Tyrosinase mutants, immobilized enzymes and enzymatic synthesis of 5,6-dihydroxyindole

Abstract

This invention belongs to the field of enzyme catalysis. It discloses a tyrosinase mutant, an immobilized enzyme, and an enzymatically catalyzed synthesis method for 5,6-dihydroxyindole. The amino acid sequence of the tyrosinase mutant is a sequence containing at least one mutation from R209H, F197W, T17P, or Q187L in the wild-type amino acid sequence shown in SEQ ID NO.1. Research in this invention shows that by mutating the specific sites mentioned above in the wild-type sequence of SEQ ID NO.1, the catalytic activity of the enzyme can be significantly enhanced. Using this enzyme in the catalytic synthesis of 5,6-dihydroxyindole can effectively improve the reaction conversion rate.

Description

Technical Field

[0001] This invention belongs to the field of enzyme catalysis, specifically relating to the enzymatic synthesis methods of tyrosinase mutants, immobilized enzymes, and 5,6-dihydroxyindole. Background Technology

[0002] In the current hair dye market, hair dyes with aromatic amines as the main ingredient are the mainstream products. These hair dyes primarily use p-phenylenediamine as the core ingredient, and also contain alternative aromatic amines such as toluene-2,5-diamine sulfate, N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate, and resorcinol. These components mainly combine with keratin in the hair to form a pigment framework, promoting pigment penetration and thus achieving the purpose of hair coloring.

[0003] The use of aromatic amine-based agents poses significant health risks. For example, p-phenylenediamine is a recognized strong allergen; contact with it can cause symptoms such as scalp redness, itching, and blisters in sensitive individuals, and in severe cases, can lead to life-threatening conditions like facial and respiratory edema. Resorcinol and p-aminophenol are highly irritating to the eyes and oral mucosa, and can exacerbate inflammation when in contact with broken scalp. A prospective cohort study published in the *British Medical Journal* showed that long-term use of permanent hair dyes is associated with an increased risk of skin cancer, breast cancer, and lymphoma. o-Phenylenediamine and toluene-3,4-diamine, due to their proven carcinogenic and teratogenic properties, have been listed as prohibited substances in cosmetics. Therefore, there is an urgent market need to develop environmentally friendly and human-friendly hair dye agents to replace existing hair dye products.

[0004] 5,6-Dihydroxyindole (DHI), CAS No. 131-52-0, molecular formula C8H7NO2, molecular weight 149.15. As a precursor for melanin biosynthesis in organisms, it participates in the pigmentation process of skin and hair and is naturally present in animals and plants. DHI is also an intermediate for some amino acids, alkaloids, and tryptamines. It has excellent staining effects on keratin fibers and can be combined with indigo as an oxidizing or co-dyeing agent in hair dyes. It can specifically bind to hair keratin fibers, penetrating into the hair medulla to form a stable pigment complex. It has excellent wash resistance and resistance to UV degradation, and its staining durability is comparable to that of p-phenylenediamines, but without the risk of carcinogenicity or sensitization, making it an ideal reagent for hair dyeing.

[0005] However, given the unique structure of DHI, namely the highly reactive bisphenol hydroxyl groups adjacent to the benzene ring, which easily lead to side reactions and thus reduce product purity, there are still significant challenges in the mass production technology of the product.

[0006] Patent application CN113816891A indicates that there are currently several methods for synthesizing DHI, which can be categorized into benzaldehyde method, phenylacetonitrile method, and phenylethylamine method. However, these methods typically require steps such as hydroxyl protection, nitration, cyclization, and deprotection, making the process difficult to control, resulting in numerous side reactions, and the overall yield is usually only 10-50%. Furthermore, the nitration step uses a concentrated nitric acid / concentrated sulfuric acid system, which is highly corrosive and generates large amounts of acidic wastewater; the reduction step often employs high-pressure hydrogenation (Pd / C catalyst), posing an explosion risk, and the recovery of heavy metal catalysts is difficult, easily causing environmental residues, making it difficult to overcome the technological bottlenecks for mass production.

[0007] In addition, existing technologies also disclose some enzyme-catalyzed synthesis methods based on tyrosinase. For example, patent document CN119799665A discloses a tyrosinase mutant, specifically describing that the tyrosinase mutant is based on the wild-type tyrosinase shown in SEQ ID NO. 1 described in the document, except that the amino acids at positions 337 and 352 are replaced by Ile instead of phe in the wild type, thus obtaining the F337I / F352I mutation.

[0008] In summary, although existing technologies have reported the use of tyrosinases and their mutants for DHI synthesis, the different wild-type sequences and mutation methods of various mutants significantly affect the overall structure and catalytic activity of the enzymes, thus substantially impacting DHI synthesis. Further development of existing enzymatic synthesis methods for DHI is needed. Summary of the Invention

[0009] To further expand the types of tyrosinase mutants, the primary objective of this invention is to provide a novel tyrosinase mutant (also referred to as a mutant in this invention), aiming to provide a novel tyrosinase mutant that combines excellent enzymatic activity and stability.

[0010] A second objective of this invention is to provide an immobilized enzyme (also referred to as a solidified enzyme) loaded with the tyrosinase mutant.

[0011] The third objective of this invention is to provide an enzymatic synthesis method for 5,6-dihydroxyindole, which aims to improve the synthesis effect of 5,6-dihydroxyindole based on tyrosinase mutants.

[0012] A tyrosinase mutant, wherein the amino acid sequence of the tyrosinase mutant is a mutated amino acid sequence containing at least one of the following mutations: R209H, F197W, T17P, and Q187L, in the wild-type amino acid sequence shown in SEQ ID NO.1.

[0013] The present invention demonstrates that by making the specific mutations at the aforementioned specific sites in the wild-type sequence of SEQ ID NO.1, the catalytic activity of the mutant enzyme can be significantly enhanced. When used for the catalysis of DHI, it can effectively improve the reaction conversion rate and stability.

[0014] In this invention, R209H refers to the mutation of arginine (R) at position 209 to histidine (H); F197W refers to the mutation of phenylalanine (F) at position 197 to tryptophan (W); T17P refers to the mutation of threonine (T) at position 17 to proline (P); and Q187L refers to the mutation of glutamine (Q) at position 187 to leucine (L).

[0015] Preferably, the amino acid sequence of the tyrosinase mutant is an amino acid sequence containing at least two or more mutations (e.g., two-point, three-point, or four-point mutations) in the wild-type amino acid sequence shown in SEQ ID NO.1. The preferred mutant of this invention can further improve the enzyme's catalytic activity and conversion rate to the substrate. Furthermore, immobilization of the mutant, while achieving excellent activity, is also expected to improve the enzyme's cyclic catalytic stability, enabling multi-batch recycling of the enzyme.

[0016] Further, the tyrosinase mutant is an amino acid sequence with double-point mutations of R209H and F197W in the wild-type amino acid sequence shown in SEQ ID NO.1 (SEQ ID NO.4); more preferably, it is an amino acid sequence with triple-point mutations of R209H, F197W, and T17P (SEQ ID NO.5), an amino acid sequence with quadruple-point mutations of R209H, F197W, T17P, and Q187L (SEQ ID NO.6), or an amino acid sequence with two-point mutations of T17P and Q187L (SEQ ID NO.7); most preferably, it is an amino acid sequence with quadruple-point mutations of R209H, F197W, T17P, and Q187L (SEQ ID NO.6). This invention shows that the 2-4 point-mutated amino acids can further optimize the physicochemical structure of the enzyme and help to further enhance the enzyme activity and stability of tyrosinase.

[0017] The present invention also provides an immobilized tyrosinase, comprising a carrier and the tyrosinase mutant thereon immobilized thereon.

[0018] In this invention, the tyrosinase mutant exhibits excellent stability; even when loaded onto a support, its active site is not significantly masked, and its enzymatic activity is not lost. Furthermore, the tyrosinase mutant described in this invention is adaptable to various immobilization formats, facilitating the first realization of continuous enzyme-catalyzed synthesis in the field of DHI enzymatic synthesis.

[0019] In this invention, the carrier can be a carrier known in the industry, such as at least one of conventional inorganic carriers, natural polymer carriers, synthetic polymer carriers, and composite carriers.

[0020] In this invention, the enzyme activity of the tyrosinase mutant in the immobilized enzyme can be reasonably adjusted as needed, for example, it can be 10~80 U / g; further, it can be 40~70 U / g.

[0021] In this invention, the immobilized enzyme can be prepared using conventional methods. For example, the vector can be activated first, followed by loading it with the tyrosinase mutant. The activation method can be, for example, glutaraldehyde activation.

[0022] This invention also provides an enzymatic synthesis method for 5,6-dihydroxyindole, wherein a dopa substrate and a cofactor are subjected to an enzymatic reaction (also known as an enzyme-catalyzed reaction) catalyzed by a tyrosinase catalyst to obtain 5,6-dihydroxyindole, wherein the tyrosinase catalyst contains the tyrosinase mutant described in this invention.

[0023] The enzyme-catalyzed synthesis method described in this invention has mild reaction conditions, high product yield, simple process, and the enzyme can be used in multiple batches, which is conducive to mass production. It has significant advantages over existing methods.

[0024] In this invention, apart from utilizing the tyrosinase mutant described in this invention, other operations and conditions can be appropriately adjusted according to the conventional principles of enzyme-catalyzed reactions.

[0025] In this invention, the cofactor is a divalent transition metal ion; further, it can be at least one of copper ions, zinc ions, cobalt ions, etc. The divalent transition metal ion is provided by a water-soluble salt of the ion, such as at least one of sulfate, acetate, and nitrate of the divalent transition metal ion.

[0026] In this invention, the dopa can be L-levodopa.

[0027] The concentration of dopa substrate in the starting solution for the enzyme-catalyzed reaction is 4–6 mg / mL; the concentration of cofactor is 1.0–1.5 mg / mL.

[0028] The solvent in the raw material solution includes water. The water may be derived from conventional buffer solutions, such as phosphate buffer.

[0029] In this invention, the temperature of the enzyme-catalyzed reaction is 30~37℃;

[0030] In this invention, the pH is 5.5~7; preferably 6.0~6.5.

[0031] In this invention, the enzyme-catalyzed reaction time can be 3 to 10 minutes, preferably 5 to 7 minutes.

[0032] In this invention, the amount of mutant enzyme can be reasonably adjusted as needed. For example, the activity concentration of mutant enzyme in the raw material solution can be 0.5 U / mL to 10 U / mL; further, it can be 3 to 8 U / mL.

[0033] In this invention, an enhancer is added during the enzyme-catalyzed reaction process;

[0034] The reinforcing agent includes at least one of component A, component B, and component C; component A is a compound of formula 1; component B is a compound of formula 2; and component C is at least one of propyl gallate, 4-hydroxybenzaldehyde thiourea derivative, acorn tannin, and kojic acid hybrid.

[0035] Formula 1;

[0036] Formula 2;

[0037] R1 is C1~C 12 Alkyl group; R2 is H, C6~C 16 The alkyl acyl or glycosidic group.

[0038] The present invention demonstrates that by using the aforementioned enhancer in conjunction with the aforementioned mutant enzyme, a synergistic effect can be achieved, thereby improving the enzyme selectivity of the substrate and further enhancing the yield and quality of DHI.

[0039] As an alternative, in Formula 1, R1 can be at the para position, and can further be methyl, ethyl, propyl, hexyl or octyl.

[0040] As an optional solution, Equation 2 is Equation 2A:

[0041] Formula 2A;

[0042] In Equation 2A, R3 is C 14 ~C 16 Straight-chain alkyl groups.

[0043] The present invention demonstrates that Formula 2A and the tyrosinase mutant have optimal site fit and can achieve better enzymatic activity.

[0044] In this invention, the enzyme-catalyzed synthesis method can be a liquid-phase enzyme-catalyzed synthesis method, the steps of which are: subjecting a raw material solution containing a substrate, a cofactor and a tyrosinase catalyst to an enzyme-catalyzed reaction, and then separating the target product from the enzyme-catalyzed reaction system.

[0045] Preferably, the concentration of the fortifying agent in the raw material solution is 1~10 mg / mL; more preferably, it can be 1~3 mg / mL.

[0046] The solvent may also contain water-soluble organic solvents, such as at least one of C1-C4 alcohols, acetone, etc. When a water-soluble solvent is included, the volume content of the organic solvent in the raw material solution may be less than 40%, and more specifically, may be 5-35%.

[0047] The enzyme-catalyzed synthesis method described in this invention can also be a solid-phase enzyme-catalyzed synthesis (also known as a continuous enzyme-catalyzed reaction), the steps of which are: filling the immobilized enzyme into a fixed reaction device, and then flowing a raw material solution containing dopa substrate and cofactors through the fixed reaction device and contacting the immobilized enzyme therein to carry out a continuous enzyme-catalyzed reaction. The immobilized enzyme includes the immobilized enzyme loaded with a tyrosinase mutant as described in this invention.

[0048] The enzyme-catalyzed synthesis method of the present invention, thanks to the combination of tyrosinase mutant and the enzyme-catalyzed synthesis conditions, can achieve good solid-phase enzyme-catalyzed synthesis.

[0049] The feed solution for the continuous enzymatic reaction preferably also contains the aforementioned enhancer. Optionally, the concentration of the dopa substrate in the feed solution is 4-6 mg / mL; the concentration of the cofactor is 1.0-1.5 mg / mL; preferably, the concentration of the enhancer is 1-10 mg / mL.

[0050] As an alternative, the continuous enzymatic reaction is carried out at a temperature of 30-37°C and a pH of 6.0-6.5.

[0051] In this invention, after the continuous enzyme-catalyzed reaction, the immobilized reaction equipment is eluted and regenerated using an eluent. Then, a new batch of raw material solution is passed through the regenerated immobilized reaction equipment and comes into contact with the immobilized enzyme therein, thereby realizing a cyclic continuous enzyme-catalyzed reaction.

[0052] The method of the present invention can also realize the cyclic continuous enzymatic reaction of DHI and has excellent cyclic enzymatic stability.

[0053] Beneficial effects

[0054] This invention provides a tyrosinase mutant with a specific amino acid sequence, which improves the enzyme's catalytic activity and stability based on the specific mutation.

[0055] This invention also provides a method for achieving DHI based on the aforementioned tyrosinase mutant, which, thanks to the innovative use of the tyrosinase mutant, can improve the yield of DHI. Furthermore, the innovative addition of the aforementioned enhancer to the enzymatic catalytic system, particularly the selection of Formula 2A as the enhancer, allows for further synergistic adaptation with the tyrosinase mutant, thereby further enhancing the enzymatic conversion rate of DHI.

[0056] This invention benefits from the tyrosinase mutant and the enzyme catalysis scheme, enabling highly stable and highly active continuous enzyme-catalyzed synthesis. Attached Figure Description

[0057] Figure 1 The HPLC chromatogram is shown for the enzymatic synthesis of DHI in Example 3, which was enhanced with vitamin C palmitate fortifier. Detailed Implementation

[0058] Example 1: Construction of recombinant tyrosinase strains and detection of DHI

[0059] The amino acid sequence of tyrosinase derived from Bacillus megaterium was downloaded from GenBank (SEQ ID NO. 1, corresponding GenBank accession number: ACC86108.1). This amino acid sequence was submitted to Beijing Qingke Biotechnology Co., Ltd. for whole-genome sequence synthesis (using E. coli-optimized codons). The synthesized gene has a His tag at the C-terminus and was constructed into the prokaryotic expression vector pET30a(+). The restriction enzyme sites of the prokaryotic expression vector are: 5' Nde I and 3' Xho I. The constructed plasmid pET30a(+)-Bmtyrc was transformed into the E. coli expression strain BL21(DE3) using the CaCl2 heat shock method. The transformed plasmid was plated on LB agar plates containing 50 µg / mL Kanamycin and incubated overnight at 37 ℃. The colonies that grew on the plates were the prokaryotic expression recombinant strain E. coli BL21(DE3) / pET30a(+)-Bmtyrc.

[0060] Using a sterile pipette tip, carefully pick a single colony of the prokaryotic expression recombinant tyrosinase strain from the above-mentioned LB solid medium plate and inoculate it into an Erlenmeyer flask containing 20 mL of LB liquid medium. Incubate overnight at 37 ℃ with shaking at 200 r / min. The next day, inoculate the shake-flask culture at a rate of 1% into an Erlenmeyer flask containing 100 mL of TB liquid medium. Incubate at 37 ℃ with shaking at 220 r / min, and measure the OD value of the culture medium every 1 h. When the OD value of the culture medium reaches 1.5, add lactose to a final concentration of 1% (m / v), and continue incubating at 25 ℃ with shaking for 4 h-6 h, then stop incubation.

[0061] Collect the above-mentioned fermented cells, wash them 2-3 times with sterile water, add physiological saline, and break the cells by ultrasound or homogenization. After solid-liquid separation, take an appropriate amount of supernatant for DHI synthesis testing. The specific reaction was as follows: 500 μL of crude tyrosinase solution was added to an EP tube, followed by 500 μL of 8 mM L-DOPA solution. The mixture was thoroughly mixed, and the reaction tube was placed in a constant temperature shaker at 37 ℃ and 220 rpm for 5 min. After reacting precisely, 2 μL of 37% concentrated hydrochloric acid and 1 mL of anhydrous ethanol were added sequentially. The mixture was then vortexed thoroughly and placed in a constant temperature shaker at 20 ℃ and 220 rpm for another 30 min. After removing the tube, the mixture was vortexed for 1–2 min and centrifuged at 13400 × g and 20 ℃ for 10 min using a high-speed refrigerated centrifuge. The supernatant was aspirated with a 1 mL syringe, filtered through a 0.22 μm organic phase filter membrane, and transferred to a liquid chromatography vial. The product DHI in the sample was qualitatively and quantitatively detected using high-performance liquid chromatography (HPLC).

[0062] HPLC detection conditions: Instrument model: Agilent 1200, UV detector wavelength: 300 nm, column: C18 (4.6 × 250 mm, 5 μm), column temperature: 20 ℃, injection volume: 10 μL, flow rate: 1.0 mL / min, mobile phase A: 0.15% phosphoric acid aqueous solution, mobile phase B: 100% chromatographic grade methanol. Gradient elution was used. From 0 to 5 min, the mobile phase ratio increased from 5% B phase to 50%; from 5 to 10 min, the mobile phase ratio remained at 50% B phase; from 10 to 13 min, the mobile phase ratio decreased from 50% B phase to 5% B phase; from 13 to 15 min, the mobile phase ratio remained at 5% B phase. After 15 min of liquid phase separation, the retention time of L-DOPA was 10 min, and the retention time of DHI was 13.5 min.

[0063] Example 2: Directed Evolution of Tyrosinase

[0064] Using the pET30a(+)-Bmtyrc recombinant plasmid as a PCR template, and conventional T7F / T7R primers (primer sequences: T7F: 5'-TAATACGACTCACTATAGGG-3', T7R: 5'-GCTAGTTATTGCTCAGCGG-3') as universal primers, error-prone PCR amplification of the Bmtyrc gene was performed. The Mg content in the PCR amplification reaction system was adjusted. 2+ Mn 2+The concentrations of dCTP and dTTP oligonucleotides were adjusted to ensure that the base mismatch rate of the mutant library was only 0.2%, guaranteeing that only 1 to 2 amino acids were mutated in each mutant. The error-prone PCR reaction system is shown in Table 1.

[0065] Table 1: Error-prone PCR reaction systems ;

[0066] Error-prone PCR reaction conditions: Pre-denaturation at 95 ℃ for 5 min; followed by denaturation at 94 ℃ for 30 s, annealing at 56 ℃ for 1 min, and extension at 72 ℃ for 1.5 min, for a total of 25 cycles; final extension at 72 ℃ for 10 min. 2 μL of the error-prone PCR product was sampled and detected by agarose gel electrophoresis. After successful detection, the product was purified using a PCR product purification kit. At 37 ℃, the purified PCR product and the prokaryotic expression vector pET30a(+) were double-digested with Nde I and Xho I restriction endonucleases, respectively. The digested products were recovered from the gel (the recovered purified 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 for overnight ligation at 16 ℃. The next day, the ligation product was transferred into E. coli BL21(DE3) by electroporation to construct an engineered bacterium, thus obtaining a large-capacity random mutant library.

[0067] The methods used in high-throughput screening are as follows:

[0068] 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, use an 8-channel pipette to transfer 50 µL of the solution into 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. Finally, centrifuge at 4000 rpm and 4 °C for 20 minutes, and collect 50 μL of the supernatant from each well. To each well, add 100 μL of reaction solution (levodopa substrate concentration: 2 mg / mL, pH 6.5–7.0 citrate-phosphate buffer) to 50 μL of supernatant and incubate at 30 °C for 5 min. After the reaction, add 2 μL of concentrated hydrochloric acid (37%) and 1 mL of anhydrous ethanol, mix well, and incubate at 20 °C, 220 rpm for 30 min. Analyze the results using a microplate reader (detection wavelength 475 nm). Wells with high absorbance values ​​are selected for further amplification and validation analysis, as well as for HPLC analysis of the product.

[0069] Through repeated screening and verification (approximately 200,000 clones), sequencing analysis, and enzyme activity assays, six mutant strains with significantly higher levodopa activity to the substrate Bmtyrc than the wild-type Bmtyrc were obtained, namely Bmtyrc-1, Bmtyrc-2, Bmtyrc-3, Bmtyrc-4, Bmtyrc-5, and Bmtyrc-6, which are summarized in Table 2 below.

[0070] Table 2: Expression strains of Bmtyrc mutants obtained from screening error-prone mutation libraries ;

[0071] As can be clearly seen from Table 2, among the positive mutant enzymes obtained through screening, Bmtyrc-1, Bmtyrc-2, Bmtyrc-3, Bmtyrc-4, Bmtyrc-5, and Bmtyrc-6 mutants showed particularly significant catalytic performance for DHI synthesis compared to the wild-type enzyme (Bmtyrc). Among them, the quadruple mutant Bmtyrc-5 exhibited a 3.75-fold increase in DHI concentration compared to the starting template, and can be considered the optimal mutant for further research and testing.

[0072] Example 3: Optimization of synthesis conditions of 5,6-dihydroxyindole catalyzed by liquid enzyme system

[0073] The optimal mutant Bmtyrc-5 was selected, and the recombinant strain was fermented and disrupted according to the conditions of Example 1. The enzyme solution was collected for optimization of DHI transformation conditions. The specific reaction conditions were as follows: The total reaction volume was 10 mL. 56 mg of L-L-DOPA was weighed and added to 1 mL of copper sulfate solution (concentration 1.4 mg / mL). 8.5 mL of 0.1 M phosphate buffer at different pH values ​​was added as a solvent. The mixture was preheated to 37 °C, and then 0.5 mL of tyrosinase (total enzyme activity: 35 U) was added. After thorough mixing, the mixture was placed in a water bath at 37 °C and 170 rpm. After the reaction, 300 μL of 6 M hydrochloric acid and 10 mL of anhydrous ethanol were added, and samples were taken for HPLC analysis.

[0074] (1) In the reaction system, different pH values ​​were set, the reaction time was controlled at 5 min, the reaction was terminated, and samples were taken for detection and analysis. The results are shown in Table 3 (enzyme dosage: 35 U):

[0075] Table 3: DHI synthesis under different pH conditions ;

[0076] (2) In the reaction system, the optimal reaction pH was determined to be 6.0. Different reaction times were set, the reaction was terminated, and samples were taken for detection and analysis. The results are shown in Table 4 (enzyme dosage: 35 U):

[0077] Table 4: Effect of different reaction times on DHI synthesis ;

[0078] (3) In the reaction system, the optimal reaction pH was determined to be 6.0, the reaction time was strictly controlled to be 7 min, different enzyme dosages were set, the reaction was terminated after the reaction was completed, and samples were taken for detection and analysis. The results are shown in Table 5:

[0079] Table 5: Effects of different enzyme dosages on DHI synthesis ;

[0080] (4) In the reaction system, the optimal reaction pH was determined to be 6.0, the reaction time was 7 min, and 0.5 mL (35 U) of enzyme solution was added. The following enhancers were also added to the initial reaction solution system. The results are shown in Table 6:

[0081] Table 6: Effects of adding different reagents to the reaction system on the synthesis of DHI ;

[0082] HPLC chromatogram of DHI synthesized by enzymatic catalysis with added vitamin C palmitate fortifier is shown below. Figure 1 .

[0083] Through optimization, the optimal parameters for the synthesis of DHI by the mutant of this invention are: pH 6.0-6.5, single reaction time 5-7 min, enzyme activity 3-8 U / mL; and the fortifying agents are vitamin C palmitate and ethanol.

[0084] Example 4: Continuous flow catalytic synthesis of 5,6-dihydroxyindole using immobilized enzymes

[0085] To further improve the conversion efficiency of 5,6-dihydroxyindole, this invention adopts a continuous flow reaction system with immobilized enzymes to achieve efficient batch-to-batch catalytic synthesis of 5,6-dihydroxyindole.

[0086] Enzyme immobilization steps:

[0087] (1) Activation of immobilized carrier: Accurately measure 30 mL of 60% (m / v) glutaraldehyde, add 4.76 g of dipotassium hydrogen phosphate (K2HPO4·3H2O) to 600 mL of deionized water, dissolve, and then make up to 1000 mL with deionized water. Adjust the pH to 8.0 with phosphoric acid solution. Add 250 g of immobilized carrier (EVF) to the above solution and activate by stirring at low speed at 25 °C for 2 h. Filter and collect the carrier, rinse 2-3 times with sterile deionized water, and then vacuum filter dry for later use.

[0088] (2) Immobilization of Bmtyrc-5 recombinant protein: Take a certain amount of the purified Bmtyrc recombinant protein, dilute it with phosphate buffer (pH 8.0, 0.1 mol / L), then add 50 g of the activated vector, and immobilize it at 25 ℃ and 120 rpm for 48 h. The immobilized enzyme is washed with deionized water 3 to 5 times, and then vacuum filtered to obtain the immobilized enzyme product (the immobilization load involved in the mutant is 50 to 55 U / g).

[0089] The reactor is a fixed-bed column reactor, and the reactor material is a glass column or a plastic column resistant to organic solvents. The reactor inner diameter is 1.0 cm, the column height is 10~20 cm, and the upper and lower ends are porous sieve plates.

[0090] Immobilized enzyme loading method:

[0091] (1) Soak the above immobilized enzyme particles in phosphate buffer (pH 8.0, 0.1 mol / L) for 10-30 min to fully swell and degas.

[0092] (2) Add buffer solution to the column to a height of 1 / 5 to 1 / 4 of the column first to remove air bubbles at the bottom. Close the outlet and keep the liquid level above the sieve plate.

[0093] (3) Mix the pretreated immobilized enzyme with buffer to form a uniform, flowable slurry without clumping or dry areas. Open the top of the column and slowly and continuously pour the slurry along the column wall. Let the enzyme particles settle naturally for 10-20 minutes. Gently replenish the top with buffer to prevent the liquid level from falling below the enzyme layer.

[0094] The reaction system is as follows:

[0095] Step 1: Reaction Phase

[0096] (1) Immobilized enzyme column packing and reactor pretreatment: rinse the enzyme column with buffer for 30 min at a flow rate of 0.3 mL / min, remove air bubbles, check the pH and conductivity of the effluent to ensure stability, confirm no enzyme leakage, and keep the temperature constant at 37 ℃.

[0097] (2) Preparation of reaction substrate solution: Prepare a raw material solution containing L-levodopa, copper sulfate, phosphate buffer at pH 6.0, vitamin C palmitate and ethanol, wherein the concentration of L-levodopa is 25 mM, the concentration of copper sulfate is 1.4 mg / mL, the concentration of vitamin C palmitate is 2 mg / mL and the ethanol is 20% of the volume of the raw material solution.

[0098] (3) Start the continuous flow reaction and set the pump flow rate to 0.2~0.4 mL / min.

[0099] (4) Collect the effluent, protect it from light and pass nitrogen gas for protection.

[0100] (5) Take samples for HPLC detection and analysis.

[0101] Step 2: De-inhibition phase (regeneration)

[0102] To remove enzyme inhibition in the column reactor, the column was washed with 0.1 M pH 6.0 phosphate buffer as solvent, approximately one column volume (25 mL), and the collected solution was analyzed by HPLC.

[0103] Step 3: Start the next batch of reaction. Steps 1 and 2 constitute one cycle batch. The reaction system and conditions are the same as in steps 1 and 2. The results are shown in Table 7.

[0104] Table 7. Effects of multi-batch cyclic processing ;

[0105] In summary, the method of the present invention can achieve excellent cyclic synthesis.

[0106] Characteristic sequences of tyrosinase:

[0107] SEQ NO.1: MSNKYRVRKNVLHLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPIKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLNALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIIHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS。

[0108] SEQ NO.2 (R209H): MSNKYRVRKNVLHLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPIKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLNALKITQYDTPPWDMTSQNSFRNQLEGFINGPQLHNRVHHWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIIHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS。

[0109] SEQ NO.3 (F197W): MSNKYRVRKNVLHLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPIKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLNALKITQYDTPPWDMTSQNSFRNQLEGWINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIIHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS。

[0110] SEQ NO.4 (R209H,F197W): MSNKYRVRKNVLHLTDTEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPIKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLNALKITQYDTPPWDMTSQNSFRNQLEGWINGPQLHNRVHHWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIIHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS。

[0111] SEQ NO.5 (R209H,F197W,T17P): MSNKYRVRKNVLHLTDPEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPIKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLNALKITQYDTPPWDMTSQNSFRNQLEGWINGPQLHNRVHHWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIIHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS。

[0112] SEQ NO.6 (R209H,F197W,T17P,Q187L): MSNKYRVRKNVLHLTDPEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPIKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLNALKITQYDTPPWDMTSLNSFRNQLEGWINGPQLHNRVHHWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIIHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS。

[0113] SEQ NO.7(T17P,Q187L) : MSNKYRVRKNVLHLTDPEKRDFVRTVLILKEKGIYDRYIAWHGAAGKFHTPPGSDRNAAHMSSAFLPWHREYLLRFERDLQSINPEVTLPYWEWETDAQMQDPSQSQIWSADFMGGNGNPIKDFIVDTGPFAAGRWTTIDEQGNPSGGLKRNFGATKEAPTLPTRDDVLNALKITQYDTPPWDMTSLNSFRNQLEGFINGPQLHNRVHRWVGGQMGVVPTAPNDPVFFLHHANVDRIWAVWQIIHRNQNYQPMKNGPFGQNFRDPMYPWNTTPEDVMNHRKLGYVYDIELRKSKRSS。

Claims

1. A tyrosinase mutant, characterized in that: The amino acid sequence of the tyrosinase mutant is an amino acid sequence with at least one mutation among R209H, F197W, T17P, and Q187L in the wild-type amino acid sequence shown in SEQ ID NO.

1.

2. The tyrosinase mutant as described in claim 1, characterized in that: The amino acid sequence of the tyrosinase mutant is an amino acid sequence with two or more mutations in the wild-type amino acid sequence shown in SEQ ID NO.

1.

3. The tyrosinase mutant as described in claim 1, characterized in that: The amino acid sequence of the tyrosinase mutant is the amino acid sequence described in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.

7.

4. An immobilized tyrosinase, characterized in that: Including the vector and the tyrosinase mutant of any one of claims 1 to 3 immobilized thereon; The enzyme activity of the tyrosinase mutant in the immobilized enzyme is 10~80 U / g.

5. An enzymatically catalyzed synthesis method for 5,6-dihydroxyindole, comprising an enzymatic reaction of a dopa substrate and a cofactor under the catalysis of tyrosinase to obtain 5,6-dihydroxyindole, characterized in that, The tyrosinase catalyst comprises the tyrosinase mutant according to any one of claims 1 to 3.

6. The enzymatically catalyzed synthesis method of 5,6-dihydroxyindole as described in claim 5, characterized in that, The cofactor is a divalent transition metal ion; The concentration of dopa substrate in the starting solution for the enzyme-catalyzed reaction is 4-6 mg / mL; the concentration of cofactor is 1.0-1.5 mg / mL. The temperature for enzyme-catalyzed reactions is 30~37℃; pH range: 5.5 to 7.

0.

7. The enzymatically catalyzed synthesis method of 5,6-dihydroxyindole as described in claim 5, characterized in that, A fortifier is added during the enzyme-catalyzed reaction; The reinforcing agent includes at least one of component A, component B, and component C; component A is a compound of formula 1; component B is a compound of formula 2; and component C is at least one of propyl gallate, 4-hydroxybenzaldehyde thiourea derivative, acorn tannin, and kojic acid hybrid. Formula 1; Formula 2; R1 is C1~C 12 Alkyl group; R2 is H, C6~C 16 The alkyl acyl or glycosidic group; And / or, the concentration of the enhancer in the starting material solution of the enzyme-catalyzed reaction is 1~10 mg / mL; And / or, the starting solution for the enzyme-catalyzed reaction may also contain a water-soluble organic solvent.

8. The enzymatically catalyzed synthesis method of 5,6-dihydroxyindole according to any one of claims 5 to 7, characterized in that, The tyrosinase catalyst comprises the immobilized enzyme of claim 4.

9. The enzymatically catalyzed synthesis method of 5,6-dihydroxyindole as described in claim 8, characterized in that, The immobilized enzyme is filled into a fixed reaction device, and then a raw material solution containing dopa substrate and cofactor is flowed through the fixed reaction device and comes into contact with the immobilized enzyme therein to carry out a continuous enzymatic reaction.

10. The enzymatically catalyzed synthesis method of 5,6-dihydroxyindole as described in claim 9, characterized in that, After the continuous enzyme-catalyzed reaction, the immobilized reaction equipment is eluted and regenerated using an eluent. Then, a new batch of raw material solution is passed through the regenerated immobilized reaction equipment and comes into contact with the immobilized enzyme therein, thus realizing a cyclic continuous enzyme-catalyzed reaction.

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