VI-type recombinant mussel foot protein and preparation method and application thereof
By constructing and purifying type VI recombinant mussel foot protein and co-catalyzing it with tyrosinase, the hydroxylation reaction was optimized, solving the problem of low in vitro hydroxylation efficiency of recombinant mussel adhesive protein and achieving efficient dopa content enhancement and functional protection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTH CHINA UNIV OF TECH
- Filing Date
- 2026-01-19
- Publication Date
- 2026-05-12
AI Technical Summary
Existing recombinant mussel adhesive proteins exhibit low in vitro hydroxylation efficiency, and excessive catalysis leads to oxidative cross-linking of dopaquinone, weakening its function. Existing modifiers increase pretreatment complexity and losses.
A plasmid for expressing the type VI recombinant mussel foot protein gene was constructed and transformed into an engineered strain. The type VI recombinant mussel foot protein was expressed and purified. It was then co-catalyzed with tyrosinase to optimize the hydroxylation reaction system and improve the hydroxylation efficiency of tyrosine residues.
It improved the hydroxylation efficiency of mussel adhesive protein from 20-25% to 40-45%, protected dopa from excessive oxidation, and simplified the pretreatment process.
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Abstract
Description
Technical Field
[0001] This invention relates to the fields of recombinant proteins and biocatalysis, specifically to the construction, expression, purification, and application of a recombinant mussel adhesive protein that can improve the in vitro hydroxylation efficiency of mussel adhesive protein. Background Technology
[0002] Mussels are able to attach themselves to solid surfaces in the high-salinity, humid marine environment constantly battered by waves. This is because various mussel foot proteins (MFPs) are produced at the byssal threads that directly interact with the surface. These proteins interact to exert an adhesive effect, hence the name mussel adhesive protein (MAP). Existing research indicates that type III and V proteins, rich in tyrosine residues, are the primary adhesive components. These tyrosine residues can be converted into the atypical amino acid 3,4-dihydroxyphenyl-L-alanine (Dopa) through hydroxylation. Dopa can participate in various non-covalent interactions, such as hydrogen bonding, cation-π interactions, and π-π interactions. Furthermore, Dopa can participate in various chemical interactions, such as forming metal-Dopa complexes through coordination chemistry. These physical and chemical interactions are involved in Dopa's underwater adhesion. In addition to its adhesive function, Dopa's high reducing properties provide the protein with antioxidant functions, making mussel adhesive protein a potential ingredient in cosmetics and medical devices. Because naturally extracted mussel adhesive protein is extremely expensive, using exogenous expression of recombinant mussel adhesive protein through microorganisms can reduce costs while significantly increasing the content of functional proteins.
[0003] However, because in vitro hydroxylated dopa is prone to over-catalysis, it produces oxidatively cross-linked dopaquinone, significantly weakening the function of recombinant mussel adhesive protein. The main modifiers in existing hydroxylation systems are sodium borate and vitamin C. Sodium borate helps disperse the protein and expose hydroxylation sites, while vitamin C protects dopa from excessive oxidation. This hydroxylation system achieves an in vitro hydroxylation efficiency of 20-25% for mussel adhesive protein. Although this is an improvement over systems without modifiers, the addition of other reagents necessitates secondary dialysis recovery after hydroxylation, increasing losses and complicating the pretreatment of recombinant mussel adhesive protein for application. Therefore, efficiently improving the hydroxylation rate of in vitro hydroxylated mussel adhesive protein is an important future research direction. Summary of the Invention
[0004] This invention aims to provide a method for preparing recombinant type VI mussel foot protein and its application. It optimizes the hydroxylation reaction system for converting tyrosine residues in recombinant mussel adhesive protein to dopa, thereby improving the efficiency of in vitro hydroxylation of tyrosine residues while protecting dopa from excessive oxidation to dopaquinone and loss of function. The method involves screening with NCBI, constructing a type VI mussel foot protein gene expression plasmid, and transforming it into an engineered strain for expression. The recombinant type VI mussel foot protein was successfully expressed and purified. When the purified recombinant type VI mussel foot protein was mixed with type V or III mussel adhesive protein and tyrosinase for catalysis, the in vitro hydroxylation efficiency was increased from 20-25% to 40-45%.
[0005] To achieve the above objectives, the present invention is carried out according to the following scheme:
[0006] This invention is based on the known natural type VI mussel foot protein sequence. Successfully expressed and purified type VI recombinant protein is mixed with type III and type V mussel adhesive proteins rich in tyrosine residues. The mixture is then hydroxylated using tyrosinase to obtain mussel adhesive proteins with high dopa content.
[0007] The type VI mussel foot protein sequence is a gene sequence rich in cysteine and glycine residues, wherein the cysteine residues provide reducing power, while the glycine-rich residues promote the self-assembly of peptides.
[0008] A type VI recombinant mussel foot protein, the amino acid sequence of which is shown in SEQ ID NO.4.
[0009] The gene sequence encoding the type VI recombinant mussel foot protein is shown in SEQ ID NO.2.
[0010] A method for preparing type VI recombinant mussel foot protein, comprising the following steps:
[0011] (1) The gene sequence encoding the type VI recombinant mussel foot protein of claim 1 is ligated to the vector plasmid, and the synthesized plasmid is introduced into the engineered bacteria;
[0012] (2) The engineered bacteria were inoculated into LB liquid medium and fermented until OD600=0.6~0.8. After induction, fermentation was carried out for 6-8 hours. After fermentation was completed, the bacterial cells were collected.
[0013] (3) After resuspending the collected bacterial cells in Buffer A, the cells were sonicated and the supernatant was collected by centrifugation and passed through a nickel column to adsorb recombinant proteins. Then, the impurities were eluted with a gradient of Buffer B containing different concentrations of imidazole and the recombinant proteins were recovered.
[0014] (4) The recovered recombinant protein was dialyzed and freeze-dried to obtain type VI recombinant mussel foot protein.
[0015] Preferably, the gene sequence described in step (1) is as shown in SEQ ID NO.2.
[0016] Preferably, the vector plasmid in step (1) is pET28a, and the engineered bacteria is Escherichia coli BL21(DE3); the fermentation conditions in step (2) are a temperature of 37±2℃ and a rotation speed of 220±50rpm.
[0017] The above-mentioned type VI recombinant mussel foot protein can be used as a modifier to improve the hydroxylation efficiency of mussel adhesive protein, including the following steps:
[0018] (1) Dissolve type VI recombinant mussel foot protein and add mussel adhesive protein;
[0019] (2) Add tyrosinase to catalyze the reaction; after the reaction is complete, add acid to terminate the reaction.
[0020] In step (1), the VI recombinant mussel foot protein is preserved by preparing freeze-dried powder using a vacuum freeze dryer and storing it at 4°C.
[0021] Preferably, the mass ratio of mussel adhesive protein to type VI recombinant mussel foot protein in step (1) is 9±3:1, and the ratio can be 12:1, 9:1, or 6:1.
[0022] Preferably, the tyrosinase in step (2) is Agaricus bisporus tyrosinase, and the amount of tyrosinase added is 1.0 ± 0.5 wt.% of mussel adhesive protein; the mussel adhesive protein is mussel adhesive protein rich in tyrosine.
[0023] Preferably, the mussel adhesive protein in step (1) is rich in tyrosine residues and is of type III or V.
[0024] Preferably, the type VI recombinant mussel foot protein in step (1) is dissolved in PBS buffer; the reaction conditions in step (2) are: temperature 25±5℃, rotation speed 220±50rpm, pH=6.5±0.5, and reaction time 90±30min.
[0025] Characterization of recombinant type VI mussel adhesive protein: The reduction performance of recombinant type VI mussel foot protein was verified using the DPPH free radical reduction experiment; the hydroxylation effect of Mcfp6 and the traditional hydroxylation system was compared by the acid-sodium borate method, and the mechanical properties of mussel adhesive protein were further characterized by a universal testing machine; the optimization of the mass ratio of mussel adhesive protein to Mcfp6 was determined by NBT; the hydroxylation time was optimized by an amino acid analyzer; and the adhesion force of mussel adhesive protein after hydroxylation under different hydroxylation systems was detected by tensile test using a universal testing machine.
[0026] Compared with the prior art, the present invention has the following advantages:
[0027] (1) This invention constructs a type VI recombinant mussel foot protein Mcfp6 that can be stably expressed and purified. The high content of cysteine residues in the Mcfp6 protein sequence can provide stable and long-lasting reductive properties.
[0028] (2) Type VI recombinant mussel foot protein Mcfp6 was added as a modifier to the hydroxylation system of Mcfp6. The mixture was hydroxylated by tyrosinase, which can increase the hydroxylation efficiency of mussel adhesive protein from 20-25% to 40-45%, thereby obtaining mussel adhesive protein with high dopa content.
[0029] (3) The recombinant mussel foot protein of type VI of the present invention is not toxic to cells and can be used as a biomedical material and cosmetic raw material. Attached Figure Description
[0030] Figure 1 This is a PCR result image of a single colony containing the recombinant plasmid pET28a-Mcfp6. Lanes 2-9 of the DL2000 DNA marker show the PCR results of a single BL21(DE3) colony containing the recombinant plasmid pET28a-Mcofp6.
[0031] Figure 2 This is a graph showing the results of recombinant protein Mcfp6 expression. Lane 1 is the marker, lane 2 is the whole liquid after disruption of engineered bacteria without IPTG induction, and lanes 3-5 are the whole liquid, supernatant, and precipitate after disruption of bacteria induced by IPTG, respectively.
[0032] Figure 3 This is a diagram showing the results of expression and purification of recombinant mussel adhesive protein Mcfp6. Lane 1 is the Maker, lane 2 is the supernatant flow-through after the engineered bacteria were broken, lane 3 is the Buffer A equilibration solution, and lanes 4-13 are the elution solutions with different concentrations of imidazole Buffer B, respectively.
[0033] Figure 4 This is a graph showing the results of the DPPH free radical scavenging experiment of recombinant protein Mcfp6. Deionized water is the negative control, and vitamin C is the positive control. The molar concentration of vitamin C is consistent with that of Mcfp6.
[0034] Figure 5 Figure 1 shows the results of recombinant protein Mcfp6 improving the hydroxylation efficiency of mussel adhesive protein. Figure A is the standard curve of DOPA concentration detected by the acid-sodium borate method; Figure B shows the hydroxylation rate of mussel adhesive protein under different hydroxylation systems.
[0035] Figure 6Figure 1 shows the results of parameter optimization and hydroxylation rate characterization of the hydroxylation system involving recombinant protein Mcfp6 as a modifier. Figure A shows the NBT staining results of different proportions of mussel adhesive protein mixed with Mcfp6; Figure B shows the optimized catalytic time results under the optimal catalytic conditions of tyrosine.
[0036] Figure 7 The universal testing machine is used to test the adhesion force of mussel adhesive protein Mfp5 under different reinforcement systems through tensile testing. Detailed Implementation
[0037] Example 1: Screening of the Mcfp6 gene, a foot protein in type VI mussels
[0038] To enhance the biological function of the expressed type VI recombinant mussel adhesive protein, this invention uses the gene sequence of the natural type VI California mussel Mcfp6 (GenBank: 6549) for design, as shown in SEQ ID NO.1. This sequence contains 121 amino acids, as shown in SEQ ID NO.3. This sequence includes 11 cysteine residues, ensuring the realization of the biological function of the recombinant Mcfp6.
[0039] Example 2: Construction, transformation and validation of recombinant Mcfp6 expression plasmid
[0040] Based on the codon preference of *E. coli*, the coding gene of Mcfp6 shown in SEQ ID NO.1 of Example 1 was codon optimized. The optimized nucleotide sequence of the coding gene is shown in SEQ ID NO.2. The optimized amino acid sequence is shown in SEQ ID NO.4. The nucleotide sequence of Mcfp6 was synthesized into the expression vector pET28a by Nanjing Genewiz Biotechnology Co., Ltd., transformed into *E. coli* BL21(DE3), and successfully verified by sequencing by Hangzhou Youkang Biotechnology Co., Ltd., resulting in *E. coli* BL21(DE3) strain containing the pET28a-Mcfp6 recombinant plasmid. The results are as follows: Figure 1 As shown.
[0041] The optimized gene sequence is as follows:
[0042] ATGCACCGTGTTCTGCACAAACACGTTCACAAACACCGTGTTCTGGGGGGAGGAAATTATAGAGGGTATTGTTCAAATAAAGGATGTAGGAGCGGGTATATATTTTATGATAATCGGGGATATTGTAAATATGGGTCGAGTACGTACAAATACGATTGTGGAAGATATGCGG GTTGTTGCCTGCCGCGTAATCCTTATGGGAACGTGAAGTATTACTGCACCAAGAAAAATGCGTGCCCGAAAGACTTCTACTTCTATAACAATAAAGGCTCCTACTACTATAAACGAAACGCATTCTACGATTGCCGCTCATATAATGGGTGTTGCTTGCGTTCAGGTTACTAA
[0043] The optimized amino acid sequence is as follows:
[0044] MHRVLHKHVHKHRVLGGGNYRGYCSNKGCRSGYIFYDNRGYCKYGSSTYKYDCGRYAGCCLPRNPYGNVKYYCTKKNACPKDFYFYNNKGSYYYKRNAFYDCRSYNGCCLRSGY
[0045] Example 2: Recombinant strain E. coil BL21(DE3) / pET28a-Mcfp6
[0046] Recombinant bacteria *E. coil BL21(DE3)* containing the recombinant plasmid pET28a-Mcfp6 were removed from glycerol tubes and streaked onto plates. Single colonies were selected and inoculated into LB broth containing 50 μg / mL kanamycin as a seed culture, and cultured overnight at 37°C and 220 rpm. The seed culture was then transferred at a 1% inoculum to LB broth containing 50 μg / mL kanamycin. When the bacterial OD600 reached 0.6–0.8, 1 mM IPTG was added to induce protein expression for 6–8 h. Protein samples were collected at 5 OD of bacterial culture and verified by SDS-PAGE. The results showed successful expression of the recombinant protein Mcfp6, with most of the protein being soluble. (Results are as follows...) Figure 2 As shown.
[0047] Example 3: Purification and preparation of recombinant protein Mcfp6
[0048] After fermentation under the conditions described in Example 2, the bacterial culture was centrifuged at 6000 rpm for 10 min. The collected bacterial cells were then purified using the following steps to obtain pure recombinant protein Mcfp6:
[0049] (1) Resuspension and disruption: After resuspending the bacterial cells in Buffer A (8M urea, 0.5M NaCl, 0.02M Tris, pH=7) at a ratio of 10 OD / mL, the resuspension was disrupted using an ultrasonic disruptor. The disruption power and time were adjusted according to the actual situation until the bacterial solution was disrupted and clear.
[0050] (2) Centrifugation: The lysate was centrifuged at 4°C and 10,000 rpm for 30 min using a high-speed centrifuge. The supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the unpurified sample.
[0051] (3) Affinity chromatography: Remove the nickel column from 4℃ and allow the preservative solution to slowly drain under gravity before adding 3-5 column volumes of ddH2O. Equilibrate the column with 3-5 column volumes of Buffer A (8M urea, 0.5M NaCl, 0.02M Tris, pH=7). Load the filtered, unpurified sample at a rate of 5 mL / time, allowing it to flow out under gravity; collect the effluent as the flow-through solution. After loading, rinse the nickel column with 3-5 column volumes of Buffer A and collect the effluent as the equilibration solution. Then, perform gradient elution with different imidazole concentrations (10%, 20%, 30%, 50%, 100%) of Buffer B (4M urea, 0.5M NaCl, 0.02M Tris, 0.5M imidazole, pH=7), collecting the eluent from each gradient. SDS-PAGE verification showed that the target protein began to elute at a 50% gradient. The results are as follows Figure 3 As shown.
[0052] (4) Dialysis desalting: The collected purified stock solution containing a single protein band was desalted in 2.5% acetic acid using a dialysis membrane with a molecular cutoff of 3.5 kDa. The solution was changed every 6-12 hours, and dialysis was performed for 24 hours.
[0053] (5) Freeze-drying: Place the purified sample after dialysis into a pre-cooled vacuum freeze dryer for freeze-drying and preservation.
[0054] Example 4: DPPH free radical scavenging experiment of recombinant protein Mcfp6
[0055] The reducing power of recombinant protein Mcfp6 was assessed using DPPH radical scavenging. 0.008 g of DPPH (98% purity) was dissolved in anhydrous ethanol and diluted to 100 mL to prepare a 0.2 mM DPPH solution. Lyophilized recombinant protein Mcfp6 was dissolved in PBS at pH 6, and the mass concentration of Mcfp6 was determined to be 0.5 mg / mL using the BCA method, converted to molar concentration. The recombinant protein Mcfp6 solution sample and a vitamin C solution sample of the same molar concentration were mixed with the reaction reagents. The specific reaction system used is as follows:
[0056] Table 1. DPPH free radical scavenging experiment
[0057]
[0058] Mix well, incubate at 37℃ in the dark for 30 min, 60 min, 90 min, and 120 min, then centrifuge at 8000 r / min for 5 or 10 min. Take the supernatant and detect the absorbance at a wavelength of 517 nm. The absorbance values of the samples at this time are A. Mcfp6 and A 维生素C The blank absorbance value is A 空白 The control absorbance value is A. 对照 Each sample was tested in triplicate. Free radical scavenging rate R DPPH The calculation is as follows:
[0059]
[0060] Calculations show that at the same molar concentration, Mcfp6 has stronger reducing power than vitamin C, and the free radical scavenging rate of Mcfp6 against DPPH gradually increases with time, indicating that the reducing properties of Mcfp6 are long-lasting. The results are as follows... Figure 4 As shown.
[0061] Example 5: Recombinant protein Mcfp6 as a modifier to improve the hydroxylation efficiency of mussel adhesive proteins
[0062] To compare the effect of Mcfp6 in significantly improving the hydroxylation efficiency of mussel adhesive proteins, the groups were divided into three groups for comparison under the same conditions:
[0063] A. PBS group: Mussel adhesive protein was hydroxylated in 1×PBS without the modifier;
[0064] B.Vc+Na2B4O7 group: mussel adhesive proteins were hydroxylated in 1×PBS containing conventional modifiers (20mM vitamin C, 25mM sodium borate);
[0065] C.Mcfp6 group: Mussel adhesive protein and recombinant protein Mcfp6 were mixed in a certain ratio, and 1×PBS was used as the hydroxylation buffer.
[0066] Hydroxylation was performed using tyrosinase, added at a ratio of 50 Units / (mg mussel adhesive protein). Hydrochloric acid was added to terminate the hydroxylation process after the reaction. Because components such as sodium borate in the reaction system of group B might interfere with the detection results, the hydroxylated mussel adhesive protein was dialyzed into a 1×PBS system using a dialysis bag with a molecular cutoff of 13.5 kDa. Based on the reactivity of the Dopa group, acid-borate differential spectroscopy was used to quantitatively assess the content of Dopa residues in mussel adhesive protein under different hydroxylation systems. The specific detection method is as follows:
[0067] (1) Establishment of the theoretical molar concentration benchmark of tyrosine
[0068] First, the concentration of the unmodified mussel adhesive protein solution, denoted as C, was quantitatively determined using the BCA method. Based on the known amino acid sequence of the unmodified mussel adhesive protein, its theoretical tyrosine molar concentration M was calculated using the following formula:
[0069]
[0070] Where N represents the predetermined number of tyrosine residues in each mussel adhesive protein, MW is its theoretical molecular weight in its unmodified state, and C is the theoretical concentration of the unmodified protein calculated from the measured concentration.
[0071] (2) Acid-borate spectral determination and dopa conversion calculation
[0072] The modified mussel adhesive protein solution was mixed with a predetermined volume of acid buffer and sodium borate solution, respectively. The specific reaction system used is as follows:
[0073] Table 2. Determination of DOPA content by acid-sodium borate method
[0074]
[0075] After mixing, immediately place the solution in an ELISA reader and measure the absorbance at 293 nm. Based on the standard curve, determine the characteristic concentration of dopa in the solution, denoted as M1. Based on the theoretical tyrosine molar concentration M obtained in step (1), calculate the percentage of tyrosine residues converted to dopa during the modification process using the following formula: Dopa conversion rate (%) = [M1 / M] × 100%
[0076] The calculation results showed that the hydroxylation rate of group A was 4.5±1%, that of group B was 21.5±1.5%, and that of group C was 48±2%, meaning the dopamine content in group A was 4.5%±1% (mol / mol Tyr), in group B it was 21.5±1.5% (mol / mol Tyr), and in group C it was 48±2% (mol / mol Tyr). The reducing properties of Mcfp6 can effectively improve the hydroxylation efficiency of type III and type V mussel adhesive proteins rich in tyrosine residues, twice that of traditional hydroxylation systems. The results are as follows... Figure 5 As shown.
[0077] Example 6: Optimization of the hydroxylation system involving recombinant protein Mcfp6 and characterization of hydroxylation rate
[0078] To minimize costs while achieving optimal hydroxylation results, the addition ratio of recombinant protein Mcfp6 to the hydroxylation system was optimized under optimal tyrosinase catalysis conditions (pH 6.5, 25℃). NBT can be reduced by dopamine to a water-insoluble blue formazan compound, and the sites where dopamine accumulates appear blue. The hydroxylation effect of mussel adhesive protein was compared using NBT staining.
[0079] The hydroxylation rate of mussel agarin was further analyzed using an amino acid analyzer. After hydroxylation of mussel agarin, 10 mL of 6M HCl and the protein sample were added to a protein hydrolysis tube. The tube was repeatedly evacuated and purged 2-3 times to maintain an anaerobic environment and avoid affecting the hydrolysis. The sealed tube was then placed in a 110℃ oven for 22-24 hours for hydrolysis. The hydrolyzed solution was filtered through filter paper into a 25 mL volumetric flask, and ddH2O was added to bring the volume to 25 mL. 1 mL of the solution was transferred from the volumetric flask to a 10 mL beaker and deacidified in a 60℃ water bath under a fume hood until only solid residue remained at the bottom. 1 mL of sample buffer was added to dissolve the deacidified sample, which was then filtered through a 0.22 μm filter and placed into a sample vial for analysis. The amino acid analysis results are as follows: Figure 6 As shown, calculations show that when the hydroxylation time is 120 min, the accurate hydroxylation rate of mussel adhesive protein is 42%, which means the dopa content is 42% (mol / mol Tyr).
[0080] Example 7: Tensile test of universal testing machine to detect the adhesive force of hydroxylated mussel adhesive protein
[0081] Mussel adhesive protein, taking mfp5 as an example, was analyzed using three protein groups: PBS group, Vc+Na2B4O7 group, and Mcfp6 group. The protein concentration in each group was controlled at 0.5 mg / mL. 10 μL of protein solution was dropped onto a clean, dried glass slide. Two slides were overlapped and fixed at the protein addition site, with an overlap area of 25 mm × 25 mm. The fixed slides were left at room temperature for 24–48 hours, followed by tensile testing using an Instron 5697 universal testing machine. Both ends of the slide were fixed to the clamps of the universal testing machine, and a 500 N sensor was used for testing. The tensile results are shown below. Figure 7 As shown in the figure. The tensile test results of the universal testing machine show that the Mcfp6 group has better adhesion than the other two groups, further demonstrating that Mcfp6 can improve the hydroxylation efficiency during the hydroxylation process.
[0082] The above embodiments are preferred experimental schemes of the present invention, but the implementation schemes of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A type VI recombinant mussel foot protein, characterized in that, The amino acid sequence of the type VI recombinant mussel foot protein is shown in SEQ ID NO.
4.
2. The gene sequence encoding the type VI recombinant mussel foot protein of claim 1, characterized in that, The gene sequence is shown in SEQ ID NO.
2.
3. A method for preparing type VI recombinant mussel foot protein, characterized in that, Includes the following steps: (1) The gene sequence encoding the type VI recombinant mussel foot protein of claim 1 is ligated to the vector plasmid, and the synthesized plasmid is introduced into the engineered bacteria; (2) The engineered bacteria were inoculated into LB liquid medium and fermented until OD600=0.6~0.
8. After induction, fermentation was carried out for 6-8 hours. After fermentation was completed, the bacterial cells were collected. (3) After resuspending the collected bacterial cells in Buffer A, the cells were sonicated and the supernatant was collected by centrifugation and passed through a nickel column to adsorb recombinant proteins. Then, the impurities were eluted with a gradient of Buffer B containing different concentrations of imidazole and the recombinant proteins were recovered. (4) The recovered recombinant protein was dialyzed and freeze-dried to obtain type VI recombinant mussel foot protein.
4. The preparation method according to claim 3, characterized in that, The gene sequence described in step (1) is shown in SEQ ID NO.
2.
5. The preparation method according to claim 3, characterized in that, The vector plasmid mentioned in step (1) is pET28a, and the engineered bacteria is Escherichia coli BL21(DE3); the fermentation conditions mentioned in step (2) are a temperature of 37±2℃ and a rotation speed of 220±50rpm.
6. The use of the type VI recombinant mussel foot protein of claim 1 or the type VI recombinant mussel foot protein prepared by the method of any one of claims 3-5 as a modifier in improving the hydroxylation efficiency of mussel adhesive protein.
7. The application according to claim 6, characterized in that, Includes the following steps: (1) Dissolve type VI recombinant mussel foot protein and add mussel adhesive protein; (2) Add tyrosinase to catalyze the reaction.
8. The application according to claim 7, characterized in that, The mass ratio of mussel adhesive protein and type VI recombinant mussel foot protein in step (1) is 9±3:
1.
9. The application according to claim 7, characterized in that, The tyrosinase mentioned in step (2) is Agaricus bisporus tyrosinase, and the amount of tyrosinase added is 1.0 ± 0.5 wt.% of mussel adhesive protein; the mussel adhesive protein is mussel adhesive protein rich in tyrosine.
10. The application according to claim 7, 8, or 9, characterized in that, The type VI recombinant mussel foot protein described in step (1) was dissolved in PBS buffer; the reaction conditions in step (2) were: temperature 25±5℃, rotation speed 220±50rpm, pH=6.5±0.5, and reaction time 90±30min.