A method for preparing a cross-linking enzyme based on a tyrosinase mutant, the cross-linking enzyme and application thereof

Abstract

The application provides a method for preparing cross-linking enzyme based on a tyrosinase mutant, the cross-linking enzyme and application thereof. The tyrosinase mutant is mixed with an ultrasonic broken solution of S-adenosylmethionine synthetase containing a tyrosine tag to obtain water-insoluble cross-linking enzyme particles, the cross-linking enzyme is collected by centrifugation or filtration and the like, and enzymatic conversion is carried out. It is found that the cross-linking enzyme of S-adenosylmethionine synthetase can be continuously and stably used for 6 batches, and the activity retention is 85%. The cross-linking enzyme particles prepared by the method have the characteristics of easy recovery, reusability and low cost, and have good industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, and relates to a method for preparing cross-linking enzymes based on tyrosinase mutants, the cross-linking enzymes and their applications. Background Technology

[0002] S-adenosyl-L-methionine (SAM) is an important metabolic intermediate in living organisms. SAM has extremely high medicinal value and can be used clinically to treat liver damage, arthritis, depression, and cholestasis. It can also be used as a health supplement to prevent cancer and slow down aging.

[0003] There are three main methods for producing S-adenosylmethionine (SAM): chemical synthesis, microbial fermentation, and enzymatic catalysis. Chemical synthesis primarily uses S-homocysteine ​​and a methyl donor, producing two isomers, only one of which is active. This method suffers from problems such as difficulty in separating impurities and high cost. Microbial fermentation mainly utilizes SAM synthase within yeast cells. By adding the precursor L-methionine to the culture medium, SAM accumulates and is synthesized within the yeast cells. This method suffers from problems such as large fermentation volume, high impurity content, and multiple purification steps. Enzymatic catalysis utilizes S-adenosylmethionine synthase (MAT) to catalyze the reaction of the substrate L-methionine and adenosine triphosphate (ATP) to produce SAM. The reaction principle is described in [link to reaction description]. Figure 1 Compared with chemical synthesis and microbial fermentation, it has advantages such as high substrate conversion rate, easy product separation and purification, short reaction cycle and environmental friendliness, and has good prospects for industrial application.

[0004] Immobilized enzymes are enzymes that are soluble in solution but insoluble, retaining their catalytic activity. Once the reaction is complete, they can be easily separated from the solution by filtration, centrifugation, etc., allowing for enzyme reuse. Currently, there are some reports on immobilized S-adenosylmethionine synthase (SAM). For example, invention patent CN201010545201.1 discloses a method for preparing immobilized SAM synthase and SAM, which uses an amino resin carrier Seplite LX-1000HA crosslinked with glutaraldehyde solution to prepare immobilized SAM synthase. The enzyme activity recovery rate is high, and it can be reused, providing a new approach for the industrial production of SAM. Yin Chunli et al. prepared immobilized SAM synthase using amino resin as a carrier, achieving an enzyme activity recovery rate of 74.5% (Yin Chunli, Cao Shanshan, Xu Le et al. Study on S-adenosylmethionine synthase immobilized with amino resin [J]. Chemical and Biological Engineering, 2014, 31(09):17-20). Yin Chunli prepared immobilized SAM synthase using sodium alginate encapsulation, with an enzyme activity recovery rate of 42% (Yin Chunli, Cao Shanshan, Niu Weining. Preparation and property study of sodium alginate immobilized S-adenosylmethionine synthase [J]. Chemical and Biological Engineering, 2012, 29(10):21-24); using chitosan as a carrier and glutaraldehyde as a cross-linking agent, the enzyme activity recovery rate of the prepared immobilized SAM synthase was 76% (Yin Chunli, Tao Guirong, Xu Le et al. Chitosan immobilized S-adenosylmethionine synthase [J]. Journal of Food and Biotechnology, 2013, 32(09):945-950). Both immobilized enzymes can be reacted in multiple batches. Hua Haoju conducted immobilization studies on SAM synthase using amino resin and epoxy resin, respectively. He found that amino resin immobilization was more effective, with a specific activity of (27.91±1.33) U / g and an activity recovery rate of (25.78±1.23)%, exhibiting good stability in continuous reactions (Hua Haoju, Wu Yong, Huang Zongqing, et al. Immobilization of thermophilic S-adenosylmethionine synthase and preparation of S-adenosylmethionine [J]. China Pharmaceutical Industry Journal, 2019, 50(04):406-410). These results indicate that the immobilized SAM synthase prepared using resin, sodium alginate, and chitosan carriers shows promising industrial application prospects in the in vitro catalytic synthesis of SAM. However, due to limitations in the stability of the immobilized enzyme and its high cost, true industrial production has not yet been achieved.

[0005] In recent years, cross-linking enzymes have emerged as a novel type of immobilized enzyme. These are typically produced using chemical cross-linking agents (such as glutaraldehyde) to covalently link enzyme proteins, which can cause chemical damage to the enzyme molecule, disrupting its structure, reducing its catalytic activity, and leaving chemical residues. Tyrosinase, a polyphenol oxidase, catalyzes the oxidation of the phenolic hydroxyl groups of tyrosine residues on polypeptide chains to generate catechol, which in turn catalyzes the formation of active benzoquinones. Benzoquinones can self-polymerize or react with lysine, histidine, cysteine ​​residues, etc., to form protein cross-links, thus becoming a novel biological cross-linking enzyme. The core of preparing cross-linking enzymes using tyrosinase is the presence of tyrosine residues, as their different positions within the protein produce differentiated effects. Existing patent 202010696371.3 discloses a method for preparing cross-linking enzymes, which involves mixing polyphenol oxidase with a glutamine transaminase solution to prepare cross-linking enzyme microparticles, which are then applied to the modification reaction of protein drug molecules with polyethylene glycol (PEG). Apart from the aforementioned patents, there are no reports on the preparation and industrial application of immobilized MAT using tyrosinase. Therefore, this invention provides a method for preparing cross-linking enzymes based on tyrosinase mutants. The immobilized MAT prepared by this method has a high enzyme activity recovery rate, good stability, and retains high activity even after multiple batches of repeated use, making it suitable for large-scale industrial production of SAM. Summary of the Invention

[0006] Given the lack of reports on the preparation of immobilized S-adenosylmethionine synthase using tyrosinase, the primary objective of this invention is to provide a method for preparing cross-linking enzymes based on tyrosinase. This method utilizes a tyrosinase mutant to perform an enzymatic cross-linking reaction with S-adenosylmethionine synthase containing a tyrosine tag to prepare active cross-linking enzyme microparticles, without the need for a carrier or purification of the S-adenosylmethionine synthase.

[0007] A method for preparing cross-linking enzymes based on tyrosinase mutants, comprising using an enzyme containing a tyrosine tag as a substrate, adding a tyrosinase mutant to obtain a cross-linking enzyme; wherein the tyrosinase mutant includes a tyrosinase mutant with mutant amino acid sites including S146G and G283D obtained by mutating the wild-type amino acid sequence shown in SEQ ID NO.1, the sequence of which is SEQ ID NO.2.

[0008] The enzyme containing a tyrosine tag is an enzyme with a tyrosine residue at the end, and more specifically, an S-adenosylmethionine synthase with a tyrosine residue at the end.

[0009] Further, there is S-adenosylmethionine synthase, which contains a tyrosine residue at its C-terminus.

[0010] S-adenosylmethionine synthase is synthesized by adding 1-6 tyrosine residues to its C-terminus; preferably, 1-3 tyrosine residues are added to its C-terminus; more preferably, 3 tyrosine residues are added to its C-terminus.

[0011] The S-adenosylmethionine synthase described herein is derived from Escherichia coli, and its original amino acid sequence is SEQ ID NO.3.

[0012] The cross-linking reaction system contains: tyrosinase mutant 8-15 U / ml; S-adenosylmethionine synthase 10-15 U / ml; 0.20-0.30 mg / ml CuSO4·5H2O; pH 7.0-8.0; temperature 30-37℃; stirring speed 150-200 r / min; and reaction time 4-6 h.

[0013] A second objective of this invention is to provide a cross-linking enzyme for S-adenosylmethionine synthase prepared by the above-described method.

[0014] A third objective of this invention is to provide the application of the above-described S-adenosylmethionine synthase cross-linking enzyme in the catalytic preparation of S-adenosylmethionine. Under suitable catalytic reaction conditions, the cross-linking enzyme particles of S-adenosylmethionine synthase can be used for continuous multi-batch production of S-adenosylmethionine.

[0015] The catalytic reaction conditions are as follows: In 50-100 ml of 0.1 mol / L Tris-HCl buffer solution with a final concentration of 100-150 mM L-methionine, 80-150 mM sodium ATP, 150-250 mM MgCl2, 50-150 mM KCl, 100-300 mM mercaptoethanol, and 100-300 mM p-toluenesulfonate, respectively, 150-300 U of S-adenosylmethionine synthase cross-linking enzyme microparticles were mixed evenly with 10 ml of buffer solution and then added to the reaction solution. The reaction was carried out at 30-37℃, pH 7.0-8.0, and 150-200 r / min for 2-4 h until the endpoint. After the reaction was completed, the cross-linking enzyme microparticles were collected by centrifugation and the reaction was continued.

[0016] A fourth objective of this invention is to provide a tyrosinase mutant, which is a mutation of the wild-type amino acid sequence shown in SEQ ID NO.1, wherein the mutated amino acid sites include S146G and G283D.

[0017] When the mutant is reacted with tyrosine as a substrate, its reactivity is increased by 8.6 times compared with that before the mutation, and it has a better cross-linking effect.

[0018] This invention uses an ultrasonically disrupted solution of S-adenosylmethionine synthase containing a tyrosine tag as a substrate, and utilizes a tyrosinase mutant for a cross-linking reaction. Water-insoluble cross-linking enzyme particles are collected by centrifugation or filtration. Under suitable catalytic reaction conditions, these cross-linking enzyme particles catalyze the reaction of the substrate methionine and ATP to produce S-adenosylmethionine, and multiple batches can be reacted continuously.

[0019] The tyrosinase described in this invention is derived from Bacillus megaterium, with the original amino acid sequence of the enzyme being SEQ ID NO.1 and the mutant amino acid sequence being SEQ ID NO.2; the S-adenosylmethionine synthase is derived from Escherichia coli, with the original amino acid sequence being SEQ ID NO.3 and the amino acid sequence with 3 tyrosine residues added to its C-terminus being SEQ ID NO.4.

[0020] The expression vector for S-adenosylmethionine synthase and tyrosinase described in this invention is pET30a(+), and the expression host bacterium is Escherichia coli BL21(DE3).

[0021] The advantages of this invention are that the S-adenosylmethionine synthase cross-linking enzyme microparticles prepared by the method for preparing cross-linking enzymes based on tyrosinase mutants provided by this invention have high enzyme activity recovery rate, conversion yield, and can be reused in multiple batches; the S-adenosylmethionine synthase with tyrosine tag added to the C-terminus has amino acid residues exposed at the terminal that can directly participate in the cross-linking reaction of tyrosinase without affecting the activity of S-adenosylmethionine synthase; the mutant tyrosinase has higher catalytic activity for tyrosine than the wild-type tyrosinase, thus having a better cross-linking effect. Attached Figure Description

[0022] Figure 1 A roadmap for SAM synthesis;

[0023] Figure 2 This is a diagram illustrating the construction of the pET30-MAT-3Y expression vector;

[0024] Figure 3 This is an HPLC chromatogram of the production of SAM catalyzed by MAT-3Y cross-linking enzyme microparticles, specifically the detection results after the completion of the 6th batch reaction. Detailed Implementation

[0025] To further illustrate the present invention, the technical solutions provided by the present invention are described in detail below with reference to embodiments. The described embodiments are only some embodiments of the present invention, and not all embodiments. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention and without creative effort are within the scope of protection of the present invention.

[0026] Example 1: Construction of gene expression engineered strains

[0027] The amino acid sequences (SEQ ID NO.1 and SEQ ID NO.3) of the tyrosinase TYRC and S-adenosylmethionine synthase MAT involved in this invention were sent to a gene synthesis company for E. coli codon optimization and artificial synthesis of the encoding genes. These genes were cloned into the NdeI and XhoI restriction sites of the prokaryotic expression vector pET30a(+), respectively, and introduced into E. coli BL21(DE3) via electroporation to obtain the corresponding enzyme gene expression engineered strains BL21(DE3) / pET30-MAT and BL21(DE3) / pET30-TYRC. Fermentation of the engineered strains was carried out using conventional fermentation medium TB, first cultured at 37℃ and 200 r / min at OD. 600 The expression was induced for 10 h at 25 °C using a final concentration of 0.5 mM IPTG or 0.1 g / L lactose. The cells were collected and lysed, and the activity was tested. The results showed that the activity of MAT in shake flask was 0.59 U / ml, and the activity of TYRC in shake flask was 2.3 U / ml.

[0028] Example 2: Construction of S-adenosylmethionine synthase fused with a tyrosine tag

[0029] Using the pET30-MAT vector constructed in Example 1 as a template, primers MAT-1Y-F / R, MAT-2Y-F / R, MAT-3Y-F / R, and MAT-6Y-F / R (Table 1) were designed for full-plasmid PCR amplification, yielding DNA fragments of approximately 6418 bp, 6424 bp, 6430 bp, and 6448 bp in length, respectively. The PCR product fragments were recovered by column chromatography using a kit, and then the template plasmid was removed by DpnI digestion. The resulting fragments were transformed into *E. coli* BL21(DE3), and an appropriate amount of bacterial cells were plated on agarose plates containing 50 μg / ml kanamycin. Then, each single-clone strain was selected and sent to a sequencing company for sequencing analysis to obtain the expression vectors pET30-MAT-1Y, pET30-MAT-2Y, and pET30-MAT-3Y (…). Figure 2 ), pET30-MAT-6Y and the corresponding BL21(DE3) expression strain.

[0030] Table 1: Primer sequences for constructing the MAT expression vector

[0031]

[0032]

[0033] Example 3: Construction of a tyrosinase mutant library

[0034] Using pET30-TYRC plasmid as a PCR template, the TYRC gene was amplified by error-prone PCR using universal T7F / R primers (primer sequences: T7F: 5'-TAATACGACTCACTATAGGG-3' and T7R: 5'-GCTAGTTATTGCTCAGCGG-3'). The Mg content in the PCR amplification reaction system was adjusted. 2+ Mn 2+ The concentrations of dCTP and dTTP oligonucleotides ensure that the base mismatch rate of the mutant library is only two per thousand, which means that only one or two amino acids in a mutant are mutated.

[0035] Error-prone PCR reaction system:

[0036]

[0037] Error-prone PCR reaction conditions: First, pre-denature at 95℃ for 5 min; then denature at 94℃ for 30 s, anneal at 56℃ for 1 min, extend at 72℃ for 1.5 min, for a total of 25 cycles; finally, extend at 72℃ for 10 min.

[0038] Two μL of the error-prone PCR product was sampled and analyzed by agarose gel electrophoresis. After verification, the product was purified using a PCR product purification kit. At 37°C, the purified PCR product and the prokaryotic expression vector pET30a(+) were double-digested with NdeI and XhoI restriction endonucleases, respectively. The digested products were recovered from the gel (the recovered PCR product fragment was approximately 1200 bp, and the recovered pET30a(+) fragment was approximately 5400 bp). The mixture was then combined with the pET30a(+) prokaryotic expression vector at a molar ratio of 3:1, and T4 DNA ligase was added. The mixture was incubated overnight at 16°C. The next day, the ligation product was transformed into *E. coli* BL21(DE3) using electroporation to construct an engineered bacterium, resulting in a large-capacity random mutant library.

[0039] Example 4: High-throughput screening of tyrosinase mutant libraries

[0040] Because tyrosine substrates, in the presence of oxygen, are oxidized by tyrosinase to form quinones, resulting in a noticeable color change, and because dopaquinone exhibits a specific absorption peak at 475 nm, a high-throughput screening method was established. The deeper the color of the reaction solution and the higher the A475 absorbance, the better the enzyme's properties and the higher its activity, thus enabling the screening of favorable clones. The specific method used for high-throughput screening is as follows:

[0041] Using sterilized toothpicks, carefully pick single colonies from the mutant library (one colony per toothpick) and inoculate them into different wells of a 96-well cell culture plate (each well already containing LB liquid medium with 50 μg / ml kanamycin). Incubate the 96-well cell culture plate at 37°C and 700 rpm for 6 hours in a constant-temperature shaker. Then, using an 8-channel pipette, transfer 50 μL of the culture to a new 96-well plate as seed culture. Add lactose to each well to a final concentration of 1% (m / v) and incubate at 25°C and 250 rpm for 8 hours. After induction, freeze the 96-well cell culture plate at -86°C for 2 hours, then allow it to stand at room temperature for half an hour. Centrifuge at 4000 rpm and 4°C for 20 minutes, and collect 50 μL of the supernatant from each well. Add 100 μL of reaction solution (Tyrosine concentration: 5-10 mg / mL, pH 7.5 Tris-HCl buffer) to 50 μL of supernatant in each well, and incubate at 37°C for 30 min. Observe the color change and analyze it using a microplate reader (detection wavelength 475 nm). Select wells with high absorbance values ​​for further analysis and verification.

[0042] Approximately 50,000 clones were screened through initial and secondary screening. Enzyme activity was then determined (the method for determining the activity of tyrosinase and its mutants is as follows: Prepare 10 mM tyrosine substrate in 0.1 mol / L, pH 7.5 Tris-HCl buffer and preheat to 30°C; in a 1 cm quartz cuvette, add 4 mL of the preheated 0.01 mol / L tyrosine solution and 0.1 mL of 0.01 mol / L copper sulfate solution, mix thoroughly, zero at 475 nm, then add 0.1 mL of enzyme solution, mix quickly, and begin the measurement. Record the absorbance (A475) every 10 seconds. Enzyme activity unit: at 30°C and pH 7.5, the absorbance at OD475 is measured in units of 10 mM. 475The amount of enzyme required when the nm change value is 0.001 is defined as one unit (U). HPLC analysis was performed using an Agient liquid chromatograph with a 5C18-AR-II column (250 mm × 4.6 mm, 5 μm); column temperature: 40 ℃; injection volume: 20 μL; detection wavelength: 280 nm; mobile phase: A: 50 mmol / L phosphate; B: acetonitrile-ethyl acetate (7:1, V / V); flow rate: 1.0 mL / min; elution program: 0-25 min, 82%-68% A phase). Sequencing analysis yielded a mutant strain with 8.6-fold increased activity against the substrate tyrosine compared to the wild-type TYRC, named TYRC-mu. This mutant strain generated two new mutation sites based on the original sequence: serine at position 146 (Ser) was mutated to glycine (Gly) and glycine at position 283 (Gly) was mutated to aspartic acid (Asp). When used for MAT protein cross-linking reactions, this mutant enzyme exhibited a better cross-linking rate than the wild-type enzyme, resulting in approximately a 1-fold increase in MAT enzyme activity recovery (Table 2).

[0043] Table 2: Enzymatic properties of tyrosinases and mutants

[0044]

[0045] Note: MAT enzyme activity recovery rate refers to the data calculated by adding equal volumes of TYRC and TYRC-mu enzyme solutions in the same reaction system.

[0046] Example 5: Determination of S-adenosylmethionine synthase activity

[0047] The reaction substrate composition was: 10 mmol / L adenosine triphosphate (ATP), 30 mmol / L L-methionine, 20 mmol / L magnesium chloride hexahydrate, 100 mmol / L pH 8.0 Tris-HCl buffer, and 30 mmol / L 1,4-butanedisulfonate.

[0048] Enzyme activity determination of fermentation broth: Take 50 mL of fermentation broth, centrifuge at 12000 r / min, discard the supernatant and collect the cells. Resuspend the cells in 50 mL of physiological saline and sonicate them (3 s for each sonication, 2 s interval, 99 sonications, 500 W power). After completion, take 2 mL of the disruption solution and add the reaction substrate (adjust the volume to 20 mL with physiological saline). Control the temperature at 30℃, stir at 150 r / min, and react for 10 min. After completion, take 1 mL of the reaction solution and determine the enzyme activity by HPLC.

[0049] Assay for immobilized enzyme activity: Accurately weigh 0.2-1 g of immobilized enzyme, add it to the reaction substrate (make up to 20 mL with physiological saline), control the temperature at 30℃, stir at 150 r / min, react for 10 min, take 1 mL of sample after completion, and determine the enzyme activity by HPLC.

[0050] The specific HPLC determination conditions are as follows:

[0051] Chromatographic column: Diamonsil C18 (250mm × 4.6mm, 5μm)

[0052] Mobile phase: Dissolve 6.8g KH2PO4 in 1000ml water, adjust the pH to 2.5 with H3PO4, take out 950ml and add 50ml methanol to obtain the mobile phase.

[0053] Standard solution: Accurately weigh 20-25 mg of S-adenosylmethionine standard, dissolve it in the mobile phase and dilute to 100 ml in a volumetric flask, shake well and filter.

[0054] Detection temperature: 30℃

[0055] Detection flow rate: 1.0 ml / min

[0056] Detection wavelength: 210nm

[0057] Injection volume: 20 μl

[0058]

[0059]

[0060] Enzyme activity unit: The amount of enzyme required to generate 1 μmol of SAM per minute at a temperature of 30℃ and a pH of 8.0 is defined as one unit (U).

[0061] W standard: Weighing of S-adenosylmethionine standard, mg;

[0062] p-label: S-adenosylmethionine standard content, %;

[0063] Sample A: Peak area of ​​S-adenosylmethionine in the sample determined by HPLC;

[0064] Standard A: Peak area of ​​S-adenosylmethionine determined by HPLC of standard;

[0065] M: S-adenosylmethionine molecular weight;

[0066] T: Reaction time, in minutes;

[0067] V: Sample volume of enzyme solution, ml;

[0068] W: Immobilized enzyme weight, g.

[0069] Example 6: Preparation of S-adenosylmethionine synthase cross-linking enzyme microparticles

[0070] S-adenosylmethionine synthase expression strains BL21(DE3) / pET30-MAT, pET30-MAT-1Y, pET30-MAT-2Y, pET30-MAT-3Y, and pET30-MAT-6Y were subjected to shake-flask fermentation. The enzyme activity of the four strains was measured according to the MAT fermentation broth assay method described in Example 5. The results are summarized below:

[0071] Table 3: Activity analysis of different MAT expression strains

[0072]

[0073] As shown in Table 3, after adding 1, 2, 3 and 6 tyrosine residues to the C-terminus of MAT, the shake-flask fermentation activity of MAT with 1-3 tyrosine residues was not affected, while the shake-flask fermentation activity of MAT with 6 tyrosine residues was reduced by about 50%, which affected the spatial structure of the protein. Therefore, MAT-3Y was selected for the preparation and production application of cross-linking enzyme microparticles in this invention.

[0074] The original strain BL21(DE3) / pET30-MAT and the tyrosine-tagged expression strain pET30-MAT-3Y were fermented in 2000 ml shake flasks respectively. The cells were collected by centrifugation, and 120 ml of a suspension with a bacterial concentration of 500 g / L (the bacterial concentration refers to the percentage of weight after centrifugation at 10000 r / min) was prepared by adding 0.1 mol / L Tris-HCl buffer at pH 7.5. The suspension was then subjected to ultrasonic disruption and centrifugation to obtain the disrupted mixed sample and the clear sample after centrifugation to remove the precipitate. The activity of the mixed sample and the clear sample was detected. The results are shown in Table 4. There was no significant difference in the total activity of the mixed sample and the clear sample after strain disruption, indicating that the precipitated protein after centrifugation was inactive, and MAT and MAT-3Y were almost entirely soluble expression. Therefore, the next cross-linking experiment selected the broken sample mixture as the research object. 12 ml (1320 U) of tyrosinase mutant TYRC-mu and 30 mg CuSO4·5H2O were added to 120 ml of the above two enzyme mixtures, and the mixture was reacted at 37℃ and 200 r / min for 4 h. After the reaction was completed, the enzyme activity of the clear liquid and the lower precipitate was detected by centrifugation (the cross-linking enzyme particles were directly formed by centrifugation precipitation). The results are shown in Table 5.

[0075] Table 4: Vitality assay of MAT and MAT-3Y shake flask concentrates

[0076]

[0077] Table 5: Preparation of MAT and MAT-3Y cross-linking enzyme microparticles

[0078] name Volume / weight vitality Yield (%) MAT Clear Liquid 115ml 7.45 U / mL 52.88 MAT precipitate (cross-linked enzyme microparticles) 30g 0.52U / g 0.96 MAT-3Y Cleansing Solution 110ml 3.32 U / mL 23.05 MAT-3Y precipitate (cross-linked enzyme microparticles) 32g 25.48 U / g 51.47

[0079] As shown in Table 5, the MAT-3Y cross-linked enzyme microparticles obtained by the cross-linking reaction had an activity of 25.48 U / g and an immobilization activity yield of 51.47%. In contrast, the original enzyme MAT had 52.88% active protein that did not undergo cross-linking. The remaining enzyme was mostly inactivated by the cross-linking action of tyrosinase, with an activity yield of only 0.96%. This indicates that adding three tyrosine residues to the C-terminus of MAT can significantly improve the cross-linking efficiency between MAT and the tyrosinase mutant, and the enzyme activity loss rate after cross-linking is relatively small.

[0080] Example 7: Application of S-adenosylmethionine synthase cross-linking enzyme microparticles in the production of SAM

[0081] In 50 ml of 0.1 mol / L, pH 8.0 Tris-HCl buffer, reaction solutions were prepared with final concentrations of 110 mM L-methionine, 104 mM sodium ATP, 184 mM MgCl2, 100 mM KCl, 200 mM mercaptoethanol, and 150 mM p-toluenesulfonate. 14.1 ml (191 U) of MAT mixture and 7.5 g (191 U) of MAT-3Y cross-linking enzyme microparticles (mixed thoroughly with 10 ml buffer) were added to the reaction solutions. The mixture was stirred at 30 °C, pH 8.0, and 200 rpm for 2–4 h until the endpoint. After the reaction, the cross-linking enzyme microparticles were collected by centrifugation and the reaction was repeated for the next batch. This process was repeated multiple times to examine the production of S-adenosylmethionine and the residual enzyme activity after each batch of reaction. The results are as follows:

[0082] Table 6: Application Validation of SAM Production from MAT-3Y Crosslinker Microparticles

[0083]

[0084] Table 7: Application Validation of SAM Production from MAT Mixtures

[0085]

[0086] As shown in Tables 6 and 7 above, the SAM yield of the uncrosslinked MAT mixture was 51.27% when converting to S-adenosylmethionine. However, after six consecutive batches of S-adenosylmethionine production, the MAT-3Y crosslinked enzyme microparticles maintained a stable conversion yield of around 51%, with 85% enzyme activity retained. This indicates that the conversion properties of the MAT-3Y crosslinked enzyme microparticles are comparable to those of the MAT liquid enzyme. The greater advantage lies in its ability to be reused in multiple batches, reducing enzyme production costs. It is expected that with a suitable extension of the reaction time, several more batches can be carried out, demonstrating significant industrial application value.

[0087] The original amino acid sequence of tyrosinase derived from Bacillus megaterium:

[0088] SEQ ID NO.1

[0089] MSNKYRVRKNVLHLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPIKDFIVDTGPFAAGRWTTIDEQGNPSGG LKRNFGATKEAPTLPTRDDVLNALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIIHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS

[0090] Amino acid sequence of tyrosinase mutant derived from Bacillus megaterium:

[0091] SEQ ID NO.2

[0092] MSNKYRVRKNVLHLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPIKDFIVDTGPFAAGRWTTIDEQGNPGGG LKRNFGATKEAPTLPTRDDVLNALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIIHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLDYVYDIELRKSKRSS

[0093] The original amino acid sequence of S-adenosylmethionine synthase from Escherichia coli:

[0094] SEQ ID NO.3

[0095] MAKHLFTSESVSEGHPDKIADQISDAVLDAILEQDPKARVACETYVKTGMVLVGGEITTSAWVDIEEITRNTVREIGYVHSDMGFDANSCAVLSAI GKQSPDINQGVDRADPLEQGAGDQGLMFGYATNETDVLMPAPITYAHRLVQRQAEVRKNGTLPWLRPDAKSQVTFQYDDGKIVGIDAVVLSTQHSE EIDQKSLQEAVMEEIIKPILPAEWLTSATKFFINPTGRFVIGGPMGDCGLTGRKIIVDTYGGMARHGGGAFSGKDPSKVDRSAAYAARYVAKNIVAAGLADRCEIQVSYAIGVAEPTSIMVETFGTEKVPSEQLTLLVREFFDLRPYGLIQMLDLLHPIYKETAAYGHFGREHFPWEKTDKAQLLRDAAGLK

[0096] The amino acid sequence of S-adenosylmethionine synthase with 3 tyrosine residues added to its C-terminus is as follows:

[0097] SEQ ID NO.4

[0098] MAKHLFTSESVSEGHPDKIADQISDAVLDAILEQDPKARVACETYVKTGMVLVGGEITTSAWVDIEEITRNTVREIGYVHSDMGFDANSCAVLSAIGKQSPDINQGVDRADPLEQGAGDQGLMFGYATNETDVLMPAPITYAHRLVQRQAEVRKNGTLPWLRPDAKSQVTFQYDDGKIVGIDAVVLSTQHSEEIDQKSLQEAVMEEIIKPILPAEWLTSATKFFINPTGRFVIGGPMGDCGLTGRKIIVDTYGGMARHGGGAFSGKDPSKVDRSAAYAARYVAKNIVAAGLADRCEIQVSYAIGVAEPTSIMVETFGTEKVPSEQLTLLVREFFDLRPYGLIQMLDLLHPIYKETAAYGHFGREHFPWEKTDKAQLLRDAAGLKYYY

Claims

1. A method for preparing cross-linking enzymes based on tyrosinase mutants, characterized in that, Using an enzyme with a tyrosine tag as a substrate, a tyrosinase mutant is added to obtain a cross-linking enzyme; the tyrosinase mutant is a tyrosinase mutant with mutant amino acid sites S146G and G283D obtained by mutating the wild-type amino acid sequence shown in SEQ ID NO.

1. The enzyme with the added tyrosine tag is an S-adenosylmethionine synthase with three tyrosine residues added to its C-terminus. The S-adenosylmethionine synthase described herein is derived from Escherichia coli, and its original amino acid sequence is SEQ ID NO.

3.

2. The method of claim 1, wherein, The cross-linking reaction system contains: tyrosinase mutant 8-15 U / ml; S-adenosylmethionine synthase 10-15 U / ml; 0.20-0.30 mg / ml CuSO4·5H2O; pH 7.0-8.0; temperature 30-37℃; stirring speed 150-200 r / min; and reaction time 4-6 h.

3. The cross-linking enzyme of S-adenosylmethionine synthase prepared by the method according to any one of claims 1-2.

4. The application of the cross-linking enzyme according to claim 3 in the catalytic preparation of S-adenosylmethionine.

5. Use according to claim 4, characterized in that, The catalytic reaction conditions are as follows: In 50-100 ml of 0.1 mol / L Tris-HCl buffer solution with a final concentration of 100-150 mM L-methionine, 80-150 mM sodium ATP, 150-250 mM MgCl2, 50-150 mM KCl, 100-300 mM mercaptoethanol, and 100-300 mM p-toluenesulfonate, respectively, 150-300 U of S-adenosylmethionine synthase cross-linking enzyme microparticles were mixed evenly with 10 ml of buffer solution and then added to the reaction solution. The reaction was carried out at 30-37℃, pH 7.0-8.0, and 150-200 r / min for 2-4 h until the endpoint. After the reaction was completed, the cross-linking enzyme microparticles were collected by centrifugation and the reaction was continued.

6. A tyrosinase mutant characterized in that it is The wild-type amino acid sequence shown in SEQ ID NO.1 was mutated at amino acid sites S146G and G283D.

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