A method for preparing regenerated cellulose fiber combined with recombinant protein

Abstract

The present invention discloses a method for preparing a regenerated cellulose fiber combined with a recombinant protein, comprising the following steps: dissolving a recombinant protein and cellulose in a solvent and mixing them evenly to obtain a spinning solution; spinning the spinning solution to obtain the regenerated cellulose fiber combined with the recombinant protein; the recombinant protein is a recombinant protein obtained by fusion expression of at least one cellulose binding domain and at least one functional protein. The recombinant functional protein containing CBM of the present invention can maintain structural and functional stability in the NMMO system, thereby providing the biological activity of the protein for the Lyocell fiber system. The composite of fluorescent protein can bring a natural fluorescent effect to the fiber, and has application prospects in the field of textile fashion. The fiber strength is increased by expressing the MaSp1 recombinant spider silk protein, and the fiber elasticity is enhanced by expressing the MaSp4 recombinant spider silk protein. At the same time, the CBM fusion protein can also carry other functional modules to give the cellulose fiber specific functions.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a method for preparing regenerated cellulose fiber combined with recombinant protein. Background Art

[0002] Lyocell fiber is a regenerated cellulose fiber obtained by dissolving cellulose-containing pulp in the organic solvent N-methylmorpholine-N-oxide (NMMO) and wet-spinning it through dry jet. It has the characteristics of high strength (3.8-4.2 cN / dtex), excellent hygroscopicity and comfort, easy dyeing and easy biodegradability. However, higher strength fibers are still needed in sports equipment and industrial applications, and fibers with specific functionality are required in daily applications. Lyocell fiber itself does not have antibacterial properties, flame retardancy, and is prone to radially detached fibrils after wet friction (called fibrillation). These shortcomings and properties limit the application of Lyocell fiber in a wider range of scenarios.

[0003] In order to give Lyocell fibers specific functionality and broaden their usage scenarios, combining cellulose with functional materials to prepare functional cellulose fibers has become the mainstream idea. The method of depositing inorganic powder particles (such as silica, alumina, etc.) on the fiber surface can make the fiber have corresponding functionality (such as antibacterial, thermal conductivity, wear resistance, etc.), but there is no interaction between inorganic powder and cellulose, and powder shedding problems will occur, affecting the durability of fiber functions; protein, as a type of natural biomass material, is also used for the modification of cellulose fibers, thus providing new ideas and methods for the preparation of green and environmentally friendly cellulose fibers. However, the current protein modification method cannot make the protein and cellulose interact with each other, but is simply a physical doping. In subsequent use, there will be problems such as protein shedding, loss, and uneven distribution, which will gradually weaken the function of the fiber.

[0004] Spider silk protein is the main component of natural spider silk, which provides spider silk with unparalleled strength, elasticity and toughness. Because its structure is rich in β-folded crystalline regions, spider silk is endowed with strength, among which the strength of the large ampulla silk is comparable to that of carbon fiber and Kevlar 49. Because its structure is rich in α-helix and β-turn, spider silk is endowed with excellent ductility, among which the elasticity of the whip gland silk is comparable to that of spandex. The unique structural characteristics and excellent water solubility of spider silk protein have laid a solid foundation for it as a fiber reinforcement material. Through genetic engineering and synthetic biology technology, specific functional protein domains can be added to spider silk protein, which can give it new functions. If functional proteins such as spider silk protein can be effectively combined with lyocell fiber, lyocell fiber can be given stronger performance and Lyocell fiber with special functions can be obtained. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing regenerated cellulose fiber combined with recombinant protein, so as to solve the problem that the combination effect of protein and cellulose in the regenerated cellulose fiber is poor.

[0006] A method for preparing regenerated cellulose fiber combined with recombinant protein comprises the following steps:

[0007] Dissolving the recombinant protein and cellulose in a solvent and mixing them evenly to obtain a spinning solution; spinning the spinning solution to obtain the recombinant protein-bound regenerated cellulose fiber;

[0008] The recombinant protein is a recombinant protein obtained by fusion expression of at least one cellulose binding domain and at least one functional protein.

[0009] Optionally, the amino acid sequence of the cellulose binding domain is as shown in SEQ ID NO.1.

[0010] The cellulose binding domain is not limited to the above-mentioned sequence, as long as it can achieve the effect of binding to cellulose, and thus it can be replaced by other cellulose binding domain amino acid sequences.

[0011] Optionally, the functional protein comprises at least one of a fluorescent protein and a spider silk protein.

[0012] Optionally, the spider silk protein is a MaSp spider silk protein, and the amino acid sequence of the spider silk protein contains at least one spider silk protein repeat region sequence;

[0013] The spider silk protein repeat region sequence is shown as SEQ ID NO.6 or SEQ ID NO.7.

[0014] Optionally, the N-terminus of the spider silk protein repeat region sequence is connected to the spider silk protein N-terminal sequence, and the C-terminus of the spider silk protein repeat region sequence is connected to the spider silk protein C-terminal sequence.

[0015] Optionally, the N-terminus and / or C-terminus of the functional protein in the recombinant protein is connected to at least one cellulose binding domain.

[0016] Optionally, the recombinant protein comprises a plurality of functional proteins, wherein at least two functional proteins are connected via at least one cellulose binding domain.

[0017] Optionally, both the N-terminus and the C-terminus of the functional protein in the recombinant protein are connected to at least one cellulose binding domain.

[0018] Optionally, the recombinant protein is prepared by the following method:

[0019] Optionally, the bacteria are collected and the recombinant protein is extracted.

[0020] Optionally, the nucleic acid sequence for expressing the recombinant protein is shown in any one of SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.12.

[0021] Optionally, the cellulose is powdered cellulose after activation treatment.

[0022] Optionally, the activated cellulose powder is obtained by:

[0023] The cellulose raw material with a cellulose content of not less than 95 wt% and an average degree of polymerization of 500 to 650 is impregnated, and then washed, dried and crushed to obtain activated cellulose powder;

[0024] The immersion treatment is carried out using a concentrated sulfuric acid solution with a concentration of 9 to 36 g / L and a temperature of 60 to 90° C. or a sodium hydroxide solution with a concentration of 100 to 300 g / L and a temperature of 40 to 80° C., and the immersion time is 2 to 10 hours;

[0025] Optionally, the cellulose raw material is wood pulp board;

[0026] Optionally, the cellulose methyl cellulose used has a content of greater than 98% and an average degree of polymerization of 620;

[0027] Optionally, the concentration of concentrated sulfuric acid used is 18 g / L, the temperature is 80° C., and the treatment time is 6 h;

[0028] Optionally, the concentration of sodium hydroxide used is 200 g / L, the temperature is 80° C., and the treatment time is 8 h.

[0029] The solvent is NMMO solvent or ionic liquid solvent;

[0030] Preferably, the ionic liquid solvent is 1-butyl-3-methylimidazolium chloride or N,N-dimethylacetamide / lithium chloride.

[0031] Optionally, the method of dissolving the recombinant protein and the cellulose raw material in a solvent and mixing them uniformly is specifically as follows:

[0032] Step 1) dissolving the recombinant protein in NMMO solvent, wherein the NMMO solvent concentration is 50-90wt%, the dissolution temperature is 25-95°C, and the dissolution time is 0.2-2h; obtaining a recombinant protein NMMO solution;

[0033] Step 2) adding cellulose to the recombinant protein NMMO solution and stirring to dissolve to obtain a spinning solution;

[0034] Optionally, the NMMO solvent concentration used in step 1) is 50wt%, the dissolution temperature is 60°C, and the dissolution time is 0.5h.

[0035] Optionally, in step 2), the cellulose concentration is 4-13wt% of the total mass of the recombinant spider silk protein / NMMO mixed spinning solution, the stirring and dissolving temperature is 80-110° C., and the dissolving time is 1-5 hours, until a transparent amber composite spinning solution is obtained.

[0036] Optionally, the cellulose concentration used is 12 wt % of the total mass of the recombinant spider silk protein / NMMO mixed spinning solution, the stirring dissolution temperature is 100° C., and the dissolution time is 3 h.

[0037] Optionally, the mass ratio of the recombinant protein to cellulose is 1:0.5-100.

[0038] Optionally, the spinning solution is degassed and filtered at high temperature, and then passed through a spinneret for dry-jet wet spinning. The spinning speed is 40-50 m / min, the spinneret aperture is 100-300 μm, the air gap length is 5-50 mm, and the spinning line is vertically stretched by air into a coagulation bath for forming. The first coagulation bath uses a 5-15wt% NMMO solution at 25-70°C, and the second coagulation bath uses an aqueous solution at 25-70°C. Then, the obtained primary fiber is washed by hot and cold water exchange, cut, oiled and dried to obtain recombinant spider silk protein lyocell fiber.

[0039] Optionally, the spinning speed is 35 m / min, the spinneret aperture is 260 μm, the air gap length is 5 mm, the first coagulation bath uses a 15 wt % NMMO solution at 25° C., and the second coagulation bath uses an aqueous solution at 25° C.

[0040] Optionally, the method of dissolving the recombinant protein and the cellulose raw material in a solvent and mixing them uniformly is specifically as follows:

[0041] The ionic liquid, cellulose and recombinant protein are mixed and stirred at 100° C. for 1 to 2 hours; then vacuum degassing is performed at 100° C. for 1 to 2 hours and filtering is performed to obtain a composite spinning solution.

[0042] Optionally, the composite spinning solution is added into a syringe for spinning, the spinning speed is 40-50 m / min, the spinning needle aperture is 100-300 μm, and the air gap length is 5-50 mm; the spinning thread is vertically stretched into a coagulation bath through air to be formed, and the coagulation bath temperature is 25-70°C; then the obtained primary fiber is washed, stretched, oiled, and dried to obtain recombinant spider silk regenerated fiber.

[0043] The present invention has the following beneficial effects:

[0044] The recombinant functional protein of CBM of the present invention can maintain the stability of structure and function in the NMMO system, thereby providing the biological activity of protein for the Lyocell fiber system. The compound of fluorescent protein can bring natural fluorescent effect to the fiber, and has application prospects in the field of textile fashion; MaSp is the most common spider silk protein, usually present in the large ampullate gland, responsible for forming the framework structure of the spider web, MaSp1 is rich in β-folding composed of alanine, this structure gives spider silk high strength, MaSp4 is rich in β-turn composed of proline and glycine, so that spider silk has excellent elasticity. The fiber strength is increased by expressing MaSp1 recombinant spider silk protein, and the fiber elasticity performance is enhanced by expressing MaSp4 recombinant spider silk protein. At the same time, CBM fusion protein can also carry other functional modules to give cellulose fiber specific functions, further broaden the use scenarios of cellulose fiber, and make important contributions in daily consumption, outdoor sports, medical health, industrial production, and military defense.

[0045] The present invention utilizes the recombinant protein with a double CBM structure to achieve directional grasping with cellulose, as well as the cross-linking between recombinant spider silk proteins to form a double cross-linked network structure, which innovatively and significantly improves the fibrillation phenomenon of Lyocell fibers, while enhancing their mechanical properties. The strength of Lyocell fibers reinforced with strong recombinant spider silk proteins can be increased by 168.48%, and the elongation at break of Lyocell fibers reinforced with elastic recombinant spider silk proteins can be increased by 189.93%. After an industrial spinning process, the strength of recombinant spider silk protein Lyocell fibers can reach 5.40 cN / dtex. The improvement in physical properties greatly broadens the application scenarios of the fibers;

[0046] The recombinant spider silk protein structure designed by the present invention is easily soluble in NMMO solution. The spinning solution prepared with high protein concentration (more than 20%) is clear and free of particle precipitation, and has high spinnability. In addition, the NMMO solvent will not be polluted during the dissolution process, and the clean recovery of the NMMO solvent will not be affected.

[0047] The protein and cellulose spinning process designed by the present invention is green and environmentally friendly, simple and efficient to operate, and the solvent system can achieve 99.5% recycling and reuse, thus avoiding the problem of chemical residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0049] Figure 1The following are pictures of the spinning solutions of Example 2 and Comparative Example 1 in a flask and under a microscope; Figure 1 A is a photo of the spinning solution in the flask of Example 2, Figure 1 B is a photo of the spinning solution of Example 2 under a microscope. Figure 1 C is a photo of the spinning solution in the flask of Comparative Example 1, Figure 1 D is a photograph of the spinning solution of Comparative Example 1 under a microscope.

[0050] Figure 2 These are the bright field images and fluorescent field images of the fibers of Example 2 and Comparative Example 2 under a fluorescent microscope; Figure 2 A is the bright field picture of comparative example 2 under a fluorescence microscope, Figure 2 B is the fluorescence field image under the fluorescence microscope of Comparative Example 2; Figure 2 C is a bright field image of the sample in Example 2 with the recombinant protein CBM-GFP added under a fluorescence microscope. Figure 2 D is a fluorescence field image of the sample added with recombinant protein CBM-GFP under a fluorescence microscope in Example 2; Figure 2 E is a bright field image of the sample in Example 2 with the recombinant protein GFP-CBM added under a fluorescence microscope. Figure 2 F is a fluorescence field image of the sample added with recombinant protein GFP-CBM under a fluorescence microscope in Example 2; Figure 2 G is a bright field image of the sample in Example 2 after multiple ultrasonic washings with the recombinant protein GFP-CBM under a fluorescence microscope. Figure 2 H is a fluorescence field image under a fluorescence microscope of the sample to which the recombinant protein GFP-CBM is added in Example 2 after multiple ultrasonic washings.

[0051] Figure 3 This is a comparison of the mechanical properties data of the fiber samples of Example 4 and Comparative Example 4.

[0052] Figure 4 This is a comparison of the mechanical properties data of the fiber samples of Example 5 and Comparative Example 5.

[0053] Figure 5 This is a comparison of the mechanical properties data of the fiber samples of Example 6 and Comparative Example 6.

[0054] Figure 6 is a picture of the control sample in Example 7 after the fibrillation test; wherein Figure 6 A is the picture before the test, where Figure 6 B is the picture after the test.

[0055] Figure 7 This is a picture of the sample of comparative example 5 after the fibrillation test; Figure 7 A is the picture before the test, where Figure 7B is the picture after the test.

[0056] Figure 8 This is a picture of the sample added with 1 wt% recombinant spider silk protein after the fibrillation test in Example 7; Figure 8 A is the picture before the test, where Figure 8 B is the picture after the test.

[0057] Fig. 9 This is a picture of the sample added with 2 wt% recombinant spider silk protein after the fibrillation test in Example 7; Fig. 9 A is the picture before the test, where Fig. 9 B is the picture after the test.

[0058] Fig.10 This is a picture of the sample added with 5 wt% recombinant spider silk protein after the fibrillation test in Example 7; Fig.10 A is the picture before the test, where Fig.10 B is the picture after the test.

[0059] Fig.11 This is a picture of the sample added with 10 wt% recombinant spider silk protein after the fibrillation test in Example 7; Fig.11 A is the picture before the test, where Fig.11 B is the picture after the test.

[0060] Fig.12 This is a picture of the sample added with 20 wt% recombinant spider silk protein after the fibrillation test in Example 7; Fig.12 A is the picture before the test, where Fig.12 B is the picture after the test.

[0061] Fig.13 This is a schematic diagram of the combination of CBM and functional protein.

[0062] Fig.14 Fiber stress / strain curves of Example 10 and Comparative Examples 7 and 8.

[0063] Fig.15 ] are the fiber stress / strain curves of Example 11 and Comparative Examples 7 and 8.

[0064] Fig.16 1 and 2 are the fiber stress / strain curves of Example 12 and Comparative Examples 9 and 10.

[0065] Fig.17 Fiber stress / strain curves of Example 13 and Comparative Examples 9 and 10.

[0066] Fig.18 is a photo of the fiber product of Example 7. Fig.18 A is the control fiber sample; Fig.18B is a fiber sample containing 5% F-01 protein.

[0067] Fig.19 The fiber product photos of Comparative Example 7 and Example 10 are shown in FIG. Fig.18 A is comparative example 7; Fig.18 B is Example 10.

[0068] Fig. 20 The fiber product photos of Comparative Example 9 and Example 12 are shown. Fig.18 A is comparative example 9; Fig.18 B is Example 12. DETAILED DESCRIPTION

[0069] Now, various exemplary embodiments of the present invention are described in detail, which should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0070] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0071] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0072] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0073] Example 1

[0074] Recombinant spider silk protein culture and protein purification

[0075] All recombinant protein nucleic acid sequences of this patent are SEQ ID NO.4 (the amino acid sequence of CBM-GFP fusion protein is shown in SEQ ID NO.2, in which the 1st to 238th positions are GFP sequence, the 238th to 251st positions are the linker connection region between CBM and GFP, the 252nd to 409th positions are CBM sequence, and the CBM sequence can also be found in SEQ ID NO.1), SEQ ID NO.5 (the amino acid sequence of GFP-CBM fusion protein is shown in SEQ ID NO.3, in which the 1st to 158th positions are CBM sequence, the 159th to 171st positions are the linker connection region between CBM and GFP, and the 172nd to 409th positions are GFP sequence), SEQ ID NO.8 (recombinant spider silk protein F-01, comprising the MaSp1 spider silk protein repeat region and connected to CBM at both ends), SEQ ID NO.9 (recombinant spider silk protein F-02, comprising the MaSp4 spider silk protein repeat region and connected to CBM at both ends), SEQ ID NO.10 (recombinant spider silk protein F-03, containing MaSp1 spider silk protein repeat region, spider silk protein N-terminus and spider silk protein C-terminus and connected to CBM at both ends), SEQ ID NO.11 (recombinant spider silk protein F-04, containing MaSp1 spider silk protein repeat region, spider silk protein N-terminus and spider silk protein C-terminus, without CBM) and SEQ ID NO.12 (recombinant spider silk protein F-05, containing CBM, MaSp1 spider silk protein repeat region and C-terminal GFP) were all synthesized by BGI. The above synthetic sequences were inserted into the pET28a commercial plasmid vector, and the insertion site was between BamHI and XhoI. The final plasmid was chemically transformed into the Escherichia coli BL21 (DE3) strain (completed by BGI service). Schematic diagram of the combination of CBM and functional protein is shown in Fig.13 .

[0076] The constructed strain was cultured by fermentation, purified and freeze-dried to obtain recombinant spider silk protein dry powder, as follows:

[0077] (1) Shake flask fermentation

[0078] After the strain is constructed, take the preserved glycerol tube and streak it on a solid LB plate (containing 50ug / ml kanamycin Kana) and culture it in a 37°C incubator overnight. Pick a single clone and grow it in 4-5mL LB liquid medium (containing 50ug / ml kanamycin Kana) at 37°C, 200rpm shaking overnight. Transfer to a 400mL shake flask for fermentation at 1%, and culture at 200rpm, 37°C for 2-3h until OD600 = 0.6. Add isopropylthiogalactoside (IPTG) at a final concentration of 0.3mM and induce at 20°C for 12-16h. Centrifuge at 12000rpm for 5min to collect the bacteria.

[0079] (2) Protein purification

[0080] Add 20mM Tris-HCl to re-dissolve the bacteria into a concentrated solution, OD600 = about 20. Ultrasonicate at 25kHz, power 70-80%, 3S per ultrasound, stop for 6S, total ultrasound for 30min, until the solution turns from turbid to clear, marked as sample WC. Centrifuge at 12000rpm, 10-15min to take the supernatant, marked as sample S. Repeat the supernatant through the packed nickel column (Shenggong, product number NO.A600657) 2-3 times, collect the flow-through liquid, and mark it as sample FT; wash the column material with 20mM Tris-HCl 3-5 times (1-3 times the volume of the column material each time) until the dripping solution is clear; wash the column material with 1-2 times the column volume of 0.5M imidazole solution at least 3 times, collect them in sequence, and record them as E1, E2, and E3; wash away the residual 0.5M imidazole solution with 5 times the column volume of 20mM Tris-HCl, and finally preserve it with 20% alcohol. The washed column material can be reused. Mix E1, E2, and E3 and dialyze them in clean water. The dialyzed sample is marked as D.

[0081] (3) SDS-PAGE electrophoresis

[0082] Prepare 10% SDS-PAGE gel (Biyuntian, product number: P0012A)

[0083] Sample preparation and detection: Take 20uL of the protein purification sample and add 5uL of loading buffer for SDS-PAGE detection.

[0084] (4) Protein freeze-drying

[0085] The dialyzed protein solution was placed in a -80°C refrigerator until the protein solution was completely solidified, and then placed in a freeze dryer to freeze-dry the protein at a freezing temperature of -55°C and a vacuum degree of 5 Pa. The freeze-dried protein was weighed to finally obtain the purified recombinant spider silk protein powder.

[0086] The present invention discloses a process for preparing recombinant spider silk protein lyocell fiber with inhibited fibrillation, wherein the preparation of the recombinant spider silk protein / cellulose composite spinning solution comprises the following steps:

[0087] 1. Pre-activation of cellulose:

[0088] A wood pulp board with a cellulose alpha content of 99wt%, an ash content of 0.2wt% and an average degree of polymerization of 700 is used as the cellulose raw material. The cellulose is then impregnated with a concentrated sulfuric acid solution of 18g / L and a temperature of 80°C or a sodium hydroxide solution of 100g / L and a temperature of 80°C for 5 hours, and then washed, dried and crushed to obtain activated powdered cellulose.

[0089] 2. Dissolution of recombinant protein in NMMO system:

[0090] 10 wt% of the recombinant protein was weighed according to the weight of the solvent, added to a 50 wt% NMMO solution system at 60° C., and stirred thoroughly for 1 h to dissolve until a clear and transparent recombinant protein / NMMO mixed solution was obtained.

[0091] 3. Preparation of recombinant protein / cellulose composite spinning solution:

[0092] A certain mass of the recombinant protein mixed solution obtained in step 2 is prepared, and 4-13wt% activated cellulose powder is added to the mixed solution, wherein the mass ratio of the recombinant protein to the activated cellulose powder is 1:0.5-100, and the mixture is stirred and dissolved under vacuum at 80-100°C to finally obtain a blended spinning solution of spider silk recombinant protein and cellulose. The blended spinning solution is subjected to a vacuum degassing operation at 90°C to obtain a recombinant protein / cellulose composite spinning solution to be spun.

[0093] The recombinant protein / cellulose composite spinning solution to be spun is added to the spinning system, and after high-temperature static degassing, filtration and other operations, the dry-jet wet spinning is carried out through the spinning needle. The spinning speed is 35m / min, the spinning needle aperture is 260μm, and the air gap length is 5mm. The spinning line is vertically drawn into the coagulation bath through the air to form. The first coagulation bath uses a 5wt% NMMO solution at 55°C, and the second coagulation bath uses a 60°C aqueous solution. Then, the obtained primary fiber is washed and dried by cold and hot water exchange to obtain the Lyocell fiber combined with the recombinant protein.

[0094] Example 2

[0095] The preparation process of a recombinant protein lyocell fiber with fluorescence excitation function in this embodiment includes the following steps:

[0096] 1) Recombinant protein was obtained in Example 1, and recombinant protein CBM-GFP (shown in SEQ ID NO.4) and recombinant protein GFP-CBM (shown in SEQ ID NO.5) were selected to prepare two recombinant protein lyocell fibers with fluorescence excitation function.

[0097] 2) Preparation of recombinant protein lyocell fiber spinning solution with fluorescence excitation function: A wood pulp board with a cellulose content of 99wt%, an ash content of 0.2wt%, and an average degree of polymerization of 700 is used as a cellulose raw material, and then immersed in a concentrated sulfuric acid solution with a concentration of 9g / L and a temperature of 60°C for 2h, and then washed, dried, and crushed to obtain activated powdered cellulose. 10wt% recombinant protein is weighed according to the weight of the solvent, added to a 50wt% NMMO solution system at 90°C, and fully stirred and dissolved for 0.2h until a clear and transparent recombinant spider silk protein / NMMO mixture is obtained. The rest of the process is the same as in Example 1. The content of recombinant protein in the fiber is controlled to be 5wt%.

[0098] 3) Preparation of recombinant protein lyocell fiber with fluorescence excitation function:

[0099] The recombinant protein lyocell fiber spinning solution with fluorescence excitation function obtained in step 2 is added to the spinning system, and after high-temperature standing degassing, filtering and other operations, dry-jet wet spinning is performed through a spinning needle, the spinning speed is 35m / min, the spinning needle aperture is 260μm, the air gap length is 5mm, and the spinning line is vertically drawn into a coagulation bath through air to form. The first coagulation bath uses a 5wt% NMMO solution at 55°C, and the second coagulation bath uses an aqueous solution at 60°C. Then, the obtained primary fiber is washed and dried by cold and hot water exchange to obtain a recombinant spider silk protein lyocell fluorescent fiber.

[0100] The obtained recombinant protein Lyocell fluorescent fiber was placed under a fluorescence microscope, excited by ultraviolet light and its fluorescence effect was observed.

[0101] Comparative Example 1

[0102] The difference between this comparative example and Example 2 is that, in the preparation process of the protein / cellulose composite spinning solution, the recombinant protein is replaced by an equal weight of casein, and the other preparation processes and testing methods are exactly the same as those in Example 2.

[0103] Different types of protein / cellulose composite spinning solutions such as Figure 1 Compared with comparative example 1, example 2 has higher transparency and uniformity, and the bubbles in the microscope detection image are significantly reduced, which shows that the dissolution effect of recombinant spider silk protein is better, indicating that recombinant spider silk protein has higher solubility and spinnability than casein.

[0104] Comparative Example 2

[0105] The only difference between this comparative example and Example 2 is that no protein is added during the preparation process, and the other preparation processes and testing methods are exactly the same as those in Example 2.

[0106] Fluorescence test results are as attached Figure 2Compared with Comparative Example 2, the recombinant spider silk protein lyocell fiber containing a fluorescent protein structure and CBM in Example 2 showed a significant fluorescence effect in the fluorescence test, while the fiber of Comparative Example 2 had no fluorescence effect, which shows that the spider silk recombinant protein can maintain its protein activity in the NMMO solution system. Figure 2 In the embodiment 2, the recombinant spider silk protein lyocell fluorescent fiber still showed a significant fluorescence effect after multiple ultrasonic washings (using an ultrasonic oscillation cleaning machine, ultrasonic power of 800 W, and ultrasonic time of 0.5 h), indicating that there is a strong interaction between the CBM binding domain and cellulose.

[0107] Example 3

[0108] The present invention provides a process for preparing high-strength recombinant spider silk protein lyocell fiber, comprising the following steps:

[0109] 1) The recombinant spider silk protein was obtained in Example 1, wherein the recombinant spider silk protein was F01 fusion protein (SEQ ID NO. 8)

[0110] 2) Preparation of high-strength recombinant spider silk protein lyocell fiber spinning solution: A wood pulp board with a cellulose content of 99wt%, an ash content of 0.2wt%, and an average degree of polymerization of 700 is used as a cellulose raw material, and then soaked in sodium hydroxide at a concentration of 300g / L and a temperature of 40°C for 10h, and then washed, dried, and crushed to obtain activated powdered cellulose. 10wt% recombinant spider silk protein is weighed according to the weight of the solvent, added to a 60wt% NMMO solution system at 25°C, and stirred and dissolved for 2h until a clear and transparent recombinant spider silk protein / NMMO mixture is obtained. The rest of the process is the same as in Example 2. The spider silk protein content in the fiber is controlled to be 5wt%.

[0111] 3) Preparation of a high-strength recombinant spider silk protein lyocell fiber:

[0112] The high-strength recombinant spider silk protein lyocell fiber spinning solution obtained in step 2 is added to the spinning system, and after high-temperature standing degassing, filtering and other operations, dry-jet wet spinning is performed through a spinning needle, the spinning speed is 35m / min, the spinning needle aperture is 260μm, the air gap length is 5mm, and the spinning line is vertically drawn into a coagulation bath through air to form, the first coagulation bath uses a 5wt% NMMO solution at 55°C, and the second coagulation bath uses a 60°C aqueous solution. Then, the obtained primary fiber is washed and dried by cold and hot water exchange to obtain a recombinant spider silk protein lyocell high-strength fiber.

[0113] The obtained recombinant spider silk protein lyocell high-strength fiber was subjected to mechanical testing, and its dry breaking strength, dry breaking elongation and other properties were tested with reference to GB / T14337-2022.

[0114] Comparative Example 3

[0115] The difference between this comparative example and Example 3 is that, in the process of preparing a high-strength recombinant spider silk protein lyocell fiber, no recombinant spider silk protein is added, and the other preparation processes and testing methods are exactly the same as those in Example 3.

[0116] Mechanical properties test results are shown in the attached Figure 3 Compared with comparative example 3, the fiber obtained in example 3 showed a significant improvement in strength in the mechanical property test, with its strength increased to 32.62 cN (168.48% higher than the control), and its elongation increased by 46.15%.

[0117] Table 1 Fiber mechanical data of Example 3 and Comparative Example 3

[0118]

[0119] Example 4

[0120] The present invention provides a process for preparing recombinant spider silk protein lyocell fibers with high elasticity, comprising the following steps:

[0121] 1) The recombinant spider silk protein was obtained in Example 1, wherein the recombinant spider silk protein was F02 fusion protein (SEQ ID NO.9)

[0122] 2) The preparation process of a spinning solution of a recombinant spider silk protein lyocell fiber with high elasticity is the same as that in Example 3. The spider silk protein content in the fiber is controlled to be 5wt%.

[0123] 3) Preparation of recombinant spider silk protein lyocell fiber with high elasticity:

[0124] The recombinant spider silk protein lyocell fiber spinning solution with high elasticity obtained in step 2 is added to the spinning system, and after high-temperature standing degassing, filtering and other operations, dry-jet wet spinning is performed through a spinning needle, the spinning speed is 35m / min, the spinning needle aperture is 260μm, the air gap length is 5mm, and the spinning line is vertically drawn into a coagulation bath through air to form, the first coagulation bath uses a 5wt% NMMO solution at 55°C, and the second coagulation bath uses a 60°C aqueous solution. Then, the obtained primary fiber is washed and dried by cold and hot water exchange to obtain a recombinant spider silk protein lyocell high elastic fiber.

[0125] The obtained recombinant spider silk protein lyocell high-elastic fiber was subjected to mechanical testing, and its dry breaking strength, dry breaking elongation and other properties were tested with reference to GB / T14337-2022.

[0126] Comparative Example 4

[0127] The difference between this comparative example and Example 4 is that, in the process of preparing a recombinant spider silk protein lyocell fiber with high elasticity, no recombinant spider silk protein is added, and the other preparation processes and testing methods are exactly the same as those in Example 4.

[0128] Mechanical properties test results are shown in the attached Figure 4 Compared with comparative example 4, the fiber obtained in example 4 showed a significant improvement in elasticity in the mechanical property test, with its elongation at break increased to 30.53% (189.93% higher than the control), and its strength increased by 51.60% compared with the control.

[0129] Table 2 Fiber mechanical data of Example 4 and Comparative Example 4

[0130]

[0131] Example 5 Preparation process of different types of recombinant spider silk protein lyocell fibers

[0132] The preparation process of the recombinant spider silk protein lyocell fiber of the present invention comprises the following steps:

[0133] 1) The recombinant spider silk protein was obtained in Example 1, wherein the recombinant spider silk protein was F03 fusion protein (SEQ ID NO. 10)

[0134] 2) The preparation process of recombinant spider silk protein lyocell fiber is the same as that in Example 2. The spider silk protein content in the fiber is controlled to be 2wt%.

[0135] 3) Preparation of recombinant spider silk protein lyocell fibers:

[0136] The recombinant spider silk protein lyocell fiber spinning solution obtained in step 2 is added to the spinning system, and after high-temperature standing degassing, filtering and other operations, dry-jet wet spinning is performed through a spinning needle, the spinning speed is 35m / min, the spinning needle aperture is 260μm, the air gap length is 5mm, and the spinning line is vertically drawn into the coagulation bath through the air to form. The first coagulation bath uses a 5wt% NMMO solution at 55°C, and the second coagulation bath uses an aqueous solution at 60°C. Then, the obtained nascent fiber is washed and dried by cold and hot water exchange to obtain a recombinant spider silk protein lyocell high-strength fiber. At the same time, a protein-free sample without adding F03 fusion protein is prepared as a control according to the above method.

[0137] The obtained recombinant spider silk protein lyocell high-strength fiber was subjected to mechanical testing, and its dry breaking strength, dry breaking elongation and other properties were tested with reference to GB / T14337-2022.

[0138] Comparative Example 5

[0139] In this embodiment, recombinant spider silk protein without CBM is used to prepare lyocell fiber, which includes the following steps:

[0140] 1) The recombinant spider silk protein was obtained in Example 1, wherein the recombinant spider silk protein was F04 recombinant protein (SEQ ID NO. 11).

[0141] 2) The preparation process of recombinant spider silk protein lyocell fiber is the same as that in Example 2. The spider silk protein content in the fiber is controlled to be 2wt%.

[0142] 3) Preparation of recombinant spider silk protein lyocell fibers:

[0143] The recombinant spider silk protein lyocell fiber spinning solution obtained in step 2 is added to the spinning system, and after high-temperature standing degassing, filtering and other operations, dry-jet wet spinning is performed through a spinning needle, the spinning speed is 35m / min, the spinning needle aperture is 260μm, the air gap length is 5mm, and the spinning line is vertically drawn into a coagulation bath through air to form. The first coagulation bath uses a 5wt% NMMO solution at 55°C, and the second coagulation bath uses a 60°C aqueous solution. Then, the obtained primary fiber is washed and dried by cold and hot water exchange to obtain a recombinant spider silk protein lyocell high-strength fiber.

[0144] The obtained recombinant spider silk protein lyocell high-strength fiber was subjected to mechanical testing, and its dry breaking strength, dry breaking elongation and other properties were tested with reference to GB / T14337-2022.

[0145] Example 6

[0146] In this embodiment, the recombinant spider silk protein containing only one end connected to the CBM structure is used to prepare the lyocell fiber, including the following steps:

[0147] 1) The recombinant spider silk protein was obtained in Example 1, wherein the recombinant spider silk protein was F-05 recombinant protein (SEQ ID NO. 12).

[0148] 2) The preparation process of recombinant spider silk protein lyocell fiber is the same as that in Example 2. The spider silk protein content in the fiber is controlled to be 2 wt%.

[0149] 3) Preparation of recombinant spider silk protein lyocell fibers:

[0150] The recombinant spider silk protein lyocell fiber spinning solution obtained in step 2 is added to the spinning system, and after high-temperature standing degassing, filtering and other operations, dry-jet wet spinning is performed through a spinning needle, the spinning speed is 35m / min, the spinning needle aperture is 260μm, the air gap length is 5mm, and the spinning line is vertically drawn into a coagulation bath through air to form. The first coagulation bath uses a 5wt% NMMO solution at 55°C, and the second coagulation bath uses a 60°C aqueous solution. Then, the obtained primary fiber is washed and dried by cold and hot water exchange to obtain a recombinant spider silk protein lyocell high-strength fiber.

[0151] The obtained recombinant spider silk protein lyocell high-strength fiber was subjected to mechanical testing, and its dry breaking strength, dry breaking elongation and other properties were tested with reference to GB / T14337-2022.

[0152] Comparative Example 6

[0153] In this embodiment, casein is used to prepare lyocell fiber, which includes the following steps:

[0154] 1) Weigh casein. Due to the lack of interaction between casein and cellulose, transglutaminase (TG enzyme) is often used in the industry to enhance the cross-linking reaction between molecules or within molecules, so TG enzyme with a casein addition amount of 0.2 wt% is weighed and added to the NMMO solution.

[0155] 2) The preparation process of casein lyocell fiber is the same as that of Example 2, except that the recombinant spider silk protein is replaced by casein. The casein content in the fiber is controlled to be 2 wt%.

[0156] 3) Preparation of casein lyocell fiber:

[0157] The casein lyocell fiber spinning solution obtained in step 2 is added to the spinning system, and after high-temperature standing degassing, filtering and other operations, dry-jet wet spinning is performed through a spinning needle, the spinning speed is 35m / min, the spinning needle aperture is 260μm, the air gap length is 5mm, and the spinning line is vertically drawn into a coagulation bath through air to form, the first coagulation bath uses a 5wt% NMMO solution at 55°C, and the second coagulation bath uses an aqueous solution at 60°C. Then, the obtained nascent fiber is washed and dried by cold and hot water exchange to obtain a casein lyocell high-strength fiber.

[0158] The obtained casein lyocell high-strength fiber was subjected to mechanical testing, and its dry breaking strength, dry breaking elongation and other properties were tested with reference to GB / T14337-2022.

[0159] The mechanical properties test results are shown in Table 3 and Figure 5As shown. Compared with Comparative Examples 5 and 6, the protein-containing fiber obtained in Example 5 showed a significant improvement in strength in the mechanical properties test, wherein the strength of the sample containing F-03 was increased to 27.37 cN (159.43% higher than the control group), and the strength of the sample containing F-04 was increased to 14.48 cN (37.72% higher than the control group), indicating that the double CBM structure is more conducive to the combination of spider silk protein and cellulose. Compared with the double CBM structure protein in Example 5, the protein fiber sample containing a single CBM structure in Example 6 has a strong improvement in fiber performance to 19.13 cN (81.33% higher than the control group), which is not as good as the protein sample containing a double CBM structure (ie, Example 5), but better than the sample without CBM-containing F-04, which also shows that the double CBM structure is more conducive to the combination of spider silk protein and cellulose than the single CBM structure. The comparative example used TG enzyme to enhance the combination of protein and cellulose, and the strength of casein fiber was increased by 54.12%, but it was far from the increase of 159.43% in Example F-03.

[0160] Table 3 Fiber mechanical data of Example 5, Example 6, Comparative Example 5 and Comparative Example 6

[0161]

[0162] Example 7

[0163] The preparation process of a high-strength recombinant spider silk protein lyocell fiber with suppressed fibrillation of the present invention comprises the following steps:

[0164] 1) The recombinant spider silk protein was obtained in Example 1, wherein the recombinant spider silk protein was protein F01 (containing a double CBM structural protein (SEQ ID NO.8)

[0165] 2) A process for preparing a spinning solution of recombinant spider silk protein lyocell fiber with inhibited fibrillation is the same as in Example 2. The spider silk protein content in the fiber is controlled to be 0wt% (control), 1wt%, 2wt%, 5wt%, 10wt%, and 20wt%, respectively.

[0166] 3) Preparation of recombinant spider silk protein lyocell fiber with inhibited fibrillation:

[0167] The recombinant spider silk protein lyocell fiber spinning solution obtained in step 2 that inhibits fibrillation is added to the spinning system, and after high-temperature standing degassing, filtering and other operations, dry-jet wet spinning is performed through a spinneret, the spinning speed is 50m / min, the spinneret aperture is 100μm, the air gap side blowing temperature is 25°C, the air gap length is 30mm, and the spinning line is vertically drawn into a coagulation bath through the air to form, the first coagulation bath uses a 25°C 15wt% NMMO solution, and the second coagulation bath uses a 25°C aqueous solution. Then, the obtained primary fiber is washed by cold and hot water exchange, cut, oiled and dried to obtain a recombinant spider silk protein lyocell high elastic fiber.

[0168] The obtained recombinant spider silk protein lyocell high-elastic fiber was subjected to mechanical testing, and its dry breaking strength, dry breaking elongation and other properties were tested with reference to GB / T14337-2022.

[0169] The obtained recombinant spider silk protein lyocell fibers were subjected to a fibrillation test according to standard FZ / T 52019, and the fibrillation phenomenon of the treated fibers was observed under a microscope.

[0170] The results of mechanical properties test are shown in Table 4. After the F-01 protein was spun by the spinneret process, the addition of 1%-20% protein significantly improved the mechanical properties, among which the content of 2% (enhanced by 29.4%), 10% (enhanced by 35.86%), and 20% (enhanced by 40.4%) was particularly significant. When the protein addition exceeded 5%, the elongation of the fiber was significantly improved, and the 10% protein content increased the elongation of the fiber by 26.2%.

[0171] Table 4 Physical properties of recombinant spider silk protein lyocell fibers produced by industrial spinneret spinning system

[0172]

[0173]

[0174] Anti-fibrosis test results Figures 6 to 12 Compared with the control group and comparative example 5, the fibers obtained by adding each recombinant protein showed excellent anti-fibrillation effect in the anti-fibrillation test, and the mechanical properties of the fibers were improved to a certain extent, which further explained the interaction between the CMB binding domain of the spider silk recombinant protein and cellulose, and effectively improved the fibrillation phenomenon of the fibers.

[0175] Example 8

[0176] Recombinant protein lyocell fibers with fluorescence excitation function are prepared according to the method in Example 2, except that the amount of recombinant protein added is increased during the preparation of the recombinant protein / cellulose composite spinning solution, so that the mass ratio of recombinant protein to activated cellulose powder in the recombinant protein / cellulose composite spinning solution is 1:0.5. The resulting spinning solution has good transparency and uniformity, can be spun normally, and the fibers obtained by spinning can emit fluorescence normally.

[0177] Example 9

[0178] Recombinant protein lyocell fibers with fluorescence excitation function were prepared according to the method in Example 3, except that the amount of recombinant spider silk protein added was increased during the preparation of the recombinant spider silk protein / cellulose composite spinning solution, so that the mass ratio of recombinant protein to activated cellulose powder in the recombinant spider silk protein / cellulose composite spinning solution was 1:1, and the obtained spinning solution had good transparency and uniformity, and could be spun normally.

[0179] Example 10

[0180] In this embodiment, ionic liquid 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) is used as a solvent for dissolving cellulose and recombinant spider silk protein to prepare recombinant spider silk regenerated fiber. The specific process is as follows:

[0181] 1) The recombinant spider silk protein was obtained in Example 1, wherein the recombinant spider silk protein was F01 fusion protein (SEQ ID NO. 8)

[0182] 2) Preparation of recombinant spider silk protein / cellulose spinning solution: A wood pulp board with a cellulose content of 99wt%, an ash content of 0.2wt%, and an average degree of polymerization of 700 is used as a cellulose raw material, and then soaked in sodium hydroxide at a concentration of 300g / L and a temperature of 40°C for 10h, and then washed, dried, and crushed to obtain activated powdered cellulose. Take 15g of ionic liquid 1-butyl-3-methylimidazolium chloride, 0.9g of cellulose powder, and 0.018g of spider silk protein powder, add them to a mechanical stirring reactor in sequence and mix them evenly, control the temperature in the reactor to 100°C and stir the reaction for 1-2h; observe the spinning solution under a polarizing microscope until there are no microfibers and solid particles, indicating that the cellulose and spider silk protein are completely dissolved. Subsequently, the dissolved slurry is vacuum degassed at 100°C for 1-2h and filtered to obtain a regenerated spider silk protein / cellulose spinning solution. Among them, the content of recombinant spider silk protein in the fiber is controlled to be 2wt%.

[0183] 3) Preparation of recombinant spider silk regenerated fiber: Add the spinning solution into a syringe for spinning, use a 5% ionic liquid solution as the coagulation bath, the coagulation bath temperature is 50°C, the spinning speed is 10m / min, the spinning needle aperture is 260μm, and the air gap length is 5mm. The spinning line is vertically drawn into the coagulation bath by air to form, and the obtained primary fiber is processed by washing, stretching, oiling, drying and other processing procedures to obtain recombinant spider silk regenerated fiber.

[0184] The obtained recombinant spider silk regenerated fiber was subjected to mechanical testing, and its dry breaking strength, dry breaking elongation and other properties were tested with reference to GB / T14337-2022.

[0185] Comparative Example 7

[0186] The difference between this comparative example and Example 10 is that, in the process of preparing the recombinant spider silk regenerated fiber, no recombinant spider silk protein is added, and the other preparation processes and testing methods are exactly the same as those of Example 10.

[0187] Comparative Example 8

[0188] The difference between this comparative example and Example 10 is that in this comparative example, recombinant spider silk regenerated fibers are prepared using recombinant spider silk protein without CBM, and ionic liquid 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) is used as a solvent for dissolving cellulose and recombinant spider silk protein, including the following steps:

[0189] 1) The recombinant spider silk protein was obtained in Example 1, wherein the recombinant spider silk protein without CBM was F-04 recombinant protein (SEQ ID NO.11).

[0190] 2) Preparation of recombinant spider silk protein / cellulose spinning solution: The spinning solution preparation process is the same as that of Example 10, except that the recombinant spider silk protein F01 fusion protein is replaced by the recombinant spider silk protein F-04 without CBM, and the recombinant spider silk protein content in the fiber is controlled to be 2 wt%.

[0191] 3) Preparation of recombinant spider silk regenerated fibers: The preparation process of recombinant spider silk regenerated fibers is the same as that of Example 10.

[0192] The obtained recombinant spider silk regenerated fiber was subjected to mechanical testing, and its dry breaking strength, dry breaking elongation and other properties were tested with reference to GB / T14337-2022.

[0193] Mechanical properties test results are shown in the attached Fig.14As shown in Table 5. Compared with Comparative Examples 10 and 11, the recombinant spider silk protein fiber obtained in Example 10 showed more obvious performance improvement in the mechanical property test, and the strength of the F-01 sample fiber was increased to 36.11 cN (43.98% higher than the control), and the elongation was increased by 185.71%. Compared with the protein-free control sample, the strength of the F-04 sample fiber of Comparative Example 11 was 32.52 cN (only increased by 29.67%), and the elongation was 7.36% (only increased by 22.26%), indicating that the double CBM structure is more conducive to the combination between spider silk protein and cellulose.

[0194] Table 5 Fiber mechanical data of Example 10 and Comparative Examples 7 and 8

[0195]

[0196] Embodiment 11

[0197] In this embodiment, ionic liquid 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) is used as a solvent for dissolving cellulose and recombinant spider silk protein to prepare recombinant spider silk regenerated fiber. The specific process is as follows:

[0198] 1) The recombinant spider silk protein was obtained in Example 1, wherein the recombinant spider silk protein was F02 fusion protein (SEQ ID NO.9)

[0199] 2) Preparation of recombinant spider silk protein / cellulose spinning solution: The spinning solution preparation process is the same as that of Example 10, except that the recombinant spider silk protein F01 fusion protein is replaced by F02 fusion protein, and the recombinant spider silk protein content in the fiber is controlled to be 2 wt%.

[0200] 3) Preparation of recombinant spider silk regenerated fibers: The preparation process of recombinant spider silk regenerated fibers is the same as that of Example 10.

[0201] The obtained recombinant spider silk regenerated fiber was subjected to mechanical testing, and its dry breaking strength, dry breaking elongation and other properties were tested with reference to GB / T14337-2022.

[0202] Mechanical properties test results are shown in the attached Fig.15 As shown in Table 6. Compared with Comparative Examples 10 and 11, the fiber obtained in Example 11 showed more obvious performance improvement in the mechanical property test, and the strength of its F-02 fiber was increased to 74.00 cN (195.06% higher than the control), and the elongation was increased by 147.84%. Compared with the protein-free control sample, the strength of the F-04 sample fiber of Comparative Example 11 was 32.52 cN (only increased by 29.67%), and the elongation was 7.36% (only increased by 22.26%), indicating that the double CBM structure is more conducive to the combination between spider silk protein and cellulose.

[0203] Table 6 Fiber mechanical data of Example 11 and Comparative Examples 7 and 8

[0204]

[0205] Example 12

[0206] In this embodiment, N,N-dimethylacetamide (DMAc) / lithium chloride (LiCl) system is used as a solvent for dissolving cellulose and spider silk protein to prepare recombinant spider silk regenerated fiber. The specific process is as follows:

[0207] 1) The recombinant spider silk protein was obtained in Example 1, wherein the recombinant spider silk protein was F01 fusion protein (SEQ ID NO. 8)

[0208] 2) Preparation of recombinant spider silk protein / cellulose spinning solution: A wood pulp board with a cellulose content of 99wt%, an ash content of 0.2wt%, and an average degree of polymerization of 700 was used as a cellulose raw material, and then soaked in sodium hydroxide at a concentration of 300g / L and a temperature of 40°C for 10h, and then washed, dried, and crushed to obtain activated powdered cellulose. Weigh 30mL DMAc, weigh 3g LiCl and add it to the DMAc solution, heat it to 100°C, add 0.018g recombinant spider silk protein, then heat it to 150°C and keep it for 10min, and the spider silk protein is completely dissolved; add 0.9g cellulose powder to the above mixed liquid and stir for 20min, then cool it to 90°C and stir for 45min, and finally cool it to 40°C, where the cellulose is completely dissolved during the cooling process; observe the spinning solution under a polarizing microscope until there are no microfibrils and solid particles, indicating that the cellulose and spider silk protein are completely dissolved.

[0209] 3) Preparation of recombinant spider silk regenerated fiber: Add the spinning solution into a syringe for spinning, use a deionized water solution as the coagulation bath, the coagulation bath temperature is 50°C, the spinning speed is 40m / min, the spinning needle aperture is 260μm, and the air gap length is 5mm. The spinning line is vertically drawn into the coagulation bath by air to form, and the obtained primary fiber is processed by washing, stretching, oiling, drying and other processing procedures to obtain recombinant spider silk regenerated fiber.

[0210] The obtained recombinant spider silk regenerated fiber was subjected to mechanical testing, and its dry breaking strength, dry breaking elongation and other properties were tested with reference to GB / T14337-2022.

[0211] Comparative Example 9

[0212] The difference between this comparative example and Example 12 is that no recombinant spider silk protein is added during the process of preparing the recombinant spider silk regenerated fiber, and the other preparation processes and testing methods are exactly the same as those of Example 12.

[0213] Comparative Example 10

[0214] The difference between this comparative example and Example 12 is that in this comparative example, recombinant spider silk regenerated fibers are prepared using recombinant spider silk protein without CBM, and N,N-dimethylacetamide (DMAc) / lithium chloride (LiCl) system is used as a solvent for dissolving cellulose and recombinant spider silk protein, including the following steps:

[0215] 1) The recombinant spider silk protein was obtained in Example 1, wherein the recombinant spider silk protein without CBM was F04 recombinant protein (SEQ ID NO.11).

[0216] 2) The preparation process and testing method of the recombinant spider silk protein fiber are the same as those in Example 12. The spider silk protein content in the fiber is controlled to be 2 wt%.

[0217] Mechanical properties test results are shown in the attached Fig.16 As shown in Table 7. Compared with Comparative Examples 12 and 13, the fiber obtained in Example 12 showed more obvious performance improvement in the mechanical property test, and the strength of its F-02 fiber was increased to 62.13 cN (72.34% higher than the control), and the elongation was increased by 78.93%. Compared with the protein-free control sample, the strength of the F-04 sample fiber of Comparative Example 13 was 39.27 cN (only increased by 8.93%), and the elongation was 9.99% (only increased by 11.37%), indicating that the double CBM structure is more conducive to the combination between spider silk protein and cellulose.

[0218] Table 7 Fiber mechanical data of Example 12 and Comparative Examples 12 and 13

[0219]

[0220] Embodiment 13

[0221] In this embodiment, N,N-dimethylacetamide (DMAc) / lithium chloride (LiCl) system is used as a solvent for dissolving cellulose and spider silk protein to prepare recombinant spider silk regenerated fiber. The specific process is as follows:

[0222] 1) The recombinant spider silk protein was obtained in Example 1, wherein the recombinant spider silk protein was F02 fusion protein (SEQ ID NO.9)

[0223] 2) Preparation of recombinant spider silk protein / cellulose spinning solution: The spinning solution preparation process is the same as that in Example 12, except that the recombinant spider silk protein F01 fusion protein is replaced by F02 fusion protein, and the spider silk protein content in the fiber is controlled to be 2 wt%.

[0224] 3) Preparation of recombinant spider silk regenerated fibers: The preparation process of recombinant spider silk regenerated fibers is the same as that of Example 12.

[0225] The obtained recombinant spider silk regenerated fiber was subjected to mechanical testing, and its dry breaking strength, dry breaking elongation and other properties were tested with reference to GB / T14337-2022.

[0226] Mechanical properties test results are shown in the attached Fig.17 As shown in Table 7. Compared with Comparative Examples 12 and 13, the fiber obtained in Example 13 showed more obvious performance improvement in the mechanical property test, and the strength of its F-02 fiber was increased to 65.41 cN (81.44% higher than the control), and the elongation was increased by 54.18%. Compared with the protein-free control sample, the strength of the F-04 sample fiber of Comparative Example 13 was 39.27 cN (only increased by 8.93%), and the elongation was 9.99% (only increased by 11.37%), indicating that the double CBM structure is more conducive to the combination between spider silk protein and cellulose.

[0227] Table 8 Fiber mechanical data of Example 13 and Comparative Examples 12 and 13

[0228]

[0229]

[0230] like Fig.18 Shown are fiber samples with and without added spider silk protein produced by an industrial spinneret; Fig.19 , 20 These are fiber samples prepared in the laboratory with and without added spider silk protein. There is no obvious difference in fiber appearance with or without the addition of protein, and it has no effect on fiber whiteness.

[0231] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing regenerated cellulose fiber combined with recombinant protein, characterized in that: The steps include: Dissolving the recombinant protein and cellulose in a solvent and mixing them evenly to obtain a spinning solution; spinning the spinning solution to obtain the recombinant protein-bound regenerated cellulose fiber; The recombinant protein is a recombinant protein obtained by fusion expression of at least one cellulose binding domain and at least one functional protein.

2. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 1, characterized in that: The amino acid sequence of the cellulose binding domain is shown in SEQ ID NO.

1.

3. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 1, characterized in that: The functional protein comprises at least one of a fluorescent protein and a spider silk protein.

4. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 3, characterized in that: The spider silk protein is a MaSp spider silk protein, and the amino acid sequence of the spider silk protein contains at least one spider silk protein repeat region sequence; The spider silk protein repeat region sequence is shown as SEQ ID NO.6 or SEQ ID NO.

7.

5. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 4, characterized in that: The N-terminus of the spider silk protein repeat region sequence is connected to the spider silk protein N-terminal sequence, and the C-terminus of the spider silk protein repeat region sequence is connected to the spider silk protein C-terminal sequence.

6. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 1, characterized in that: The N-terminus and / or C-terminus of the functional protein in the recombinant protein is connected to at least one cellulose binding domain.

7. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 1, characterized in that: The recombinant protein comprises a plurality of functional proteins, wherein at least two functional proteins are connected via at least one cellulose binding domain.

8. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 1, characterized in that: The N-terminus and the C-terminus of the functional protein in the recombinant protein are both connected to at least one cellulose binding domain.

9. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 1, characterized in that: The recombinant protein was prepared by the following method: The nucleic acid sequence for expressing the recombinant protein is transferred into the strain to construct an engineered bacterium; the engineered bacterium is cultured, the bacterial cells are collected and the recombinant protein is extracted.

10. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 9, characterized in that: The nucleic acid sequence used to express the recombinant protein is shown in any one of SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.

12.

11. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 1, characterized in that: The cellulose is activated cellulose in powder form.

12. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 11, characterized in that: The activated cellulose powder is obtained by the following method: The cellulose raw material with a cellulose content of not less than 95 wt% and an average degree of polymerization of 500 to 650 is impregnated, and then washed, dried and crushed to obtain activated cellulose powder; The immersion treatment is carried out using a concentrated sulfuric acid solution with a concentration of 9 to 36 g / L and a temperature of 60 to 90° C. or a sodium hydroxide solution with a concentration of 100 to 300 g / L and a temperature of 40 to 80° C. The immersion time is 2 to 10 hours.

13. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 1, characterized in that: The solvent is a NMMO solvent or an ionic liquid solvent.

14. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 13, characterized in that: The ionic liquid solvent is 1-butyl-3-methylimidazolium chloride or N,N-dimethylacetamide / lithium chloride.

15. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 1, characterized in that: The method of dissolving the recombinant protein and the cellulose raw material in the solvent and mixing them uniformly is specifically as follows: Step 1) dissolving the recombinant protein in NMMO solvent, wherein the NMMO solvent concentration is 50-90wt%, the dissolution temperature is 25-95°C, and the dissolution time is 0.2-2h; obtaining a recombinant protein NMMO solution; Step 2) adding cellulose to the recombinant protein NMMO solution and stirring to dissolve to obtain a spinning solution.

16. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 15, characterized in that: In the step 2), the cellulose concentration is 4-13wt% of the total mass of the recombinant spider silk protein / NMMO mixed spinning solution, the stirring and dissolving temperature is 80-110°C, and the dissolving time is 1-5h, until a transparent amber composite spinning solution is obtained.

17. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 1, characterized in that: The mass ratio of the recombinant protein to the cellulose is 1:0.5-100.

18. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 15, characterized in that: The spinning solution is subjected to high-temperature static degassing and filtration, and then is subjected to dry-jet wet spinning through a spinneret, the spinning speed is 40-50 m / min, the spinneret aperture is 100-300 μm, the air gap length is 5-50 mm, and the spinning line is vertically drawn through the air into a coagulation bath for forming, the first coagulation bath uses a 5-15wt% NMMO solution at 25-70°C, and the second coagulation bath uses an aqueous solution at 25-70°C; the obtained primary fiber is then subjected to cold and hot water exchange washing treatment, cutting, oiling and drying to obtain recombinant spider silk protein lyocell fiber.

19. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 1, characterized in that: The method of dissolving the recombinant protein and the cellulose raw material in the solvent and mixing them uniformly is specifically as follows: The ionic liquid, cellulose and recombinant protein are mixed and stirred at 100° C. for 1 to 2 hours to obtain a composite spinning solution.

20. The method for preparing regenerated cellulose fiber combined with recombinant protein according to claim 19, characterized in that: The composite spinning solution is placed at high temperature for degassing and filtration, and then added into a syringe for spinning, the spinning speed is 40-50 m / min, the spinning needle aperture is 100-300 μm, and the air gap length is 5-50 mm; the spinning line is vertically drawn into a coagulation bath through air to be formed, and the coagulation bath temperature is 25-70°C; then the obtained primary fiber is washed, stretched, oiled, and dried to obtain the recombinant spider silk regenerated fiber.

21. Regenerated cellulose fiber bound to a recombinant protein obtained by the method according to any one of claims 1 to 20.

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