A heterologously expressed mussel protein precursor and its application

Abstract

The present invention discloses a heterologously expressed mussel protein precursor and its application, relating to the fields of genetic engineering, fermentation engineering and applied biology. The mussel protein (Mfp2-6) precursor has an amino acid sequence shown in any one of SEQ ID NO.1-5, and the expression strain of the mussel protein precursor is Saccharomyces cerevisiae, Pichia pastoris or Escherichia coli. The preparation method of a heterologously expressed mussel protein in the present invention is easier to prepare than other physical, chemical and biosynthetic technologies, has a low preparation cost, is more economical for large-scale application, and the enzyme preparations and processing aids added during the whole process are safe and harmless.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering, fermentation engineering and applied biology, and particularly relates to a heterologously expressed mussel protein precursor and its application. Background Art

[0002] Mussel protein, as a natural adhesive substance derived from nature, has excellent adhesion, flexibility and good biocompatibility, and has many excellent properties such as low toxicity and no induction of human immune response. Mussels fix themselves on solid surfaces such as rocks, ship hulls, cables, and drift bottles underwater through the mussel adhesive protein secreted by their byssus, forming a water-resistant bond and withstanding the scouring of wind and waves. Through the separation and identification of the byssus formed after the curing of mussel protein, currently four major categories and a total of 11 proteins have been obtained, namely mussel byssus proteins: six kinds of mfp-1 to 6, three kinds of pre-collagen (Pre-Collagen), one proximal matrix thread protein (PMTP), and polyphenol oxidase enzyme.

[0003] Mussel protein has a broad application field. However, the large-scale production of mussel protein remains an unsolved problem. In the medical field, mussel protein can be used for the repair of skin injuries such as burns and after laser surgery, and the adhesion of corneal, conjunctival, and small bones, etc.; as a biological coating to make metal materials have better biocompatibility; in the field of daily cosmetics, mussel protein can form a microscopic nano-level protective film on the skin surface. Under the action of physical barrier, antioxidant, and local anti-inflammatory effects, it can treat acne pitted scars, quickly repair the wound skin, inhibit itching and melanin deposition, and keep the skin away from PM2.5 particles; in the field of anti-corrosion coatings, mussel protein is seawater and salt spray resistant and can protect metal and microelectronic devices.

[0004] At present, there are three main methods for producing mussel protein: 1. Extraction and purification from mussel foot glands to obtain a single protein product, which is also the main method for producing mussel protein products. However, due to the extremely low secretion of mussel foot silk protein, 10,000 mussels can extract 1 mg of mussel foot silk protein, which has high production costs and low extraction efficiency, resulting in the price of this directly extracted product being as high as more than 2,000 yuan per milligram; 2. In vitro culture of foot gland cells. However, due to the difficulty in culturing invertebrate cells, their cultivation is still a global problem, and the subculture and establishment of mussel foot gland cells have not been successful; 3. Genetic engineering. At present, recombinant expression using Escherichia coli and Saccharomyces cerevisiae has been achieved, but since the genetic engineering approach cannot perform strict and complex glycosylation modifications, although glycosylation modifications can be performed in vitro, the properties of recombinant proteins are far inferior to natural proteins. Therefore, a mussel protein precursor transformed in vitro after heterologous expression and its application are proposed. Summary of the invention

[0005] The purpose of the present invention is to solve the problem of high price and low yield of mussel protein obtained by the current bioconversion production method. The present invention provides a heterologously expressed mussel protein precursor and its application, by using Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli as genetic engineering starting strains, and the engineered bacteria modified by genetic engineering are fermentation strains, and the composite conversion enzymes involved include oxidase and dehydrogenase. The process system involves an aqueous phase / non-aqueous phase catalytic system. The two-step method mainly includes the construction of engineering strains, fermentation expression of mussel protein precursors and enzymatic conversion and purification of fermentation products.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] A heterologously expressed mussel protein precursor, the mussel protein precursor having an amino acid sequence as shown in any one of SEQ ID NO.1-5, and the strain in which the mussel protein precursor is heterologously expressed is Saccharomyces cerevisiae, Pichia pastoris, or Escherichia coli.

[0008] The present invention also provides a use of the heterologously expressed mussel protein precursor in the preparation of mussel protein.

[0009] A further improvement is that the preparation of mussel protein specifically comprises the following steps:

[0010] (1) According to the codon preference of the mussel protein precursor expression strain, the coding gene sequences of the mussel protein precursor amino acid sequences shown in SEQ ID NO. 1-5 were designed respectively. The gene sequences were ligated to the PET28a vector and transferred into Escherichia coli DH5α. After screening and amplification culture, the plasmid was extracted and verified by double digestion, and then recovered to obtain the target sequence. The target sequence was ligated to the protein expression vector plasmid, and the receptor bacterium was transformed to obtain the engineering strain;

[0011] (2) The engineering strain was activated and amplified, and then fermented for expression. After the fermentation was completed, the bacterial cells were disrupted, and the fermentation product was purified using an affinity chromatography column to obtain the purified product;

[0012] (3) The purified product was added to the composite invertase system for enzymatic conversion. After the enzymatic conversion was completed, the conversion product was separated and purified using an affinity chromatography column. The purified product was freeze-dried and dissolved in 1% citric acid solution to obtain the mussel protein.

[0013] Further improvement lies in that the solvent in the system in step (3) is one of water, ethyl acetate, dimethyl sulfoxide, and glycerol.

[0014] Further improvement lies in that the composite invertase in step (3) is added to the purified product at a mass-volume concentration of 0.1‰-20% in the system, and the enzymatic conversion time is 0.4-18 h.

[0015] Further improvement lies in that the composite invertase in step (3) includes oxidase and dehydrogenase.

[0016] Further improvement lies in that the oxidase includes human ceruloplasmin, vitamin C oxidase, glucose oxidase, manganese peroxidase, lignin peroxidase, tyrosinase, laccase, and horseradish peroxidase.

[0017] Further improvement lies in that the pH of the system in step (3) is 3.5-8.5, and the temperature is 35-65 °C.

[0018] Further improvement lies in that the flow rate of the mobile phase during the purification process in step (2) is 2.0-3.0 mL / s / kg.

[0019] The present invention has the following beneficial effects:

[0020] Compared with other physical, chemical, and biological synthesis technologies, the mussel protein in the method of the present invention is easier to prepare, has a low preparation cost, is more economical for large-scale application, and the enzyme preparations and processing aids added during the whole process are safe and harmless. Description of the Drawings

[0021] Figure 1This is the technical roadmap for the two-step production of mussel protein in the present invention;

[0022] Figure 2 It is a schematic diagram of the pathway for the production of catechol catalyzed by oxidase;

[0023] Figure 3 It is an electron paramagnetic resonance spectrum of the free radical reaction mechanism for the ortho-addition of phenol catalyzed by oxidase;

[0024] Figure 4 It is a three-dimensional structure comparison diagram of the binding affinity and bond energy between the enzyme and the substrate and free radical intermediate. Detailed implementation manners

[0025] The following further describes the present application in conjunction with the accompanying drawings. It is necessary to point out here that the following detailed implementation manners are only used to further illustrate the present application and cannot be construed as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0026] 1. Materials

[0027] All reagents used in this experiment are conventional reagents without special instructions and are prepared with deionized water. All instruments used are conventional laboratory instruments.

[0028] 2. Methods

[0029] 2.1 Use Saccharomyces cerevisiae as the starting strain for genetic engineering to prepare mussel protein

[0030] 2.1.1 Construction of engineering strains

[0031] (1) Gene sequence amplification

[0032] Design a DNA target sequence (GenBank: AST36139.1) with an amino acid sequence of SEQ ID NO.1 according to the codon preference of Saccharomyces cerevisiae and synthesize it.

[0033] Using Saccharomyces cerevisiae as the host, pPIC9k as the cloning vector and expression vector, design primers, and select EcoR1 and Not1 downstream of the AOXI promoter as the restriction enzyme sites. Synthesize the following primer sequences:

[0034] Primer sequences of SEQ ID NO.1 sequence:

[0035] Upstream primer sequence: 5'-GACGAATTCTTGGCGAGTGTAATTCCTGATGTAG-3 ’ ;

[0036] Downstream primer sequence: 5'-AAGGAAAAAAGCGGCCGCGTGGTGGTGGTGGTGGTGAAATCCACAGCTGGATACAT-3';

[0037] PCR amplify the target sequence. The amplification system is as follows:

[0038]

[0039] Put it into the PCR instrument and set the program as follows:

[0040]

[0041] After the PCR program ends, add 6 μL of DNA loading Buffer to the PCR tube, and use a pipette to aspirate all of it and load it into the large-pore agarose gel. Load Marker into another lane and perform constant-voltage electrophoresis for 30 min.

[0042] Put on the protective clothing, take out the agarose gel, put it into the gel imager to compare the size of the target band with that of the Marker to check whether the size of the target band is correct. Under the ultraviolet light, use a sterilized and clean blade to cut off the gel with the target fragment, and perform gel extraction according to the operation instructions of the kit.

[0043] (2) Recombinant plasmid construction

[0044] Perform double digestion on the target sequence and the pPIC9k vector respectively. The digestion system is as follows:

[0045]

[0046] Add the above systems to the PCR tube in sequence, put it into a 37°C water bath for gentle digestion for 2 - 3 h, and then take it out.

[0047] According to the operation requirements of the kit, perform rapid recovery on the digestion products. Aspirate 3 μL of each of the recovered fragment and plasmid, add 1 μL of DNA Loading Buffer to each, load them into the agarose gel, run at constant voltage for 20 min, and then put it into the gel imager to observe the brightness of the bands.

[0048] Determine the addition amount of each in the ligation reaction according to the brightness of the bands of the fragment and the plasmid respectively. The ligation system is as follows:

[0049]

[0050] Put it into the PCR instrument and set the program as follows:

[0051]

[0052] After the program ends, put the ligation product and DH5α competent cells together into an ice box filled with ice. After standing for 5 min, suck all the ligation product into the melted competent cells in a laminar flow hood, then quickly put it back into the ice box and let it stand for 30 min. Heat shock at 42 °C for 90 s, and quickly put it back into the ice box and let it stand for 5 min. Add 600 μL of antibiotic-free LB liquid medium to it in the laminar flow hood, and culture it in a shaker at 37 °C and 180 rpm for 45 - 60 min.

[0053] (3) Resistance screening

[0054] Centrifuge at 12000g for 1 min, retain about 100 μL of the culture medium, aspirate the excess part in the laminar flow hood, resuspend the cell pellet by pipetting up and down with a pipette tip, then aspirate it onto an LB solid plate containing ampicillin, and gently spread it with a spreader until it is dry and as even as possible. Incubate it upside down at 37 °C for 12 - 16 h.

[0055] (4) Colony PCR and scale-up culture

[0056] Pick all the single colonies and add them to 1 ml of LB liquid medium containing 1‰ ampicillin, and culture them at 37 °C and 180 rpm for 12 h.

[0057] Perform bacterial liquid PCR, and the reaction system is as follows:

[0058]

[0059]

[0060] The PCR program is as follows:

[0061]

[0062] After the program is completed, perform agarose gel electrophoresis. Expand the culture of the bacterial liquid with bands. Aspirate 20 μL of the bacterial liquid and add it to 5 ml of LB liquid medium containing 1‰ ampicillin, and culture it at 37 °C and 180 rpm for 12 - 16 h.

[0063] Aspirate the expanded bacterial liquid into a 2 ml Ep tube, centrifuge at 12000g for 1 min, pour off the supernatant, and extract the recombinant plasmid according to the operation requirements of the plasmid extraction kit for sequencing verification. Correct sequencing indicates successful construction of the recombinant plasmid, and the remaining bacterial liquid is frozen and retained.

[0064] 2.1.2 Fermentation expression of mussel protein precursor and enzymatic conversion and purification of fermentation products

[0065] (1) Fermentation expression of mussel protein precursor

[0066] Extract the recombinant plasmid and transfer the recombinant plasmid into the cell wall - removed Saccharomyces cerevisiae competent cells according to the transformation steps in the construction of the engineering bacteria above. Gently spread it onto the YPD solid plate containing ampicillin with a spreader and incubate it upside - down in an incubator at 30 °C for 12 - 16 h.

[0067] Pick some single colonies and add them to 5 ml of YPD liquid medium containing 1‰ ampicillin, and place them in a shaker at 30 °C and 180 rpm for 12 h.

[0068] Take 50 μL of the bacterial liquid and add it to 100 ml of liquid YPD medium (add 1‰ ampicillin), and culture it in a shaker at 30 °C until the OD 600 is in the range of 0.6 - 0.8, and take the sample before induction.

[0069] Add 25 μL of methanol to it in a laminar flow hood, place it in a shaker at 16 °C and 180 rpm for 20 hours, and take the sample after induction.

[0070] Pour the bacterial liquid after induction into a centrifuge tube, accurately balance it with a balance, centrifuge at 4000 rpm and 4 °C for 20 min; pour off the supernatant, resuspend the bacterial cells in a tube with sterile water, centrifuge at 5000 rpm and 4 °C for 30 min; pour off the supernatant, and resuspend the bacterial cells with 35 - 45 ml of Buffer (20 mM Tris - HCl, 500 mM NaCl, pH 7.5).

[0071] Insert the centrifuge tube into a beaker filled with ice and perform ultrasonic disruption. Set the ultrasonic time to 2 s, pause for 3 s, power 300 W, 40%. After the solution becomes clear and transparent, take it out, pour it into a round - bottom centrifuge tube, centrifuge at 12000 rpm and 4 °C for 1 hour. Separate the supernatant from the precipitate, resuspend the precipitate with the same volume of Buffer as the supernatant, and take samples of the supernatant and the precipitate.

[0072] Nickel - column affinity chromatography: Wash the nickel column three times with sterile water, and then wash it three times with the same Buffer, about 10 column volumes each time; add the samples before induction, after induction, 50 ml of Buffer, supernatant (pass through twice), precipitate, flow - through, 50 ml of 20 mM imidazole, 50 ml of 50 mM imidazole, 30 ml of 100 mM imidazole, 20 ml of 200 mM imidazole, 10 ml of 300 mM imidazole, and 10 ml of 500 mM imidazole into the nickel column respectively, take samples of them respectively, and collect the effluent into an ice box respectively;

[0073] Add an equal volume of protein loading buffer to the sample, mix it evenly and then heat it in a microwave oven for 1 - 2 min to denature the protein. Then, sequentially load the denatured protein samples into the gel wells, load the protein marker, and perform electrophoresis at a constant voltage for 50 min;

[0074] After the electrophoresis was completed, the gel was peeled off from the gel plate, Coomassie Brilliant Blue staining solution was added to cover the gel, and it was placed in a microwave oven for staining for 2 min. Then it was taken out and placed on a gel imager. It was observed that the protein was mainly in the supernatant and the protein content in the precipitate was less, indicating that the protein was mainly expressed in a soluble form. In addition, in imidazole with different concentrations, the protein was mainly eluted at 100 mM and 200 mM, and there was almost no target protein in low-concentration imidazole. The eluents of 100 mM and 200 mM imidazole were collected and placed in a suitable concentrator tube, and centrifuged at 3000 rpm for 30 - 60 min;

[0075] The concentrated protein was aspirated from the concentrator tube into an Ep tube, and strictly balanced with a balance, and centrifuged at low speed and frozen for 30 min;

[0076] Gel filtration chromatography: A 2-ml sample loop was selected. First, 10 ml of ultrapure water was aspirated with a syringe and injected into the sample hole, then 10 ml of Buffer was aspirated and injected. Finally, the protein solution was aspirated with a syringe with a needle and injected into the sample hole. The flow rate was adjusted to 1 ml / min, and the Load mode was adjusted to the Inject mode. After waiting for 30 min, the sample was collected

[0077] An Ep tube was placed in the sample collection hole in advance, the sample collection was adjusted to the Load mode, and it was set to collect 1.5 ml per tube. The sample was collected after about 40 min.

[0078] According to the peak size and time, it was judged which peak was the target protein. All the samples before and after the peak were collected into Ep tubes, numbered, and placed in an ice box to obtain a mussel protein precursor with higher purity.

[0079] (2) Enzymatic conversion and purification of fermentation products

[0080] Ten volumes of water were added to the purified product, and the addition amount of the composite enzyme was 1‰ of the system, and the ratio was VC oxidase:glucose oxidase:SOD enzyme = 2:2:1. The conversion was carried out at 30 °C and 100 r / min for 8 h.

[0081] After the enzymatic conversion was completed, it was purified using an affinity chromatography column. During the treatment process, the flow rate of the mobile phase was controlled at 2.0 mL / s / kg according to the amount of the packing.

[0082] 2.2 Preparation of mussel protein using Pichia pastoris as the starting strain for genetic engineering

[0083] 2.2.1 Construction of engineering strains

[0084] (1) Gene sequence

[0085] According to the codon preference of Pichia pastoris, the DNA target sequence with the amino acid sequence of SEQ ID NO.4 (GenBank: ABE01084.1) was designed and synthesized.

[0086] Using Pichia.pastoris as the host and pPIC9k as the cloning and expression vector, primers were designed. The restriction enzyme sites selected were EcoR1 and Not1 downstream of the AOXI promoter. The following primer sequences were synthesized:

[0087] Primer sequence of SEQ ID NO.4:

[0088] Forward primer sequence: 5'-AAATCTAGATACTTCGATTCAACG-3'

[0089] Reverse primer sequence: 5'-AAACATATGACCGCCTCCGGAGGA-3';

[0090] (2) Construction of engineering bacteria

[0091] According to the method in 2.1.1, the engineering bacteria were constructed and verified. The positive transformants were cultured at 30 °C and 180 rpm for 12 - 16 h to expand the culture until the OD 600 was in the range of 0.6 - 0.8.

[0092] 2.2.2 Fermentation expression of mussel protein precursor and enzymatic transformation and purification of fermentation products

[0093] (1) Fermentation expression of mussel protein precursor

[0094] In a laminar flow hood, 1‰ methanol of the system was added thereto, and it was placed in a shaker at 16 °C and 180 rpm for induction for 20 hours.

[0095] The induced bacterial solution was poured into a centrifuge tube, accurately balanced with a balance, centrifuged at 4000 rpm and 4 °C for 20 min; the supernatant was discarded, and the bacterial cells were resuspended in a tube with sterile water, centrifuged at 5000 rpm and 4 °C for 30 min; the supernatant was discarded, and 35 - 45 ml of Buffer (20 mM Tris-HCl, 500 mM NaCl, pH 7.5) was added to resuspend the bacterial cells.

[0096] The centrifuge tube was inserted into a beaker filled with ice for ultrasonic disruption. The ultrasonic settings were 2 s, intermittent for 3 s, power 300 W, 40%. After the solution became clear and transparent, it was taken out, poured into a round-bottom centrifuge tube, and centrifuged at 12000 rpm and 4 °C for 1 hour. The supernatant and the precipitate were separated, and the precipitate was resuspended with Buffer of the same volume as the supernatant.

[0097] According to the steps in 2.1.2, the fermentation products were purified.

[0098] (2) Enzymatic transformation and purification of fermentation products

[0099] Add anhydrous dimethyl sulfoxide in a volume ten times that of the purified product, and the addition amount of the composite enzyme is 2‰ of the system, with the ratio of manganese catalase:SOD enzyme:tyrosinase = 1:2:2. Carry out the conversion at 35 °C and 100 r / min for 16 h.

[0100] After the enzymatic conversion is completed, purify using an affinity chromatography column. During the treatment process, the flow rate of the mobile phase is controlled at 2.0 mL / s / kg according to the amount of the packing material.

[0101] 2.3 Preparation of mussel protein using Escherichia coli as the starting strain for genetic engineering

[0102] 2.3.1 Construction of engineering plasmid

[0103] According to the codon preference of Escherichia coli, DNA target sequences with amino acid sequences of SEQ ID NO.2 (Danner E W, Kan Y, Hammer M U et al. Adhesion of mussel foot protein Mefp-5 to mica: an underwater superglue[J]. Biochemistry, 2012, 51(33): 6511-6518), SEQ ID NO.3 (GenBank: ABC84185.1), and SEQ ID NO.5 (GenBank: ABC84188.1) were designed and synthesized respectively.

[0104] Using Escherichia coli as the host and pET28a as the cloning vector and expression vector, primers were designed. The restriction enzyme sites were selected as Xba1 and Nco1 downstream of the T7 promoter, and the following primer sequences were synthesized.

[0105] Primer sequences for SEQ ID NO.2 sequence:

[0106] Upstream primer sequence: 5, -AAATCTAGACGACTGATAATGCCA-3

[0107] Downstream primer sequence: 5, -AAACATATGGCACCTTTCATGACC-3;

[0108] Primer sequences for SEQ ID NO.3 sequence:

[0109] Upstream primer sequence: 5, -AAATCTAGATACTTCGCTCCTGAT-3

[0110] Downstream primer sequence: 5, -AAACATATGATGCGTATGCCTCTC-3;

[0111] Primer sequences of SEQ ID NO.5:

[0112] Forward primer sequence: 5'-AAATCTAGATACGATAAGTCAAAGAAC-3'

[0113] Reverse primer sequence: 5'-AAACCATGGTAGTTCTCCTTCTTATG-3'.

[0114] Use the above primers to amplify the gene sequence, and then successively complete double digestion, gel extraction, ligation, transformation of Escherichia coli DH5α, coating of LB(A+) plates, picking of single colonies, colony PCR, and sequencing according to the steps in 2.1.1.

[0115] 2.3.2 Fermentation expression of mussel protein and enzymatic conversion and purification of fermentation products

[0116] (1) Fermentation expression of mussel protein

[0117] After the plasmid construction is correct, transform Escherichia coli BL21(DE3) according to the steps of transforming DH5α.

[0118] Pick some single colonies and add them to 5 ml of LB liquid medium containing 1‰ kanamycin, and place them in a shaker at 37°C and 180 rpm for 12 h.

[0119] Take 2 ml of the bacterial solution and add it to 100 ml of liquid LB medium (add 1‰ kanamycin), and culture it in a shaker at 37°C until the OD 600 is in the range of 0.6 - 0.8.

[0120] Add 25 μL of IPTG to it in a laminar flow hood, place it in a shaker at 16°C and 180 rpm for 20 hours, and take the induced sample.

[0121] Pour the induced bacterial solution into a centrifuge tube, centrifuge at 4000 rpm and 4°C for 20 min; discard the supernatant. Resuspend the bacterial cells in a tube with sterile water, centrifuge at 5000 rpm and 4°C for 30 min; discard the supernatant. Add 35 - 45 ml of Buffer (20 mM Tris-HCl, 500 mM NaCl, pH 7.5) to resuspend the bacterial cells.

[0122] Insert the centrifuge tube into a beaker filled with ice, perform ultrasonic disruption, set the ultrasound for 2 s, stop for 3 s, power 300 W, 40%. After the solution becomes clear and transparent, take it out, pour it into a round-bottom centrifuge tube, and centrifuge at 12000 rpm and 4°C for 1 hour.

[0123] Separate the supernatant and the precipitate, resuspend the precipitate with Buffer of the same volume as the supernatant, and take samples of the supernatant and the precipitate.

[0124] Purify the fermentation product according to the steps in 2.1.2.

[0125] (2) Purification of the fermentation product by enzymatic conversion

[0126] Add ethyl acetate anhydride with a volume 15 times that of the purified product. The complex conversion enzyme system can be added at 0.1‰ - 20% of the mass - volume concentration of the system, and the enzymatic conversion time is 0.4 - 18 h. In this example, the addition amount of the complex conversion enzyme system is 0.1‰ of the system, and the mass ratio is horseradish peroxidase: manganese catalase: glucose oxidase = 2:1:3. The conversion conditions are 35 °C and 150 r / min for 12 h.

[0127] After the enzymatic conversion is completed, purify using an affinity chromatography column. During the treatment process, the flow rate of the mobile phase is controlled at 3.0 mL / s / kg according to the amount of the packing.

[0128] 2.4 Results

[0129] Use SDS - PAGE method to determine the protein purity, HPLC method to determine the DOPA content, and universal testing machine to determine the bio - adhesiveness, so as to detect the influence of different genetically engineered starting strains on the properties of the mussel protein finished product. The results are as follows:

[0130] Table 1 Test results of mussel protein properties under different strains and different conversion conditions

[0131]

[0132] As shown in the appendix Figures 1-4 and the above table, among them, Figure 3 shows the electron paramagnetic spectrum of the free - radical reaction mechanism of the oxidase - catalyzed ortho - addition of phenol. Figure 4Show the affinity comparison of neutral substrate, phenolic hydroxyl radical, aldehyde radical and furan acetal; after the above two-step treatment, using the amino acid sequence of mussel protein precursor of SEQ ID NO.1 and Saccharomyces cerevisiae as the starting strain to prepare mussel protein, the protein content of the recombinant mussel protein after affinity chromatography purification is 98.4%, the purity is 99.7%, its DOPA (dopa, 3,4-dihydroxyphenylalanine) content is 31.8 pmol / g, and the biological viscosity is 3116 nN; using the amino acid sequence of mussel protein precursor of SEQ ID NO.4 and Pichia pastoris as the starting strain to prepare mussel protein, the protein content of the recombinant mussel protein after affinity chromatography purification is 98.6%, the purity is 99.3%, its DOPA (dopa, 3,4-dihydroxyphenylalanine) content is 30.6 pmol / g, and the biological viscosity is 3019 nN; using the amino acid sequence of mussel protein precursor of SEQ ID NO.5 and Escherichia coli as the starting strain to prepare mussel protein, the protein content of the recombinant mussel protein after affinity chromatography purification is 96.6%, the purity is 99.3%, its DOPA (dopa, 3,4-dihydroxyphenylalanine) content is 28.6 pmol / g, and the biological viscosity is 2919 nN. Among them, the mussel protein prepared using the amino acid sequence of mussel protein precursor of SEQ ID NO.1 and Saccharomyces cerevisiae as the starting strain has the highest protein content, and the DOPA content and biological viscosity are both the best, making the mussel protein easier to prepare.

[0133] The above has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of a heterologously expressed mussel protein precursor in the preparation of mussel protein, characterized in that, the method for preparing mussel protein using the heterologously expressed mussel protein precursor includes the construction of engineering strains, the fermentation expression of mussel protein precursor, and the enzymatic conversion and purification of fermentation products; the mussel protein precursor has an amino acid sequence shown in any one of SEQ ID NO.1-5, and the strains for heterologous expression of the mussel protein precursor are Saccharomyces cerevisiae, Pichia.pastoris, and Escherichia coli; The preparation of mussel protein specifically includes the following steps: (1) According to the codon preference of the mussel protein precursor expression strain, the coding gene sequences of the mussel protein precursor amino acid sequences shown in SEQ ID NO.1-5 are respectively designed, the gene sequences are ligated to the PET28a vector and transferred into Escherichia coli DH5α, after screening and amplification culture, the plasmid is extracted and verified by double digestion, recovered, the target sequence is obtained, the target sequence is ligated to the protein expression vector, and the receptor bacterium is transformed to obtain an engineering strain; (2) After activating and expanding the culture of the engineering strain, fermentation expression is carried out. After fermentation, the bacterial cells are broken, and the fermentation product is purified using an affinity chromatography column to obtain a purified product; (3) Add the purified product to a composite conversion enzyme system for enzymatic conversion. After the enzymatic conversion is completed, use an affinity chromatography column to separate and purify the conversion product, freeze-dry the purified product, and dissolve it in a 1% citric acid solution to obtain mussel protein; among them, when the strain for heterologous expression of the mussel protein precursor is Saccharomyces cerevisiae, the composite conversion enzyme is VC oxidase, glucose oxidase, and SOD enzyme. When the strain for heterologous expression of the mussel protein precursor is Pichia.pastoris, the composite conversion enzyme is manganese catalase, SOD enzyme, and tyrosinase. When the strain for heterologous expression of the mussel protein precursor is Escherichia coli, the composite conversion enzyme is horseradish peroxidase, manganese catalase, and glucose oxidase.

2. The application according to claim 1, characterized in that, the solvent in the system in step (3) is one of water, ethyl acetate, dimethyl sulfoxide, and glycerol.

3. The application according to claim 1, characterized in that, the composite conversion enzyme in step (3) is added to the purified product at a mass-volume concentration of 0.1‰-20% of the system, and the enzymatic conversion time is 4-18h.

4. The application according to claim 1, characterized in that, the pH of the system in step (3) is 3.5-8.5, and the temperature is 35-65°C.

5. The application according to claim 1, characterized in that, the flow rate of the mobile phase during the purification process in step (2) is 2.0-3.0 mL / s / kg.

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