A composite biological enzyme system and a method for efficiently catalyzing synthesis of melanin and application thereof

Abstract

This invention belongs to the field of metabolic engineering technology, specifically relating to a composite enzyme system and its efficient catalytic synthesis method and application of melanin. Using catechol and pyruvate as substrates, this invention obtains a highly efficient composite enzyme system through molecular modification techniques involving triple site-directed mutagenesis of tyrosinase and laccase glycosylation-surface charge dual modification. Simultaneously, a segmented temperature-controlled catalytic process combined with dynamic substrate replenishment, adapted to the characteristics of the modified enzyme, is designed to synergistically solve the technical challenges of high-concentration substrate inhibition, low enzyme catalytic efficiency, and product polymerization lag in existing enzyme catalysis technologies at both the molecular and process levels. The modified composite enzyme system, combined with an optimized process, achieves a melanin concentration as high as 120 g / L, with a substrate conversion rate of 57.14%, representing a yield increase of over 130% compared to the unmodified enzyme catalytic system. The synthesis method of this invention features mild reaction conditions and strong process controllability, meeting the large-scale demand for high-concentration melanin in multiple fields and possessing promising industrial application prospects.

Description

Technical Field

[0001] This invention belongs to the field of metabolic engineering technology, specifically relating to a composite biological enzyme system and its efficient catalytic synthesis of melanin and its application. Background Technology

[0002] Melanin is a biological pigment widely found in human skin, retina, pia mater, and mucous membranes. It plays several important physiological roles in the body, primarily involving photoprotection, visual regulation, and immune regulation. Melanin not only absorbs UVB (280-315nm) and UVA (315-400nm), reducing direct damage to skin cells from ultraviolet radiation, but it also reduces the formation of thymine dimers by absorbing and scattering UV rays, thereby decreasing the risk of skin cancers such as melanoma. Furthermore, melanin possesses excellent semiconductor properties and biocompatibility, showing great potential in functional materials and biomedicine.

[0003] Melanin is formed by the oxidative polymerization of tyrosine as a precursor through a series of enzymatic reactions. Currently, melanin can be produced using chemical synthesis, microbial fermentation, and enzyme catalysis. Among these methods, enzyme catalysis offers advantages such as mild reaction conditions, green sustainability, high product specificity and superior performance, good biocompatibility, low safety risks, and short batch times.

[0004] However, existing enzyme catalysis methods suffer from two main drawbacks: low catalytic efficiency and substrate inhibition in high-concentration substrate environments, which suppresses enzyme activity and limits efficient melanin production; and a mismatch between process and enzyme characteristics, as traditional isothermal processes cannot simultaneously address the needs of both oxidation and polymerization reactions, further restricting melanin yield increases. Therefore, improving enzyme performance through molecular modification, combined with appropriate process design, has become crucial for significantly raising the barrier to melanin production. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a composite bio-enzyme system and its method and application for the efficient catalytic synthesis of melanin. This invention uses catechol and pyruvate as substrates, and achieves efficient melanin synthesis through a composite bio-enzyme system.

[0006] To achieve the above objectives, the technical solution adopted by the present invention includes: In a first aspect, the present invention provides a method for preparing a complex bio-enzyme system for the efficient catalytic synthesis of melanin, comprising the following steps: (1) Site-directed mutagenesis of amino acid sites was performed on the original tyrosinase to obtain recombinant mutant tyrosinase; the site-directed mutagenesis sites were selected from any one or more of the following: tyrosinase 302nd lysine was mutated to arginine, 423rd serine was mutated to threonine, and 253rd glutamic acid was mutated to aspartic acid. (2) The laccase was subjected to a dual chemical modification treatment of glycosylation and surface charge to obtain glycosylated modified laccase; (3) The recombinant mutant tyrosinase obtained in step (1) is combined with polystyrene-Ni 2+ Mesoporous silica is mixed and coupled to obtain enzyme coupling mixture I; the glycosylated modified laccase obtained in step (2) is mixed and immobilized with chitosan to obtain enzyme immobilization mixture II; mixture I and mixture II are mixed to obtain the composite biological enzyme system.

[0007] This invention addresses existing problems such as low catalytic efficiency in melanin synthesis, easy accumulation of intermediate products inhibiting enzyme activity, and poor adaptability of single-enzyme catalytic systems. It constructs a composite biological enzyme system from three dimensions: enzyme molecule modification, enzyme chemical modification, and immobilization coupling and compounding. Each technical link has a clear division of labor and synergistic effect, which comprehensively optimizes the catalytic system for melanin synthesis.

[0008] In the site-directed mutagenesis of tyrosinase, this invention combines the spatial structural characteristics of Streptomyces tyrosinase and precisely selects key residues in the substrate binding pocket, Lys302 (corresponding to lysine at position 302 in the original tyrosinase), Ser423 (serine at position 423), an auxiliary site in the active center, and Glu253 (glutamate at position 253), a proton transfer-related site, as mutation sites. Using overlap extension PCR technology, Lys302 is mutated to arginine (K302R), Ser423 to threonine (S423T), and Glu253 to aspartic acid (E253D), to optimize the spatial conformation of the enzyme molecule, enhance the binding ability of the enzyme to catechol substrates, and improve the regulation of the catalytic process and the proton transfer efficiency of reaction intermediates. This significantly improves the catalytic activity and reaction rate of tyrosinase from the enzyme molecule itself, solving the core problems of weak substrate affinity and insufficient catalytic efficiency of natural tyrosinase.

[0009] In the dual modification of laccase by glycosylation and surface charge, the surface charge characteristics of laccase are altered after modification. The electrostatic repulsion with quinone intermediates is transformed into weak electrostatic attraction, doubling the concentration of quinone intermediates at the active site and effectively accelerating the conversion rate of intermediate products. This modification method allows for precise matching of the catalytic rhythm of laccase with the oxidation rate of mutant tyrosinase, completely avoiding the enzyme activity inhibition caused by the large accumulation of intermediate products during melanin synthesis, and ensuring the stable progress of continuous catalytic reactions.

[0010] In the enzyme immobilization and compounding process, two functional enzymes were directionally immobilized using different carriers: a recombinant mutant tyrosinase was coupled with polystyrene-Ni²⁺ mesoporous silica, leveraging the structural advantages of the carrier to maintain the enzyme's spatial conformation and catalytic stability; laccase was immobilized with chitosan to further enhance its environmental tolerance and reusability. The two types of immobilized enzymes were then mixed to obtain a composite bioenzyme system, achieving synergistic function between the two modified enzymes. This integrated the advantages of highly active tyrosinase and highly adaptable laccase, forming a coherent, efficient, and stable integrated catalytic system.

[0011] Overall, this invention overcomes the technical bottlenecks of traditional single-enzyme and natural enzyme-catalyzed melanin synthesis through a combination of molecular mutation, chemical modification, and carrier immobilization. This not only significantly improves overall catalytic efficiency and eliminates product inhibition, but also enhances the practical stability of the enzyme preparation and reduces usage costs. The preparation method of this composite bio-enzyme system has a clear process route and strong targeting, providing a reliable enzyme preparation solution for the industrial-scale, efficient synthesis of melanin.

[0012] Preferably, the site-directed mutation sites are selected from: mutating lysine at position 302 of tyrosinase to arginine, serine at position 423 to threonine, and glutamic acid at position 253 to aspartic acid.

[0013] Experimental studies have revealed that, compared to single-site mutations or two-site combination mutations, simultaneous site-directed mutations at three key sites can work synergistically to create a superimposed synergistic effect, which can maximize substrate conversion and significantly increase the final melanin yield. This is a key limiting condition for ensuring the optimal catalytic performance of the composite biological enzyme system.

[0014] Preferably, in step (2), the glycosylation-surface charge dual chemical modification treatment includes: first, dissolving laccase in a buffer solution, adding maltodextrinase, and carrying out a glycosylation reaction under pH 8-9 conditions to obtain glycosylated laccase; then, mixing the glycosylated laccase with 2-aminoethanesulfonic acid to adjust the isoelectric point of the enzyme molecule to 6-7 to complete the modification.

[0015] This invention preferably utilizes maltodextrinase to glycosylate laccase under pH 8-9 conditions, followed by surface charge modification with 2-aminoethanesulfonic acid, and adjusts the isoelectric point to 6-7. This pH range is the optimal environment for the glycosylation reaction, ensuring that the glycosylation modification acts fully and uniformly on the laccase molecules, and the subsequent charge modification can also precisely control the surface charge of the enzyme. If the pH of the glycosylation reaction deviates from this range, it will directly weaken the modification effect, disrupt the interaction between laccase and quinone intermediates, and ultimately cause a decline in the substrate conversion rate of the complex enzyme system and a significant reduction in melanin production.

[0016] Preferably, the mass ratio of laccase to maltosidase is 1:(1-2); the glycosylation reaction is carried out at 28-32℃ for 3.5-4.5 h; and the glycosylated laccase and 2-aminoethanesulfonic acid are reacted at 22-28℃ for 1.5-2.5 h.

[0017] Preferably, in step (3), the recombinant mutant tyrosinase and polystyrene-Ni 2+ The mass ratio of mesoporous silica is 1:(6-10); the mass ratio of the glycosylated modified laccase to chitosan is 1:(8-12); and the volume ratio of mixture I to mixture II is (3-7):1.

[0018] More preferably, the recombinant mutant tyrosinase and polystyrene-Ni 2+ The mass ratio of mesoporous silica is 1:8; the mass ratio of the glycosylated modified laccase to chitosan is 1:10; and the volume ratio of mixture I to mixture II is 5:1.

[0019] Experimental research revealed that the above-mentioned component ratio is the optimal ratio for the binding of each enzyme with the carrier and the synergistic reaction of the two-enzyme system. This ratio can achieve uniform loading of enzyme molecules on the carrier, conformational stability, and full utilization of catalytic activity, while also ensuring precise matching of the catalytic rhythms of the two types of enzymes. If the ratio is deviated from the above ratio, it will cause uneven enzyme loading, masking of active sites, or imbalance of enzyme component ratio, directly reducing the substrate conversion rate of the complex enzyme system and resulting in a decrease in melanin production.

[0020] Preferably, in step (1), the encoding nucleotide sequence of the recombinant mutant tyrosinase is as shown in SEQ ID NO.1.

[0021] Secondly, the present invention provides a composite biological enzyme system prepared by the aforementioned preparation method.

[0022] Thirdly, the present invention provides the application of the aforementioned complex bioenzyme system in the synthesis of melanin.

[0023] Fourthly, the present invention provides a method for highly efficient catalytic synthesis of melanin, comprising the following steps: (1) Add catechol, pyruvic acid, and composite cosolvent to a pH-responsive buffer solution, and then add the composite biological enzyme system described in claim 5 to prepare an enzyme-catalyzed reaction system. (2) The reaction is carried out by segmented temperature-controlled catalytic process. The temperature is controlled at 36-38℃ in the first stage and the reaction is carried out for 4 hours. The temperature is controlled at 41-43℃ in the second stage and the reaction is carried out for another 8-10 hours.

[0024] Preferably, in step (2), catechol, pyruvic acid and composite cosolvent are added to the reaction system at the 6th and 8th hours of the reaction, respectively.

[0025] This invention employs a segmented temperature-controlled catalytic process combined with a dynamic substrate replenishment strategy. First, the catechol oxidation reaction is efficiently completed at 36-38℃, matching the optimal temperature of the mutant tyrosinase. Then, the temperature is increased to 41-43℃ to activate and modify the laccase, thereby increasing the polymerization rate and accelerating the polymerization process of quinones. Simultaneously, the substrate and composite cosolvent are replenished at regular intervals, effectively avoiding the reaction interruption caused by rapid substrate depletion. This fully leverages the catalytic performance of the adapted and modified composite enzyme system, significantly improving the efficiency of melanin synthesis.

[0026] Preferably, in step (2), the rotation speed is controlled at 400-500 r / min in the first stage, the aeration rate is 1.0-1.5 L / min, and the pH of the reaction system is maintained at 7-7.3 (to match the surface charge of the enzyme molecule); in the second stage, the rotation speed is controlled at 500-600 r / min, the aeration rate is 1.2-1.8 L / min, and the pH of the reaction system is maintained at 7.2-7.9 (to optimize the quinone binding efficiency).

[0027] Preferably, in step (1), the initial concentration of catechol in the reaction system is 200-240 g / L, the initial concentration of pyruvic acid is 190-210 g / L, the concentration of the composite cosolvent is 10-20 g / L, and the concentration of the composite enzyme system is 4-8 mg / L.

[0028] More preferably, in step (1), the initial concentration of catechol in the reaction system is 220-240 g / L, the initial concentration of pyruvic acid is 210 g / L, the concentration of the composite cosolvent is 15 g / L, and the concentration of the composite enzyme system is 6 mg / L.

[0029] Preferably, the composite cosolvent includes β-cyclodextrin, Tween 80 and PEG400, and the mass ratio of β-cyclodextrin, Tween 80 and PEG400 is (2-4):1:(1-3).

[0030] Preferably, the pH-responsive buffer solution is a 0.3-0.5 mol / L Tris-HCl-citric acid buffer solution, which also contains 3-6 g / L PVP and 1-2 g / L ascorbate glucoside.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Significantly improved enzyme molecular catalytic performance: This invention performs triple site mutation on tyrosinase and double modification on laccase, thereby optimizing the enzyme structure at the molecular level and effectively improving the overall catalytic efficiency. This lays the core technical foundation for high production and high production intensity of melanin. Compared with the unmodified natural enzyme, the catalytic effect is significantly improved.

[0032] (2) High compatibility between process and modified enzyme: The present invention adopts a process mode of segmented temperature control combined with substrate replenishment, which can accurately match the enzymatic characteristics of the modified enzyme, effectively solve the drawbacks of poor compatibility between traditional production process and enzyme performance, and ensure that the catalytic reaction proceeds stably and orderly.

[0033] (3) Excellent production efficiency and product yield: This invention combines the modified composite bio-enzyme system with the optimized catalytic process, which greatly shortens the production cycle and can ultimately achieve a melanin yield of 100-120g / L, achieving the production goal of high yield and high production intensity, and has good industrial application value. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the enzyme-catalyzed synthesis of melanin in this invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Furthermore, the raw materials used in each parallel experiment are the same.

[0037] The melanin-producing strain MEL-09 / pET28a-melC2-melC1 used in the following examples is derived from patent 2025116078536 and was provided by Nanjing Hegu Life Biotechnology Co., Ltd. The original white-rot fungus laccase was purchased from Guangdong Mingtong Biotechnology Co., Ltd.

[0038] The culture medium components involved in the following examples are as follows: LB (Luria Bertani) liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, sterilized at 121°C for 20 min.

[0039] LB (Luria Bertani) solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar powder. Sterilize at 121°C for 20 min. Cool the solid medium to about 50°C, add the required antibiotics, pour the plate, and let it solidify. Then, place it at 4°C for later use.

[0040] In the following examples, unless otherwise specified, the method for transforming plasmids into competent E. coli cells is electroporation. Electroporation includes the following steps: adding the target fragment to the corresponding engineered bacterial cells, gently mixing and adding it to an electroporation cuvette, incubating on ice for 50 min, electroporating at 2.5 kV, quickly adding pre-cooled LB liquid medium, incubating on a shaker at 37°C for 2 h, spreading 100 μL of bacterial culture onto LB solid medium, and culturing at 30°C for 24 h.

[0041] The method for preparing electrocompetent cells is as follows: (1) Pick a single colony with a pipette tip and put it into a 50 mL centrifuge tube containing 10 mL of LB liquid medium (at the same time, make a blank control for the medium and pipette tip). (2) Incubate at 37℃ and 220rpm for 14-16h. Inoculate the bacterial culture into 50mL LB liquid medium at a ratio of 1:100. Incubate at 37℃ and 220rpm for 2-3h. Measure the OD every half hour. Stop the incubation when the OD value reaches 0.6-0.8. (3) Pre-cool the bacterial solution on ice for 30 min, then aliquot the bacterial solution into 50 mL pre-cooled centrifuge cups, centrifuge at 4℃ and 4500 rpm for 10 min, discard the supernatant, suspend the precipitate in sterile water, centrifuge at 4℃ and 4500 rpm for 10 min, discard the supernatant, suspend the precipitate in 10% sterile glycerol, centrifuge at 4℃ and 4500 rpm for 10 min, discard the supernatant, add 300 μL of 10% glycerol to each centrifuge cup to suspend the precipitate, aliquot the bacterial solution into 1.5 mL centrifuge tubes at 100 μL / tube, and store in a refrigerator at 80℃.

[0042] Unless otherwise specified, the tyrosinase used in the following examples was purified using the following specific steps: (1) Preparation of crude enzyme solution: After the expression was completed, the fermentation broth of the engineered strain MEL-09 containing mutant tyrosinase was collected. After centrifugation at 4℃ and 8000r / min for 20min, the cell pellet was collected. Five volumes of lysis buffer (0.05mol / L Tris-HCl, pH 8.0, containing 1mmol / L PMSF, 5mmol / L β-mercaptoethanol, and 0.1% Triton X-100) were added to the cell pellet. The mixture was sonicated under ice bath conditions (600W power, 3s working and 5s stopping, total time 30min). After the mixture was centrifuged at 4℃ and 12000r / min for 30min, the supernatant was collected as the crude enzyme solution. (2) Ni²⁺ affinity chromatography purification: ① Preparation of chromatography column: Use HiTrap Chelating HP chromatography column (5mL, GE Healthcare), and equilibrate 5 column volumes with equilibration buffer (0.05mol / L Tris-HCl, pH 8.0, containing 50mmol / L imidazole and 0.5mol / L NaCl) at a flow rate of 10mL / min until the baseline is stable; ② Sample loading and elution of contaminating proteins: After filtering the crude enzyme solution through a 0.22 μm filter membrane, the sample was loaded at a flow rate of 5 mL / min. After the sample loading was completed, the sample was eluted with equilibration buffer and the breakthrough peak (containing contaminating proteins) was collected until the absorbance of the UV detector (280 nm) dropped below 0.05. ③ Target enzyme elution: Gradient elution was used. Elution buffer A was the equilibration buffer, and elution buffer B was 0.05 mol / L Tris-HCl (pH 8.0, containing 50 mmol / L imidazole and 0.5 mol / L NaCl). The gradient was set to increase the percentage of solution B from 0% to 30% within 0-30 min, and from 30% to 100% within 30-40 min. The flow rate was 5 mL / min, and one fraction was collected every 2 mL. The fraction containing a single target band (molecular weight approximately 62 kDa) was collected by SDS-PAGE (12% separating gel). (3) Desalting and concentration: The target component was loaded onto a HiPrep 26 / 10 Desalting column (GE Healthcare), and desalted with 0.05 mol / L Tris-HCl buffer (pH 8.0) at a flow rate of 15 mL / min. The protein peak was collected. Then, the protein was concentrated to an enzyme concentration of 10-15 mg / mL by centrifugation at 4°C and 4000 r / min using an Amicon Ultra-15 centrifuge tube (molecular weight cutoff 30 kDa, Millipore) to obtain purified mutant tyrosinase.

[0043] The steps for detecting melanin concentration are as follows: Take 1 mL of fermentation broth, centrifuge at 12000 r / min at 4℃ for 10 min, dilute the supernatant with 0.1 mM NaOH, filter through a 0.22 μm aqueous filter membrane, and then inject the sample. HPLC analysis is then performed, with the standard curve y = 10.311x + 0.011 (R0). 2 =0.9983) to obtain the melanin concentration. y is the liquid phase peak area / 1000, and x is the melanin concentration.

[0044] The detection conditions were: Agilent C18 (150mm × 4.6mm, 5μm), column temperature 25℃, and detection wavelength 254nm.

[0045] HPLC analysis procedure: 0-7 min, mobile phase A - 100%; 7-11 min, mobile phase A - 70%; 11-14 min, mobile phase A - 40%; 14-19 min, mobile phase A - 100%; Preparation method of mobile phase A: Add 15.2g of anhydrous sodium acetate to 1850mL of water, adjust the pH to 6.5 with glacial acetic acid, filter through a 0.22μm water membrane, and finally add 140mL of acetonitrile and sonicate for 30min.

[0046] Mobile phase B: 80% acetonitrile.

[0047] The standard curve preparation method is as follows: High-purity melanin standard was accurately weighed and diluted to volume with ultrapure water to prepare a 0.1 g / L standard stock solution. This stock solution was then serially diluted to nine concentration gradients: 0.01, 0.02, 0.03, 0.04, and 0.09 g / L. HPLC analysis was performed under the aforementioned conditions. The standard curve was obtained by plotting the standard concentration on the x-axis and the corresponding peak area / 1000 on the y-axis: y = 10.311x + 0.011 (R² + π / 4)². 2 =0.9983).

[0048] Substrate conversion rate calculation method: It is calculated based on the ratio of melanin concentration to pyruvate concentration in the fermentation broth.

[0049] The primers used in the following examples are shown in Table 1.

[0050] Table 1 Example 1 This embodiment provides a genetically engineered bacterium MEL-09 / pET28a-melC2-melC1 mut The construction method and purification method for the mutant tyrosinase are as follows: The engineered bacteria MEL-09 / pET28a-melC2-melC1 mut A strain of Escherichia coli MEL-09 / pET28a-melC2-melC1 (BL21-09) capable of producing 5.6 g / L melanin in a shake flask was used as the starting strain. MEL-09 / pET28a-melC2-melC1 is derived from the patent: A genetically engineered bacterium producing melanin and its construction method and application (application number: 202511607853.6), which was constructed by Nanjing Hegu Life Biotechnology Co., Ltd.

[0051] Overlap extension PCR was used to mutate Lys302 to arginine using primers K302R upstream and downstream (K302R); Ser423 to threonine using primers S423T upstream and downstream (S423T); and Glu253 to aspartic acid using primers E253D upstream and downstream (E253D). After successful mutation, the mutated *Streptomyces* tyrosinase gene (nucleotide sequence shown in SEQ ID NO.1) was cloned into the expression vector pET28a. The mutant plasmids were then transformed into BL21-09 competent cells via electroporation. The transformation products were plated on LB agar plates containing 50 μg / mL streptomycin and incubated at 37°C for 14 h. Single colonies were then picked, and after sequencing by a biotechnology company, the genetically engineered strain was identified as MEL-09 / pET28a-melC2-melC1. mut Successfully constructed. The successfully constructed engineered strain MEL-09 was cultured in LB medium containing 50 μg / mL streptomycin resistance at 30°C with shaking for 72 h to obtain the fermentation broth of engineered strain MEL-09 containing mutant tyrosinase. The mutant tyrosinase was then purified according to the enzyme purification method described above.

[0052] Example 2 This embodiment provides a method for preparing a complex biological enzyme system, including the following steps: (1) Glycosylation modification: White rot fungus laccase was dissolved in 0.1 mol / L NaHCO3 buffer, pH was controlled at 8.5, 1.5 times the mass of maltosidase was added, and the reaction was carried out at 30℃ for 4 h. Free sugar was removed by Sephadex G-75 chromatography to obtain glycosylated laccase. (2) Surface charge adjustment: 0.2 mol / L 2-aminoethanesulfonic acid was added to the glycosylated laccase solution and reacted at 25°C for 2 h to raise the isoelectric point (pI) of the enzyme molecule to 6.5, thus obtaining glycosylated modified laccase; (3) Preparation of enzyme conjugate: The mutant tyrosinase obtained in Example 1 was coupled with polystyrene-Ni²⁺ mesoporous silica at a mass ratio of 1:8 to obtain enzyme coupling mixture I; the glycosylated modified laccase obtained in step (2) was fixed with chitosan at a mass ratio of 1:10 to obtain enzyme immobilization mixture II; mixture I and mixture II were mixed at a volume ratio of 5:1 to obtain the composite biological enzyme system.

[0053] Example 3 This embodiment provides a method for preparing a complex biological enzyme system, including the following steps: (1) Glycosylation modification: White rot fungus laccase was dissolved in 0.1 mol / L NaHCO3 buffer, pH was controlled at 8, 1 mass of maltosidase was added, and the reaction was carried out at 28℃ for 4.5 h. Free sugar was removed by Sephadex G-75 chromatography to obtain glycosylated laccase. (2) Surface charge adjustment: 0.2 mol / L 2-aminoethanesulfonic acid was added to the glycosylated laccase solution and reacted at 22℃ for 2.5 h to raise the isoelectric point (pI) of the enzyme molecule to 6, thus obtaining glycosylated modified laccase; (3) Preparation of enzyme conjugate: The mutant tyrosinase obtained in Example 1 was coupled with polystyrene-Ni²⁺ mesoporous silica at a mass ratio of 1:6 to obtain enzyme coupling mixture I; the glycosylated modified laccase obtained in step (2) was fixed with chitosan at a mass ratio of 1:8 to obtain enzyme immobilization mixture II; mixture I and mixture II were mixed at a volume ratio of 3:1 to obtain the composite biological enzyme system.

[0054] Example 4 This embodiment provides a method for preparing a complex biological enzyme system, including the following steps: (1) Glycosylation modification: White rot fungus laccase was dissolved in 0.1 mol / L NaHCO3 buffer, pH was controlled at 9, 2 times the mass of maltodextrinase was added, and the reaction was carried out at 32℃ for 3.5 h. Free sugar was removed by Sephadex G-75 chromatography to obtain glycosylated laccase. (2) Surface charge adjustment: 0.2 mol / L 2-aminoethanesulfonic acid was added to the glycosylated laccase solution and reacted at 28℃ for 1.5 h to raise the isoelectric point (pI) of the enzyme molecule to 7, thus obtaining glycosylated modified laccase; (3) Preparation of enzyme conjugate: The mutant tyrosinase obtained in Example 1 was coupled with polystyrene-Ni²⁺ mesoporous silica at a mass ratio of 1:10 to obtain enzyme coupling mixture I; the glycosylated modified laccase obtained in step (2) was fixed with chitosan at a mass ratio of 1:12 to obtain enzyme immobilization mixture II; mixture I and mixture II were mixed at a volume ratio of 7:1 to obtain the composite biological enzyme system.

[0055] Example 5 This embodiment provides a highly efficient method for catalytic synthesis of melanin, wherein the composite enzyme system prepared in Example 2 is used for catalytic synthesis of melanin, and includes the following steps: (1) Add 200 g / L catechol, 190 g / L pyruvate, 10 g / L composite solubilizer (β-cyclodextrin: Tween 80: PEG400 in a mass ratio of 3:1:2), 10 g / L pH-responsive buffer and 6 mg / L complex enzyme system to a 50 L fermenter for reaction; the pH-responsive buffer is 0.5 mol / L Tris-HCl-citric acid buffer (containing 5 g / L PVP and 1.5 g / L ascorbate glucoside); (2) Set the initial reaction parameters as follows: rotation speed 500 r / min, aeration rate 1.5 L / min, pH of the reaction system automatically maintained at 7.1, temperature controlled at 36.5℃ by automatic temperature control, and reaction under these conditions for 4 hours; (3) After 4 hours, the reaction parameters were set to 550 r / min rotation speed, 1.5 L / min aeration rate, and the pH of the reaction system was automatically maintained at 7.5. The temperature was controlled at 42℃ by automatic temperature control. The reaction was carried out under these conditions for 8 hours. (4) Manually add 40 g / L catechol, 20 g / L pyruvic acid, and 10 g / L composite cosolvent at 6 h and 8 h of reaction respectively; (5) The reaction period is 12 hours. After the reaction is completed, the melanin production is measured.

[0056] Example 6 This embodiment provides a highly efficient method for catalytic synthesis of melanin, wherein the composite enzyme system prepared in Example 2 is used for catalytic synthesis of melanin, and includes the following steps: (1) Add 220 g / L catechol, 210 g / L pyruvate, 15 g / L composite solubilizer (β-cyclodextrin: Tween 80: PEG400 in a mass ratio of 3:1:2), 10 g / L pH-responsive buffer and 6 mg / L composite enzyme system to a 50 L fermenter for reaction; the pH-responsive buffer is 0.5 mol / L Tris-HCl-citric acid buffer (containing 5 g / L PVP and 1.5 g / L ascorbate glucoside); (2) Set the initial reaction parameters as follows: rotation speed 500 r / min, aeration rate 1.5 L / min, pH of the reaction system automatically maintained at 7.1, temperature controlled at 36.5℃ by automatic temperature control, and reaction under these conditions for 4 hours; (3) After 4 hours, the reaction parameters were set to 550 r / min rotation speed, 1.5 L / min aeration rate, and the pH of the reaction system was automatically maintained at 7.5. The temperature was controlled at 42℃ by automatic temperature control. The reaction was carried out under these conditions for 8 hours. (4) Manually add 40 g / L catechol, 20 g / L pyruvic acid, and 10 g / L composite cosolvent at 6 h and 8 h of reaction respectively; (5) The reaction period is 12 hours. After the reaction is completed, the melanin production is measured.

[0057] Example 7 This embodiment provides a highly efficient method for catalytic synthesis of melanin, wherein the composite enzyme system prepared in Example 2 is used for catalytic synthesis of melanin, and includes the following steps: (1) Add 240 g / L catechol, 210 g / L pyruvate, 15 g / L composite solubilizer (β-cyclodextrin: Tween 80: PEG400 mass ratio of 3:1:2), 10 g / L pH-responsive buffer and 6 mg / L composite enzyme system to a 50 L fermenter for fermentation; the pH-responsive buffer is 0.5 mol / L Tris-HCl-citric acid buffer (containing 5 g / L PVP and 1.5 g / L ascorbate glucoside); (2) Set the initial reaction parameters as follows: rotation speed 500 r / min, aeration rate 1.5 L / min, pH of the reaction system automatically maintained at 7.1, temperature controlled at 36.5℃ by automatic temperature control, and reaction under these conditions for 4 hours; (3) After 4 hours, the reaction parameters were set to 550 r / min rotation speed, 1.5 L / min aeration rate, and the pH of the reaction system was automatically maintained at 7.5. The temperature was controlled at 42℃ by automatic temperature control. The reaction was carried out under these conditions for 8 hours. (4) Manually add 40 g / L catechol, 20 g / L pyruvic acid and 10 g / L composite cosolvent at 6 h and 8 h of fermentation, respectively; (5) The reaction period is 12 hours. After the reaction is completed, the melanin production is measured.

[0058] Example 8 This embodiment provides a highly efficient catalytic synthesis method for melanin, which differs from Example 7 only in that: no additional catechol, pyruvic acid, and composite cosolvent are added during the fermentation reaction, while the rest of the operation process is the same as in Example 7.

[0059] Example 9 This embodiment provides a highly efficient method for catalytic synthesis of melanin, wherein the composite enzyme system prepared in Example 3 is used for catalytic synthesis of melanin, and includes the following steps: (1) Add 240 g / L catechol, 210 g / L pyruvate, 15 g / L composite solubilizer (β-cyclodextrin: Tween 80: PEG400 in a mass ratio of 2:1:3), 10 g / L pH-responsive buffer and 6 mg / L complex enzyme system to a 50 L fermenter for reaction; the pH-responsive buffer is 0.3 mol / L Tris-HCl-citric acid buffer (containing 3 g / L PVP and 2 g / L ascorbate glucoside); (2) Set the initial reaction parameters as follows: rotation speed 400 r / min, aeration rate 1 L / min, pH of the reaction system automatically maintained at 7, temperature controlled at 36℃ by automatic temperature control, and reaction under these conditions for 4 h. (3) After 4 hours, the reaction parameters were set to 500 r / min rotation speed, 1.2 L / min aeration rate, and the pH of the reaction system was automatically maintained at 7.2. The temperature was controlled at 41℃ by automatic temperature control. The reaction was carried out under these conditions for 8 hours. (4) Manually add 40 g / L catechol, 20 g / L pyruvic acid, and 10 g / L composite cosolvent at 6 h and 8 h of reaction respectively; (5) The reaction period is 12 hours. After the reaction is completed, the melanin production is measured.

[0060] Example 10 This embodiment provides a highly efficient method for catalytic synthesis of melanin, wherein the composite enzyme system prepared in Example 4 is used for catalytic synthesis of melanin, and includes the following steps: (1) Add 240 g / L catechol, 210 g / L pyruvate, 15 g / L composite solubilizer (β-cyclodextrin: Tween 80: PEG400 in a mass ratio of 4:1:1), 10 g / L pH-responsive buffer and 6 mg / L complex enzyme system to a 50 L fermenter for reaction; the pH-responsive buffer is 0.5 mol / L Tris-HCl-citric acid buffer (containing 6 g / L PVP and 1 g / L ascorbate glucoside); (2) Set the initial reaction parameters as follows: rotation speed 500 r / min, aeration rate 1 L / min, pH of the reaction system automatically maintained at 7.3, temperature controlled at 38℃ by automatic temperature control, and reaction under these conditions for 4 hours; (3) After 4 hours, the reaction parameters were set to 600 r / min rotation speed, 1.8 L / min aeration rate, and the pH of the reaction system was automatically maintained at 7.9. The temperature was controlled at 43℃ by automatic temperature control. The reaction was carried out under these conditions for 8 hours. (4) Manually add 40 g / L catechol, 20 g / L pyruvic acid, and 10 g / L composite cosolvent at 6 h and 8 h of reaction respectively; (5) The reaction period is 12 hours. After the reaction is completed, the melanin production is measured.

[0061] Example 11 This embodiment provides a method for synthesizing melanin, which differs from Example 7 only in that the composite enzyme system used is different. In this comparative example, the mutant tyrosinase used in the composite enzyme system undergoes only a single-point mutation at K302R, while the S423 and E253 sites remain wild-type, and the S423T and E253D mutations are not performed. The preparation process of the remaining composite enzyme system is the same as in Example 2, and the fermentation synthesis process of melanin is the same as in Example 7.

[0062] Example 12 This embodiment provides a method for synthesizing melanin, which differs from Example 7 only in that the composite enzyme system used is different. In this comparative example, the mutant tyrosinase used in the composite enzyme system is only subjected to the K302R+E253D double-point mutation and the S423T mutation is missing. The preparation process of the rest of the composite enzyme system is the same as that of Example 2, and the fermentation synthesis process of melanin is the same as that of Example 7.

[0063] Comparative Example 1 This comparative example provides a method for synthesizing melanin, which differs from Example 7 only in that the same amount of unmodified original tyrosinase and white rot fungus laccase are used to replace the composite bio-enzyme for fermentation.

[0064] Comparative Example 2 This comparative example provides a method for synthesizing melanin, which differs from Example 7 only in that: it does not use segmented temperature control for fermentation. During the fermentation reaction of the fermentation system described in this comparative example, the temperature is kept constant at 40°C throughout the process, the pH is maintained at 7.3 throughout the process, the rotation speed is 500 r / min, the aeration rate is 1.5 L / min, and the reaction cycle is 12 h. At 6 h and 8 h of the reaction, 40 g / L catechol, 20 g / L pyruvic acid and 10 g / L composite cosolvent are manually added, respectively.

[0065] Comparative Example 3 This comparative example provides a method for synthesizing melanin, which differs from Example 7 only in that the composite enzyme system used is different. In this comparative example, when preparing the composite enzyme system, the buffer solution for laccase glycosylation modification is replaced with PBS buffer solution, and the pH is controlled at 7. The preparation process of the remaining composite enzyme system is the same as that of Example 2, and the fermentation synthesis process of melanin is the same as that of Example 7.

[0066] Comparative Example 4 This comparative example provides a method for synthesizing melanin, which differs from Example 7 only in that the composite enzyme system used is different. In the preparation of the composite enzyme system in this comparative example, the mutant tyrosinase is coupled with polystyrene-Ni²⁺ mesoporous silica at a mass ratio of 1:4, and mixture I and mixture II are mixed at a volume ratio of 2:1. The preparation process of the remaining composite enzyme system is the same as that in Example 2, and the fermentation synthesis process of melanin is the same as that in Example 7.

[0067] Example of effect This effect example is based on the synthesis method described in Examples 5-12 and Comparative Examples 1-4. The melanin content in each group of samples was determined, and the substrate conversion rate of the reaction system was further calculated. The test results are shown in Table 2.

[0068] Table 2 This invention enhances catalytic efficiency at the molecular level through a triple mutation site on tyrosinase and a dual modification process using laccase, providing core technological support for high yield and high production intensity. The catalytic efficiency is significantly improved compared to the unmodified enzyme, with substrate conversion increasing by 130% after mutation. Furthermore, by precisely matching the modified enzyme characteristics through segmented temperature control and substrate replenishment, the problem of mismatch between traditional processes and enzyme performance is solved. Ultimately, by combining the above methods, a melanin yield of 120 g / L is achieved after 12 hours of reaction. Compared to traditional methods (such as Streptomyces fermentation or traditional enzyme catalysis), the engineered bacteria of this invention have significant advantages such as a short fermentation cycle (12 hours), significantly increased yield, strong process controllability, and environmental friendliness, providing an efficient and feasible solution for the large-scale production of melanin.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a complex bio-enzyme system for the efficient catalytic synthesis of melanin, characterized in that, Includes the following steps: (1) Site-directed mutagenesis of amino acid sites was performed on the original tyrosinase to obtain recombinant mutant tyrosinase; the site-directed mutagenesis sites were selected from any one or more of the following: tyrosinase 302nd lysine was mutated to arginine, 423rd serine was mutated to threonine, and 253rd glutamic acid was mutated to aspartic acid. (2) The laccase was subjected to a dual chemical modification treatment of glycosylation and surface charge to obtain glycosylated modified laccase; (3) The recombinant mutant tyrosinase obtained in step (1) is combined with polystyrene-Ni 2+ Mesoporous silica is mixed and coupled to obtain enzyme coupling mixture I; the glycosylated modified laccase obtained in step (2) is mixed and immobilized with chitosan to obtain enzyme immobilization mixture II; mixture I and mixture II are mixed to obtain the composite biological enzyme system.

2. The preparation method according to claim 1, characterized in that, In step (2), the glycosylation-surface charge dual chemical modification treatment includes: first, dissolving laccase in buffer solution, adding maltodextrinase, and carrying out glycosylation reaction under pH 8-9 conditions to obtain glycosylated laccase; then, mixing glycosylated laccase with 2-aminoethanesulfonic acid to adjust the isoelectric point of the enzyme molecule to 6-7 to complete the modification.

3. The preparation method according to claim 1, characterized in that, In step (3), the recombinant mutant tyrosinase and polystyrene-Ni 2+ The mass ratio of mesoporous silica is 1:(6-10); the mass ratio of the glycosylated modified laccase to chitosan is 1:(8-12); and the volume ratio of mixture I to mixture II is (3-7):

1.

4. The preparation method according to claim 1, characterized in that, In step (1), the encoding nucleotide sequence of the recombinant mutant tyrosinase is shown in SEQ ID NO.

1.

5. The composite bio-enzyme system prepared by the preparation method according to any one of claims 1-4.

6. The application of the complex bioenzyme system as described in claim 5 in the synthesis of melanin.

7. A method for highly efficient catalytic synthesis of melanin, characterized in that, Includes the following steps: (1) Add catechol, pyruvic acid, and composite cosolvent to a pH-responsive buffer solution, and then add the composite biological enzyme system described in claim 5 to prepare an enzyme-catalyzed reaction system. (2) The reaction is carried out by segmented temperature-controlled catalytic process. The temperature is controlled at 36-38℃ in the first stage and the reaction is carried out for 4 hours. The temperature is controlled at 41-43℃ in the second stage and the reaction is carried out for another 8-10 hours.

8. The method as described in claim 7, characterized in that, In step (1), the initial concentration of catechol in the reaction system is 200-240 g / L, the initial concentration of pyruvic acid is 190-210 g / L, the concentration of the composite cosolvent is 10-20 g / L, and the concentration of the composite enzyme system is 4-8 mg / L.

9. The method as described in claim 7, characterized in that, The composite cosolvent includes β-cyclodextrin, Tween 80 and PEG400, and the mass ratio of β-cyclodextrin, Tween 80 and PEG400 is (2-4):1:(1-3).

10. The method as described in claim 7, characterized in that, In step (2), catechol, pyruvic acid and composite cosolvent are added to the reaction system at the 6th and 8th hour of fermentation, respectively.

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