Tyrosine phenol lyase recombinant vector and application thereof in synthesis of levodobar

Abstract

The invention discloses a tyrosine phenol lyase recombinant vector and application of the tyrosine phenol lyase recombinant vector in synthesis of levodobaba.The tyrosine phenol lyase recombinant vector is characterized in that any one or more of a first base T, a second base A, a fourth base T and a fifth base A of a T7 promoter in a vector pET28a are replaced with other bases, so that the strength and activity of the promoter are improved; therefore, the engineering bacteria with high tyrosine phenol lyase activity in the levodopa enzymatic synthesis process are obtained. The engineering strain can efficiently convert catechol in a catalytic reaction system and improve the yield of levodopa, and the yield of the levodopa is far higher than that of an engineering strain corresponding to an unmodified T7 promoter. On the basis, the dosage of the biocatalyst can be reduced, and the economical efficiency of the levodopa enzymatic synthesis process is improved.

Description

Technical Field

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

[0002] Parkinson's disease is a movement dysfunction 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 lower before the age of 50, but this trend is changing. 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. It decarboxylates to form dopamine after passing through the blood-brain barrier, solving the problem that dopamine cannot reach the brain directly. In 1967, Levodopa was finally approved by the US FDA for the treatment of Parkinson's disease. Levodopa is a white or off-white crystalline powder, slightly soluble in water. Its CAS number is 59-92-7 and its molecular formula is C9H 11 NO4, molecular weight is 197 g / mol, density is 1.5 g / cm 3 , boiling point 448 ℃.

[0003] At present, there are three main methods for producing L-DOPA: 1. Extraction method: Extracted from plants such as cat beans and quinoa beans, with few raw material sources, low yields, and high production costs; 2. Chemical method: The process is complicated, and the conversion rate and optical rotation are both low, and it is easy to produce a mixture of D-LOPA and L-LOPA, among which D-LOPA can cause toxic reactions in the human body; 3. Bioenzyme method: Enzyme-catalyzed synthesis mainly includes tyrosinase synthesis method, tyrosine phenol lyase synthesis method, transaminase synthesis method, etc. Compared with the three, the bioenzyme method is specific and efficient, and can greatly simplify the manufacturing process. Among the bioenzyme methods, tyrosine phenol lyase is currently the most important biocatalyst. Tyrosine phenol lyase (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 four 50 kDa subunits, and each subunit tetramer is combined with four PLP molecules.

[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 L-DOPA. The existing technology often focuses on the coding sequence of tyrosine phenol lyase, and performs directed evolution or rational design to improve the enzyme activity. The protein engineering workload is large, and the effect is often unsatisfactory. In comparison, promoter engineering is a key means to achieve efficient and accurate gene expression regulation at the transcription level. At present, the strategy of promoter engineering mainly involves the construction of promoter libraries, replacement of promoters, modification of promoters, and reasonable design of hybrid promoters. Mutation modification of promoters can significantly increase the strength of promoters and improve promoter activity, thereby improving the expression activity of tyrosine phenol lyase, and then improving the enzymatic synthesis process of L-DOPA. Summary of the invention

[0005] Objectives of the invention: The objective of the present invention is to provide a recombinant vector of tyrosine phenol-lyase; another objective of the present invention is to provide a host cell comprising the aforementioned recombinant vector of tyrosine phenol-lyase; another objective of the present invention is to provide an 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 an application of a recombinant pET T7 promoter obtained based on site-directed mutagenesis in the preparation of high-conversion tyrosine phenol-lyase; another objective of the present invention is to provide a method for preparing levodopa, a method for preparing levodopa by biocatalytically using a bacterial solution cultured with the aforementioned host cell comprising the recombinant vector of tyrosine phenol-lyase as a catalyst and sodium pyruvate and catechol as substrates.

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

[0007] The tyrosine phenol lyase TPL described in the present invention is derived from Erwinia herbicola, and its amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO: 2. Those skilled in the art may improve the aforementioned sequence as necessary without departing from the principles of the invention of the present application. As a preferred embodiment, the nucleotide sequence of the tyrosine phenol lyase encoding gene has a homology of not less than 98% compared with SEQ ID NO: 2. As an optional embodiment of the present invention, the nucleotide sequence of the tyrosine phenol lyase encoding gene is shown in SEQ ID NO: 2.

[0008] Further, the site-directed mutagenesis of any one of the pET T7 promoters provided by the present invention is experimentally demonstrated 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 mutagenesis scheme is any one of the three mutation combinations of T4C / T1A, T4C / A2T, and T4C / A5C. As the optimal solution 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 of the present invention is derived from pET28a, which includes Nco I and Hind III sites for connecting the tyrosine phenol lyase encoding gene, thereby obtaining the recombinant plasmid pET28a-TPL.

[0010] Furthermore, the recombinant plasmid pET28a-TPL was 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, wherein the site-directed mutation of the pET T7 promoter plays an important role in improving the substrate catalytic efficiency and levodopa yield.

[0011] The invention provides a method for preparing levodopa, which comprises taking catechol and sodium pyruvate as substrates, adding tyrosine phenol lyase enzyme solution, EDTA, ammonium acetate, sodium sulfite and 5-pyridoxal phosphate, and carrying out a catalytic reaction in a reaction system of pH 7-9 and 20-30°C to obtain the levodopa; the tyrosine phenol lyase enzyme solution is obtained by using the aforementioned host cells through amplification culture, fermentation and cell wall breaking.

[0012] Furthermore, the amount of tyrosine phenol lyase 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.

[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 added at regular intervals to control the final feed amount of catechol 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, 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.

[0015] The present invention transforms the T7 promoter of the vector pET28a, and the corresponding engineered bacteria obtained have improved catalytic activity in the enzymatic synthesis of levodopa. In particular, when the engineered strain E. coli BL21 (DE3) / pET28a-TPL-T4C / A2T is used as a biocatalyst, in a catalytic reaction system containing catechol and sodium pyruvate as substrates, 99.52 g / L of levodopa can be generated at 25 ° C and pH 8.3 for 8 hours, while the levodopa concentration converted by the engineered strain corresponding to the unmodified T7 promoter is only 71.91 g / L. It can be seen that the yield of levodopa after the promoter transformation is increased by 38.4%, which is helpful to reduce the amount of catalyst used in the enzymatic synthesis of levodopa and improve the economy of the enzymatic synthesis of levodopa process. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the synthesis of L-DOPA catalyzed by tyrosine phenol lyase;

[0017] Figure 2 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;

[0018] Figure 3 It is the standard curve for the colorimetric detection of sodium pyruvate concentration;

[0019] Figure 4 is the HPLC chromatogram of the substrate catechol standard;

[0020] Figure 5 is the HPLC chromatogram of the product levodopa standard;

[0021] Figure 6 It is the agarose gel electrophoresis pattern of the PCR product obtained by the T7 promoter transformation in the recombinant plasmid pET28a-TPL; Lane M is a marker; Lanes 1-18 are: 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 wet bacteria induced by TPL expression under different promoters; Lane M, Marker; Lanes 1-19 are: 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 The figure is the plasmid map of the preferred recombinant vector pET28a-TPL-T4C / A2T. 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] 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 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). The synthetic route of this method is as follows Figure 1 The amino acid sequence of tyrosine phenol lyase TPL is shown in SEQ ID NO.1, and the nucleotide sequence of the codon-optimized TPL gene is shown in SEQ ID NO.2, which is 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 2As 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 genetically engineered 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 under different promoters

[0036] Tyrosine phenol lyase E. coli BL21 (DE3) / pET28a-TPL 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 and add it to 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%, and placed in a shaker at 37 ° C and 200 rpm to cultivate the bacterial concentration OD600 to 0.6-0.8. Add the prepared IPTG solution to a final concentration of 0.3 mM, and induce expression at 24 ° C and 150 rpm for 12-16 h. After induction, transfer the bacterial solution to a centrifuge cup and centrifuge at 4°C and 8000 rpm for 10 min, discard the supernatant; resuspend the bacterial precipitate in 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 future use.

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

[0038] According to the method described in Example 2, wet bacteria expressing tyrosine phenol lyase TPL under different promoters were prepared, 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 T7 promoter 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. Add the reagents in Table 2 in sequence, mix well, leave at room temperature for 2 h, and detect 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 were treated with alkali according to the above steps to obtain a standard curve of sodium pyruvate. OD465 is proportional to the concentration of sodium pyruvate. The standard curve is shown in the following figure. Figure 3 As 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 4 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 5 As shown, the peak time is 18.652 min.

[0051] Example 6 Single point mutation of T7 promoter in recombinant plasmid pET28a-TPL

[0052] The T7 promoter transformation scheme is to make the following substitutions based on 5'-TAATACGACTCACTATAG-3':

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

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

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

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

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

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

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

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

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

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

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

[0064] (12) Replace the sixth 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 eighth 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] Plasmid pET28a-TPL was used as an amplification template and inverse PCR technology was used to construct vectors with different promoter sequences. The primers are shown in Table 3 and the PCR amplification system is shown in Table 4.

[0072] 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.

[0073] Table 3 T7 promoter mutation primers

[0074]

[0075] Table 4 PCR system for full plasmid amplification

[0076]

[0077] 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 6 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.

[0078] 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 base substitution. For example, when the base mutation is T1A, the genetically engineered bacteria is named E. coli BL21(DE3) / pET28a-TPL-T1A.

[0079] The method of Example 2 was used to induce expression under different promoters 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 7 As shown, the protein molecular weight of TPL is about 50 kDa, the protein bands expressed by each strain are obviously thickened and the band position is around 50 kDa, and the size is correct.

[0080] In order to verify the activity of different mutants, the method in Example 2 was used to induce expression under different promoters to obtain wet cells of engineered bacteria, and the method in Example 3 was used to catalyze the T7 promoter and its base substitution, and the reaction liquids of 0 min, 20 min, 40 min, and 1 h were taken, respectively, and the method described in Example 4 was used for colorimetric verification, and the results are shown in Table 5. Compared with the T7 promoter, base substitutions such as T1A, A2T, T4C, T4G, and A5C resulted in a faster decrease in the concentration of the substrate sodium pyruvate, and were confirmed to be beneficial mutations.

[0081] Table 5 Colorimetric verification of the catalytic results of engineered bacteria obtained under different promoters

[0082] Note: * indicates beneficial mutations

[0083] In order to further verify the situation that each beneficial mutation improves the catalytic activity of the engineered bacteria, the method of Example 5 was used to perform liquid phase detection analysis on the catalytic results of the engineered bacteria under the T7 promoter and its base substitution, and the catalytic effect was 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 6. Compared with the T7 promoter, multiple mutations led to a significant increase in catalytic activity, namely T1A, A2T, T4C, T4G, and A5C. The liquid phase detection results were basically consistent with the results obtained by the colorimetric method and could be mutually verified. Among these mutants, T4C performed best. After 1 h of reaction, the substrate consumption increased by 47% compared with TPL, and the product production increased by 30% compared with TPL.

[0084] Table 6 Liquid phase detection of T7 promoter and the catalytic results of engineered bacteria under base substitution

[0085]

[0086] Example 7 Combined mutation of T7 promoter in recombinant plasmid pET28a-TPL

[0087] Plasmid pET28a-TPL-T4C was used as an amplification template and the inverse PCR technique was used to construct a combined mutant strain according to the method described in Example 6, wherein the following bases were replaced:

[0088] (1) The fourth base T in the T7 promoter was replaced with C, and the first base T was replaced with A to obtain the mutant strain E. coli BL21(DE3) / pET28a-TPL-T4C / T1A;

[0089] (2) The fourth base T in the T7 promoter was replaced with C, and the second base A was replaced with T to obtain the mutant strain E. coli BL21(DE3) / pET28a-TPL-T4C / A2T;

[0090] (3) The fourth base T in the T7 promoter was replaced with C, and the fifth base A was replaced with C to obtain the mutant strain E. coli BL21(DE3) / pET28a-TPL-T4C / A5C.

[0091] The wet cells of the engineered bacteria were obtained by inducing expression under different promoters using the method of Example 2, and the wet cells were used for catalytic reaction using the method of Example 3. The liquid phase analysis of the reaction liquid obtained by cell catalysis was performed using the method of Example 5. The consumption of substrate catechol and the production of product levodopa in the 1 h catalytic reaction liquid were compared to compare the cell catalytic effect. The results are shown in Table 7. Compared with the T7-promoter, these combined mutations significantly improved the catalytic activity, among which T4C / A2T performed the best. After 1 h of reaction, the substrate consumption increased by 81% and the product production increased by 56%. The corresponding plasmid diagram is shown in Table 7. Figure 8 shown.

[0092] Table 7 Liquid phase detection of T7 promoter and the catalytic results of engineered bacteria under base substitution

[0093]

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

[0095] 1. Seed liquid culture

[0096] Take out strains E. coli BL21(DE3) / pET28a-TPL and strains E. coli BL21(DE3) / pET28a-TPL-T4C / A2T from the -80 ℃ refrigerator, 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, when the OD600 is around 5.0.

[0097] 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.

[0098] 2. High-density fermentation

[0099] 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 hours after the start of feeding, slowly cool down to 30-33 ℃. When OD600 reaches 20-25, cool down to 25 ℃. Add 0.3 mM IPTG to induce for 14-16 hours before releasing the tank.

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

[0101]

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

[0103]

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

[0105] 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.

[0106] Example 9 Scale-up of the synthesis of L-DOPA catalyzed by tyrosine phenol lyase

[0107] 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-T4C / A2T, 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.

[0108] 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-T4C / A2T catalyzed the reaction for 8 h, and the yield of L-DOPA was 99.52 g / L, which increased the yield of L-DOPA by 38.4%. 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-T4C / A2T was lower, only 7.52 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.

[0109] Table 10 The residual amount of catechol and the amount of L-DOPA produced during the catalytic reaction of different strains

[0110]

[0111] 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 recombinant vector, characterized in that: The recombinant vector is derived from a pET vector, and includes a T7 promoter with a site-directed mutation and a tyrosine phenol lyase encoding gene, wherein the site-directed mutation is selected from any one or more combinations of T1A, A2T, T4C, T4G, and A5C; the nucleotide sequence of the tyrosine phenol lyase encoding gene has a homology of not less than 95% compared with SEQ ID NO:

2.

2. A tyrosine phenol lyase recombinant vector according to claim 1, characterized in that: The site-directed mutation is selected from any one of the following mutation combinations: T4C / T1A, T4C / A2T, T4C / A5C.

3. A tyrosine phenol lyase recombinant vector according to claim 1 or 2, characterized in that: The recombinant vector comprises Nco I and Hind III sites for connecting the tyrosine phenol lyase encoding gene.

4. A host cell comprising a tyrosine phenol lyase recombinant vector according to any one of claims 1 to 3.

5. Use of a tyrosine phenol lyase recombinant vector as claimed in any one of claims 1 to 3 in catalytic synthesis of levodopa.

6. Use of a recombinant pET T7 promoter in the preparation of a high-conversion rate tyrosine phenol lyase, characterized in that: The pET T7 promoter is subjected to site-directed mutagenesis, wherein the site-directed mutagenesis is selected from any one or more combinations of T1A, A2T, T4C, T4G, and A5C.

7. A method for preparing levodopa, characterized in that: The tyrosine phenol lyase enzyme solution is obtained by using catechol and sodium pyruvate as substrates, adding tyrosine phenol lyase 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 enzyme solution is obtained by using the host cell according to claim 4, 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 solution added to the reaction system is 20-60 g / L based on the wet bacterial cell count.

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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