Method for preparing antioxidant active polypeptide based on tyrosinase modified collagen, silk fibroin and mussel protein

Abstract

The invention discloses a method for preparing antioxidant active polypeptide based on tyrosinase modified collagen, silk fibroin and mussel protein, and belongs to the technical field of bioengineering. According to the invention, high-activity tyrosinase protein is screened and prepared, and the prepared tyrosinase is used for dopa modification of antioxidant protein. The dopa modification degree of the antioxidant protein prepared by the method disclosed by the invention is remarkably improved, and the DPPH free radical scavenging power and superoxide anion free radical scavenging power of the antioxidant protein are enhanced. The method is suitable for various proteins such as collagen, silk fibroin and mussel protein, can be used in the fields of functional food, cosmetics, biological materials and the like, and has good adaptability and wide application prospects.

Description

TECHNICAL FIELD

[0001] The application relates to a method for preparing an antioxidant active polypeptide based on tyrosinase modification of collagen, silk fibroin and mussel protein, and belongs to the technical field of bioengineering. BACKGROUND

[0002] Tyrosinase, also known as polyphenol oxidase, is a copper-containing oxidoreductase widely existing in animals, plants, microorganisms and human bodies, and is a key enzyme for melanin synthesis. Tyrosinase also has tyrosine hydroxylase activity, which catalyzes the conversion of tyrosine residues into dopa residues, realizes the modification of biological macromolecules by dopa and improves the cross-linking and antioxidant properties of molecules. In recent years, the application of tyrosinase in the fields of food, daily chemicals, medicine and the like has attracted much attention. At present, tyrosinase on the market mainly depends on mushroom extraction, and is limited in source and expensive in price.

[0003] Antioxidant polypeptides mainly include collagen peptides, silk fibroin and mussel protein. Collagen peptides are the products of collagen hydrolyzed by protease. Due to the small molecular weight, the absorption rate of collagen peptides is better than that of plant proteins and macromolecular collagen. In addition, due to its unique biological activity, collagen peptides have been widely used in various fields. Hydrolyzed silk fibroin polypeptide obtained by hydrolysis of silk fibroin is often used as a raw material for functional daily-use cosmetics. Silk fibroin has tyrosine residues that can be modified, and after modification by dopa, it has good antioxidant function and is expected to play a role in the preparation of silk fibroin-based cross-linked hydrogel. Mussel foot silk protein is mainly derived from marine mussels and is a multifunctional protein secreted by the foot structure of mussels. It mainly includes foot silk fiber protein that plays a role in structural support and connection, and foot silk disc protein that plays a role in adhesion and fixation. Mussel protein has excellent calming, soothing, moisturizing, antioxidant, wound healing, adhesion, cross-linking and other functions, and shows great application potential in the fields of biomedicine, material science and environmental protection.

[0004] The use of tyrosinase to catalyze the monophenol structure of tyrosine residues in polypeptides can introduce an additional hydroxyl group to convert it into a dopa residue. This catalytic process is called dopa modification. This catalytic process was first discovered in the analysis of marine mussel foot silk protein. The presence of dopa is believed to have a dual role in interface binding and adhesion protein solidification. According to reports, through the study of substrate and oxidation-dependent adhesion of dopa, it is proved that dopa has high affinity and multiple chemical actions, and can bind to different medium surfaces through the formation of reversible non-covalent or irreversible covalent interactions. At the same time, dopa modification can also improve the antioxidant activity of polypeptides. However, in the natural state, most antioxidant polypeptides do not have the conditions for dopa modification, so it is urgent to develop a dopa modification method for antioxidant polypeptides to improve their antioxidant activity. SUMMARY

[0005] The application provides a recombinant Escherichia coli, which is expressed with pETDuet as an expression vector, and expresses a tyrosinase as shown in SEQ ID NO. 2.

[0006] In an embodiment, the Escherichia coli is Escherichia coli BL21 (DE3).

[0007] The application also provides an application of the tyrosinase with an amino acid sequence as shown in SEQ ID NO. 2 in modifying an antioxidant protein.

[0008] In an embodiment, the protein includes but is not limited to one or more of collagen, silk fibroin and mussel protein.

[0009] In an embodiment, the collagen includes but is not limited to human-derived collagen IV, VII and XVII collagen peptide; and the mussel protein includes but is not limited to Mfp-3, Mfp-5, Mfp-6, TMP, PTMP and other short mussel protein peptides.

[0010] In an embodiment, the application is to react the protein with the tyrosinase in a reaction system containing metal ions.

[0011] In an embodiment, the concentration of the antioxidant protein in the reaction system is 2-4 g / L, preferably 4 g / L.

[0012] In an embodiment, the buffer in the reaction system is selected from one or more of 10-100 mM Tris-HCl (pH 6.0-8.0), 10%-50% (v / v) acetic acid and 5 mM sodium carbonate.

[0013] In an embodiment, the salt for providing the metal ions includes but is not limited to one or more of magnesium sulfate, sodium sulfate, copper sulfate and zinc sulfate.

[0014] In an embodiment, the concentration of the metal ions is 0.1-1 mM.

[0015] In an embodiment, the concentration of the tyrosinase is 0.1-1 mg / mL, preferably 0.1 mg / mL.

[0016] In an embodiment, the reaction is performed at 20-50°C.

[0017] In an embodiment, the reaction is performed for 2-240 min, preferably 4 min.

[0018] The application also provides a method for improving the antioxidant property of an antioxidant peptide, which is to perform dopamine modification on the antioxidant peptide with the tyrosinase as shown in SEQ ID NO. 2.

[0019] In an embodiment, the antioxidant peptide includes, but is not limited to, collagen, silk fibroin or mussel protein.

[0020] In an embodiment, the antioxidant peptide is reacted with the tyrosinase in a reaction system containing metal ions at a final concentration of 2-8 g / L and a final concentration of 0.1-0.25 mg / L for 2-8 min.

[0021] In an embodiment, the salt for providing the metal ions includes, but is not limited to, magnesium sulfate, sodium sulfate, copper sulfate or zinc sulfate.

[0022] The application also provides collagen, silk fibroin or mussel protein with dopa modification prepared by the method.

[0023] In an embodiment, the protein has a dopa modification degree of ≥60%.

[0024] In an embodiment, the collagen has a dopa modification degree of 76.72%-97.25%.

[0025] In an embodiment, the silk fibroin has a dopa modification degree of 79.25%-97.82%.

[0026] The application also provides application of the method or the antioxidant protein in the fields of biology, medicine, materials and the like.

[0027] Advantages:

[0028] 1. The application provides a catalytic preparation system for preparing antioxidant active polypeptides by modifying collagen, silk fibroin and mussel protein and the like by using recombinant bacterial tyrosinase. The tyrosine residue hydroxylation reaction catalyzed by the recombinant tyrosinase realizes a dopa modification degree of up to 97.25%, 97.82% and 91.50% for collagen, silk fibroin and mussel protein respectively in vitro.

[0029] 2. The application optimizes the reaction system and reaction time and the like, so that the dopa modification degree of collagen is up to 97.23%, realizing high-level dopa modification of collagen peptide. Under the conditions of high substrate concentrations of 16 g / L and 48 g / L, the reaction system can still catalyze silk fibroin and collagen peptide to realize high-level dopa modification.

[0030] 3. The antioxidant activity of the collagen peptide after dopa modification is determined, and the total Fe 3+ reducing power, DPPH clearance rate and superoxide anion radical clearance rate of the collagen peptide after dopa modification are respectively increased by 121.2%, 524.5% and 232.1% compared with before modification, indicating that the antioxidant activity of the antioxidant peptide is obviously enhanced after dopa modification. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The plate color development of recombinant tyrosinases from different sources, wherein 1-7 are pETDuet empty negative control, tyrosinase recombinant bacteria from Rhizobium leguminosarum, tyrosinase recombinant bacteria from Ralstonia solanacearum, tyrosinase recombinant bacteria from Bacillus megaterium, tyrosinase recombinant bacteria from Micrococcus variabilis, pETDuet empty negative control and tyrosinase recombinant bacteria from Mycobacterium mediterraneum, respectively.

[0032] Figure 2 SDS-PAGE analysis of recombinant expression of tyrosinases from different sources.

[0033] Figure 3 Purification analysis of recombinant bacterial tyrosinases.

[0034] Figure 4 Dopa modification degree of collagen peptide and mussel protein.

[0035] Figure 5 Effect of enzyme addition amount on the degree of dopa modification.

[0036] Figure 6 Effect of reaction time on the degree of dopa modification.

[0037] Figure 7 Dopa modification degree of high concentration substrate.

[0038] Figure 8 Fe 3+ Total reducing power scavenging ability.

[0039] Figure 9 DPPH free radical scavenging ability of collagen peptide and dopa modified collagen peptide.

[0040] Figure 10 Superoxide anion radical scavenging ability of collagen peptide and dopa modified collagen peptide. DETAILED DESCRIPTION

[0041] (I) Materials

[0042] The method for obtaining mussel protein is described in the paper "Multi-strategy combination optimization of efficient expression of Mytilus coruscus byfusiform protein Mcofp-3 in E. coli".

[0043] Collagen and silk fibroin were purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.

[0044] (II) Culture medium:

[0045] LB medium: 10 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract.

[0046] TB medium, 2.31 g / L KH2PO4, 12.54 g / L K2HPO4, 12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol.

[0047] Tyrosinase activity rapid identification medium: on the basis of LB solid medium, add CuSO4 with a final concentration of 0.2 mM and IPTG with a final concentration of 1 mM.

[0048] (III) Detection method:

[0049] Tyrosinase enzyme activity determination method: tyrosinase can catalyze L-tyrosine to be oxidized to L-DOPA, and further oxidized to dopachrome which is color visible. In this study, the enzyme activity of tyrosinase is defined as 1 μM dopachrome generated per minute with L-tyrosine as substrate, that is, the average increase of A475 per minute is 0.001, which is 1 enzyme activity unit, recorded as 1 U. Prepare a buffer solution of a specific pH, add L-tyrosine and CuSO4 to it, and prepare a reaction solution containing 2 mM L-tyrosine (the final concentration of CuSO4 is 0.2 mM), and shake mix for 1-5 min to increase the oxygen content. Take a 96-well plate, add 290 μL of reaction solution and 10 μL of enzyme solution to be tested to each well, and continue to shake the reaction at the corresponding temperature. Use an enzyme marker to determine the change in absorbance at 475 nm after 0 min, 5 min and 10 min of reaction, and calculate the enzyme activity of the corresponding enzyme solution.

[0050] Example 1: Screening of tyrosinase

[0051] The recombinant strains are Escherichia coli expressing tyrosinase from Bacillus megaterium (TYR-Bm, amino acid sequence as shown in SEQ ID NO. 1), Variovorax paradoxus (TYR-Vs, amino acid sequence as shown in SEQ ID NO. 2), Ralstonia solanacearum (TYR-Rs, amino acid sequence as shown in SEQ ID NO. 3), Rhizobium leguminosarum (TYR-Re, amino acid sequence as shown in SEQ ID NO. 4), Mycobacterium marinum (TYR-Mm, amino acid sequence as shown in SEQ ID NO. 5). After codon optimization, the nucleotide sequences as shown in SEQ ID NO. 6-10 are synthesized, the genes are ligated to pETDuet plasmid, the constructed recombinant plasmid is transformed into Escherichia coli BL21 (DE3), a single colony of the constructed recombinant E. coli is picked and cultured in LB medium at 37°C for 12 h to prepare seed liquid, an inoculation loop is used to dip the overnight cultured seed liquid, and streaked on LB color developing solid plate containing corresponding antibiotic, and incubated at 37°C for 40 h, and the color developing plate is black, indicating that the corresponding recombinant strain has tyrosinase activity. As shown in FIG. 1, the color developing plates of TYR-Bm and TYR-Vs are black, indicating that the two strains have tyrosinase activity. Figure 1

[0052] Example 2: Preparation of tyrosinase

[0053] A single colony of the recombinant E. coli expressing TYR-Bm and TYR-Vs constructed in Example 1 is picked and cultured in LB medium to prepare seed liquid, which is transferred to TB medium at a 2% inoculation amount, and cultured at 37°C at a rotation speed of 220 rpm for 1.5 h, then induced with 1 mM IPTG, and cultured at 37°C for another 8 h. The fermentation liquid is centrifuged at 4°C (8000 rpm, 10 min), and the supernatant is discarded, the bacterial body is resuspended with 20 mM Tris-HCl (pH = 7.5) buffer for 2-3 times, and then broken by ultrasonic cell disrupter and centrifuged at high speed at 12000 rpm for 20 min to obtain crude enzyme liquid. The enzyme activity of the crude enzyme liquid is (16.1 ± 0.2) U·mL -1 and (48.6 ± 0.9) U·mL -1 , respectively. The crude enzyme liquid is filtered with 0.45 μm filter membrane, and the filtered supernatant is loaded onto a nickel column through AKTA purification system, eluted with eluent containing 20 mM Tris-HCl buffer, 500 mM imidazole and 500 mM NaCl to obtain purified enzyme liquid (as shown in FIG. 2 and FIG. 3) for catalyzing the modification of dopamine on polypeptide. Figure 2 Figure 3

[0054] ​​​Example 3: In vitro Dopa modification of collagen peptide, silk fibroin and mussel protein

[0055] Collagen peptide, silk fibroin and mussel protein aqueous solution with a final concentration of 4 g / L were used as substrates, respectively, and 0.2 mM CuSO4 with a final concentration was added before reaction as reaction solution. 0.5 mg / L of recombinant tyrosinase TYR-Vs pure enzyme prepared in Example 2 was added to the reaction solution, and the reaction was carried out at 37°C, 220 rpm for 15 min. After the reaction was completed, 50 μL of catalytic product was taken to determine the Dopa modification degree.

[0056] The determination method was as follows: A solution was prepared: 0.5 M HCl; B solution: 1% sodium molybdate, 1% sodium nitrite, room temperature, avoid light; C solution: 1 M NaOH. Take 96-well plate, add 25 μL of sample to be tested in single well, first add 50 μL of A solution, mix well, then add 75 μL of B solution, mix well, stand for 30 s, finally add 100 μL of C solution, mix well, immediately measure the absorbance value at 510 nm. 0.8 mg·mL -1 L-dopa solution as standard solution, after gradient dilution, the corresponding absorbance value was determined to draw a standard curve, and the Dopa modification degree of collagen peptide was calculated to be 75.27%, and the modification degrees of silk fibroin and mussel protein were 95.25% and 91.50%, respectively.

[0057] Example 4: Optimization of in vitro Dopa modification system

[0058] On the basis of Example 3, the addition amount of recombinant tyrosinase TYR-Vs in the Dopa modification system and the reaction time were further optimized. Collagen peptide aqueous solution with a final concentration of 4 g / L was used as substrate, and 0.2 mM CuSO4 with a final concentration was added before reaction as reaction solution. Recombinant tyrosinase TYR-Vs was prepared according to the method of Example 2, and 0.1 mg / L, 0.25 mg / L, 0.5 mg / L, 0.75 mg / L and 1 mg / L of tyrosinase pure enzyme with a final concentration were added, respectively, and the reaction was carried out at 37°C, 220 rpm for 15 min, and the catalytic product was separated by centrifugation. According to the Dopa modification degree determination result Figure 5 ), the Dopa modification degree can reach 76.72% when the addition amount of tyrosinase TYR-Vs pure enzyme is 0.1 mg / L.

[0059] The reaction time was further optimized, and 4 g / L of collagen peptide aqueous solution was used as substrate, and 0.1 mg / L of pure enzyme was added for Dopa modification reaction, and the Dopa modification degree of the product was determined after 2, 4, 6, 8, 10, 12 and 16 min, respectively. The results showed that Figure 6), the modification degree of dopamine gradually increased and reached the highest value of 97.23% at 4 min, and the optimal reaction time was 4 min. With the extension of the modification time, the modified dopamine residues may spontaneously oxidize to form dopamine quinone, resulting in a decrease in the measured modification degree.

[0060] Example 5: In vitro expansion of dopamine modification substrate concentration and improvement of catalytic intensity

[0061] To further improve the catalytic intensity, the concentrations of collagen peptide and silk fibroin in the reaction system were increased to 16 g / L and 48 g / L, respectively, for dopamine modification reaction. The recombinant tyrosinase TYR-Vs prepared according to the method of Example 2 was added at a final concentration of 0.1 mg / mL, and the dopamine modification degree of the product was measured at 5, 10, 15, 20, 25, 30, and 35 min, respectively. The results showed that the modification degree of 16 g / L collagen peptide aqueous solution reached 97.26% at 25 min, and the modification degree of 48 g / L collagen peptide aqueous solution reached 97.25% at 35 min; the modification degree of 16 g / L silk fibroin aqueous solution reached 97.82% at 15 min; the modification degree of 48 g / L silk fibroin aqueous solution was 79.25% at 5 min, and the modification degree reached 97.61% at 30 min.

[0062] Example 6: Effect of dopamine modification on total iron reducing power

[0063] The Fe 3+ reducing ability of the collagen peptide modified with dopamine was tested. Distilled water was used as a blank control, and vitamin C was used as a positive control. Vitamin C, collagen peptide aqueous solution, and dopamine-modified collagen peptide aqueous solution were prepared at concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 mg / mL, respectively. The reducing capacity of the samples on Fe 3+ was determined by the amount of Prussian blue precipitate generated after the reaction. A 0.2 M phosphate buffer solution (pH 6.6), 10 g / L potassium ferricyanide solution, 100 g / L trichloroacetic acid, and 1 g / L ferric chloride solution were prepared. 100 μL of the sample to be tested was taken into a 2 mL EP tube, and 250 μL of the phosphate buffer solution and 250 μL of the potassium ferricyanide solution were added in sequence. After mixing well, the mixture was incubated in a 50°C water bath for 20 min. After incubation, the mixture was quickly cooled, 250 μL of trichloroacetic acid was added, and the mixture was mixed well. After centrifugation at 10,000 rpm for 5 min, the supernatant was taken and added to a 96-well plate. Then, 100 μL of distilled water and 15 μL of ferric chloride solution were added in sequence, and the absorbance at 700 nm was measured. As shown in Figure 8 , the collagen peptide itself has certain antioxidant properties, and the dopamine modification can improve the antioxidant properties of the collagen peptide. 3+The reducing power was improved to some extent, and the A700 nm absorbance of the collagen peptide modified by dopamine increased by 0.036 compared with the unmodified collagen under the action of 2 g / L.

[0064] Example 7: DPPH radical scavenging ability detection of dopamine-modified collagen

[0065] The DPPH radical scavenging ability of the dopamine-modified collagen peptide with a modification degree of 76.72% obtained in Example 4 was detected. During the detection, distilled water was used as a blank control, and vitamin C was used as a positive control. 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 mg / mL vitamin C, collagen peptide, and dopamine-modified collagen peptide aqueous solutions were prepared, respectively. Anhydrous ethanol was used to prepare a DPPH solution with a final concentration of 0.4 mM, which was stored in the dark. 500 μL of the sample to be tested was taken into a 2 mL centrifuge tube, 500 μL of the DPPH solution was added, and the mixture was mixed and placed in the dark for 30 min. After centrifugation at 10,000 rpm for 5 min, 200 μL of the supernatant was taken into a 96-well plate, and the absorbance D1 at 517 nm was measured. According to the same operation method, distilled water was used instead of the sample to be tested, and the absorbance at 517 nm was measured as the blank control D0. The DPPH radical scavenging rate was calculated according to the following formula: As shown in Figure 9 , the DPPH radical scavenging rate of the collagen peptide was only 15.5% under the action of 2 mg / mL, and the DPPH radical scavenging rate of the dopamine-modified collagen peptide increased to 81.3%.

[0066] Example 8: Superoxide anion radical scavenging ability detection of dopamine-modified collagen

[0067] The superoxide anion radical scavenging capacity of the collagen peptide modified by dopa with a modification degree of 76.72% in Example 4 was detected. In the detection, distilled water was used as a blank control, vitamin C was used as a positive control, and 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 mg / mL of the aqueous solutions of vitamin C, collagen peptide, and dopa-modified collagen peptide were respectively prepared. 0.1M Tris-HCl buffer (pH 8.2) was prepared, 0.75 mM of an aqueous solution of o-phenanthroline was prepared using 0.1M HCl solution and was stored in the dark. A 96-well plate was taken, 15 μL of the sample to be tested was mixed with 170 μL of the Tris-HCl buffer in a single well, and the mixture was uniformly mixed and incubated at 37°C for 20 min. Then, 15 μL of the o-phenanthroline solution was added, and the mixture was mixed and immediately measured for the kinetics at 325 nm using an enzyme marker. The sampling time interval was 30 s, the total time was 3 min, and the measurement was performed for 7 times. The slope of the absorbance value obtained was the auto-oxidation rate V1 of o-phenanthroline (ΔA325 / min). According to the same operation method, the sample to be tested was replaced with distilled water, and the auto-oxidation rate of o-phenanthroline was measured as a blank control V0. The superoxide anion radical scavenging rate C V The superoxide anion radical scavenging rate C was calculated by Formula 2.3: The superoxide anion radical scavenging rate of the collagen peptide was up to 40% at the action concentration of 2 mg / mL, and the DPPH scavenging rate of the dopa-modified collagen peptide was up to 97.3%.

[0068] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. Application of tyrosinase with amino acid sequence as shown in SEQ ID NO.2 in the modification of antioxidant proteins.

2. The application according to claim 1, characterized in that, The proteins include, but are not limited to, one or more of collagen, silk fibroin, and mussel protein.

3. A method for preparing an antioxidant protein with dopa modification, characterized in that, Antioxidant proteins are reacted with tyrosinase in a reaction system containing metal ions; the antioxidant proteins include, but are not limited to, one or more of collagen, silk fibroin, and mussel protein; the amino acid sequence of the tyrosinase is shown in SEQ ID NO.

2.

4. The method according to claim 3, characterized in that, The salts of the metal ions include, but are not limited to, one or more of magnesium sulfate, sodium sulfate, copper sulfate, and zinc sulfate.

5. The method according to claim 4, characterized in that, The reaction parameters are selected from at least one of the following (a) to (d): (a) The concentration of antioxidant protein in the reaction system is 2–4 g / L; (b) The buffer solution in the reaction system is selected from 10-100 mM Tris-HCl, 10%-50% acetic acid or 5 mM sodium carbonate; (c) The concentration of metal ions in the reaction system is 0.1–1 mM; (d) The concentration of tyrosinase in the reaction system is 0.1–1 mg / mL; (e) React at 20–50°C for 2–240 min.

6. An antioxidant protein with dopa modification, characterized in that, The protein includes, but is not limited to, one or more of collagen, silk fibroin, and mussel protein; the protein has a dopa modification degree of ≥60%.

7. The antioxidant protein according to claim 6, characterized in that, The collagen has a dopa modification degree of 76.72% to 97.25%; the silk fibroin has a dopa modification degree of 79.25% to 97.82%.

8. A method for improving the antioxidant activity of antioxidant peptides, characterized in that, The method involves using the tyrosinase shown in SEQ ID NO.2 to modify antioxidant peptides with dopa; the antioxidant peptides include, but are not limited to, collagen, silk fibroin, or mussel protein.

9. The method according to claim 8, characterized in that, The antioxidant peptide was reacted with the tyrosinase at a final concentration of 0.1–0.25 mg / L in a reaction system containing metal ions for 2–8 min.

10. The application of the method according to any one of claims 3 to 5, 8 to 9 or the antioxidant protein according to any one of claims 6 to 7 in the fields of biology, medicine or materials.

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