A bioenzyme catalyst for preparing mussel mucin and preparation method thereof

Abstract

The present invention relates to the technical field of bioenzyme catalysts, and more particularly to a bioenzyme catalyst for preparing mussel mucin and a preparation method thereof, comprising the following steps: S1, adding N-isopropylacrylamide and vinylimidazole to a reactor, adding azobisisobutyronitrile, and reacting to obtain a copolymer; S2, adding chitosan to concentrated sulfuric acid, dripping fuming sulfuric acid, and reacting to obtain a chitosan derivative; S3, adding transglutaminase and tyrosinase to a phosphate buffer, adding a copolymer and a chitosan derivative, adjusting the reaction conditions, stirring at room temperature, centrifuging or filtering and separating after standing, and obtaining a bioenzyme catalyst. The bioenzyme catalyst prepared by the present invention can improve its purification efficiency and DOPA content in the preparation of mussel mucin, and promotes the efficient purification of recombinant mussel mucin.

Description

Technical Field

[0001] The invention relates to the technical field of bio-enzyme catalysts, and in particular to a bio-enzyme catalyst for preparing mussel mucin and a preparation method thereof. Background Art

[0002] Mussel mucin is a natural bioadhesive with high strength, high toughness and good waterproofness. It is widely used in many fields such as biomedicine, marine engineering, surface chemistry and daily chemical products. The super strong interfacial adhesion ability of mussel mucin in wet environment depends on its own complex solidification regulation process, including the post-translational modification reaction of mucin catalyzed by tyrosinase in glandular cells, protein storage after the reaction, antioxidant protection of DOPA (3,4-dihydroxyphenylalanine) of mature protein in vesicles, delayed oxidation of protein at the adhesion interface after secretion, and pH change in water environment. The traditional method of extracting mussel mucin mainly relies on extraction from mussel byssus, but this method is limited by raw materials and extraction process, with low efficiency and yield, and it is difficult to meet the needs of large-scale application. Therefore, in recent years, genetic engineering technology and bioenzyme catalysis have been used for the fermentation production of recombinant mussel mucin.

[0003] In the prior art, tyrosinase is a commonly used bio-enzyme catalyst that can oxidize tyrosine in the protein sequence into DOPA, further forming a membrane or a mesh scaffold, so that the protein can be more tightly and firmly attached to the human body surface. The invention patent with the patent publication number CN117247441A discloses a recombinant mussel mucin and its expression system and application. By mixing the recombinant mussel mucin with tyrosinase, a recombinant mussel mucin with multiple functions of whitening, anti-oxidation, promoting cell repair and promoting wound repair is obtained. However, the activity of tyrosinase is affected by pH value and temperature. Tyrosinase usually exhibits the highest catalytic activity in a near-neutral aqueous solution and is sensitive to temperature changes. Once the environmental conditions exceed its optimal range, the enzyme activity will significantly decrease or even be lost, thereby affecting the yield and quality of the recombinant mussel mucin. In addition, the activity of tyrosinase is also easily inhibited in the presence of high concentrations of substrates or products. Substrate inhibition can be alleviated by controlling the low level of substrate concentration by continuous dripping, but product inhibition is more difficult to solve, thereby affecting the final functional properties of the recombinant mussel mucin. At the same time, it has high stability in aqueous solution, but its activity will be significantly reduced in non-aqueous media. Summary of the invention

[0004] In order to solve the problems mentioned in the above background technology, the present invention provides a bio-enzyme catalyst for preparing mussel mucin and a preparation method thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for preparing a bio-enzyme catalyst for preparing mussel mucin comprises the following steps:

[0007] S1. Add N-isopropylacrylamide and vinylimidazole to a reactor pre-filled with solvent dimethylformamide, stir until completely dissolved to form a transparent solution, place in an oil bath for preheating, pass nitrogen to deoxygenate for 15-25 minutes to ensure that the reaction system is oxygen-free, quickly add azobisisobutyronitrile, continue to pass nitrogen for 5-15 minutes, seal the reactor, keep warm and stir, after the reaction is completed, cool to room temperature to obtain a copolymer solution, pour the copolymer solution into ethanol, stir for 30-60 minutes, and then stand for 2-3 hours to form a precipitate, filter and collect the precipitate, wash with ethanol 3-5 times, and vacuum dry at 40-50° C. to constant weight to obtain a poly (N-isopropylacrylamide-polyvinylimidazole) copolymer;

[0008] S2, adding chitosan powder to an acid solution (1-2% HCl solution), stirring for 2-4 hours, filtering to remove insoluble matter, collecting the filtrate, adding the filtrate to an alkaline solution (1-2% NaOH solution), adjusting the pH value to neutral, filtering to collect the precipitate again, washing the precipitate with deionized water to neutrality, vacuum drying at 60-70°C to constant weight, obtaining chitosan, adding chitosan to concentrated sulfuric acid, stirring until the chitosan is completely dissolved, slowly dropping fuming sulfuric acid, maintaining the reaction temperature, stirring the reaction, after the reaction is completed, cooling to room temperature, slowly pouring the reaction solution into ice water, standing for 2-8 hours to form a precipitate, filtering to collect the precipitate, washing with deionized water to neutrality, and drying at 60-70°C to constant weight to obtain a modified chitosan derivative;

[0009] S3. Add transglutaminase and tyrosinase to phosphate buffer, gently stir at room temperature for 30-40 minutes, add copolymer and modified chitin derivative, adjust reaction conditions, stir at room temperature for 2-4 hours, let stand for 12-24 hours, separate by centrifugation or filtration, and store in a refrigerator at 4°C to obtain a bio-enzyme catalyst.

[0010] Furthermore, the preheating temperature in the oil bath in step S1 is 70-80°C.

[0011] Furthermore, in step S1, the insulation temperature is 70-80° C., the stirring speed is 100-200 rpm, and the reaction time is 24-48 h.

[0012] Furthermore, in step S1, the mass ratio of N-isopropylacrylamide, vinylimidazole, dimethylformamide and azobisisobutyronitrile is (5.1-5.4):(5.5-5.9):(18-20):(0.1-0.3).

[0013] Furthermore, in step S2, the mass ratio of chitosan, concentrated sulfuric acid and fuming sulfuric acid is (6-7): (20-20.5): (10.2-10.6).

[0014] Furthermore, in step S2, the reaction temperature is 80-90° C., the stirring speed is 100-200 rpm, and the reaction time is 4-6 h.

[0015] Furthermore, in step S3, the mass ratio of transglutaminase, tyrosinase, copolymer, chitosan derivative and phosphate buffer is (0.1-0.2):(0.05-0.1):(1-1.5):(1.8-2):20.

[0016] Furthermore, in step S3, the reaction conditions are adjusted as follows: the pH value is adjusted to 6.5-7.5, the temperature is adjusted to 25-30° C., and a saturated NaCl solution is added to adjust the ionic strength to 0.1-0.2M.

[0017] According to another aspect of the present invention, a bio-enzyme catalyst for preparing mussel mucin prepared by the above preparation method is provided.

[0018] Beneficial effects of the present invention:

[0019] 1. In the technical solution of the present invention, transglutaminase and tyrosinase are combined with substrates (copolymers and chitin derivatives) through their active centers to form enzyme-substrate complexes, so that the substrate can better approach the active center of the enzyme. Poly (N-isopropylacrylamide-polyvinyl imidazole) copolymers have unique temperature sensitivity and can undergo phase changes at specific temperatures, so that the copolymers can precisely regulate the activity of transglutaminase and tyrosinase at different temperatures, and optimize the catalytic conditions by changing the conformation or microenvironment of the enzymes, thereby improving the preparation efficiency of mussel mucin. The copolymers effectively stabilize the conformations of transglutaminase and tyrosinase through non-covalent interactions with the enzymes, preventing the inactivation of the enzymes during the catalytic process. At the same time, the copolymers may also act as auxiliary factors of the enzymes, directly participating in the catalytic process, further improving the catalytic efficiency of the enzymes and accelerating the synthesis of mussel mucin.

[0020] 2. In the technical solution of the present invention, chitosan derivatives, by virtue of their specific functional groups and structural characteristics, may not only directly participate in the catalytic reaction as substrates of transglutaminase and tyrosinase, but also regulate the catalytic activity of the enzyme by affecting the active center structure of the enzyme.

[0021] 3. After the catalytic reaction is completed, the enzyme can quickly separate the generated mussel mucin from the substrate molecules, restore them to their original state, and prepare to react again. In this process, the copolymer and chitin derivative play a role in stabilizing the enzyme conformation or promoting product release, ensuring the continuity and efficiency of the catalytic process. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the raw materials used in the present invention are all conventional products purchased from the market. Example 1

[0024] A method for preparing a bio-enzyme catalyst for preparing mussel mucin comprises the following steps:

[0025] S1. Add 5.1 g of N-isopropylacrylamide and 5.5 g of vinylimidazole to a reactor pre-filled with 18 g of dimethylformamide solvent, stir until completely dissolved to form a transparent solution, preheat in a 70°C oil bath, pass nitrogen to deoxygenate for 15 min to ensure that the reaction system is oxygen-free, quickly add 0.1 g of azobisisobutyronitrile, continue to pass nitrogen for 5 min, seal the reactor, keep warm at 70°C and stir at 100 rpm for 24 h, after the reaction is completed, cool to room temperature to obtain a copolymer solution, pour the copolymer solution into ethanol, stir for 30 min and then stand for 2 h to form a precipitate, collect the precipitate by filtration, wash with ethanol 3 times, and vacuum dry at 40°C to constant weight to obtain a poly-N-isopropylacrylamide-polyvinylimidazole copolymer;

[0026] S2, adding chitosan powder to an acid solution (1% HCl solution), stirring for 2 hours, filtering and collecting the filtrate, adding the filtrate to an alkaline solution (1% NaOH solution), adjusting the pH value to neutral, filtering and collecting the precipitate again, washing the precipitate with deionized water to neutrality, and vacuum drying at 60°C to constant weight to obtain chitosan, adding 6g of chitosan to 20g of 98% concentrated sulfuric acid, stirring until the chitosan is completely dissolved, slowly adding 10.2g of fuming sulfuric acid, maintaining the reaction temperature at 80°C, stirring the reaction at a speed of 100rpm for 4 hours, after the reaction is completed, cooling to room temperature, slowly pouring the reaction solution into ice water, standing for 2 hours to form a precipitate, filtering and collecting the precipitate, washing with deionized water to neutrality, and drying at 60°C to constant weight to obtain a modified chitosan derivative;

[0027] S3. Add 0.1g transglutaminase and 0.05g tyrosinase to 20g phosphate buffer, gently stir at room temperature for 30min, add 1g copolymer and 1.8g modified chitin derivative, adjust the pH to 6.5 and the temperature to 25°C, add saturated NaCl solution to adjust the ionic strength to 0.1M, stir at room temperature for 2h, let stand for 12h, centrifuge, and store in a 4°C refrigerator to obtain a bio-enzyme catalyst. Example 2

[0028] A method for preparing a bio-enzyme catalyst for preparing mussel mucin comprises the following steps:

[0029] S1. Add 5.2 g of N-isopropylacrylamide and 5.7 g of vinyl imidazole to a reactor pre-filled with 19 g of dimethylformamide solvent, stir until completely dissolved to form a transparent solution, place in a 75°C oil bath for preheating, pass nitrogen for deoxygenation for 20 min to ensure that the reaction system is oxygen-free, quickly add 0.2 g of azobisisobutyronitrile, continue to pass nitrogen for 10 min, seal the reactor, keep warm at 75°C and stir at 150 rpm for 36 h. After the reaction is completed, cool to room temperature to obtain a copolymer solution, pour the copolymer solution into ethanol, stir for 45 min, and then stand for 2.5 h to form a precipitate, collect the precipitate by filtration, wash with ethanol 4 times, and vacuum dry at 45°C to constant weight to obtain a poly (N-isopropylacrylamide-polyvinyl imidazole) copolymer;

[0030] S2, adding chitosan powder to an acid solution (1.5% HCl solution), stirring for 3 hours, filtering to remove insoluble matter, collecting the filtrate, adding the filtrate to an alkaline solution (1.5% NaOH solution), adjusting the pH value to neutral, filtering to collect the precipitate again, washing the precipitate with deionized water to neutrality, and vacuum drying at 65°C to constant weight to obtain chitosan, adding 6.5g chitosan to 20.3g 98% concentrated sulfuric acid, stirring until the chitosan is completely dissolved, slowly adding 10.4g fuming sulfuric acid, maintaining the reaction temperature at 85°C, stirring at a speed of 150rpm for 5 hours, after the reaction is completed, cooling to room temperature, slowly pouring the reaction solution into ice water, standing for 6 hours to form a precipitate, filtering to collect the precipitate, washing with deionized water to neutrality, and drying at 65°C to constant weight to obtain a modified chitosan derivative;

[0031] S3. Add 0.15g transglutaminase and 0.07g tyrosinase to 20g phosphate buffer, gently stir at room temperature for 35min, add 1.2g copolymer and 1.9g modified chitin derivative, adjust the pH value to 7.0 and the temperature to 28°C, add saturated NaCl solution to adjust the ionic strength to 0.15M, stir at room temperature for 3h, let stand for 18h, centrifuge, and store in a refrigerator at 4°C to obtain a bio-enzyme catalyst. Example 3

[0032] A method for preparing a bio-enzyme catalyst for preparing mussel mucin comprises the following steps:

[0033] S1, adding 5.4g N-isopropylacrylamide and 5.9g vinylimidazole to a reactor pre-filled with 20g dimethylformamide solvent, stirring until completely dissolved to form a transparent solution, placing it in an 80°C oil bath for preheating, passing nitrogen to deoxygenate for 25min to ensure that the reaction system is oxygen-free, quickly adding 0.3g azobisisobutyronitrile, continuing to pass nitrogen for 15min, sealing the reactor, keeping warm at 80°C and stirring at a speed of 200rpm for 48h, after the reaction is completed, cooling to room temperature to obtain a copolymer solution, pouring the copolymer solution into ethanol, stirring for 60min and then standing for 3h to form a precipitate, filtering and collecting the precipitate, washing with ethanol 5 times, and vacuum drying at 50°C to constant weight to obtain a poly-N-isopropylacrylamide-polyvinylimidazole copolymer;

[0034] S2, adding chitosan powder to an acid solution (2% HCl solution), stirring for 4 hours, filtering to remove insoluble matter, collecting the filtrate, adding the filtrate to an alkaline solution (2% NaOH solution), adjusting the pH value to neutral, filtering to collect the precipitate again, washing the precipitate with deionized water to neutrality, and vacuum drying at 70°C to constant weight to obtain chitosan, adding 7g chitosan to 20.5g 98% concentrated sulfuric acid, stirring until the chitosan is completely dissolved, slowly adding 10.6g fuming sulfuric acid, maintaining the reaction temperature at 90°C, stirring the reaction at a speed of 200rpm for 6 hours, after the reaction is completed, cooling to room temperature, slowly pouring the reaction solution into ice water, standing for 8 hours to form a precipitate, filtering to collect the precipitate, washing with deionized water to neutrality, and drying at 70°C to constant weight to obtain a modified chitosan derivative;

[0035] S3. Add 0.2g transglutaminase and 0.1g tyrosinase to 20g phosphate buffer, gently stir at room temperature for 40min, add 1.5g copolymer and 2g modified chitin derivative, adjust the pH value to 7.5, the temperature to 30°C, add saturated NaCl solution to adjust the ionic strength to 0.2M, stir at room temperature for 4h, let stand for 24h, centrifuge, and store in a 4°C refrigerator to obtain a bio-enzyme catalyst.

[0036] Comparative Example 1

[0037] The difference between this comparative example and Example 1 is that no copolymer is added, and the remaining steps are the same as those of Example 1.

[0038] Comparative Example 2

[0039] The difference between this comparative example and Example 2 is that no modified chitosan derivative is added, and the remaining steps are the same as those in Example 2.

[0040] Comparative Example 3

[0041] The difference between this comparative example and Example 3 is that the copolymer and the modified chitosan derivative are not added, and the remaining steps are the same as those in Example 3.

[0042] Preparation of recombinant mussel mucin:

[0043] (I) Recombinant mussel mucin was fermented and cultured in Pichia pastoris GS115 using a 100L pilot fermentation system. The fermentation method was based on the operation method in the "Pichia Fermentation Process Guidelines" of Invitrogen Life Technologies. The pH value during fermentation was 4.8, the temperature was 28±0.1℃, and the dissolved oxygen content was >30%. When the wet bacterial weight of the fermentation liquid reached 200g / L, methanol induction was performed for 48h, and the fermentation liquid was collected.

[0044] (II) The fermentation broth was separated by centrifugation, and the filtrate was collected and microfiltered with a 0.22 μm microfiltration membrane and ultrafiltered with an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain a retentate with a conductivity of less than 0.25 mS / cm. The feed liquid was concentrated to 1 / 10 of the original volume and divided into six equal parts. 20 mmol / L phosphate buffer (pH 7.6) and 20 U of the bio-enzyme catalyst prepared in Example 1-3 and Comparative Example 1-3 were added to the concentrate respectively, and the mixture was shaken and cultured at a speed of 100 rpm for 3 h at room temperature, and glacial acetic acid was added to adjust the pH to 3.2.

[0045] (III) The SPFF cation exchange chromatography column was used for column elution and purification, wherein the loading buffer was an aqueous solution of acetic acid with a mass fraction of 0.1% (pH value was 3.5), the impurity washing mobile phase was 0.1% acetic acid containing 0.19 mol / L NaCl to elute the impurities, and the elution mobile phase was 0.1% acetic acid containing 0.35 mol / L NaCl to elute the recombinant mussel mucin. The eluate was then desalted and concentrated using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, the retentate was collected, and freeze-dried at -4°C to obtain six recombinant mussel mucin freeze-dried powders, which were recorded as experimental groups 1-3 and comparative groups 1-3, corresponding to Example 1-3 and Comparative Example 1-3, respectively.

[0046] The Kjeldahl method was used to determine the pure protein content and purification yield of the six groups of recombinant mussel mucins. The results are shown in Table 1:

[0047] Table 1. Pure protein content and purification yield results of experimental groups 1-3 and comparative groups 1-3

[0048] serial number Pure protein content (%) Purification yield (%) Experimental Group 1 92.35 40.42 Experimental Group 2 92.39 40.46 Experimental Group 3 92.41 40.49 Comparison group 1 90.01 37.30 Comparison group 2 89.58 36.11 Comparison group 3 89.14 36.23

[0049] The DOPA content of six groups of recombinant mussel mucins was determined by the modified Arnow method (see WAITE JH, BENEDICT CV. Assay of dihydroxyphenylalanine (dopa) in invertebrate structural proteins [J]. MethEnzymol, 1984, 107: 397-413.). The results are shown in Table 2:

[0050] Table 2. Results of DOPA content in experimental groups 1-3 and control groups 1-3

[0051] serial number DOPA content (%) Experimental Group 1 1.73 Experimental Group 2 1.76 Experimental Group 3 1.76 Comparison group 1 0.60 Comparison group 2 0.69 Comparison group 3 0.71

[0052] As shown in Table 1, the pure protein content and purification yield of experimental groups 1-3 (corresponding to Examples 1-3) are significantly higher than those of comparative groups 1-3 (corresponding to Comparative Examples 1-3). It shows that in the examples, the addition of copolymers (poly N-isopropylacrylamide-polyvinyl imidazole copolymers) and modified chitin derivatives significantly improves the purification efficiency and purity of recombinant mussel mucin. Poly N-isopropylacrylamide-polyvinyl imidazole copolymers may have interactions with recombinant mussel mucin such as electrostatic attraction and hydrogen bonding, thereby facilitating the selective adsorption and elution of proteins during the purification process, thereby improving the purification efficiency and purity. Modified chitin derivatives may provide additional adsorption sites or change the physicochemical properties of the solution such as viscosity and polarity, further optimizing the purification environment of the protein, making it easier to separate the protein from a complex mixture. Copolymers and modified chitin derivatives may have a synergistic effect, jointly promoting the efficient purification of recombinant mussel mucin.

[0053] As shown in Table 2, the DOPA content of experimental groups 1-3 was also significantly higher than that of control groups 1-3. DOPA is a key amino acid in mussel mucin and has an important influence on the adhesion properties of the protein. The addition of copolymers and modified chitin derivatives may have improved the catalytic efficiency of transglutaminase and tyrosinase, allowing more DOPA residues to be introduced into the recombinant mussel mucin. During the purification process, copolymers and modified chitin derivatives may have played a role in protecting DOPA residues from oxidation or degradation, thereby retaining more DOPA content.

[0054] In summary, the bio-enzyme catalysts prepared in Examples 1-3 can improve the purification efficiency and DOPA content in the preparation of mussel mucin, and promote the efficient purification of recombinant mussel mucin.

[0055] In the description of the specification, the description with reference to the terms "embodiment", "various embodiments" and the like means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or preparation example are included in at least one embodiment or preparation example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or preparation example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or preparation examples in a suitable manner.

[0056] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a bio-enzyme catalyst for preparing mussel mucin, characterized in that: The following steps are involved: S1, adding N-isopropylacrylamide and vinylimidazole to a reactor pre-filled with a solvent of dimethylformamide, stirring until completely dissolved, preheating in an oil bath, passing nitrogen to deoxygenate for 15-25 minutes, adding azobisisobutyronitrile, continuing to pass nitrogen for 5-15 minutes, sealing the reactor, keeping warm and stirring to react, obtaining a copolymer solution, precipitating, filtering, washing, and drying to obtain a copolymer; S2, washing the crude chitosan powder with acid and alkali to obtain chitosan, adding chitosan to concentrated sulfuric acid, stirring until completely dissolved, slowly adding fuming sulfuric acid dropwise, maintaining the reaction temperature, stirring the reaction, cooling to room temperature, pouring into ice water, standing for 2-8h, filtering and collecting the precipitate, washing, and drying to obtain a chitosan derivative; S3, adding transglutaminase and tyrosinase to phosphate buffer, stirring at room temperature for 30-40 minutes, adding copolymer and chitosan derivative, adjusting reaction conditions, continuing stirring at room temperature for 2-4 hours, standing for 12-24 hours, centrifuging or filtering, and storing in a refrigerator at 4°C to obtain a bio-enzyme catalyst; Wherein, the mass ratio of N-isopropylacrylamide, vinylimidazole, dimethylformamide and azobisisobutyronitrile in step S1 is (5.1-5.4): (5.5-5.9): (18-20): (0.1-0.3); The mass ratio of chitosan, concentrated sulfuric acid and fuming sulfuric acid in step S2 is (6-7): (20-20.5): (10.2-10.6); In step S3, the mass ratio of transglutaminase, tyrosinase, copolymer, chitin derivative and phosphate buffer is (0.1-0.2):(0.05-0.1):(1-1.5):(1.8-2):

20.

2. The method for preparing a biological enzyme catalyst for preparing mussel mucin according to claim 1, characterized in that: The preheating temperature in the oil bath in step S1 is 70-80°C.

3. The method for preparing a biological enzyme catalyst for preparing mussel mucin according to claim 1, characterized in that: In step S1, the insulation temperature is 70-80° C., the stirring speed is 100-200 rpm, and the reaction time is 24-48 hours.

4. The method for preparing a biological enzyme catalyst for preparing mussel mucin according to claim 1, characterized in that: In step S2, the reaction temperature is 80-90° C., the stirring speed is 100-200 rpm, and the reaction time is 4-6 h.

5. The method for preparing a biological enzyme catalyst for preparing mussel mucin according to claim 1, characterized in that: In step S3, the reaction conditions are adjusted as follows: the pH value is adjusted to 6.5-7.5, the temperature is adjusted to 25-30° C., and a saturated NaCl solution is added to adjust the ionic strength to 0.1-0.2M.

6. A biological enzyme catalyst for preparing mussel mucin obtained by the preparation method according to any one of claims 1 to 5.