carried out at 24 °C and 150 rpm for 12 - 16 h. After the induction was completed, the bacterial solution was transferred to a centrifuge tube and centrifuged at 4 °C and 8000 rpm for 10 min, and the supernatant was discarded; the cell pellet was resuspended in 100 mM PBS buffer (pH 7.0) and centrifuged again at 4 °C and 8000 rpm for 10 min, and the supernatant was discarded. The obtained pellet was the wet cells and was stored in a -20 °C refrigerator for later use.

[0037] Example 3 Construction of catalytic reaction system for induced expression of tyrosine phenol lyase TPL under different promoters

[0038] Wet cells of tyrosine phenol lyase TPL induced expression under different promoters were prepared according to the method described in Example 2, and a 15 mL reaction system was constructed and reacted in a shaker at 30 °C and 200 rpm. The addition amounts of each component are shown in Table 1.

[0039] Table 1 Reaction system for catalytic synthesis of L-dopa by tyrosine phenol lyase TPL

[0040]

[0041] Example 4 Rapid screening of T7 promoter mutant library by detecting pyruvate using colorimetry

[0042] In a strong alkaline solution, sodium pyruvate reacts with salicylaldehyde to form a colored product, 1,5-bis(2-hydroxyphenyl)-1,4-pentadienone, which has 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. The reagents in Table 2 are added sequentially, mixed evenly, and left at room temperature for 2 h. Then, it is detected with a microplate 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. The OD465 is proportional to the concentration of sodium pyruvate. The standard curve is as shown in Figure 3 shown, and its standard curve is y = 0.0289x + 0.2363, with R 2 = 0.9995.

[0043] When detecting the process of enzymatic synthesis of L-dopa by colorimetry, the concentration of the substrate sodium pyruvate decreases as the reaction proceeds. Therefore, the faster the concentration of sodium pyruvate decreases within the same reaction time, the higher the activity of tyrosine phenol lyase in the genetically engineered bacteria.

[0044] Table 2 Reaction system for detecting sodium pyruvate by colorimetry

[0045]

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

[0047] 1. Treatment of the reaction solution

[0048] According to the method of Example 3, a whole-cell catalytic reaction system is constructed. After the reaction ends, 500 μL of the reaction solution is taken, 500 μL of acetic acid and 250 μL of acetonitrile are added for extraction, centrifuged at 10000 rpm for 2 min, and the supernatant is taken. The supernatant is filtered through a membrane and used for sample injection.

[0049] 2. High-performance liquid chromatography analysis method

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

[0051] Example 6 Single Point Mutation of T7 Promoter in Recombinant Plasmid pET28a-TPL

[0052] The T7 promoter modification scheme is as follows: replace the bases on the basis of 5’-TAATACGACTCACTATAG-3’

[0053] (1)Replace the 1st base T in the T7 promoter with A;

[0054] (2)Replace the 1st base T in the T7 promoter with C;

[0055] (3)Replace the 1st base T in the T7 promoter with G;

[0056] (4)Replace the 2nd codon A in the T7 promoter with T;

[0057] (5)Replace the 3rd base A in the T7 promoter with T;

[0058] (6)Replace the 4th base T in the T7 promoter with A;

[0059] (7)Replace the 4th base T in the T7 promoter with C;

[0060] (8)Replace the 4th base T in the T7 promoter with G;

[0061] (9)Replace the 5th base A in the T7 promoter with C;

[0062] (10)Replace the 5th base A in the T7 promoter with T;

[0063] (11)Replace the 6th base C in the T7 promoter with G;

[0064] (12)Replace the 6th base C in the T7 promoter with T;

[0065] (13)Replace the 7th base G in the T7 promoter with A;

[0066] (14)Replace the 8th base A in the T7 promoter with T;

[0067] (15)Replace the 12th base A in the T7 promoter with T;

[0068] (16)Replace the 13th base C in the T7 promoter with G;

[0069] (17)Replace the 16th base T in the T7 promoter with A;

[0070] (18)Replace the 17th base A in the T7 promoter with T.

[0071] Using plasmid pET28a-TPL as the amplification template, different promoter sequence vector constructs were made using inverse PCR technology. The primers set are shown in Table 3, and the PCR amplification system is shown in Table 4.

[0072] PCR reaction program: pre-denaturation: 95 °C, 5 min; complete denaturation: 95 °C, 15 s; annealing: 58 °C, 15 s; extension: 72 °C, 90 s; 30 cycles; final extension: 72 °C, 5 min; cool down to 4 °C for incubation.

[0073] Table 3 Primers for T7 promoter mutation

[0074]

[0075] Table 4 PCR system for whole plasmid amplification

[0076]

[0077] Appropriately sized PCR products were taken for agarose gel electrophoresis verification. After electrophoresis, a bright band was visible between 5000 - 8000 bp under ultraviolet light, which was consistent with the theoretical value of the plasmid. The results of agarose gel electrophoresis are as Figure 6 shown. Immediately, 1 μL of Dpn I restriction endonuclease was added to the PCR product and reacted at 37 °C for 1 h to remove the methylated template. Then, a DNA gel extraction and purification kit was used to recover and purify the PCR product. It was stored at -20 °C in a low-temperature refrigerator for future use.

[0078] The obtained mutant target fragment was directly transformed into the host bacterium Escherichia coli BL21(DE3): 50 μL of E. coli BL21(DE3) competent cells were taken and thawed on ice. 5 μL of the mutant PCR product was added and incubated on ice for 30 min. After the ice bath, the competent cells were heat-shocked at 42 °C for 90 s and then immediately placed on ice for 3 - 5 min. Subsequently, 1 mL of antibiotic-free LB medium was added and incubated in a shaker at 37 °C and 200 rpm for 1 h. After incubation, the culture solution was centrifuged at 4 °C and 4500 rpm for 5 min, 900 μL of the supernatant was discarded, and the remaining bacteria were fully resuspended. 100 - 150 μL of the bacterial solution was spread on an LB solid plate containing kanamycin resistance. It was incubated upside down in a 37 °C constant temperature incubator for 12 - 16 h to obtain the genetically engineered bacterium Escherichia coli BL21(DE3) / pET28a-TPL-M, where M represents base substitution. For example, when the base mutation is T1A, the genetically engineered bacterium is named Escherichia coli BL21(DE3) / pET28a-TPL-T1A.

[0079] The wet cells of the engineered bacteria were obtained by inducing expression under different promoters using the method of Example 2, and the expression effect was verified by SDS-PAGE electrophoresis. The results of the electrophoresis experiment are as Figure 7 shown. The molecular weight of the TPL protein is about 50 kDa. The protein bands expressed by each strain are significantly thickened and the band positions are around 50 kDa, with the correct size.

[0080] To verify the activities of different mutants, the wet cells of the engineered bacteria were obtained by inducing expression under different promoters using the method of Example 2. The catalytic reactions of the T7 promoter and its base substitutions were carried out using the method of Example 3. The reaction solutions at 0 min, 20 min, 40 min, and 1 h were taken respectively, and the colorimetric method described in Example 4 was used for verification. The results are shown in Table 5. Compared with the T7 promoter, base substitutions such as T1A, A2T, T4C, T4G, and A5C led to a faster decrease rate of the concentration of the substrate pyruvate, and were confirmed as beneficial mutations.

[0081] Table 5 Catalytic results of the engineered bacteria obtained under different promoters verified by colorimetric method

[0082] Note: *Marked as beneficial mutations

[0083] To further verify the improvement of the catalytic activity of the engineered bacteria by each beneficial mutation, the catalytic results of the engineered bacteria under the T7 promoter and its base substitutions were analyzed by liquid phase detection using the method of Example 5. The catalytic effects were compared by comparing the consumption of the substrate catechol and the production of the product L-dopa in the 1 h catalytic reaction solution. The results are shown in Table 6. Compared with the T7 promoter, multiple mutations led to a significant increase in catalytic activity, namely T1A, A2T, T4C, T4G, and A5C. The results obtained by this liquid phase detection were basically consistent with those obtained by the colorimetric method and could be mutually verified. Among these mutants, T4C showed the best performance. After reacting for 1 h, the substrate consumption increased by 47% compared with TPL, and the product production increased by 30% compared with TPL.

[0084] Table 6 Catalytic results of the engineered bacteria under the T7 promoter and its base substitutions detected by liquid phase

[0085]

[0086] Example 7 Combinatorial mutations of the T7 promoter in the recombinant plasmid pET28a-TPL

[0087] Using the plasmid pET28a-TPL-T4C as the amplification template, combinatorial mutant strains were constructed using the inverse PCR technique according to the method described in Example 6, and the following bases were substituted:

[0088] (1) Replace the 4th base T with C and the 1st base T with A in the T7 promoter to obtain the mutant strain E. coli BL21(DE3) / pET28a-TPL-T4C / T1A;

[0089] (2) Replace the 4th base T with C and the 2nd base A with T in the T7 promoter to obtain the mutant strain E. coli BL21(DE3) / pET28a-TPL-T4C / A2T;

[0090] (3) Replace the 4th base T with C and the 5th base A with C in the T7 promoter to obtain the mutant strain E. coli BL21(DE3) / pET28a-TPL-T4C / A5C.

[0091] Using the method of Example 2, induce the expression of the engineered bacteria to obtain wet bacterial cells under different promoters. Using the method of Example 3, use the wet bacterial cells to catalyze the reaction. Using the method of Example 5, perform liquid-phase analysis on the reaction solution catalyzed by the bacterial cells. Compare the consumption of the substrate catechol and the production of the product L-dopa in the 1 h catalytic reaction solution to compare the catalytic effects of the bacterial cells. The results are shown in Table 7. Compared with the T7-promoter, these combined mutations significantly improved the catalytic activity. Among them, T4C / A2T showed the best performance. After reacting for 1 h, the substrate consumption increased by 81%, and the product production increased by 56%. The corresponding plasmid map is as Figure 8 shown.

[0092] Table 7 Catalytic results of the engineered bacteria under the T7 promoter and its base substitutions

[0093]

[0094] Example 8 High-density fermentation of tyrosine phenol lyase and preparation of enzyme solution

[0095] 1. Seed culture

[0096] Take out the strains E. coli BL21(DE3) / pET28a-TPL and E. coli BL21(DE3) / pET28a-TPL-T4C / A2T from the -80 °C refrigerator. Take the glycerol tube and streak it on the test tube slant containing 100 μg / mL kanamycin, and culture it at 37 °C for 13 - 15 h. Add 6 mL of sterile water to the cultured test tube slant, and use an inoculation loop to scrape and wash the bacteria on the slant into the sterile water. Inoculate it into the LB medium at an inoculation amount of 1%, and culture it at 37 °C and 220 rpm for 10 - 11 h. At this time, the OD600 is about 5.0.

[0097] Composition of LB liquid medium: 5 g / L of yeast extract, 10 g / L of tryptone, 10 g / L of NaCl, with distilled water as the solvent, pH 7.0 - 7.5. LB solid medium is prepared by adding 20 g / L of agar to LB liquid medium.

[0098] 2. High - density fermentation

[0099] Prepare 5 L of fermentation medium with the formula shown in Table 8. Prepare 1 L of feeding medium with the formula shown in Table 9. The fermentation medium and the feeding medium are sterilized at 121 °C for 30 min. After sterilization, when the temperature drops to 60 °C, kanamycin is added to the fermentation medium at a concentration of 100 μg / L. When the temperature drops to 37 °C, the seed liquid is inoculated into the fermentation medium at an inoculation amount of 2.0% (i.e., 100 mL / 5 L) and cultured at 37 °C. The aeration rate is initially 1 vvm and increases to 1.2 vvm after 3 h. The tank pressure is maintained at 0.05 - 0.06 Mpa. The stirring speed is 500 - 700 rpm. Control the dissolved oxygen to be not less than 20% at least. The dissolved oxygen drops rapidly after 2 h of fermentation and rises rapidly around 5 h. At this time, start adding the feeding medium to control the dissolved oxygen at about 30%. The pH may rise slowly in the early stage of fermentation and drops after feeding. Thereafter, the pH can be adjusted with ammonia water and controlled at 6.7 - 6.9. Slowly cool down to 30 - 33 °C 1 - 2 h after starting the feeding. When OD600 reaches 20 - 25, cool down to 25 °C again. Add 0.3 mM of IPTG and induce for 14 - 16 h, then the fermentation can be ended.

[0100] Table 8 Fermentation medium formula (5 L)

[0101]

[0102] Table 9 Feeding medium formula (1 L)

[0103]

[0104] 3. Preparation of cell - wall - breaking enzyme solution

[0105] Before discharging the fermentation broth, use the cooling system of the fermenter to cool the fermentation broth to 10 - 15 °C in advance. The fermentation broth passes through a ceramic membrane and is rinsed twice with tap water to ensure that the fermentation broth is washed off as much as possible, and is concentrated to a cell content of 200 g / L. Heat is generated during the membrane - passing process and needs to be cooled below 20 °C. The cooled cell suspension is broken by a high - pressure homogenizer at a pressure of 60 - 80 kg / cm². Heat is generated during the cell - wall - breaking process, and ensure that the temperature does not exceed 30 °C during this period. The cell - wall - breaking cycle is 2 times. The pH of the enzyme solution needs to be controlled throughout the whole cell - wall - breaking process, and the pH is adjusted to about 7.0 - 7.2 with sodium hydroxide aqueous solution. After the cell - wall - breaking is completed, cool down to 0 °C, aliquot the enzyme solution, and store it frozen in a cold storage for later use.

[0106] Example 9 Amplification Process of L-DOPA Catalytic Synthesis by Tyrosine Phenol Lyase

[0107] To further confirm the viability difference between strain E. coli BL21(DE3) / pET28a-TPL and strain E. coli BL21(DE3) / pET28a-TPL-T4C / A2T, using the method of Example 8, high-density fermentation production was carried out on the two strains to obtain the corresponding enzyme solutions. Subsequently, a 200 mL catalytic reaction system was constructed, adding 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), and adding 60 mL of the enzyme solution prepared in Example 9 (60 g / L based on wet cell mass), and making up to 200 mL of the catalytic reaction system with pure water, setting the pH to 8.3 and the temperature to 25 °C. After the start of the reaction, 2.4 g of sodium pyruvate and 2.0 g of catechol were added every 1 h, with a total of 6 additions (sampling was completed before each addition), and after the addition was completed, the reaction continued for 2 h, and the final feeding amount of catechol was 75 g / L.

[0108] Taking the reaction solutions at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, and 8 h, using the method of Example 5, liquid phase detection and analysis were carried out on the catalytic results of strain E. coli BL21(DE3) / pET28a-TPL and strain E. coli BL21(DE3) / pET28a-TPL-T4C / A2T to monitor the residual amount of catechol and the production amount of L-DOPA during the reaction process. The residual amount of catechol and the production amount of L-DOPA during the reaction process are shown in Table 10. The L-DOPA production of the tyrosine phenol lyase TPL enzyme solution prepared from strain E. coli BL21(DE3) / pET28a-TPL after 8 h of catalytic reaction was 71.91 g / L; the L-DOPA production of the tyrosine phenol lyase TPL enzyme solution prepared from strain E. coli BL21(DE3) / pET28a-TPL-T4C / A2T after 8 h of catalytic reaction was 99.52 g / L, and the L-DOPA production increased by 38.4%. In addition, compared with the enzyme solution prepared from strain E. coli BL21(DE3) / pET28a-TPL, the residual catechol of the enzyme solution prepared from strain E. coli BL21(DE3) / pET28a-TPL-T4C / A2T during catalysis was lower, only 7.52 g / L. The pH of the reaction solution was adjusted to slightly acidic with an acid solution, and the crude crystals were collected by centrifugation or filtration, and then the acid was adjusted again for re-dissolution, activated carbon was added, and the temperature was raised and stirred for decolorization. The filtered clear liquid was adjusted to alkaline for crystallization, filtered, and dried to obtain L-DOPA crystalline powder.

[0109] Table 10 Residual amount of catechol and production amount of levodopa during the catalytic reaction process of different strains

[0110]

[0111] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these 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 appended claims.

Claims

1. A recombinant vector of tyrosine phenol lyase, characterized in that: The recombinant vector is derived from the pET28a vector and includes a site-directed mutagenesis T7 promoter and a tyrosine phenol lyase encoding gene. The site-directed mutagenesis is selected from any one of the mutation combinations T4C / T1A, T4C / A2T, and T4C / A5C; the nucleotide sequence of the tyrosine phenol lyase encoding gene is as shown in SEQ ID NO:

2.

2. The recombinant vector of tyrosine phenol lyase according to claim 1, characterized in that: The recombinant vector includes Nco I and Hind III sites for ligating the tyrosine phenol lyase encoding gene.

3. A host cell selected from E. coli BL21(DE3) and containing the recombinant vector of a tyrosine phenol-lyase according to claim 1 or 2.

4. Use of a tyrosine phenol lyase recombinant vector according to claim 1 or 2 in the catalytic synthesis of levodopa.

5. Use of a recombinant pET T7 promoter in a pET28a vector for preparing tyrosine phenol lyase with high conversion rate, characterized in that, Perform site-directed mutagenesis on the pET T7 promoter, and the site-directed mutagenesis is selected from any one of the mutation combinations T4C / T1A, T4C / A2T, and T4C / A5C; the vector includes a tyrosine phenol lyase encoding gene and is transferred into a host cell E. coli BL21(DE3).

6. A preparation method of levodopa, characterized in that: It is obtained by carrying out a catalytic reaction in a reaction system with catechol and sodium pyruvate as substrates, adding a tyrosine phenol lyase enzyme solution, EDTA, ammonium acetate, sodium sulfite, and pyridoxal 5-phosphate at pH 7-9 and 20-30 °C; the tyrosine phenol lyase enzyme solution is obtained by using the host cell described in claim 3, through scale-up culture, fermentation, and cell disruption.

7. A method for preparing levodopa according to claim 6, characterized in that: The addition amount of the tyrosine phenol lyase enzyme solution in the reaction system is 20-60 g / L based on the wet cell mass.

8. A method for preparing levodopa according to claim 7, characterized in that: 10-18 g / L of sodium pyruvate and 8-16 g / L of catechol are added to the reaction system; after the reaction starts, sodium pyruvate and catechol are replenished at regular intervals to control the final feeding amount of catechol to be 75 g / L.

9. The preparation method of levodopa according to claim 8, 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. Then, it is adjusted to be acidic again for redissolution, activated carbon is added, and the temperature is raised for stirring and decolorization. The filtered clear liquid is adjusted to be alkaline for crystallization, filtered, and then dried to obtain levodopa crystalline powder.

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