A bioconversion production method for mussel protein

Abstract

The present invention discloses a bioconversion production method for mussel protein, which relates to the fields of genetic engineering, fermentation engineering, and applied biology. The specific steps are: (1) constructing an engineered strain containing a mussel protein precursor; (2) activating and expanding the engineered strain to perform protein fermentation expression; after the fermentation is completed, the bacterial cells are crushed and the fermentation product is purified using an affinity chromatography column to obtain a purified product; (3) the purified product is added to a composite invertase system for enzymatic conversion; after the enzymatic conversion is completed, an affinity chromatography column is used to separate and purify the conversion product; the purified product is freeze-dried and dissolved in an acidic solution to obtain mussel protein. During the entire mussel protein synthesis and recombination process, the eukaryotic expression level is high, the concentration of the target product in the fermentation broth is high, and the biological activity of the enzymatically recombined mussel protein is similar to that of natural mussel protein.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering, fermentation engineering and applied biology, and in particular to a bioconversion production method of mussel protein. Background Art

[0002] Mussel protein, a natural sticky substance derived from nature, has excellent adhesion, flexibility, good biocompatibility, low toxicity, and does not trigger human immune responses, among other excellent properties. Mussels use mussel mucin secreted by their byssus to fix themselves to solid surfaces such as rocks, ship hulls, cables, and drifting bottles under the sea, forming a water-resistant bond that can withstand the erosion of wind and waves. Through the separation and identification of the byssus formed after the solidification of mussel protein, 11 proteins totaling four categories have been obtained. They are mussel byssus proteins: six types of mfp-1 to 6, three types of collagen precursor proteins (Pre-Collagen, Precol), a proximal matrix thread protein (PMTP), and polyphenol oxidase enzyme.

[0003] Mussel protein has a wide range of applications, but large-scale production remains an unresolved challenge. In medicine, mussel protein can be used to repair skin injuries such as burns and those following laser surgery, and to bond the cornea, conjunctiva, and small bones. As a bio-coating, it can improve the biocompatibility of metal materials. In the cosmetics sector, mussel protein can form a microscopic, nanoscale protective film on the skin surface. Through its physical barrier, antioxidant, and local anti-inflammatory properties, it can treat acne scars, rapidly repair wound skin, inhibit itching and melanin deposition, and protect the skin from PM2.5 particles. In the field of anti-corrosion coatings, mussel protein is resistant to seawater and salt spray, and can protect metals and microelectronic devices.

[0004] Currently, there are three main methods for producing mussel protein: 1. Extraction and purification from the mussel foot gland 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 byssus protein, it takes 10,000 mussels to extract 1 mg of mussel byssus protein. This results in high production costs and low extraction efficiency, resulting in a price of over 2,000 yuan per mg for this directly extracted product; 2. In vitro culturing of foot gland cells. However, due to the difficulty of culturing invertebrate cells, their cultivation remains a global challenge, and the subculture and establishment of mussel foot gland cells have been unsuccessful; 3. Genetic engineering. Currently, recombinant expression using Escherichia coli and Saccharomyces cerevisiae has been achieved. However, because genetic engineering approaches cannot perform rigorous and complex glycosylation modifications, although glycosylation modifications can be performed in vitro, the properties of the recombinant protein are far inferior to those of the native protein. Therefore, a biotransformation production method for mussel protein was proposed. Summary of the Invention

[0005] The object of the present invention is to provide a method for producing mussel protein by bioconversion, in view of the high price and low yield of mussel protein obtained by current bioconversion production methods, using yeast (Saccharomyces) as a genetically engineered starting strain, genetically engineered engineered bacteria as a fermentation strain, and then cooperating with a composite enzyme conversion system to obtain mussel protein with natural properties, which is a two-step method for producing mussel protein. The composite conversion enzyme system of the present invention comprises human ceruloplasmin, vitamin C oxidase, glucose oxidase, manganese peroxidase, lignin peroxidase, tyrosinase, laccase, horseradish peroxidase, SOD and alcohol dehydrogenase. The process system of the present invention relates to an aqueous phase catalytic system and a non-aqueous phase catalytic system. The two-step method mainly includes the construction of an engineered strain, fermentation expression of mussel protein and enzymatic conversion and purification of the fermentation product.

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

[0007] A bioconversion production method for mussel protein specifically comprises the following steps:

[0008] (1) Designing the coding gene sequence of the amino acid sequence of the mussel protein precursor according to the codon preference of the mussel protein precursor expression strain, connecting the gene sequence to the PET28a vector and transferring it into Escherichia coli DH5α, screening and amplifying, extracting the plasmid and performing double enzyme digestion verification and recovery to obtain the target sequence, connecting the target sequence to the vector plasmid, transforming the recipient bacteria, and obtaining the engineered strain;

[0009] (2) activating and expanding the engineered strain and then fermenting and expressing it. After the fermentation is completed, the bacteria are broken and the fermentation product is purified using an affinity chromatography column to obtain a purified product.

[0010] (3) adding the purified product to a composite invertase system for enzymatic conversion; after the enzymatic conversion is completed, using an affinity chromatography column to separate and purify the conversion product; freeze-drying the purified product; and dissolving it in a citric acid solution to obtain mussel protein.

[0011] A further improvement is that the mussel protein precursor expression strain is yeast (Saccharomyces), and the mussel protein precursor amino acid sequence is shown as SEQ ID NO.1.

[0012] A further improvement is that the system in step (3) is an aqueous phase conversion system or a non-aqueous phase conversion system.

[0013] A further improvement is that the non-aqueous phase conversion system includes: an inorganic phase and a composite conversion enzyme; wherein the inorganic phase includes ethyl acetate, dimethyl sulfoxide, and glycerol.

[0014] A further improvement is that the aqueous phase conversion system includes: water and a composite invertase.

[0015] A further improvement is that the composite converting enzyme in step (3) includes human ceruloplasmin, vitamin C oxidase, glucose oxidase, manganese peroxidase, lignin peroxidase, tyrosinase, laccase, horseradish peroxidase, SOD and alcohol dehydrogenase.

[0016] A further improvement is that the composite invertase system comprises any one of the following in terms of mass ratio:

[0017] (1) Horseradish peroxidase: glucose oxidase: SOD enzyme = 1:1:2;

[0018] (2) manganese peroxidase: SOD enzyme: tyrosinase = 1:2:2;

[0019] (3) VC oxidase: glucose oxidase: SOD enzyme = 2:2:1;

[0020] (4) Horseradish peroxidase: manganese peroxidase: glucose oxidase = 2:1:3.

[0021] A further improvement is that in step (3), the composite converting enzyme is added at a concentration of 0.1‰-20% of the system mass volume, and the enzymatic conversion time is 0.4-18h.

[0022] A further improvement is that in step (3), the pH of the system is 3.5 to 8.5 and the temperature is 35 to 65°C.

[0023] A further improvement is that the purified product in step (3) is freeze-dried and then dissolved in 1% citric acid solution.

[0024] The present invention has the following beneficial effects:

[0025] The method of the present invention uses a yeast eukaryotic expression system, which is technically convenient and highly controllable. During the entire mussel protein synthesis and recombination process, the eukaryotic expression level is high, the concentration of the target product in the fermentation broth is high, and the biological activity of the mussel protein after enzymatic recombination is similar to that of natural mussel protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the technical roadmap for producing mussel protein by the two-step method of the present invention;

[0027] Figure 2 Schematic diagram of the pathway for the production of catechol catalyzed by oxidase;

[0028] Figure 3The electron paramagnetic spectrum shows the free radical reaction mechanism of oxidase-catalyzed ortho-addition of phenol;

[0029] Figure 4 It is a diagram showing the co-crystallization and bond energy three-dimensional structure of the enzyme, substrate and free radical intermediate. DETAILED DESCRIPTION

[0030] The present application is described in further detail below in conjunction with the accompanying drawings. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technicians in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0031] 1. Materials

[0032] All reagents used in this experiment are conventional reagents unless otherwise specified. They are all prepared with deionized water, and all instruments used are conventional laboratory instruments.

[0033] 2. Methods

[0034] 2.1 Construction of engineered strains

[0035] (1) Gene sequence amplification

[0036] Based on the amino acid sequence of mussel protein precursor SEQ ID NO.1 (GenBank: AST36139.1) and its codon preference principle in the yeast expression system, the target sequence was designed and the DNA sequence was synthesized. Primers were designed using yeast (Saccharomyces) as the host and pPIC9k as the cloning and expression vectors. The restriction enzyme sites EcoR1 and Not1 downstream of the AOXI promoter were selected.

[0037] The following primer sequences were synthesized:

[0038] Upstream primer sequence: 5′-GACGAATTCTTGGCGAGTGTAATTCCTGATGTAG-3′;

[0039] Downstream primer sequence: 5′-AAGGAAAAAAGCGGCCGCGTGGTGGTGGTGGTGGTGGTGAAATCCACAGCTGGATACAT-3′;

[0040] PCR amplification of the target sequence, the amplification system is:

[0041]

[0042] Place in PCR instrument and set the program as follows:

[0043]

[0044] After the PCR program is completed, add 6 μL DNA loading buffer to the PCR tube, use a pipette to aspirate all of it and spot it into the macroporous agarose gel, spot a marker in another lane, and perform constant voltage electrophoresis for 30 minutes.

[0045] Put on protective clothing, take out the agarose gel, put it into the gel imaging instrument and compare the target band with the marker size to check whether the target band size is correct. Under ultraviolet light, use a sterilized and clean blade to cut the gel with the target fragment and recover the gel according to the operating instructions of the kit.

[0046] (2) Construction of engineered bacteria

[0047] The target sequence and pPIC9k vector were double-digested separately, and the enzyme digestion system was as follows:

[0048]

[0049]

[0050] Add the above system into the PCR tube in sequence, place it in a 37℃ water bath for gentle enzyme digestion for 2-3 hours, and then take it out.

[0051] According to the operating requirements of the kit, the enzyme digestion products were quickly recovered. 3 μL of the recovered fragments and plasmids were aspirated, 1 μL of DNA Loading Buffer was added to each, and the samples were spotted into the agarose gel. After running at constant pressure for 20 minutes, the gel was placed in a gel imaging instrument to observe the brightness of the bands.

[0052] The amount of each added in the ligation reaction is determined based on the brightness of the fragment and plasmid bands. The ligation system is:

[0053]

[0054] Place in PCR instrument and set the program as follows:

[0055]

[0056] After the procedure is complete, place the ligation product and DH5α competent cells in an ice box. Let it sit for 5 minutes. Then, in a clean bench, pipette the ligation product into the thawed competent cells. Quickly return the cells to the ice box and let them sit for 30 minutes. Heat shock the cells at 42°C for 90 seconds, then quickly return them to the ice box and let them sit for 5 minutes. In the clean bench, add 600 μL of antibiotic-free LB medium and incubate the cells in a shaker at 37°C and 180 rpm for 45-60 minutes.

[0057] (3) Resistance screening

[0058] Centrifuge at 12000g for 1 min, retain about 100 μL of culture medium, aspirate the excess in a clean bench, repeatedly blow and aspirate with a pipette to resuspend the bacteria, then aspirate onto a solid LB plate containing ampicillin, gently spread it with a spreader until it is dry and as evenly as possible, and culture it upside down at 37℃ for 12-16 hours.

[0059] (4) Expanded cultivation

[0060] All single colonies were picked and added to 1 ml of LB liquid medium containing 1‰ ampicillin, and cultured at 37°C and 180 rpm for 12 h.

[0061] Perform bacterial liquid PCR using the following system:

[0062]

[0063] The PCR procedure is as follows:

[0064]

[0065] After the program is completed, perform agarose gel electrophoresis, expand the bacterial solution with bands, and take 20 μL of bacterial solution and add it to 5 ml of LB liquid culture medium containing 1‰ ampicillin, and culture it at 37°C and 180 rpm for 12-16 hours.

[0066] Transfer the expanded bacterial suspension to a 2 ml Eppendorf tube and centrifuge at 12,000 g for 1 minute. Discard the supernatant and extract the recombinant plasmid according to the instructions of the plasmid extraction kit for sequencing. Correct sequencing indicates successful recombinant plasmid construction. Freeze the remaining bacterial suspension.

[0067] Extract the recombinant plasmid and transfer it into the yeast competent cells with the wall removed according to the transformation steps in the construction of the engineering bacteria. Use a spreader to gently spread it onto a YPD solid plate containing ampicillin and invert it in a 30°C incubator for 12-16 hours.

[0068] 2.2 Fermentation expression of mussel protein precursor

[0069] Pick some single colonies and add them into 5 ml of YPD liquid culture medium containing 1‰ ampicillin, and culture in a shaking incubator at 30°C and 180 rpm for 12 h.

[0070] Take 50 μL of bacterial solution and add it to 100 ml of liquid YPD medium (add 1‰ ampicillin), and culture it in a shaking incubator at 30°C until the OD 600 In the range of 0.6-0.8, take the sample before induction.

[0071] In a clean bench, 25 μL of methanol was added, and the cells were cultured in a shaker at 16°C and 180 rpm for 20 hours, and samples were taken after induction.

[0072] Pour the induced bacterial liquid into a centrifuge tube, accurately balance it with a balance, and centrifuge it at 4000 rpm and 4°C for 20 min; discard the supernatant, resuspend the bacteria in a tube with sterilized water, and centrifuge it 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 bacteria.

[0073] Insert the centrifuge tube into an ice-filled beaker and sonicate for 2 seconds, 3 seconds on, at 300W power and 40%. Once the solution is clear and translucent, remove it from the tube and transfer it to a round-bottom centrifuge tube. Centrifuge at 12,000 rpm and 4°C for 1 hour. Separate the supernatant from the precipitate and resuspend the precipitate in an equal volume of buffer. Sample the supernatant and precipitate.

[0074] Nickel column affinity chromatography: Wash the nickel column three times with sterile water and then three times with the same buffer, each time for approximately 10 column volumes. Add the pre-induction and post-induction solutions, 50 ml of buffer, supernatant (passed 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 to the nickel column, sample each column, and collect the effluent in an ice box.

[0075] Add an equal volume of protein loading buffer to the sample, mix thoroughly, and heat in a microwave for 1-2 minutes to denature the protein. Then, apply the denatured protein sample to the gel wells, add the protein marker, and perform electrophoresis at constant voltage for 50 minutes.

[0076] After electrophoresis, peel the gel from the plate, add Coomassie Brilliant Blue staining solution to cover the gel, place in a microwave oven for 2 minutes, remove and place on a gel imaging instrument. Observe that the protein is mainly in the supernatant, and the protein content in the precipitate is relatively low, indicating that the protein is mainly expressed in a soluble form. In addition, at different concentrations of imidazole, the protein is mainly eluted at 100mM and 200mM, and there is almost no target protein at low concentrations of imidazole. Collect the eluate of 100mM and 200mM imidazole, place in a suitable concentrator, and centrifuge at 3000rpm for 30-60min;

[0077] The concentrated protein was transferred from the concentrator tube to the Eppendorf tube, the balance was strictly balanced, and low-speed refrigerated centrifugation was performed for 30 minutes;

[0078] Gel filtration chromatography: Use a 2ml sample loop, first use a syringe to draw 10ml of ultrapure water into the sample well, then draw 10ml of buffer into it, and finally use a syringe with a needle to draw the protein solution into the sample well, adjust the flow rate to 1ml / min, adjust the Load mode to Inject mode, wait 30 minutes before loading the sample

[0079] Place the Eppendorf tube in the sample receiving hole in advance, switch the sample receiving mode to Load mode, set each tube to receive 1.5 ml, and receive the sample after about 40 minutes.

[0080] Determine which peak is the target protein based on the peak size and time, collect all samples before and after the peak into Ep tubes, write numbers on them, and put them in an ice box to obtain mussel protein precursor with higher purity.

[0081] 2.3 Enzyme conversion

[0082] The mussel protein precursor was added to the composite invertase system, and the enzymatic conversion process was carried out in the aqueous phase system and the non-aqueous phase system, respectively. The composition of the composite invertase system is shown in Table 1, wherein the aqueous phase conversion system includes: water, the composite invertase system, and the recombinant mussel protein; the non-aqueous phase conversion system includes: an inorganic phase, the composite invertase system, and the recombinant mussel protein, and the inorganic phase includes ethyl acetate, dimethyl sulfoxide, and glycerol.

[0083] Table 1 Ratios of different reaction systems and enzymes

[0084]

[0085] The composite invertase system can be added at a concentration of 0.1‰ to 20% of the system mass volume, and the enzymatic conversion time is 0.4 to 18 hours. In this example, the composite invertase system was added at a concentration of 0.1‰ of the system mass volume, and the conversion was carried out for 8 hours at 30°C and 100 rpm. After the enzymatic conversion, the product was purified using an affinity chromatography column. The mobile phase flow rate during the treatment was controlled at 2.0 mL / s / kg, depending on the amount of filler.

[0086] 2.4 Results

[0087] The protein purity was determined by SDS-PAGE, the DOPA content was determined by HPLC, and the bioadhesion was determined by a universal testing machine. The effects of different reaction systems on the properties of the mussel protein product were tested. The results are as follows:

[0088] Table 2 Test results of mussel protein properties under different reaction systems

[0089]

[0090]

[0091] like Figure 1-4As shown in the above table, Figure 3 Electron paramagnetic spectrum showing the free radical reaction mechanism of oxidase-catalyzed ortho-addition of phenol, Figure 4 The affinity comparison of neutral substrates, phenolic hydroxyl radicals, aldehyde radicals and furan acetals is shown; the present invention adopts yeast (Saccharomyces) as a genetic engineering starting strain, and the genetically engineered engineered bacteria is used as a fermentation strain. After purification and fermentation, a mussel protein precursor is obtained, and the mussel protein precursor is enzymatically converted in an aqueous catalytic system and a non-aqueous catalytic system, and finally the mussel protein is obtained after separation and purification; wherein the aqueous catalytic system adopts horseradish peroxidase: manganese peroxidase: glucose oxidase in a ratio of 2:1:3, and the obtained mussel protein has a protein purity of 99.6% after affinity chromatography purification, and its DO The PA (DOPA, 3,4-dihydroxyphenylalanine) content is 32.4 pmol / g, and the bioadhesion is 3205 nN; in the non-aqueous phase catalytic system, horseradish peroxidase: glucose oxidase: SOD enzyme in a ratio of 1:1:2 is used. After affinity chromatography purification, the obtained mussel protein has a protein purity of 99.2%, its DOPA (DOPA, 3,4-dihydroxyphenylalanine) content is 33.2 pmol / g, and the bioadhesion is 3295 nN; the preparation method uses a yeast eukaryotic expression system, which is technically convenient and highly controllable; during the entire mussel protein synthesis and recombination process, the eukaryotic expression level is high.

[0092] The above shows and describes 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 to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A bioconversion production method for mussel protein, characterized in that: The specific steps include: (1) Designing a gene sequence encoding the amino acid sequence of a mussel protein precursor according to the codon preference of a mussel protein precursor expression strain, wherein the mussel protein precursor expression strain is a yeast ( Saccharomyces ), the amino acid sequence of the mussel protein precursor is shown in SEQ ID NO.1 in the sequence listing, the gene sequence is connected to the PET28a vector and transformed into Escherichia coli DH5α, after screening and expansion, the plasmid is extracted and double enzyme digestion is performed for verification and recovery to obtain the target sequence, which is then connected to the vector plasmid and transformed into the recipient bacteria to obtain an engineered strain; (2) activating and expanding the engineered strain and then fermenting and expressing it. After the fermentation is completed, the bacteria are broken and the fermentation product is purified using an affinity chromatography column to obtain a purified product; (3) adding the purified product to a composite conversion enzyme system for enzymatic conversion, wherein the composite conversion enzyme system comprises any one of the following in a mass ratio: (a) horseradish peroxidase: glucose oxidase: SOD enzyme = 1:1:2; (b) manganese peroxidase: SOD enzyme: tyrosinase = 1:2:2; (c) VC oxidase: glucose oxidase: SOD enzyme = 2:2:1; (d) horseradish peroxidase: manganese peroxidase: glucose oxidase = 2:1:

3. After the enzymatic conversion is completed, an affinity chromatography column is used to separate and purify the conversion product, the purified product is freeze-dried, and dissolved in a 1% citric acid solution to obtain mussel protein.

2. The bioconversion production method of mussel protein according to claim 1, characterized in that: The system in step (3) is an aqueous phase conversion system or a non-aqueous phase conversion system.

3. The bioconversion production method of mussel protein according to claim 2, characterized in that: The non-aqueous phase conversion system comprises: an inorganic phase and a composite conversion enzyme; wherein the inorganic phase comprises ethyl acetate, dimethyl sulfoxide and glycerol.

4. The bioconversion production method of mussel protein according to claim 2, characterized in that: The aqueous phase conversion system comprises water and a composite conversion enzyme.

5. The bioconversion production method of mussel protein according to claim 1, characterized in that: In step (3), the composite converting enzyme is added at a mass volume concentration of 0.1‰-20% of the system, and the enzymatic conversion time is 0.4-18h.

6. The bioconversion production method of mussel protein according to claim 1, characterized in that: In step (3), the pH of the system is 3.5-8.5, and the temperature is 35-65°C.

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