4-hydroxyphenylpyruvate synthase method based on magnetic nanomaterial immobilized mutant strain

Abstract

The application discloses a 4-hydroxyphenylpyruvic acid synthesis method based on a mutant strain fixed by magnetic nanometer materials and belongs to the technical field of biological catalysis synthesis. Pmy The LAAD gene is subjected to site-directed mutation screening, and mutant strains M2 and M4 capable of efficiently catalyzing L-tyrosine to generate 4-hydroxyphenylpyruvic acid (4-HPP) are obtained; when M4 is used to catalyze the synthesis of 4-HPP, the yield of 4-HPP can reach 19.01 g / L; the immobilized biological catalyst prepared by using M4 can be recycled for more than 9 times and still retains more than 50% activity; tap water is used to replace a buffer and lactose is used to replace toxic IPTG in the reaction system; the catalyst can be quickly separated by a magnetic field, operation is simple, downstream processing is simple, and the catalyst is suitable for industrial large-scale production, and has important application prospects in the fields of medicines, fine chemicals and the like.

Description

Technical Field

[0001] This invention relates to a method for synthesizing 4-hydroxyphenylpyruvic acid based on a fixed mutant strain using magnetic nanomaterials, belonging to the field of biocatalytic synthesis technology. Background Technology

[0002] 4-Hydroxyphenylpyruvic acid (4-HPP) is a key intermediate in tyrosine metabolism and an important raw material for the synthesis of related drugs in the pharmaceutical field. It is also used in the research of metabolic diseases such as alkaptonuria. Simultaneously, as a fine chemical intermediate, it can participate in the synthesis of various high-value-added compounds, possessing significant practical application value. Currently, the synthesis of 4-hydroxyphenylpyruvic acid mainly relies on chemical synthesis and biocatalysis. Chemical synthesis suffers from problems such as harsh reaction conditions, numerous byproducts, and severe environmental pollution. Traditional biocatalysis methods often use wild-type enzymes or free engineered bacteria as catalysts, which have drawbacks such as low catalytic efficiency, poor enzyme stability, difficulty in catalyst recovery, and the presence of buffer ions and toxic inducers in the reaction system, limiting its industrial application.

[0003] L-amino acid deaminase (LAAD) catalyzes the deamination of L-amino acids to produce the corresponding α-keto acids, making it an ideal biocatalyst for the synthesis of 4-hydroxyphenylpyruvic acid. Among these, those derived from *Proteus mirabilis*... Pmy LAAD has broad substrate specificity, but wild-type Pmy LAAD suffers from problems such as limited substrate channel conformational flexibility and weak affinity for FAD cofactors, resulting in low catalytic efficiency.

[0004] Meanwhile, the industrial application of whole-cell biocatalysis faces challenges such as high catalyst costs, complex downstream processing, and poor recyclability. Magnetic nanoparticle immobilization technology can achieve rapid separation and recovery of catalysts, which is an effective means to solve the above problems. However, existing magnetic carriers have problems such as low cell adsorption efficiency and poor retention of enzyme activity after immobilization. In addition, traditional biocatalysis systems rely on buffer solutions such as phosphates and Tris-HCl, and use IPTG as an inducer. IPTG has cytotoxicity, which limits its large-scale application.

[0005] Therefore, improving the catalytic efficiency of L-amino acid deaminases, reducing the production cost of whole-cell catalysts, and achieving greening of the reaction system are of great research significance. Summary of the Invention

[0006] Wild-type 4-HPP biocatalytic synthesis is currently available. PmyThe low catalytic efficiency of LAAD, the inability to recycle free engineered bacteria, the poor adsorption and separation effects of traditional immobilized carriers, the dependence of reaction systems on toxic inducers and buffer solutions, and the lack of industrial-scale scaling-up solutions all hinder the achievement of large-scale green production requirements. To address these issues, this invention integrates enzyme engineering, magnetic nano-immobilization, and a green system to achieve highly efficient... Pmy The LAAD mutant was used to construct a highly adsorbent Fe3O4@PDA@PEI support and a stable immobilized catalyst, achieving green catalysis without buffer buffer or toxicity induction, and realizing high yield of 4-HPP and efficient catalyst recycling.

[0007] The first objective of this invention is to provide an L-amino acid deaminase mutant, wherein the L-amino acid deaminase mutant is formed by mutating leucine at position 315 of the L-amino acid deaminase shown in SEQ ID NO. 1 to alanine; Alternatively, the glutamine at position 73 of the L-amino acid deaminase, as shown in SEQ ID NO. 1, may be mutated to asparagine; Alternatively, the threonine at position 415 of the L-amino acid deaminase, as shown in SEQ ID NO. 1, may be mutated to cysteine; Alternatively, the glutamine at position 73 of the L-amino acid deaminase, as shown in SEQ ID NO. 1, may be mutated to asparagine, and the leucine at position 315 may be mutated to alanine. Alternatively, the leucine at position 315 of the L-amino acid deaminase, as shown in SEQ ID NO. 1, may be mutated to alanine, and the threonine at position 415 may be mutated to cysteine. Alternatively, the amino acid sequence of the L-amino acid deaminase shown in SEQ ID NO. 1 may be mutated by changing glutamine at position 73 to asparagine, leucine at position 315 to alanine, and threonine at position 415 to cysteine. Alternatively, the amino acid sequence of the L-amino acid deaminase shown in SEQ ID NO 1 may be modified by mutating glutamine at position 73 to asparagine, leucine at position 315 to alanine, threonine at position 415 to cysteine, and arginine at position 289 to tryptophan. Alternatively, the amino acid sequence of the L-amino acid deaminase shown in SEQ ID NO .1 may be mutated as follows: glutamine at position 73 is replaced with asparagine, leucine at position 315 is replaced with alanine, threonine at position 415 is replaced with cysteine, and tyrosine at position 71 is replaced with arginine. Alternatively, the amino acid sequence of the L-amino acid deaminase shown in SEQ ID NO .1 may be modified by mutating glutamine at position 73 to asparagine, leucine at position 315 to alanine, threonine at position 415 to cysteine, and leucine at position 252 to tyrosine. Alternatively, the amino acid sequence of the L-amino acid deaminase shown in SEQ ID NO .1 may be modified by mutating glutamine at position 73 to asparagine, leucine at position 315 to alanine, threonine at position 415 to cysteine, arginine at position 289 to tryptophan, tyrosine at position 71 to arginine, and leucine at position 252 to tyrosine.

[0008] In one embodiment, the mutant is obtained by mutating glutamine at position 73 to asparagine, leucine at position 315 to alanine, and threonine at position 415 to cysteine, as shown in SEQ ID NO. 1. Pmy The amino acid sequence of LAAD-L315A / Q73N / T415C (M2) is shown in SEQ ID NO. 3.

[0009] In one embodiment, the mutant is obtained by mutating glutamine at position 73 to asparagine, leucine at position 315 to alanine, threonine at position 415 to cysteine, arginine at position 289 to tryptophan, tyrosine at position 71 to arginine, and simultaneously mutating leucine at position 252 to tyrosine. Pmy The amino acid sequence of LAAD-L315A / Q73N / T415C / R289W / Y71R / L252Y (M4) is shown in SEQ ID NO. 4.

[0010] A second object of the present invention is to provide an enzyme catalyst, which is in any of the following forms: (1) Cultivate recombinant expression transformants containing the L-amino acid deaminase mutant described above, and isolate transformant cells containing recombinant L-amino acid deaminase mutant enzyme; (2) Cultivate recombinant expression transformants containing the L-amino acid deaminase mutant described above, isolate transformant cells containing recombinant L-amino acid deaminase mutant enzyme, and break the transformant cells containing recombinant L-amino acid deaminase mutant enzyme to obtain cell lysate. (3) Cultivate recombinant expression transformants containing the L-amino acid deaminase mutant described above, isolate transformant cells containing recombinant L-amino acid deaminase mutant enzyme, break the transformant cells containing recombinant L-amino acid deaminase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of recombinant L-amino acid deaminase mutant enzyme to obtain lyophilized enzyme powder.

[0011] A third objective of this invention is to provide a recombinant vector carrying a gene containing the L-amino acid deaminase mutant described above.

[0012] A fourth objective of this invention is to provide a genetically engineered bacterium that expresses the L-amino acid deaminase mutant described above.

[0013] In one embodiment, the genetically engineered bacteria include Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and yeast.

[0014] The fifth objective of this invention is to provide an immobilized biocatalyst, obtained by immobilizing any of the genetically engineered bacteria described above using a magnetic nanocarrier, comprising the following steps: (1) Inoculate the seed culture of any of the above-mentioned genetically engineered bacteria into the fermentation medium at an inoculation rate of 1-5% (v / v) and incubate at 30-37℃ and 200-220 rpm until OD. 600 The concentration was 0.6~0.8, then an inducer was added and fermented at 16~20℃ for 12~24 h. The engineered bacterial cells were then collected by centrifugation and washing. (2) The engineered bacteria prepared in step (1) are resuspended in OD. 600 After mixing with magnetic nanocarriers at a volume ratio of 1:(1~3) at a concentration of 0.5~1 mg / mL, the mixture was bound for 1~3 h. The bound products were collected by magnetic separation and washed to obtain Fe3O4@PDA@PEI-engineered bacteria immobilized biocatalyst.

[0015] In one embodiment, the inducer may be IPTG or lactose. Optionally, the final concentration of IPTG in the fermentation medium is 5-8 mM, and the final concentration of lactose is 10-15 g / L.

[0016] In one embodiment, the centrifugation is performed at 8000-10000 rpm for 5-10 minutes.

[0017] In one embodiment, the magnetic nanocarrier is a Fe3O4@PDA@PEI magnetic nanocarrier, and the preparation method of the Fe3O4@PDA@PEI magnetic nanocarrier is as follows: Fe3O4 nanoparticles were dispersed in Tris-HCl buffer and sonicated to obtain a Fe3O4 suspension. Dopamine solution was added to the Fe3O4 suspension, and after stirring, magnetic separation and washing were performed to obtain Fe3O4@PDA intermediate. The Fe3O4@PDA intermediate was dispersed in deionized water, mixed with polyethyleneimine solution, stirred, magnetically separated, and washed to obtain Fe3O4@PDA@PEI magnetic nanocarrier.

[0018] In one embodiment, the Fe3O4@PDA@PEI magnetic nanocarrier is prepared as follows: Nanoparticles were mixed with Tris-HCl buffer at a ratio of 8–12 mg: 8–12 mL and sonicated for 120–160 min to obtain Fe3O4 suspension. Fe3O4 suspension was mixed with 0.5–0.8 mg / mL dopamine solution at a volume ratio of 1–1.5: 1–1.5 and stirred at 20–30 °C and 250–350 rpm for 6–9 h. After magnetic separation and washing, Fe3O4@PDA intermediate was obtained. Fe3O4@PDA intermediate was dispersed in water to prepare a 1–1.5 g / mL Fe3O4@PDA intermediate aqueous solution and mixed with 20–25% (w / v) PEI solution at a volume ratio of 1–1.5: 1–1.5 and stirred for 8–12 h. After magnetic separation and washing, Fe3O4@PDA@PEI magnetic nanocarrier was obtained.

[0019] The sixth objective of this invention is to provide a method for preparing 4-hydroxyphenylpyruvic acid, using L-tyrosine as a substrate, and employing whole cells of any of the genetically engineered bacteria described above or their cell lysates, or any of the immobilized biocatalysts described above to catalyze the preparation of 4-hydroxyphenylpyruvic acid. The specific method is as follows: using water or buffer solution as the reaction medium, add L-tyrosine at a final concentration of 20-50 g / L as the substrate to the reaction medium, and add whole cells of any of the above-mentioned genetically engineered bacteria or their cell lysate at a final concentration of 20-50 g / L or any of the above-mentioned immobilized biocatalysts as the catalyst to carry out the catalytic reaction, and centrifuge to obtain a supernatant containing 4-hydroxyphenylpyruvic acid.

[0020] In one embodiment, the catalytic conditions are pH 6.5-7.5, temperature 25-30°C, reaction at 200-220 rpm for 24-94 h.

[0021] A seventh object of the present invention is to provide the use of any of the genetically engineered bacteria or methods described above in the preparation of 4-hydroxyphenylpyruvic acid or products containing 4-hydroxyphenylpyruvic acid.

[0022] Beneficial effects (1) This invention, through the application of... PmySite-directed mutagenesis screening of the LAAD gene yielded mutant strains M2 and M4, which can efficiently catalyze the production of 4-hydroxyphenylpyruvic acid (4-HPP) from L-tyrosine. When M4 was used to catalyze the synthesis of 4-HPP, the yield of 4-HPP reached 19.01 g / L.

[0023] (2) The immobilized biocatalyst prepared by M4 in this invention can achieve an initial conversion rate of 82% when catalyzing the synthesis of 4-HPP, and can be recycled more than 9 times while still retaining more than 50% of its activity; the reaction system uses tap water instead of buffer solution and lactose instead of toxic IPTG, which is green, environmentally friendly and low cost; the catalyst can be quickly separated by magnetic field, the operation is simple, the downstream treatment is simple, and it is suitable for large-scale industrial production, and has important application prospects in the fields of medicine and fine chemicals. Attached Figure Description

[0024] Figure 1 The graph shows the yield of 4-HPP synthesized by wild-type (WT) and various mutant strains (M2, M4).

[0025] Figure 2 The graph shows the yield of 4-HPP synthesized by mutant strain M4 after induction with IPTG and lactose, respectively.

[0026] Figure 3 This is a flowchart illustrating the fabrication process of Fe3О4@PDA@PEI magnetic nanomaterials.

[0027] Figure 4 Flowchart for the preparation of Fe3O4@PDA@PEI-engineered bacteria immobilized biocatalyst.

[0028] Figure 5 Transmission electron microscope image of Fe3O4@PDA@PEI magnetic nanocarrier.

[0029] Figure 6 Potential characterization diagrams for Fe3O4, Fe3O4@PDA intermediate, and Fe3O4@PDA@PEI magnetic nanocarriers.

[0030] Figure 7 The image shows the Fourier transform infrared spectroscopy results, where (a) represents Fe3O4, (b) represents the Fe3O4@PDA intermediate, and (c) represents the Fe3O4@PDA@PEI magnetic nanocarrier.

[0031] Figure 8 The image shows the results of the saturation magnetization measurement of Fe3O4@PDA@PEI magnetic nanocarriers.

[0032] Figure 9 The diagram shows the number of times the immobilized biocatalyst can be recycled and the effect of activity retention.

[0033] Figure 10 The number of times the catalyst can be recycled and the catalytic activity retained by the M4 mutant strain are determined. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, where specific conditions are not specified, are generally performed under conventional conditions in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0035] In this invention, % (w / v) refers to the percentage of mass to liquid volume (g / L).

[0036] In the examples, solutions without a specified solvent are water; operations without a specified temperature refer to room temperature, which is 15-35°C; and percentages without a specified meaning refer to mass percentages.

[0037] In the examples: the PCR reaction was carried out in a 50 μL system under the following conditions: pre-denaturation at 94 °C for 4 min; followed by 30 cycles: denaturation at 98 °C for 10 s, annealing at 60 °C for 5 s, extension at 72 °C for 2 min; and finally extension at 72 °C for 10 min, followed by incubation at 4 °C.

[0038] The PCR reaction system in the example was: 22 μL deionized water, 1 μL template, 1 μL upstream primer, 1 μL downstream primer, and 25 μL high-fidelity enzyme.

[0039] The sources of raw materials involved in the following embodiments: Fe3O4 nanoparticles were purchased from Sigma-Aldrich (Shanghai).

[0040] Dopamine was purchased from Sigma-Aldrich (Shanghai).

[0041] PEI (polyethyleneimine) was purchased from Sigma-Aldrich (Shanghai).

[0042] The culture media involved in the following examples: LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, natural pH.

[0043] TB medium: glycerol 5 g / L, peptone 12 g / L, yeast extract 24 g / L, potassium dihydrogen phosphate 2.31 g / L, dipotassium hydrogen phosphate 12.54 g / L, natural pH.

[0044] The detection methods involved in the following embodiments: High performance liquid chromatography (HPLC) for the determination of 4-HPP content: An Agilent 1260 HPLC system was used, featuring a Bio-Rad Aminex HPX-87H organic acid analytical column (300 mm × 7.8 mm, 9 μm). The mobile phase was 5 mmol / L dilute sulfuric acid aqueous solution (H₂SO₄), with a flow rate of 0.6 mL / min, a column temperature of 35 ℃, a detection wavelength of 210 nm, an injection volume of 10 μL, and a single chromatographic run time of 45 min.

[0045] Determination of 4-HPP content by salicylaldehyde-sodium hydroxide colorimetric method: Accurately prepare a 0.2% (w / v) anhydrous ethanol salicylaldehyde solution and a 75 g / L sodium hydroxide aqueous solution. Add 20 μL of the supernatant to be tested, 100 μL of salicylaldehyde reagent, and 80 μL of sodium hydroxide solution sequentially to a 96-well plate high-throughput system. After thorough shaking and mixing, let it stand at room temperature in the dark for 20 min (after optimization, the R² of this colorimetric time can reach 0.9992 for the standard curve). Measure the absorbance at a wavelength of 465 nm. A standard curve was established using α-keto acid standards at concentrations ranging from 1 to 10 g / L, with blank solutions used for blank correction. Sample concentrations were calculated based on absorbance and dilution factor. For sample pretreatment, the catalyst solution was centrifuged at 8900 × g for 5 min, and the clear supernatant was collected. If the product concentration was too high, it could be appropriately diluted. Screening experiments included a negative control of empty vector bacteria and a positive control of wild-type bacteria. A... 465 The initial screening threshold is ≥ the mean of positive controls + 3 times the standard deviation. Positive samples in the initial screening are then precisely verified by HPLC. This colorimetric system is effective for 4-HPP α-keto acids. The colorimetric reagent must be stored away from light. The reaction should avoid temperature rise and the measurement should be performed on time to prevent color decay.

[0046] Whole-cell specific productivity assay: Whole-cell specific production capacity is expressed as a unit OD. 600 The formula for calculating the 4-HPP yield of the bacteria is: Whole-cell specific production capacity (g) L -1 OD600 -1 = 4-HPP mass concentration (g / L) / bacterial OD of the reaction system 600 Numerical value.

[0047] Conversion rate determination: Substrate conversion rate (%) = ΔC 4-HPP / ΔC Tyr ×100% Where, ΔC 4-HPPThe change in 4-HPP concentration before and after the catalytic reaction; ΔC Tyr The change in L-tyrosine concentration before and after the catalytic reaction is shown.

[0048] Determination of cell adsorption efficiency: OD values ​​of bacterial suspensions before and after immobilization were measured separately. 600 Value, based on OD 600 The formula for calculating the proportion of bacterial cells adsorbed by the magnetic carrier is as follows: Cell adsorption efficiency = (initial OD) 600 OD of supernatant after adsorption 600 ) / Initial OD 600 ×100% Determination of bacterial load: The mass of wet bacterial cells adsorbed by a unit mass of Fe3O4@PDA@PEI magnetic nanocarrier was determined and characterized in mg cells / g carrier. The calculation formula is: bacterial load (mg / g) = (total adsorbed bacterial mass mg converted from bacterial suspension concentration) / dry mass of added magnetic carrier (g).

[0049] Example 1: Pmy Construction of LAAD mutants (1) Construction of pET28a-PmyLAAD L-amino acid deaminase derived from Proteus mirabilis Pmy LAAD (amino acid sequence as shown in SEQ ID NO. 1, nucleotide sequence as shown in SEQ ID NO. 2) was ligated to plasmid pET28a. EcoRI , HindIII plasmid pET28a- was obtained at the site (which can be sent to Suzhou Genewiz for synthesis). Pmy LAAD.

[0050] (2) Construction of single-mutant recombinant plasmids Will Pmy Molecular docking and structural bioinformatics analysis were performed on the LAAD gene (PDB ID: 5FJM) and L-tyrosine residues to identify the key region Loop1 (L309~L315) of the substrate (L-tyrosine) channel. Pmy LAAD's 309th to 315th are Pmy Mutation sites for LAAD directed evolution.

[0051] Through protein ( Pmy LAAD) Ligand (FAD) interaction analysis elucidated the FAD coenzyme binding mode, screened key residues that directly form hydrogen bonds with the FAD isorhozoline redox core ring, and identified Q73 and T415 as the core sites regulating FAD binding affinity and structural stability. Q73 and T415 were further identified as the key sites for this interaction. Pmy Mutation sites for LAAD directed evolution.

[0052] pET28a- Pmy Using LAAD as a template, site-directed saturation mutagenesis was performed on all sites in the L309-L315 region, as well as Q73 and T415, using the NNK codon degeneracy strategy. After PCR amplification, [the results were analyzed]. Dpn The template plasmid was degraded by enzyme I to obtain a recombinant plasmid containing a single mutation. The mutation site and the corresponding primer sequences are shown in Table 1; where N represents one of the four nucleotides ATCG, and K represents one of the two nucleotides GT.

[0053] Table 1. Mutant primer sequences

[0054] (3) Construction of recombinant strains containing mutants Plasmid pET28a- Pmy LAAD and recombinant plasmids containing mutants were transformed into, respectively, […]. E. coli BL21(DE3) was prepared to obtain strain E. coli BL21(DE3) / pET28a- Pmy LAAD (WT) and recombinant strains containing mutants.

[0055] (4) Screening of recombinant strains Single colonies of the recombinant strain containing the mutant were inoculated into 24-well LB medium and cultured at 37°C for 12 h. Then, the colonies were transferred to TB medium at a 2% v / v inoculation rate and cultured at 37°C for 2 h. After adding 5 mM IPTG and inducing expression at 16°C for 24 h, the bacterial cells were collected, resuspended in PBS buffer, and OD24 was prepared. 600 A bacterial suspension with a concentration of 10 was used for subsequent assays.

[0056] In a 10 mL system, using PBS as the reaction medium, L-tyrosine at a final concentration of 40 g / L and bacterial suspension at 10 g / L were added to the reaction medium at pH 7. The mixture was reacted at 35 °C and 220 rpm for 96 h to achieve whole-cell transformation. Subsequent screening was performed using the salicylaldehyde-sodium hydroxide colorimetric method (A...). 465(≥ positive control mean + 3 standard deviations) The strains after initial screening were further analyzed by HPLC to determine the 4-HPP content. High-yielding mutant strains were screened by calculating the whole-cell specific productivity. The results are shown in Table 2. The strains with better sequencing results were sequenced, and finally, the mutant recombinant strains were screened. E. coli BL21(DE3) / pET28a- Pmy LAAD-L315A (M1) E. coli BL21(DE3) / pET28a- Pmy LAAD-Q73N, E. coli BL21(DE3) / pET28a- Pmy LAAD-T415C.

[0057] Table 2

[0058] Example 2: Construction of Combinatorial Mutations (1) Construction of double-mutant recombinant strains With plasmid pET28a- Pmy Using LAAD-L315A as a template, site-directed PCR was performed using primers Q73N-F and Q73N-R as shown in Table 1 to obtain the recombinant plasmid pET28a- Pmy LAAD-L315A / Q73N; With plasmid pET28a- Pmy Using LAAD-L315A as a template, site-directed PCR was performed using primers T415C-F and T415C-R as shown in Table 1 to obtain the recombinant plasmid pET28a- Pmy LAAD-L315A / T415C; With plasmid pET28a- Pmy Using LAAD-Q73N as a template, site-directed PCR was performed using primers T415C-F and T415C-R as described in Table 1 to obtain the recombinant plasmid pET28a- Pmy LAAD-Q73N / T415C; With plasmid pET28a- Pmy Using LAAD-L315A / Q73N as a template, site-directed PCR was performed using primers T415C-F and T415C-R as described in Table 1 to obtain the recombinant plasmid pET28a- Pmy LAAD-L315A / Q73N / T415C; The recombinant plasmids were transformed into [the appropriate transfection sites]. E. coli BL21 (DE3) was used to prepare a mutant recombinant strain. E. coli BL21(DE3) / pET28a- PmyLAAD-L315A / Q73N, E. coli BL21(DE3) / pET28a- Pmy LAAD-L315A / T415C E. coli BL21(DE3) / pET28a- Pmy LAAD-Q73N / T415C, E. coli The whole-cell specific production capacity of BL21(DE3) / pET28a-PmyLAAD-L315A / Q73N / T415C(M2) was determined using the following methods: Single colonies of the recombinant strain were picked and inoculated into 24-well LB medium and cultured at 37°C for 12 h. Then, the colonies were transferred to TB medium at a 2% v / v inoculation rate and cultured at 37°C for 2 h. 5 mM IPTG was added, and expression was induced at 16°C for 24 h. The bacterial cells were then collected, resuspended in PBS buffer, and OD200 was prepared. 600 The bacterial suspension with a concentration of 10 was analyzed by HPLC to determine the 4-HPP content, and the total cell production capacity was calculated.

[0059] The test results are shown in Table 3. The results indicate that the strain... E. coli The whole-cell specific production capacity of 4-HPP produced by BL21(DE3) / pET28a-PmyLAAD-L315A / Q73N / T415C(M2) can reach 1.79 g·L. -1 ·OD 600 -1 .

[0060] Table 3

[0061] (2) Construction of multiple mutant strains Will Pmy The LAAD-L315A / Q73N / T415C gene and L-tyrosine were subjected to semi-flexible molecular docking using AutoDock Vina, limiting the substrate binding pocket to a 5 Å range. This allowed for the screening of L-tyrosine-binding compounds. The key residues for close interaction with tyrosine are Y71, L252, and R289, which have been identified as... Pmy The mutation site of LAAD-L315A / Q73N / T415C directed evolution.

[0062] With plasmid pET28a- Pmy Using LAAD-L315A / Q73N / T415C as a template, saturation mutations were performed at the Y71, L252, and R289 sites using the NNK codon degeneracy strategy. After PCR amplification, [the results were analyzed]. DpnI. The template plasmid was degraded by enzyme digestion to obtain a recombinant plasmid containing the mutant (primers are shown in Table 4).

[0063] The mutated recombinant plasmids were transformed into [various methods]. E. coli BL21 (DE3) was used to obtain a recombinant strain, and high-yield mutant strains were screened using the method in step (4) of Example 1. The screening results are shown in Table 5. Finally, the recombinant strain was obtained. E. coli BL21(DE3) / pET28a-M2-R289W, E. coli BL21(DE3) / pET28a-M2-Y71R、 E. coli BL21(DE3) / pET28a-M2-L252Y.

[0064] Furthermore, using plasmid pET28a- Pmy Using LAAD-L315A / Q73N / T415C as a template, site-directed PCR was performed using primers R289W-F and R289W-R as shown in Table 4 to obtain the recombinant plasmid pET28a- Pmy LAAD-L315A / Q73N / T415C / R289W; With plasmid pET28a- Pmy Using LAAD-L315A / Q73N / T415C / R289W as a template, site-directed PCR was performed using primers Y71R-F and Y71R-R as shown in Table 4 to obtain the recombinant plasmid pET28a- Pmy LAAD-L315A / Q73N / T415C / R289W / Y71R; With plasmid pET28a- Pmy Using LAAD-L315A / Q73N / T415C / R289W / Y71R as a template, site-directed PCR was performed using primers L252Y-F and L252Y-R as shown in Table 4 to obtain the recombinant plasmid pET28a- Pmy LAAD-L315A / Q73N / T415C / R289W / Y71R / L252Y.

[0065] Plasmid pET28a- Pmy LAAD-L315A / Q73N / T415C / R289W / Y71R / L252Y (M4) conversion to E. coli BL21 (DE3) yielded a recombinant strain. E. coli BL21(DE3) / pET28a- PmyThe whole-cell specific production capacity of LAAD-L315A / Q73N / T415C / R289W / Y71R / L252Y (M4) was determined using the method in step (1) of Example 2 (Table 5). The results showed that the whole-cell specific production capacity of 4-HPP strain M4 could reach 2.16 g·L⁻¹. -1 ·OD600 -1 .

[0066] Table 4. Mutant Primer Sequences

[0067] Table 5

[0068] Example 3: Synthesis of 4-HPP catalyzed by free engineered bacteria The synthesis of 4-HPP catalyzed by free engineered bacteria includes the following steps: 1. Culture and Induction Expression of Engineered Bacteria Single colonies of engineered bacteria were picked from LB plates and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin. The culture was carried out at 37°C and 200 rpm for 12 h to obtain seed culture. The seed culture was inoculated at a rate of 1% (v / v) into 100 mL of TB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C and 200 rpm until OD. 600 =0.8, then add 10 g / L lactose as an inducer, lower the culture temperature to 16℃, and continue to culture at 200 rpm for 24 h; centrifuge at 6000 r / min and 4℃ for 10 min, collect the engineered bacterial cells, wash twice with 0.85% physiological saline, and set aside for later use.

[0069] 2. Synthesis of 4-HPP catalyzed by free engineered bacteria 10 mL reaction system: Using tap water as the reaction medium, 40 g / L L-tyrosine and 40 g / L (wet weight) engineered bacteria were added to the tap water at a final concentration of 7. The reaction was catalyzed at 25℃ and 220 rpm for 24 h. After catalysis, the supernatant was collected by centrifugation. The supernatant was filtered through a 0.22 μm filter membrane, and the yield of 4-HPP was detected by HPLC.

[0070] The above methods were used to determine the yield and conversion rate of 4-HPP synthesized by wild-type fungus (WT), M2 (L315A / Q73N / T415C), and M4 (L315A / Q73N / T415C / R289W / Y71R / L252Y), respectively. The results are as follows: Figure 1As shown in Table 6, the results indicate that the yield of 4-HPP catalyzed by engineered bacteria M4 to L-tyrosine can reach 19.01 g / L.

[0071] Table 6

[0072] Example 4: Effect of buffer system on the catalytic synthesis of 4-HPP by recombinant cells The yield of 4-HPP synthesized by mutant strain M4 was determined using the method of Example 3, except that the tap water in step 2 of Example 3 was replaced with a phosphate buffer system (PBS, pH 7.0), while the other steps remained the same.

[0073] The results showed that there was no statistically significant difference in 4-HPP yield between the tap water system (21.17 g / L) and the buffer system (18.64 g / L) (P>0.05), proving that tap water can completely replace traditional buffer solutions.

[0074] Example 5: Preparation of bacterial cells using IPTG as an inducer for the catalytic synthesis of 4-HPP. The yield of 4-HPP synthesized by mutant strain M4 was determined using the method of Example 3, except that the 10 g / L lactose in step 2 of Example 3 was replaced with 0.5 mM IPTG, while the other steps remained the same.

[0075] The measurement results are as follows Figure 2 As shown, the results indicate that lactose-induced engineered bacteria Pmy There was no significant difference in the yield of 4-HPP catalyzed by LAAD and IPTG-induced engineered bacteria (P>0.05). The yield of 4-HPP catalyzed by lactose-induced engineered bacteria was 16.30 g / L, while the yield of 4-HPP catalyzed by IPTG-induced engineered bacteria was 18.39 g / L.

[0076] Example 6: Immobilized biocatalytic synthesis of 4-HPP The immobilized biocatalytic synthesis of 4-HPP includes the following steps: (1) Preparation of Fe3O4@PDA@PEI magnetic nanocarriers Take 10 mg of Fe3O4 nanoparticles and disperse them in 10 mL of 10 mM Tris-HCl buffer solution at pH 8.5. Disperse the mixture by sonication at 100 W for 30 min at room temperature to obtain a uniform Fe3O4 suspension. 10 mL of 0.7 mg / mL dopamine solution was added to the Fe3O4 suspension, and the mixture was stirred at 25 °C and 300 rpm for 7 h, during which the pH of the system was kept stable at 8.5. The product was collected by magnetic separation under an external magnetic field (ordinary magnet), and washed three times each with deionized water and anhydrous ethanol to obtain the Fe3O4@PDA intermediate. The Fe3O4@PDA intermediate was redispersed in 30 mL of deionized water, and 30 mL of 25% (w / v) PEI (polyethyleneimine) solution was added. The mixture was stirred at 180 rpm for 10 h at room temperature. The product was collected by magnetic separation (using a common magnet) and washed with deionized water until the supernatant was neutral to obtain the purified product. The purified product was resuspended in deionized water (1 mg / mL) and stored at 4℃ in the dark to obtain the Fe3O4@PDA@PEI magnetic nanocarrier. The specific preparation process is as follows: Figure 3 As shown.

[0077] The prepared Fe3O4@PDA@PEI magnetic nanocarriers were characterized as follows: The nanocarrier structure was observed using transmission electron microscopy (TEM), and the TEM image showed it to be an irregular sphere. Figure 5 (The average particle size is about 100 nm, and it has a clear core-shell structure;) The zeta potential of Fe3O4, Fe3O4@PDA intermediate, and Fe3O4@PDA@PEI magnetic nanocarrier was analyzed using a zeta potential analyzer. The zeta potential was +31.2 mV. Figure 6 ); Fourier transform infrared spectroscopy (FT-IR) confirmed the successful modification of PDA (polydopamine) and PEI. Figure 7 The vibrating sample magnetometer (VSM) showed a saturation magnetization of 25 emu / g, indicating superparamagnetism, and capable of separation within seconds under an applied magnetic field. Figure 8 ).

[0078] (2) Culture and induction expression of engineered bacteria Single colonies of engineered bacteria were picked from LB plates and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin. The culture was carried out at 37°C and 200 rpm for 12 h to obtain seed culture. The seed culture was inoculated into 100 mL of TB liquid medium containing 50 μg / mL kanamycin at a 1% v / v inoculation rate, and cultured at 37°C and 200 rpm until OD. 600 =0.8, then add 10 g / L lactose as an inducer, lower the culture temperature to 16℃, and continue to culture at 200 rpm for 24 h; centrifuge at 6000 r / min and 4℃ for 10 min, collect the engineered bacterial cells, wash twice with 0.85% physiological saline, and set aside for later use.

[0079] (3) Magnetic immobilization of engineered bacteria: The engineered bacterial cells collected in step (2) were resuspended in 0.85% physiological saline, and the OD of the bacterial suspension was adjusted. 600 =0.77; Take 100 mL of the above bacterial suspension and mix it with 100 mL of the Fe3O4@PDA@PEI magnetic nanocarrier (1 mg / mL) prepared in step 1. Stir and incubate at 100 rpm at room temperature for 1 h. The binding products were magnetically separated under an external magnetic field and collected. The products were then washed twice with 0.85% physiological saline to prepare the Fe3O4@PDA@PEI-engineered bacteria immobilized biocatalyst. The specific preparation process is as follows: Figure 4 As shown; Detection of OD in supernatant 600 The calculated cell adsorption efficiency was 50.1%, and the bacterial load was 665 mg cells / gram carrier.

[0080] (4) Synthesis of 4-HPP catalyzed by immobilized biocatalyst Using 40 g / L L-tyrosine as a substrate, the Fe3O4@PDA@PEI-engineered bacteria immobilized biocatalyst prepared in step (3) was added to tap water at a dosage of 20 g / L to construct a 50 mL buffer-free catalytic system. The catalytic system was placed in a shaker at pH 7.0 and 30℃ and the catalytic reaction was carried out at 220 rpm for 24 h. After the reaction was completed, the immobilized biocatalyst was collected by magnetic separation under an external magnetic field. The supernatant was filtered through a 0.22 μm filter membrane, and the conversion rate of 4-HPP was determined.

[0081] The results showed that the conversion rate of M4-catalyzed synthesis of 4-HPP in the tap water system could reach 82.37%.

[0082] Example 7: Catalyst Recycling 1. Recycling of immobilized biocatalysts Taking the Fe3O4@PDA@PEI-M4 immobilized biocatalyst as an example, the cycle stability of the immobilized biocatalyst was further determined using the following method: The immobilized biocatalyst (Fe3O4@PDA@PEI-M4 immobilized biocatalyst) obtained in step (4) of Example 6 was washed twice with 0.85% physiological saline, and then added back into tap water containing 40 g / L L-tyrosine. Step (4) of Example 6 was repeated for nine consecutive catalytic cycles. After each reaction, the yield of 4-HPP was measured, and the catalytic activity retention rate was calculated (with the first catalytic activity as 100%).

[0083] The formula for calculating the catalytic activity retention rate is: (Current catalytic yield / First catalytic yield) × 100% 2. Recycling of free engineered bacterial catalysts The centrifuged product collected after catalysis in Example 3 was washed twice with 0.85% saline and then added back into tap water containing 40 g / L L-tyrosine for four cycles of catalysis. The yield of 4-HPP was measured after each reaction, and the catalytic activity retention rate was calculated (with the first catalytic activity as 100%).

[0084] The measurement results are shown in Table 7. Figure 9 as well as Figure 10 As shown, the results indicate that after 9 cycles of catalysis, the immobilized biocatalyst retained 52% of its initial catalytic activity, and there was no significant cell leakage in each cycle (OD of supernatant). 600 The catalytic activity of the free engineered bacteria (method of Example 3) decreased to 45% after 2 cycles and to 8% after 4 cycles, indicating that the immobilized biocatalyst of the present invention has excellent cycle stability.

[0085] Table 7

[0086] The sequences involved in the embodiments are as follows: Pmy The amino acid sequence of LAAD is shown in SEQ ID NO. 1: MVRRDGKFVESKSRALFVESTEGALPSESDVVIIGGGIQGIMTAINLAERGMSVTILEKGEVAGEQSGRAYSQIISYQTSPEIFPLHHYGKILWRGMNEKIGADTSYRTQGR VEALADEKALDRAQEWIKTAKETAGFDVPLNTRIIKGEELSNRLVGAQTPWTVAAFEEDSGSVDPETGTPTLARYAKQIGVKIYTHCAVRGIETAGGKISDVVTEKGAIRTS NVVLAGGIWSRLFMGNMGVDLPTLNVYLSQQRVSGVPGAPRGNVHLPNGIHFREQADGTYAVAPRIFTSSIVKDSFLLGPKFMHLLGGELPLEFSIGEDLFNSFKMPTSWK LDEKSPFEQYRIATATQNTEHLDAVFQRMKTEFPVFEKSQIVERWGAVVSPTFDELPIISEVKEYPGLVINTATVWGMTEGPAAGEVTADIVTGKKPVIDPTPFSLDRFKK* Pmy The nucleotide sequence of LAAD is shown in SEQ ID NO. 2. mutant Pmy The amino acid sequence of LAAD-L315A / Q73N / T415C (M2) is shown in SEQ ID NO. 3: MVRRDGKFVESSKSRALFVESTEGALPSESDVVIIGGGIQGIMTAINLAERGMSVTILEKGEVAGEQSGRAYSNIISYQTSPEIFPLHHYGKILWRGMNEKIGADTSYRTQGR VEALADEKALDRAQEWIKTAKETAGFDVPLNTRIIKGEELSNRLVGAQTPWTVAAFEEDSGSVDPETGTPTLARYAKQIGVKIYTHCAVRGIETAGGKISDVVTEKGAIRTS NVVLAGGIWSRLFMGNMGVDLPTLNVYLSQQRVSGVPGAPRGNVHLPNGIHFREQADGTYAVAPRIFTSSIVKDSFLLGPKFMHLLGGGEAPLEFSIGEDLFNSFKMPTSWK LDEKSPFEQYRIATATQNTEHLDAVFQRMKTEFPVFEKSQIVERWGAVVSPTFDELPIISEVKEYPGLVINTATVWGMCEGPAAGEVTADIVTGKKPVIDPTPFSLDRFKK* mutant Pmy The amino acid sequence of LAAD-L315A / Q73N / T415C / R289W / Y71R / L252Y (M4) is shown in SEQ ID NO. 4: MVRRDGKFVESSKSRALFVESTEGALPSESDVVIIGGGIQGIMTAINLAERGMSVTILEKGEVAGEQSGRARSNIISYQTSPEIFPLHHYGKILWRGMNEKIGADTSYRTQGR VEALADEKALDRAQEWIKTAKETAGFDVPLNTRIIKGEELSNRLVGAQTPWTVAAFEEDSGSVDPETGTPTLARYAKQIGVKIYTHCAVRGIETAGGKISDVVTEKGAIRTS NVVLAGGIWSRLFMGNMGVDLPTLNVYYSQQRVSGVPGAPRGNVHLPNGIHFREQADGTYAVAPWIFTSSIVKDSFLLGPKFMHLLGGGEAPLEFSIGEDLFNSFKMPTSWK LDEKSPFEQYRIATATQNTEHLDAVFQRMKTEFPVFEKSQIVERWGAVVSPTFDELPIISEVKEYPGLVINTATVWGMCEGPAAGEVTADIVTGKKPVIDPTPFSLDRFKK* Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An L-amino acid deaminase mutant, characterized in that, The L-amino acid deaminase mutant is The leucine at position 315 of the L-amino acid deaminase, as shown in SEQ ID NO. 1, is mutated to alanine. Alternatively, the glutamine at position 73 of the L-amino acid deaminase, as shown in SEQ ID NO. 1, may be mutated to asparagine; Alternatively, the threonine at position 415 of the L-amino acid deaminase, as shown in SEQ ID NO. 1, may be mutated to cysteine; Alternatively, the glutamine at position 73 of the L-amino acid deaminase, as shown in SEQ ID NO. 1, may be mutated to asparagine, and the leucine at position 315 may be mutated to alanine. Alternatively, the leucine at position 315 of the L-amino acid deaminase, as shown in SEQ ID NO. 1, may be mutated to alanine, and the threonine at position 415 may be mutated to cysteine. Alternatively, the amino acid sequence of the L-amino acid deaminase shown in SEQ ID NO. 1 may be modified by mutating glutamine at position 73 to asparagine, leucine at position 315 to alanine, and threonine at position 415 to cysteine. Alternatively, the amino acid sequence of the L-amino acid deaminase shown in SEQ ID NO 1 may be modified by mutating glutamine at position 73 to asparagine, leucine at position 315 to alanine, threonine at position 415 to cysteine, and arginine at position 289 to tryptophan. Alternatively, the amino acid sequence of the L-amino acid deaminase shown in SEQ ID NO .1 may be modified by mutating glutamine at position 73 to asparagine, leucine at position 315 to alanine, threonine at position 415 to cysteine, and tyrosine at position 71 to arginine. Alternatively, the amino acid sequence of the L-amino acid deaminase shown in SEQ ID NO .1 may be modified by mutating glutamine at position 73 to asparagine, leucine at position 315 to alanine, threonine at position 415 to cysteine, and leucine at position 252 to tyrosine. Alternatively, the amino acid sequence of the L-amino acid deaminase shown in SEQ ID NO .1 may be modified by mutating glutamine at position 73 to asparagine, leucine at position 315 to alanine, threonine at position 415 to cysteine, arginine at position 289 to tryptophan, tyrosine at position 71 to arginine, and leucine at position 252 to tyrosine.

2. An enzyme catalyst, characterized in that, It is any of the following forms: (1) Cultivate recombinant expression transformants containing the L-amino acid deaminase mutant as described in claim 1, and isolate transformant cells containing the recombinant L-amino acid deaminase mutant enzyme; (2) Cultivate recombinant expression transformants containing the L-amino acid deaminase mutant as described in claim 1, isolate transformant cells containing recombinant L-amino acid deaminase mutant enzyme, and break the transformant cells containing recombinant L-amino acid deaminase mutant enzyme to obtain cell lysate. (3) Cultivate recombinant expression transformants containing the L-amino acid deaminase mutant as described in claim 1, isolate transformant cells containing recombinant L-amino acid deaminase mutant enzyme, break the transformant cells containing recombinant L-amino acid deaminase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of recombinant L-amino acid deaminase mutant enzyme to obtain lyophilized enzyme powder.

3. A recombinant vector carrying a gene containing the L-amino acid deaminase mutant of claim 1.

4. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria expressed the L-amino acid deaminase mutant as described in claim 1.

5. The genetically engineered bacterium according to claim 4, characterized in that, The genetically engineered bacteria include Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and yeast.

6. An immobilized biocatalyst, characterized in that, The genetically engineered bacteria described in any one of claims 4-5 are obtained by immobilization on a magnetic nanocarrier, comprising the following steps: (1) The seed culture of the genetically engineered bacteria according to any one of claims 4 to 5 is inoculated into the fermentation medium at an inoculation rate of 1 to 5% v / v and cultured at 30 to 37°C and 200 to 220 rpm until OD. 600 The concentration was 0.6~0.8, then an inducer was added and fermented at 16~20℃ for 12~24 h. The engineered bacterial cells were then collected by centrifugation and washing. (2) The engineered bacteria prepared in step (1) are resuspended in OD. 600 After mixing with magnetic nanocarriers at a volume ratio of 1:(1~3) at a concentration of 0.5~1 mg / mL, the mixture was bound for 1~3 h. The bound products were collected by magnetic separation and washed to obtain Fe3O4@PDA@PEI-engineered bacteria immobilized biocatalyst.

7. The immobilized biocatalyst according to claim 6, characterized in that, The magnetic nanocarrier is a Fe3O4@PDA@PEI magnetic nanocarrier, and the preparation method of the Fe3O4@PDA@PEI magnetic nanocarrier is as follows: Fe3O4 nanoparticles were dispersed in Tris-HCl buffer and sonicated to obtain a Fe3O4 suspension. Dopamine solution was added to the Fe3O4 suspension, and after stirring, magnetic separation and washing were performed to obtain Fe3O4@PDA intermediate. The Fe3O4@PDA intermediate was dispersed in deionized water, mixed with polyethyleneimine solution, stirred, magnetically separated, and washed to obtain Fe3O4@PDA@PEI magnetic nanocarrier.

8. A method for preparing 4-hydroxyphenylpyruvic acid, characterized in that, Using L-tyrosine as a substrate, 4-hydroxyphenylpyruvic acid is obtained by catalysis with whole cells of the genetically engineered bacteria described in any one of claims 4-5 or their cell lysates, or by immobilized biocatalysts described in any one of claims 6-7. The specific method is as follows: using water or buffer solution as the reaction medium, L-tyrosine with a final concentration of 20-50 g / L is added to the reaction medium as a substrate, and whole cells of the genetically engineered bacteria of any one of claims 4-5 or their cell lysate or immobilized biocatalyst of any one of claims 6-7 are added as catalysts to carry out the catalytic reaction, and the supernatant containing 4-hydroxyphenylpyruvic acid is obtained by centrifugation.

9. The method according to claim 8, characterized in that, The catalytic conditions are pH 6.5-7.5, temperature 25-30℃, reaction at 200-220 rpm for 24-94 h.

10. The use of the genetically engineered bacteria of any one of claims 4 to 5 or the method of any one of claims 8 to 9 in the preparation of 4-hydroxyphenylpyruvic acid or products containing 4-hydroxyphenylpyruvic acid.

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