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

Abstract

The present invention discloses a tyrosine phenol lyase mutant and its application in the synthesis of levodopa. The tyrosine phenol lyase mutant is obtained by site-directed mutagenesis based on wild-type tyrosine phenol lyase. The mutant obtained by transformation can convert more substrates, and the yield is also significantly improved, which is suitable for industrial production of levodopa.

Description

Technical Field

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

[0002] Parkinson's disease is a movement disorder disease, and 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 the treatment of Parkinson's disease. L-DOPA, CAS: 59-92-7, C9H 11 NO4, molecular weight: 197 g / mol, density 1.5 g / cm 3 , boiling point 448 °C, white or off-white crystalline powder, slightly soluble in water.

[0003] Currently, there are mainly three methods for producing L-DOPA: extraction method, chemical method and biocatalytic method. The enzyme conversion method mainly uses aminoacylase, tyrosinase, tyrosine phenol lyase, 4-hydroxyphenylacetic acid 3-hydroxylase as catalysts to synthesize L-DOPA. Among them, the synthesis of L-DOPA by tyrosine phenol lyase method has the advantages of high yield, high conversion rate, good enantioselectivity, simple reaction steps, mild conditions, etc., and has become more and more popular in recent years. Tyrosine phenol lyase (TPL, E.C.4.1.99.2), also known as β-tyrosinase, can catalyze catechol, sodium pyruvate and ammonium acetate to produce L-DOPA, 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. It should be noted that when the concentration of the reaction substrate catechol is higher than 10 g / L, it will inhibit the enzyme activity of tyrosine phenol lyase, and the substrate feeding-batch mode is often required to overcome the inhibition of the high-concentration 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 exhibits 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). In order to overcome the inhibition 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 sites is the main technical means that those skilled in the art expect to improve 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] Object of the Invention: The object of the present invention is to provide a tyrosine phenol-lyase mutant based on site-directed mutagenesis; another object of the present invention is to provide a gene fragment encoding the tyrosine phenol-lyase mutant; another object of the present invention is to provide a recombinant vector containing the gene fragment encoding the tyrosine phenol-lyase, and a host cell containing the recombinant vector; another object of the present invention is to provide the application of the foregoing host cell in the catalytic synthesis of L-dopa; another object of the present invention is to provide a method for preparing L-dopa, a method for biocatalytically preparing L-dopa using a bacterial solution expressing mutant tyrosine phenol-lyase as a catalyst and sodium pyruvate and catechol as substrates.

[0006] Technical solution: A tyrosinase phenol lyase mutant provided by the present invention is derived from the tyrosinase phenol lyase (Erwinia herbicola) TPL of wild-type Erwinia herbicola. The amino acid sequence has a length of 456, as shown in SEQ ID NO: 11, and the protein molecular weight is about 50 kDa; the nucleotide sequence encoding this enzyme is as shown in SEQ ID NO: 12. Based on this wild-type TPL, mutations are made at any one or more of the 142nd, 157th, and 520th positions, which can significantly improve the conversion efficiency of the expressed tyrosinase phenol lyase to pyruvate. Specifically, the site-directed mutations are selected from any one or any combination of R142C, N157D, and I420V. Among them, the mutants with the amino acid sequences shown in SEQ ID NOs: 1-5 have obvious improvement effects, and their corresponding mutants are as follows:

[0007]

[0008] As a preferred embodiment of the present invention, the mutants obtained by combining any two of the above mutation sites have better conversion efficiency. Among them, the two mutants TPL-I420V / R142C and TPL-I420V / N157D represented by the amino acid sequences shown in SEQ ID NO: 4 or SEQ ID NO: 5 are preferred.

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

[0010] The present invention further provides a recombinant vector containing the aforementioned gene fragment. The recombinant vector is derived from pET28a, and this vector includes the Nco I and Hind III sites for ligating the tyrosinase phenol lyase encoding gene, thereby obtaining the recombinant plasmid pET28a-TPL.

[0011] The present invention further provides a host cell containing the aforementioned recombinant vector, which is obtained by transferring the recombinant vector into the competent host cell E. coli BL21(DE3), resulting in the recombinant strain E. coli BL21(DE3) / pET28a-TPL. This genetically engineered strain can express tyrosinase phenol lyase and is used for biocatalytic preparation of levodopa from catechol and pyruvate.

[0012] Using the above host cell as a genetic engineering strain for catalyzing the synthesis of levodopa 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, and adds tyrosine phenol lyase mutant enzyme solution, EDTA, ammonium acetate, sodium sulfite, and pyridoxal 5'-phosphate, and carries out a catalytic reaction in a reaction system at pH 7-9 and 20-30 °C; the tyrosine phenol lyase mutant enzyme solution is obtained by using the aforementioned host cell through scale-up culture, fermentation, and cell disruption.

[0013]

[0014] Furthermore, the addition amount of the tyrosine phenol lyase mutant enzyme solution in the reaction system is 20-60 g / L in terms of wet cells. The obtaining of the wet cells at least includes: inoculating a genetic engineering strain carrying a tyrosine phenol lyase encoding gene into an LB liquid medium containing kanamycin, adding IPTG for induction culture, then centrifuging to discard the supernatant, and collecting the wet cells.

[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 replenished at regular intervals, and the final feeding amount of catechol is controlled 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, then the acid is adjusted again to dissolve, activated carbon is added, and the temperature is raised and stirred for decolorization. The filtered clear liquid is adjusted to be alkaline for crystallization, filtered, and then dried to obtain levodopa crystalline powder.

[0017] The present invention performs molecular modification on the tyrosine phenol lyase TPL encoding gene derived from Erwinia herbicola, obtaining 5 mutants with significantly improved enzyme activities. These mutants all exhibit better catalytic efficiency in the enzymatic synthesis of L-DOPA. When the tyrosine phenol lyase mutant TPL-I420V / N157D is used as a biocatalyst, a catalytic system is constructed with catechol (75 g / L), sodium pyruvate, ammonium acetate, pyridoxal 5'-phosphate (PLP), Na2SO3, and EDTA. The reaction is carried out at 25 °C and pH 8.3 for 8 h, and the highest amount of L-DOPA that can be produced is 130.44 g / L, while the concentration of L-DOPA corresponding to the wild-type tyrosine phenol lyase TPL is only 71.91 g / L. Compared with before the modification of the tyrosine phenol lyase encoding gene, the yield of L-DOPA has increased by 81.4%, indicating that the tyrosine phenol lyase mutant TPL-I420V / N157D has better catalytic efficiency and is helpful for further improving the economic efficiency of the enzymatic synthesis of L-DOPA process in industrial applications. Description of the Drawings

[0018] Figure 1 Agarose gel electrophoresis pattern for constructing the genetic engineering 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;

[0019] Figure 2 Standard curve for detecting the concentration of sodium pyruvate by colorimetry;

[0020] Figure 3 High-performance liquid chromatography (HPLC) chromatogram of the substrate catechol standard;

[0021] Figure 4 High-performance liquid chromatography (HPLC) chromatogram of the product L-DOPA standard;

[0022] Figure 5 Agarose gel electrophoresis pattern of the PCR products obtained by mutating key sites in the recombinant plasmid pET28a-TPL; Lane M is the marker; Lanes 1-10 are in sequence: 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 cells induced by TPL and its mutants; Lane M, Marker; Lanes 1-10 are in turn: TPL-E30Q, TPL-T49I, TPL-T83K, TPL-Q115K, TPL-T126A, TPL-R142C, TPL-N157D, TPL-Y212F, TPL-S397R, TPL-I420V. Detailed implementation manners

[0024] To make the technical solution of the present invention clearer, the following further describes the present invention in detail with reference to specific embodiments in conjunction with the accompanying drawings.

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

[0026] The amino acid sequence of tyrosine phenol lyase TPL derived 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. 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 performed using primers TPL-F and TPL-R to obtain the tyrosine phenol lyase TPL coding gene. PCR amplification was performed using primers pET28a-F and pET28a-R to obtain the pET28a linearized vector fragment, and the results of agarose gel electrophoresis are as Figure 1 shown. Using the "one-step cloning" kit, the obtained tyrosine phenol lyase TPL coding 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. After the constructed genetically engineered bacterium was verified to be correct by plasmid extraction and sequencing, it was shown 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 stored 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 bacterium 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: yeast extract 5 g / L, tryptone 10 g / L, NaCl 10 g / L, the solvent is distilled water, pH 7.0 - 7.5. LB solid medium is LB liquid medium added with 20 g / L agar.

[0035] Example 2 Induced 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 incubated inverted in a constant temperature incubator at 37 °C for 12 - 16 h. Single colonies were picked and cultured in 50 mL LB medium containing 100 μg / mL kanamycin at 37 °C and 200 rpm for 12 - 16 h to obtain seed cultures. The seed cultures were transferred to 150 mL LB liquid medium with the same kanamycin concentration at an inoculation volume ratio of 2% and cultured in a shaker at 37 °C and 200 rpm until the cell concentration OD 600 reached 0.6 - 0.8. IPTG solution was added to a final concentration of 0.2 mM and induced at 24 °C and 150 rpm for 12 - 16 h. After induction, 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 stored at -20 °C in the refrigerator for later use.

[0037] Example 3 Construction of the catalytic reaction system for 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 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 the catalytic synthesis of levodopa by tyrosine phenol lyase TPL

[0040]

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

[0042] Pyruvate reacts with salicylaldehyde in a strong alkaline solution to form a colored 1,5-bis(2-hydroxyphenyl)-1,4-pentadienone product, which has an absorbance value at a wavelength of 465 nm. As the concentration of pyruvate decreases, the color changes from orange-red to light yellow. The reaction detection system is 1 mL. The reagents in Table 2 were added in sequence, mixed evenly, and left at room temperature for 2 h, and then 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 pyruvate were treated with alkali according to the above steps to obtain the standard curve of pyruvate. OD 465 is proportional to the concentration of pyruvate, and the standard curve is as shown in Figure 2As shown, its standard curve is y = 0.0289x + 0.2363, R 2 = 0.9995.

[0043] When detecting the process of enzymatic synthesis of L-DOPA by colorimetry, the concentration of pyruvate as the substrate decreases as the reaction proceeds. Therefore, the faster the concentration of 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 pyruvate by colorimetry

[0045]

[0046] Example 5 Liquid chromatography analysis of tyrosine phenol lyase-catalyzed synthesis of L-DOPA

[0047] 1. Treatment of reaction solution

[0048] Construct a whole-cell catalytic reaction system according to the method of Example 3. After the reaction is completed, take 500 μL of the reaction solution, add 500 μL of acetic acid and 250 μL of acetonitrile for extraction, centrifuge at 10000 rpm for 2 min, take the supernatant, and the supernatant is filtered through a membrane for sample loading.

[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 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), detected 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 chromatography spectrum of the substrate catechol standard is as Figure 3 shown, and the peak emergence time is 4.635 min. The liquid chromatography spectrum of the product L-DOPA standard is as Figure 4 shown, and the peak emergence time is 18.652 min.

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

[0052] Through homology modeling and molecular docking analysis of the tyrosine phenol lyase TPL gene, 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] Using plasmid pET28a-TPL as the amplification template, mutants were constructed by reverse PCR technology. The mutant types and primers are shown in Table 3, and the PCR amplification system is shown in Table 4.

[0054] PCR reaction procedure: Pre-denaturation: 95 °C, 5 min; Complete denaturation: 95 °C, 15 s; Annealing: 58 °C, 15 s; Extension: 72 °C, 90 s; 30 cycles; Re-extension: 72 °C, 5 min; Cool down to 4 °C for incubation.

[0055] Table 3 Single-point mutation primers of tyrosine phenol lyase TPL

[0056]

[0057] Table 4 PCR system for whole plasmid amplification

[0058]

[0059] Absorb an appropriate amount of PCR product for verification by agarose gel electrophoresis. After electrophoresis, place it under ultraviolet light and observe that there is a bright band between 5000 - 8000 bp, which is consistent with the theoretical value of the plasmid. The results of agarose gel electrophoresis are as Figure 5 shown. Immediately add 1 μL of Dpn I restriction endonuclease to the PCR product and react at 37 °C for 1 h to remove the methylated template. Then use a DNA gel extraction and purification kit to recover and purify the PCR product. Store it in a -20 °C low-temperature refrigerator for later use.

[0060] Directly transform the obtained mutant target fragment into the host bacterium Escherichia coli BL21(DE3): Take 50 μL of E. coli BL21(DE3) competent cells and place them on ice to melt. Add 5 μL of the mutant PCR product and place it on ice for an ice bath for 30 min. After the ice bath, heat shock the competent cells at 42 °C for 90 s, and then immediately place them on ice for 3 - 5 min. Subsequently, add 1 mL of antibiotic-free LB medium and incubate it in a shaker at 37 °C and 200 rpm for 1 h. After the culture, centrifuge the culture solution at 4 °C and 4500 rpm for 5 min, discard 900 μL of the supernatant, resuspend the remaining bacteria thoroughly, and take 100 - 150 μL of the bacterial solution and spread it on an LB solid plate containing kanamycin resistance. Incubate it inverted 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 amino acid substitution. For example, when the 30th glutamate in the amino acid sequence is replaced by glutamine, the genetically engineered bacterium is named Escherichia coli BL21(DE3) / pET28a-TPL-E30Q.

[0061] Use the method of Example 2 to induce the expression of tyrosine phenol lyase and its mutants to obtain wet bacterial cells of the engineered bacteria, and verify the expression effect by SDS-PAGE electrophoresis. The results of the electrophoresis experiment are as Figure 6As shown, the protein molecular weight of TPL and its mutants is approximately 50 kDa. The protein bands expressed by each mutant strain are significantly thickened and the band positions are around 50 kDa, with the correct size.

[0062] To verify the activities of different mutants, wet bacterial cells of TPL and its mutants were prepared using the method of Example 2, and the catalytic reactions of TPL and its mutants were carried out using the method of Example 3. Reaction solutions at 0 min, 20 min, 40 min, and 1 h were respectively taken and verified by colorimetry using 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 led to a faster decline rate of the concentration of the substrate pyruvate, and were confirmed as 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 Catalytic results of verifying TPL and its mutants by colorimetry

[0064]

[0065] To further verify the improvement of the catalytic activities of engineered bacteria by each beneficial mutation, 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 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 tyrosine phenol lyase TPL, multiple mutations led to a significant increase in catalytic activity, namely TPL-R142C, TPL-N157D, and TPL-I420V. The results of this liquid phase detection were basically consistent with those obtained by colorimetry and could verify each other. Among these mutations, TPL-I420V showed the best performance. After reacting for 1 h, the substrate consumption increased by 87% compared with TPL, and the product production increased by 71% compared with TPL.

[0066] Table 6 Catalytic results of detecting TPL and its mutants by liquid phase

[0067]

[0068] Example 7 Combinatorial mutations of tyrosine phenol lyase TPL

[0069] Using the plasmid pET28a-TPL-I420V as the amplification template, combinatorial mutant strains were constructed using the inverse PCR technique according to the method described in Example 6, and the amino acid substitutions were as follows:

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

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

[0072] Prepare the wet cells of TPL and its mutants by the method of Example 2, use the wet cells to catalyze the reaction by the method of Example 3, and perform liquid-phase detection and analysis on the catalytic results of the cells by the method of Example 5. Compare the catalytic effects of each mutant by comparing the consumption of the substrate catechol and the production of the product L-dopa in the catalytic reaction solution for 1 h. The results are shown in Table 7. Compared with tyrosine phenol lyase TPL, these combined mutations significantly improved the catalytic activity. Among them, TPL-I420V / N157D showed the best performance. After reacting for 1 h, the substrate consumption increased by 146% and the product production increased by 128%.

[0073] Table 7 Catalytic results of TPL and its mutant engineering bacteria detected by liquid phase

[0074]

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

[0076] 1. Seed culture

[0077] Take out the strains E. coli BL21(DE3) / pET28a-TPL and E. coli BL21(DE3) / pET28a-TPL-I420V / N157D from the -80 °C refrigerator. Take the glycerol tube and streak it on a test tube slope 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 slope, and scrape the bacteria on the slope into the sterile water with an inoculation loop. 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, OD 600Around 5.0.

[0078] 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.

[0079] 2. High - density fermentation

[0080] 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 around 30%. The pH may rise slowly in the early stage of fermentation and then drop 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 OD 600 reaches 20 - 25, then cool down to 25 °C. Add 0.3 mM IPTG and induce for 14 - 16 h to harvest the broth.

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

[0082]

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

[0084]

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

[0086] Before discharging the fermentation broth, use the cooling system of the fermenter to cool the temperature of the fermentation broth to 10 - 15 °C in advance. Pass the fermentation broth through a ceramic membrane and rinse it twice with tap water to ensure that the fermentation broth is washed away as much as possible. Concentrate it to a cell content of 200 g / L. Heat will be generated during the membrane passing process, and it needs to be cooled to below 20 °C. Cool the cell suspension and break the cell wall with a high-pressure homogenizer at a pressure of 60 - 80 kg. The temperature will rise during the cell wall breaking process, and ensure that the temperature does not exceed 30 °C during this period. Perform the cell wall breaking cycle twice. The pH of the enzyme solution needs to be controlled throughout the cell wall breaking process, and adjust the pH to about 7.0 - 7.2 with an aqueous sodium hydroxide solution. After the cell wall breaking is completed, cool it to 0 °C, dispense the enzyme solution, and put it into the cold storage for freezing for later use.

[0087] Example 9 Amplification process of catalytic synthesis of L-DOPA by tyrosine phenol lyase

[0088] To further confirm the activity difference between strain E. coli BL21(DE3) / pET28a-TPL and strain E. coli BL21(DE3) / pET28a-TPL-I420V / N157D, use the method of Example 8 to perform high-density fermentation production on the two strains and prepare the corresponding enzyme solutions. Subsequently, construct a 200 mL catalytic reaction system, add 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), add 60 mL of the enzyme solution prepared in Example 9 (60 g / L based on wet cells), and make up to 200 mL of the catalytic reaction system with pure water. Set the pH to 8.3 and the temperature to 25 °C. After starting the reaction, add 2.4 g of sodium pyruvate and 2.0 g of catechol every 1 h, with a total of 6 additions (sampling is completed before each addition). After the addition is completed, continue the reaction for 2 h, and the final feeding amount of catechol is 75 g / L.

[0089] The reaction solutions at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, and 8 h were taken, and the catalytic results of the strain E. coli BL21(DE3) / pET28a-TPL and the 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 production amount of levodopa during the reaction process. The residual amount of catechol and the production amount of levodopa during the reaction process are shown in Table 10. The tyrosinase phenol lyase TPL enzyme solution prepared from the strain E. coli BL21(DE3) / pET28a-TPL was used for the catalytic reaction for 8 h, and the production amount of levodopa was 71.91 g / L; the tyrosinase phenol lyase TPL enzyme solution prepared from the strain E. coli BL21(DE3) / pET28a-TPL-I420V / N157D was used for the catalytic reaction for 8 h, and the production amount of levodopa was 125.44 g / L, and the production amount of levodopa increased by 74.4%. In addition, compared with the enzyme solution prepared from the strain E. coli BL21(DE3) / pET28a-TPL, the residual amount of catechol during the catalysis of the enzyme solution prepared from the strain E. coli BL21(DE3) / pET28a-TPL-I420V / N157D was lower, only 1.23 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. Then, the acid was adjusted again to dissolve, activated carbon was added, and the mixture was stirred and decolorized by heating. The filtered clear liquid was adjusted to alkaline for crystallization, filtered, and dried to obtain levodopa crystalline powder.

[0090] Table 10 Residual amount of catechol and production amount of levodopa during the catalytic reaction process of different strains

[0091]

[0092] The tyrosinase phenol lyase mutant provided by the present invention was catalyzed in a reaction system with 10 - 18 g / L of sodium pyruvate and 8 - 16 g / L of catechol, and 12 g / L of sodium pyruvate and 10 g / L of catechol were added dropwise every 1 h after the reaction, so that the residual amount of catechol did not exceed 1 g / L in the first 4 hours of the reaction and did not exceed 2.5 g / L within 8 hours, which reflected the excellent enzyme conversion efficiency of the mutant and also avoided the inhibition of the enzyme activity of tyrosinase phenol lyase by the substrate concentration.

[0093] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting 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 deformations 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 should be subject to the appended claims.

Claims

1. A tyrosine phenol lyase mutant, characterized in that: The amino acid sequences of the mutants are as shown in SEQ ID NO: 1-5.

2. The tyrosine phenol lyase mutant according to claim 1, wherein: 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 as shown in SEQ ID NO: 6-10.

4. 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 described in claim 5 in the catalytic synthesis of L-DOPA.

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

8. A method for preparing levodopa according to claim 7, characterized in that: The addition amount of the enzyme solution of the tyrosine phenol lyase mutant in the reaction system is 20-60 g / L in terms of wet cells.

9. A 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; 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.

10. A 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, then the solution is adjusted to be acidic again for redissolution, activated carbon is added, and the temperature is raised and stirred for decolorization. The filtered clear solution is adjusted to be alkaline for crystallization, filtered, and then dried to obtain the L-DOPA crystalline powder.

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