Method for preparing regenerated cellulose fiber incorporating recombinant protein

Abstract

Disclosed in the present application is a method for preparing a regenerated cellulose fiber incorporating a recombinant protein, the method comprising the following steps: dissolving a recombinant protein and cellulose in a solvent for uniform mixing, so as to obtain a spinning solution; and using the spinning solution to perform spinning, so as to obtain a regenerated cellulose fiber incorporating the recombinant protein, the recombinant protein being a recombinant protein obtained by fusion expression of at least one cellulose binding domain and at least one functional protein. In the present application, CBM-containing recombinant functional proteins can maintain structural and functional stability in NMMO systems, so as to provide the biological activity of proteins for Lyocell fiber systems; incorporation of fluorescent proteins can impart natural fluorescence to fibers, which have application prospects in the field of textiles and fashion; expression of MaSp1 recombinant spider silk proteins enhances the fiber strength, and expression of MaSp4 recombinant spider silk proteins improves the elastic properties of fibers; and in addition, CBM fusion proteins can also carry other functional modules, so as to endow cellulose fibers with specific functionalities.

Description

Method for preparing regenerated cellulose fibers combined with recombinant proteins

[0001] Cross-reference to related applications

[0002] This application claims priority to the Chinese patent application No. 202410631928.3, filed on May 20, 2024, and entitled “Method for preparing regenerated cellulose fibers combined with recombinant proteins”, and to the Chinese patent application No. 202411135526.0, filed on August 16, 2024, and entitled “Method for preparing regenerated cellulose fibers combined with recombinant proteins”, the contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application belongs to the technical field of biotechnology, and specifically relates to a method for preparing regenerated cellulose fibers combined with recombinant proteins. BACKGROUND

[0004] Lyocell fiber is a kind of regenerated cellulose fiber obtained by dissolving cellulose-containing pulp in an organic solvent N-methyl morpholine-N-oxide (NMMO) and then dry-jet wet spinning. It has high strength (3.8-4.2 cN / dtex), excellent moisture absorption and comfort, easy dyeing, and easy biodegradation. However, in sports equipment and industrial applications, higher strength fibers are still needed, and in daily applications, fibers need to have specific functionality. Lyocell fiber itself does not have antibacterial properties, flame retardancy, and is prone to produce radial fibrils (called fibrillation phenomenon) after wet friction. These shortcomings and properties limit the application of lyocell fiber in a wider range of scenarios.

[0005] In order to endow lyocell fiber with specific functionality and broaden its use scenarios, combining cellulose with functional materials to prepare functional cellulose fibers has become the mainstream approach. Depositing inorganic powder particles (such as silicon dioxide, aluminum oxide, etc.) on the surface of the fiber 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, which can cause powder to fall off, affecting the durability of the fiber function; proteins, as a class of natural biomass materials, have also been used for the modification of cellulose fibers, providing new ideas and methods for the preparation of green and environmentally friendly cellulose fibers. However, the current protein modification method cannot produce interaction force between the protein and the cellulose, but only simple physical doping, which can cause protein to fall off, lose, and unevenly distribute, gradually weakening the function of the fiber.

[0006] Spider silk proteins are the main components of natural spider silk, which provide spider silk with unparalleled strength, elasticity and toughness. Due to the rich β-sheet crystal region in the structure, spider silk is endowed with strength, and the strength of major ampullate silk can be comparable to that of carbon fiber and Kevlar 49. Due to the rich α-helix and β-turn in the structure, spider silk is endowed with excellent ductility, and the elasticity of flagelliform silk can be comparable to that of spandex. The unique structural characteristics and excellent water solubility of spider silk proteins lay a solid foundation for them as fiber reinforcing materials. Through genetic engineering and synthetic biology technology, specific functional protein domains can be added to spider silk proteins, which can endow them with new functions. If functional proteins such as spider silk proteins can be effectively combined with lyocell fibers, the lyocell fibers can be endowed with stronger performance and special functional lyocell fibers can be obtained. SUMMARY

[0007] The purpose of the present application is to provide a preparation method of regenerated cellulose fibers combined with recombinant proteins, so as to solve the problem of poor combination effect of proteins and cellulose in regenerated cellulose fibers.

[0008] A preparation method of regenerated cellulose fibers combined with recombinant proteins, comprising the following steps:

[0009] dissolving the recombinant protein and cellulose in a solvent to mix uniformly to obtain a spinning solution; and spinning the spinning solution to obtain the regenerated cellulose fibers combined with the recombinant protein.

[0010] The recombinant protein is a recombinant protein obtained by fusion expression of at least one cellulose binding domain and at least one functional protein.

[0011] Optionally, the amino acid sequence of the cellulose binding domain is shown in SEQ ID NO. 1.

[0012] The cellulose binding domain is not limited to the above sequence, as long as it can achieve the effect of binding cellulose, and therefore can be replaced by other cellulose binding domain amino acid sequences.

[0013] Optionally, the functional protein comprises at least one of a fluorescent protein and a spider silk protein.

[0014] Optionally, the spider silk protein is a MaSp type spider silk protein, and the amino acid sequence of the spider silk protein comprises at least one spider silk protein repeat region sequence.

[0015] The spider silk protein repeat region sequence is shown in SEQ ID NO. 6 or SEQ ID NO. 7.

[0016] Optionally, the N terminus of the spider silk protein repeat region sequence is connected with a spider silk protein N terminus sequence, and the C terminus of the spider silk protein repeat region sequence is connected with a spider silk protein C terminus sequence.

[0017] 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.

[0018] Optionally, the recombinant protein comprises a plurality of functional proteins; wherein at least two functional proteins are connected by at least one cellulose binding domain.

[0019] Optionally, the N-terminus and C-terminus of the functional protein in the recombinant protein are both connected to at least one cellulose binding domain.

[0020] Optionally, the recombinant protein is prepared by the following method:

[0021] Optionally, the bacterial cells are collected and the recombinant protein is extracted.

[0022] Optionally, the nucleic acid sequence for expressing the recombinant protein is as 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, SEQ ID NO. 12.

[0023] Optionally, the cellulose is a powder-like cellulose after activation treatment.

[0024] Optionally, the powder-like cellulose after activation treatment is obtained by the following method:

[0025] The cellulose raw material with a content of alpha cellulose not less than 95wt% and an average degree of polymerization of 500-650 is subjected to immersion treatment, and then is subjected to water washing, drying, and crushing to obtain the powder-like cellulose after activation treatment;

[0026] The immersion treatment is performed by using a concentrated sulfuric acid solution with a concentration of 9-36g / L and a temperature of 60-90℃ or a sodium hydroxide solution with a concentration of 100-300g / L and a temperature of 40-80℃ for 2-10h;

[0027] Optionally, the cellulose raw material is wood pulp board.

[0028] Optionally, the content of alpha cellulose is greater than 98% and the average degree of polymerization is 620.

[0029] Optionally, the concentration of the concentrated sulfuric acid is 18g / L, the temperature is 80℃, and the treatment time is 6h.

[0030] Optionally, the concentration of the sodium hydroxide is 200g / L, the temperature is 80℃, and the treatment time is 8h.

[0031] The solvent is an NMMO solvent or an ionic liquid solvent.

[0032] Optionally, the ionic liquid solvent is 1-butyl-3-methylimidazolium chloride or N,N-dimethylacetamide / lithium chloride.

[0033] Optionally, the method of dissolving the recombinant protein and the cellulose raw material in the solvent and mixing them uniformly comprises the following steps:

[0034] Step 1): dissolving the recombinant protein in NMMO solvent, wherein the concentration of the NMMO solvent is 50-90 wt%, the dissolving temperature is 25-95°C, and the dissolving time is 0.2-2 h; and obtaining a recombinant protein NMMO solution;

[0035] Step 2): adding cellulose to the recombinant protein NMMO solution and stirring to dissolve, to obtain a spinning solution;

[0036] Optionally, in step 1), the concentration of the NMMO solvent is 50 wt%, the dissolving temperature is 60°C, and the dissolving time is 0.5 h.

[0037] Optionally, in step 2), the concentration of the cellulose is 4-13 wt% 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 h, until a transparent amber composite spinning solution is obtained.

[0038] Optionally, the concentration of the cellulose is 12 wt% of the total mass of the recombinant spider silk protein / NMMO mixed spinning solution, the stirring and dissolving temperature is 100°C, and the dissolving time is 3 h.

[0039] Optionally, the mass ratio of the recombinant protein to the cellulose is 1:0.5-100.

[0040] Optionally, after the spinning solution is subjected to high-temperature standing and defoaming and filtration, dry-jet wet spinning is performed through a spinneret, the spinning speed is 40-50 m / min, the spinneret hole diameter is 100-300 μm, the air gap length is 5-50 mm, the spun thread is vertically drawn into a coagulation bath through air, the first coagulation bath is a 5-15 wt% NMMO solution at 25-70°C, and the second coagulation bath is an aqueous solution at 25-70°C; then, the obtained nascent fiber is subjected to cold-hot water exchange washing, cutting, oiling, and drying, to obtain recombinant spider silk protein lyocell fiber.

[0041] Optionally, the spinning speed is 35 m / min, the spinneret hole diameter is 260 μm, the air gap length is 5 mm, the first coagulation bath is a 15 wt% NMMO solution at 25°C, and the second coagulation bath is an aqueous solution at 25°C.

[0042] Optionally, the method of dissolving the recombinant protein and the cellulose raw material in the solvent and mixing them uniformly comprises the following steps:

[0043] The ionic liquid, cellulose and recombinant protein are mixed and stirred at 100℃ for 1-2h; then vacuum degassing at 100℃ for 1-2h and filtering to obtain the composite spinning solution.

[0044] Optionally, the composite spinning solution is added into a syringe for spinning, the spinning speed is 40-50m / min, the spinning needle aperture is 100-300μm, and the air gap length is 5-50mm; the spinning line is vertically drawn into a coagulation bath through air to form a shape, and the coagulation bath temperature is 25-70℃; then the obtained primary fiber is washed, stretched, oiled and dried to obtain the recombinant spider silk regenerated fiber.

[0045] The application has the following beneficial effects:

[0046] The recombinant functional protein of CBM in the application can maintain the stability of structure and function in the NMMO system, thereby providing the bioactivity of protein for the lyocell fiber system. The fluorescence protein composite can bring natural fluorescence effect to the fiber, which has application prospect in the field of textile fashion; MaSp is the most common spider silk protein, which usually exists in large ampullate glands and is responsible for the framework structure of spider webs, MaSp1 is rich in beta-fold composed of alanine, which endows spider silk with high strength, and MaSp4 is rich in beta-turn composed of proline and glycine, which makes spider silk have excellent elasticity. The expression of MaSp1 recombinant spider silk protein increases the fiber strength, and the expression of MaSp4 recombinant spider silk protein enhances the fiber elasticity. At the same time, the CBM fusion protein can also carry other functional modules to endow cellulose fiber with specific functions, further widening the use scenarios of cellulose fiber, and making important contributions in the fields of daily consumption, outdoor sports, medical health, industrial production and military defense.

[0047] The application realizes directional grabbing between the recombinant protein with double CBM structure and cellulose, and cross-linking between the recombinant spider silk proteins, forming a double cross-linking network structure, which innovatively significantly improves the fibrillation phenomenon of lyocell fiber and enhances its mechanical properties. The lyocell fiber enhanced by the strength recombinant spider silk protein can be improved by 168.48%, and the lyocell fiber enhanced by the elasticity recombinant spider silk protein can be improved by 189.93%, and the strength of the recombinant spider silk protein lyocell fiber can reach 5.40cN / dtex through the industrial spinning process. The improvement of the physical properties greatly widens the application scenarios of the fiber;

[0048] The recombinant spider silk protein structure designed in the application is easily soluble in NMMO solution, the spinning solution prepared by high-concentration protein (more than 20%) is clear without particle precipitation, and the spinnability is high. Moreover, the dissolution process does not pollute the NMMO solvent, and does not affect the clean recovery of the NMMO solvent;

[0049] The protein designed in the application and the cellulose spinning process preparation process are green and environmentally friendly, simple and efficient to operate, and the solvent system can be recycled and reused by 99.5%, avoiding the problem of chemical residues. BRIEF DESCRIPTION OF DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the specific embodiments or prior art of the present application, the drawings needed in the specific embodiment or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0051] Figure 1 is a picture of the spinning solution of Example 2 and Comparative Example 1 under the microscope in a flask; wherein Figure 1A is a picture of the spinning solution of Example 2 in a flask, Figure 1B is a picture of the spinning solution of Example 2 under the microscope, Figure 1C is a picture of the spinning solution of Comparative Example 1 in a flask, and Figure 1D is a picture of the spinning solution of Comparative Example 1 under the microscope.

[0052] Figure 2 is a bright field picture and a fluorescence field picture of the fibers of Example 2 and Comparative Example 2 under the fluorescence microscope; wherein Figure 2A is a bright field picture of Comparative Example 2 under the fluorescence microscope, Figure 2B is a fluorescence field picture of Comparative Example 2 under the fluorescence microscope; Figure 2C is a bright field picture of the sample added with recombinant protein CBM-GFP in Example 2 under the fluorescence microscope, Figure 2D is a fluorescence field picture of the sample added with recombinant protein CBM-GFP in Example 2 under the fluorescence microscope; Figure 2E is a bright field picture of the sample added with recombinant protein GFP-CBM in Example 2 under the fluorescence microscope, Figure 2F is a fluorescence field picture of the sample added with recombinant protein GFP-CBM in Example 2 under the fluorescence microscope; Figure 2G is a bright field picture of the sample added with recombinant protein GFP-CBM in Example 2 after multiple ultrasonic washing under the fluorescence microscope, and Figure 2H is a fluorescence field picture of the sample added with recombinant protein GFP-CBM in Example 2 after multiple ultrasonic washing under the fluorescence microscope.

[0053] Figure 3 is a comparison of the mechanical property data of the fiber samples of Example 4 and Comparative Example 4.

[0054] Figure 4 is a comparison of the mechanical property data of the fiber samples of Example 5 and Comparative Example 5.

[0055] Figure 5 is a comparison of the mechanical property data of the fiber samples of Example 6 and Comparative Example 6.

[0056] Figure 6 is a picture of the control sample after fibrillation test in Example 7; wherein Figure 6A is a picture before the test, and Figure 6B is a picture after the test.

[0057] Figure 7 is a picture of the sample of Comparative Example 5 after fibrillation testing; where Figure 7A is a picture before testing and Figure 7B is a picture after testing.

[0058] Figure 8 is a picture of the sample of Example 7 with 1 wt% recombinant spider silk protein after fibrillation testing; where Figure 8A is a picture before testing and Figure 8B is a picture after testing.

[0059] Figure 9 is a picture of the sample of Example 7 with 2 wt% recombinant spider silk protein after fibrillation testing; where Figure 9A is a picture before testing and Figure 9B is a picture after testing.

[0060] Figure 10 is a picture of the sample of Example 7 with 5 wt% recombinant spider silk protein after fibrillation testing; where Figure 10A is a picture before testing and Figure 10B is a picture after testing.

[0061] Figure 11 is a picture of the sample of Example 7 with 10 wt% recombinant spider silk protein after fibrillation testing; where Figure 11A is a picture before testing and Figure 11B is a picture after testing.

[0062] Figure 12 is a picture of the sample of Example 7 with 20 wt% recombinant spider silk protein after fibrillation testing; where Figure 12A is a picture before testing and Figure 12B is a picture after testing.

[0063] Figure 13 is a schematic of CBM in combination with functional proteins.

[0064] Figure 14 is a fiber stress / strain curve of Example 10 and Comparative Examples 7, 8.

[0065] Figure 15 is a fiber stress / strain curve of Example 11 and Comparative Examples 7, 8.

[0066] Figure 16 is a fiber stress / strain curve of Example 12 and Comparative Examples 9, 10.

[0067] Figure 17 is a fiber stress / strain curve of Example 13 and Comparative Examples 9, 10.

[0068] Figure 18 is a picture of fiber products of Example 7. Where Figure 18A is a control fiber sample; Figure 18B is a fiber sample containing 5% F-01 protein.

[0069] Figure 19 is a picture of fiber products of Comparative Example 7 and Example 10. Where Figure 19A is Comparative Example 7; Figure 19B is Example 10.

[0070] Figure 20 is a picture of fiber products of Comparative Example 9 and Example 12. Where Figure 20A is Comparative Example 9; Figure 20B is Example 12. DETAILED DESCRIPTION

[0071] The following detailed description of various example embodiments of the application should not be considered to be limiting of the application, but rather a description of certain example aspects, features and embodiments of the application. It is understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application.

[0072] In addition, for numerical ranges that are expressly recited herein, it is to be understood that every intervening value between the upper and lower limits of the range is also specifically disclosed. In addition, each smaller range that falls within the integer ranges expressly recited or the range recited is also included in the application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0073] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described.

[0074] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0075] Example 1

[0076] Recombinant spider silk protein culture and protein purification

[0077] The patent all recombinant protein nucleic acid sequence SEQ ID NO. 4 (CBM-GFP fusion protein amino acid sequence see SEQ ID NO. 2, the sequence of the first-238th is the GFP sequence, the 238th-251th is the CBM and the intermediate Linker connection area of GFP, the 252th-409th is the CBM sequence, the CBM sequence can also be seen in SEQ ID NO. 1), SEQ ID NO. 5 (GFP-CBM fusion protein amino acid sequence see SEQ ID NO. 3, the first-158th is the CBM sequence, the 159th-171th is the intermediate Linker connection area of CBM and GFP, the 172th-409th is the GFP sequence), SEQ ID NO. 8 (recombinant spider silk protein F-01, comprising MaSp1 spider silk protein repeat region and connecting CBM at both ends), SEQ ID NO. 9 (recombinant spider silk protein F-02, comprising MaSp4 spider silk protein repeat region and connecting CBM at both ends), SEQ ID NO. 10 (recombinant spider silk protein F-03, comprising MaSp1 spider silk protein repeat region, spider silk protein N terminal and spider silk protein C terminal and connecting CBM at both ends), SEQ ID NO. 11 (recombinant spider silk protein F-04, comprising MaSp1 spider silk protein repeat region, spider silk protein N terminal and spider silk protein C terminal, without CBM) and SEQ ID NO. 12 (recombinant spider silk protein F-05, comprising CBM, MaSp1 spider silk protein repeat region and C terminal GFP) are synthesized by Huada Gene, and the above synthesized sequences are inserted into a commercial pET28a plasmid vector, the insertion site is between BamHI and XhoI, and finally the plasmid is transformed into E. coli BL21 (DE3) strain by chemical transformation method (completed by Huada Gene Service). The schematic diagram of the combination of CBM and functional protein is shown in FIG. 13.

[0078] The constructed strain is fermented, purified and freeze-dried to obtain a recombinant spider silk protein dry powder, as follows:

[0079] (1) Bacterial shake flask fermentation culture

[0080] The constructed strain is inoculated on a solid LB (containing 50 ug / ml kanamycin Kana) plate with a preserved glycerol tube, and cultured in a 37℃ incubator overnight. A single colony is picked and inoculated in 4-5 mL LB (containing 50 ug / ml kanamycin Kana) liquid medium, and cultured at 37℃, 200 rpm on a shaker overnight. 1% of the culture is inoculated into a 400 mL shake flask for fermentation, and cultured at 200 rpm, 37℃ for 2-3 h until OD600 = 0.6. Isopropyl thiogalactoside (IPTG) is added at a final concentration of 0.3 mM, and induced at 20℃ for 12-16 h. The bacterial cells are collected by centrifugation at 12000 rpm for 5 min.

[0081] (2) Protein purification

[0082] Resuspend the bacteria in 20 mM Tris-HCl to form a concentrated bacterial solution with OD600 = about 20. Ultrasonic treatment at 25 kHz and power of 70-80% for 3 s, stop for 6 s, and repeat for 30 min. The solution changes from turbid to clear, which is marked as sample WC. Centrifuge at 12000 rpm for 10-15 min to obtain the supernatant, which is marked as sample S. Repeat the loading of the nickel column (Shanghai Generay Biotech Co., Ltd., product number NO. A600657) with the supernatant for 2-3 times, collect the flow-through, and mark it as sample FT. Wash the column with 20 mM Tris-HCl for 3-5 times (1-3 times the volume of the column) until the solution drops is clear. Wash the column with 1-2 times the volume of 0.5 M imidazole solution for at least 3 times, and collect the eluate sequentially as E1, E2, and E3. Wash the residual 0.5 M imidazole solution with 5 times the volume of 20 mM Tris-HCl, and finally store it with 20% alcohol. The washed column can be reused. Mix E1, E2, and E3, and dialyze them in water. The dialyzed sample is marked as D.

[0083] (3) SDS-PAGE electrophoresis

[0084] Prepare a 10% concentration SDS-PAGE gel (Biyun Tian, product number: P0012A).

[0085] Sample preparation and detection: take 20 uL of the protein purification process sample and add 5 uL of loading buffer for SDS-PAGE detection.

[0086] (4) Protein freeze-drying

[0087] After dialysis, store the protein solution in a -80°C refrigerator until the protein solution is completely frozen. Then, place the protein solution in a freeze-dryer with a freezing temperature of -55°C and a vacuum degree of 5 Pa. Weigh the freeze-dried protein, and finally obtain the purified recombinant spider silk protein powder.

[0088] The preparation process of the recombinant spider silk protein lyocell fiber for inhibiting fibrillation provided in the application includes the following operation steps for preparing the recombinant spider silk protein / cellulose composite spinning solution:

[0089] 1. Pre-activation treatment of cellulose

[0090] Use wood pulp board with 99 wt% content of alpha cellulose, 0.2 wt% content of ash, and average polymerization degree of 700 as the cellulose raw material. Then, immerse the cellulose in a concentrated sulfuric acid solution with a concentration of 18 g / L and a temperature of 80°C or in a sodium hydroxide solution with a concentration of 100 g / L and a temperature of 80°C for 5 h. Then, perform water washing, drying, and crushing to obtain the activated cellulose in the form of powder.

[0091] 2. Dissolution of the recombinant protein in NMMO system:

[0092] 10 wt% of the recombinant protein was weighed by solvent weight and added to a 50 wt% NMMO solution system at 60°C for 1 h of stirring until a clear and transparent recombinant protein / NMMO mixture was obtained.

[0093] 3. Preparation of recombinant protein / cellulose composite spinning solution:

[0094] A certain amount of the recombinant protein mixture obtained in step 2 was prepared, 4-13 wt% of activated cellulose powder was added to the mixture, wherein the mass ratio of the recombinant protein to the activated cellulose powder was 1:0.5-100, and the mixture was stirred and dissolved under vacuum at 80-100°C to obtain a blended spinning solution of recombinant protein and cellulose. The blended spinning solution was subjected to vacuum degassing at 90°C to obtain a recombinant protein / cellulose composite spinning solution to be spun.

[0095] The recombinant protein / cellulose composite spinning solution to be spun was added to a spinning system, subjected to high-temperature standing degassing, filtration, and other operations, and then dry-jet wet spinning was performed through a spinning needle with a spinning speed of 35 m / min, a spinning needle aperture of 260 μm, and an air gap length of 5 mm. The spun thread was vertically drawn into a coagulation bath through air, and the first coagulation bath was a 5 wt% NMMO solution at 55°C, and the second coagulation bath was a water solution at 60°C. Then, the obtained nascent fiber was subjected to cold-hot water exchange washing and drying to obtain lyocell fibers combined with recombinant protein.

[0096] Example 2

[0097] The preparation process of the recombinant protein lyocell fiber with fluorescence excitation function in this embodiment includes the following operation steps:

[0098] 1) The recombinant protein was obtained in Example 1, and two recombinant protein lyocell fibers with fluorescence excitation function were prepared by selecting recombinant protein CBM-GFP (as shown in SEQ ID NO. 4) and recombinant protein GFP-CBM (as shown in SEQ ID NO. 5), respectively.

[0099] 2) Preparation of recombinant protein lyocell fiber liquid with fluorescence excitation function: Wood pulp board with 99wt% cellulose content, 0.2wt% ash content, and an average degree of polymerization of 700 was used as the cellulose raw material. The wood pulp board was then immersed in a concentrated sulfuric acid solution with a concentration of 9g / L and a temperature of 60°C for 2h. After that, the wood pulp board was washed with water, dried, and crushed to obtain the activated cellulose powder. 10wt% of the recombinant protein was weighed according to the weight of the solvent and added to a 50wt% NMMO solution system at 90°C. The solution was stirred for 0.2h until a clear and transparent recombinant spider silk protein / NMMO mixture was obtained. The remaining process was the same as in Example 1. The recombinant protein content in the fiber was controlled to be 5wt%.

[0100] 3) Preparation of recombinant protein lyocell fiber with fluorescence excitation function:

[0101] The recombinant protein lyocell fiber liquid with fluorescence excitation function obtained in step 2 was added to the spinning system. After high-temperature standing, degassing, filtration, and other operations, dry-jet wet spinning was performed through the spinning needle. The spinning speed was 35m / min, the spinning needle aperture was 260μm, the air gap length was 5mm, and the spinning line was vertically drawn into the coagulation bath through air. The first coagulation bath was a 5wt% NMMO solution at 55°C, and the second coagulation bath was a water solution at 60°C. Then, the obtained nascent fiber was washed with cold and hot water and dried to obtain the recombinant spider silk protein lyocell fluorescent fiber.

[0102] The obtained recombinant protein lyocell fluorescent fiber was placed under a fluorescence microscope, and the fluorescence effect was observed under ultraviolet light excitation.

[0103] Comparative Example 1

[0104] The difference between this comparative example and Example 2 is that in the preparation process of the protein / cellulose composite spinning liquid, the recombinant protein is replaced by an equal weight of casein, and the other preparation processes and test methods are exactly the same as in Example 2.

[0105] Different types of protein / cellulose composite spinning liquids are shown in Figure 1. Compared with Comparative Example 1, Example 2 has higher transparency and uniformity, and the number of bubbles in the microscope detection image is significantly reduced, which indicates that the solubility of the recombinant spider silk protein is better, and the recombinant spider silk protein has higher solubility and spinnability than casein.

[0106] Comparative Example 2

[0107] The difference between this comparative example and Example 2 is only that no protein is added in the preparation process, and the other preparation processes and test methods are exactly the same as in Example 2.

[0108] The fluorescence test results are shown in Figure 2. Compared with Comparative Example 2, the recombinant spider silk protein lyocell fiber containing fluorescent protein structure and CBM in Example 2 showed significant fluorescence effect in the fluorescence test, while the fiber of Comparative Example 2 had no fluorescence effect, which indicated that the spider silk recombinant protein could maintain its protein activity in the NMMO solution system. At the same time in Figure 2, the recombinant spider silk protein lyocell fluorescent fiber of Example 2 still showed significant fluorescence effect after multiple ultrasonic washing (using an ultrasonic washing machine, ultrasonic power 800W, ultrasonic time 0.5h), indicating that the CBM binding domain has strong interaction with cellulose.

[0109] Example 3

[0110] The preparation process of a recombinant spider silk protein lyocell fiber with high strength according to the present application comprises the following operation steps:

[0111] 1) The recombinant spider silk protein is obtained from Example 1, wherein the recombinant spider silk protein is F01 fusion protein (SEQ ID NO. 8)

[0112] 2) Preparation of a recombinant spider silk protein lyocell fiber spinning solution with high strength: wood pulp board with cellulose content 99wt%, ash content 0.2wt%, and average degree of polymerization 700 is used as cellulose raw material, and then treated with 300g / L concentration, 40℃ temperature sodium hydroxide for 10h, followed by water washing, drying, and crushing to obtain activated cellulose powder. 10wt% recombinant spider silk protein is weighed according to the weight of the solvent and added to a 60wt% NMMO solution system at 25℃ for 2h of stirring and dissolution until a clear and transparent recombinant spider silk protein / NMMO mixed solution is obtained. The rest of the process is the same as Example 2. The content of spider silk protein in the fiber is controlled to be 5wt%.

[0113] 3) Preparation of a recombinant spider silk protein lyocell fiber with high strength:

[0114] The recombinant spider silk protein lyocell fiber spinning solution with high strength obtained in step 2 is added to a spinning system, and then subjected to high-temperature standing defoaming, filtration, and other operations, and then 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 thread is vertically drawn into the coagulation bath through air to form a shape, the first coagulation bath uses 5wt% NMMO solution at 55℃, and the second coagulation bath uses water solution at 60℃. Then, the obtained nascent fiber is subjected to cold-hot water exchange washing treatment and drying to obtain a recombinant spider silk protein lyocell high-strength fiber.

[0115] The obtained recombinant spider silk protein lyocell high-strength fiber is subjected to mechanical test, and the dry breaking strength, dry breaking elongation rate and other properties are tested according to GB / T14337-2022.

[0116] Comparative Example 3

[0117] The difference between this comparative example and Example 3 is that no recombinant spider silk protein is added in the preparation process of the recombinant spider silk protein lyocell fiber with high strength, and the other preparation process and test method are exactly the same as Example 3.

[0118] The mechanical property test results are shown in Figure 3. Compared with Comparative Example 3, the fiber obtained in Example 3 showed a significant strength increase in the mechanical property test, with a strength increase of 32.62 cN (an increase of 168.48% compared with the control), and an elongation increase of 46.15%.

[0119] Table 1 Mechanical data of fibers of Example 3 and Comparative Example 3

[0120] Example 4

[0121] The preparation process of the recombinant spider silk protein lyocell fiber with high elasticity according to the present application comprises the following operation steps:

[0122] 1) The recombinant spider silk protein is obtained from Example 1, wherein the recombinant spider silk protein is F02 fusion protein (SEQ ID NO. 9)

[0123] 2) The preparation process of the recombinant spider silk protein lyocell fiber spinning solution with high elasticity is the same as Example 3. The content of spider silk protein in the fiber is controlled to be 5wt%.

[0124] 3) Preparation of the recombinant spider silk protein lyocell fiber with high elasticity:

[0125] 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 defoaming, filtration and other operations, dry-jet wet spinning is carried out through the spinning needle, the spinning speed is 35 m / min, the spinning needle aperture is 260 μm, the air gap length is 5 mm, and the spinning line is vertically drawn into the coagulation bath through air to form, the first coagulation bath uses 5wt% NMMO solution at 55℃, and the second coagulation bath uses water solution at 60℃. Then, the as-spun fiber is treated by cold-hot water exchange washing and drying to obtain the recombinant spider silk protein lyocell high-elasticity fiber.

[0126] The obtained recombinant spider silk protein lyocell high-elasticity fiber is subjected to mechanical test, and the properties such as dry breaking strength and dry breaking elongation are tested according to GB / T14337-2022.

[0127] Comparative Example 4

[0128] The difference between the present comparative example and Example 4 is only that no recombinant spider silk protein is added in the preparation process of the recombinant spider silk protein lyocell fiber with high elasticity, and the other preparation process and test method are exactly the same as those of Example 4.

[0129] The mechanical property test results are shown in FIG. 4. Compared with Comparative Example 4, the fiber obtained in Example 4 shows obvious improvement in elasticity in the mechanical property test, with the elongation at break increased to 30.53% (189.93% higher than the control), and the strength increased by 51.60% compared with the control.

[0130] Table 2 Mechanical data of fibers of Example 4 and Comparative Example 4

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

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

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

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

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

[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 defoaming, filtration and other operations, dry-jet wet spinning is carried out through the spinning needle, the spinning speed is 35 m / min, the spinning needle aperture is 260 μm, the air gap length is 5 mm, and the spinning line is vertically drawn into the coagulation bath through air to form, the first coagulation bath adopts 5wt% NMMO solution at 55℃, and the second coagulation bath adopts water solution at 60℃. Then, the obtained as-spun fiber is subjected to cold-hot water exchange washing treatment and drying to obtain recombinant spider silk protein lyocell high-strength fiber. At the same time, a protein-free sample without F03 fusion protein is prepared according to the above method as a control.

[0137] The obtained recombinant spider silk protein lyocell high-strength fiber is subjected to mechanical test, and the properties such as dry breaking strength and dry breaking elongation are tested according to GB / T14337-2022.

[0138] Comparative Example 5

[0139] In the present example, CBM-free recombinant spider silk protein is used to prepare lyocell fiber, which comprises the following operation steps:

[0140] 1) Recombinant spider silk protein obtained in Example 1, wherein the recombinant spider silk protein is 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 content of spider silk protein in the fiber is controlled to be 2wt%.

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

[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 defoaming, filtering and other operations, dry-jet wet spinning is carried out through the spinning needle, the spinning speed is 35 m / min, the spinning needle aperture is 260 μm, the air gap length is 5 mm, and the spinning line is vertically drawn into the coagulation bath through air to form, the first coagulation bath adopts 5wt% NMMO solution at 55℃, and the second coagulation bath adopts water solution at 60℃. Then, the obtained primary fiber is treated by cold-hot water exchange washing and drying to obtain recombinant spider silk protein lyocell high-strength fiber.

[0144] The obtained recombinant spider silk protein lyocell high-strength fiber is subjected to mechanical test, and the dry breaking strength, dry breaking elongation and other properties are tested according to GB / T14337-2022.

[0145] Example 6

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

[0147] 1) Recombinant spider silk protein obtained in Example 1, wherein the recombinant spider silk protein is 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 content of spider silk protein in the fiber is controlled to be 2wt%.

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

[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 defoaming, filtering and other operations, dry-jet wet spinning is carried out through the spinning needle, the spinning speed is 35 m / min, the spinning needle aperture is 260 μm, the air gap length is 5 mm, and the spinning line is vertically drawn into the coagulation bath through air to form, the first coagulation bath adopts 5wt% NMMO solution at 55℃, and the second coagulation bath adopts water solution at 60℃. Then, the obtained primary fiber is treated by cold-hot water exchange washing and drying to obtain 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 in accordance with GB / T14337-2022.

[0152] Comparative Example 6

[0153] In this embodiment, casein was used to prepare lyocell fiber, including the following operation steps:

[0154] 1) Take casein. Since there is a lack of interaction between casein and cellulose, the industry often uses transglutaminase (TG enzyme) to enhance the intermolecular or intramolecular cross-linking reaction, so 0.2wt% TG enzyme is added to the NMMO solution.

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

[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 defoaming, filtration and other operations, dry-jet wet spinning is carried out through the 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 air to form. The first coagulation bath uses 5wt% NMMO solution at 55℃, and the second coagulation bath uses water solution at 60℃. Then, the obtained as-spun fiber is subjected to cold-hot water exchange washing treatment and drying to obtain 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 in accordance with GB / T14337-2022.

[0159] The mechanical property test results are shown in Table 3 and Figure 5. Compared with Comparative Examples 5 and 6, the protein-containing fiber obtained in Example 5 showed a significant increase in strength in the mechanical property test, in which the sample containing F-03 increased in strength to 27.37 cN (an increase of 159.43% compared with the control group), and the sample containing F-04 increased in strength to 14.48 cN (an increase of 37.72% compared with the control group), indicating that the double CBM structure is more conducive to the combination between the 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 had a strength of 19.13 cN (an increase of 81.33% compared with the control group) in the fiber performance, which was not as good as the protein sample containing a double CBM structure (i.e., Example 5) and better than the sample containing no CBM F-04, which also indicated that the double CBM structure is more conducive to the combination between the spider silk protein and cellulose than the single CBM structure. The use of TG enzyme in the comparative example enhanced the combination between the protein and cellulose, and the strength of the 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] Example 7

[0162] A preparation process of a high-strength recombinant spider silk protein lyocell fiber for inhibiting fibrillation according to the present application comprises the following operation steps:

[0163] 1) The recombinant spider silk protein is obtained in Example 1, wherein the recombinant spider silk protein is protein F01 (a double CBM structure protein (SEQ ID NO. 8)

[0164] 2) A preparation process of a recombinant spider silk protein lyocell fiber spinning solution for inhibiting fibrillation is the same as that in Example 2. The content of the spider silk protein in the fiber is controlled to be 0 wt% (control), 1 wt%, 2 wt%, 5 wt%, 10 wt% and 20 wt%, respectively.

[0165] 3) A preparation of a recombinant spider silk protein lyocell fiber for inhibiting fibrillation:

[0166] The recombinant spider silk protein lyocell fiber spinning solution obtained in step 2 is added to a spinning system, and after high-temperature standing defoaming, filtration and other operations, dry-jet wet spinning is performed through a spinneret, the spinning speed is 50 m / min, the spinneret hole diameter is 100 μm, the air gap side blowing temperature is 25 ℃, the air gap length is 30 mm, and the spinning line is vertically drawn into the coagulation bath through air to form, the first coagulation bath uses 15 wt% NMMO solution at 25 ℃, and the second coagulation bath uses water solution at 25 ℃. Then, the obtained primary fiber is washed by cold-hot water exchange, cut, oiled and dried to obtain recombinant spider silk protein lyocell high-elasticity fiber.

[0167] The obtained recombinant spider silk protein lyocell high-elasticity fiber is subjected to mechanical test, and the dry breaking strength, dry breaking elongation and other properties thereof are tested according to GB / T14337-2022.

[0168] The obtained recombinant spider silk protein lyocell fiber is subjected to fibrillation test according to standard FZ / T 52019, and the treated fiber is observed under a microscope for fibrillation phenomenon.

[0169] The mechanical property test results are shown in Table 4. After the F-01 protein is spun through the spinneret process, the protein addition amount of 1%-20% can significantly improve the mechanical properties, especially the content of 2% (enhanced by 29.4%), 10% (enhanced by 35.86%) and 20% (enhanced by 40.4%). When the protein addition amount exceeds 5%, the elongation of the fiber is significantly improved, and the protein content of 10% improves the elongation of the fiber by 26.2%.

[0170] Table 4 Physical properties of recombinant spider silk protein lyocell fiber spun through industrial spinning hole system

[0171] The results of the anti-fibrillation test are shown in Figures 6-12. Compared with the control group and Comparative Example 5, the fibers obtained by adding each amount of recombinant protein all show excellent anti-fibrillation effect in the anti-fibrillation test, and the mechanical properties of the fibers are also improved to a certain extent, which further indicates the interaction between the CMB binding domain of the spider silk recombinant protein and cellulose, and effectively improves the fibrillation phenomenon of the fiber.

[0172] Example 8

[0173] The recombinant protein lyocell fiber with fluorescence excitation function is prepared according to the method in Example 2, except that the addition amount of recombinant protein is increased during the preparation of 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 obtained spinning solution has good transparency and uniformity, and can normally perform spinning. The fiber spun can normally emit fluorescence.

[0174] Example 9

[0175] The recombinant protein lyocell fiber with fluorescence excitation function was prepared according to the method in Example 3, except that the amount of added recombinant spider silk protein 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. The transparency and uniformity of the obtained spinning solution were good, and the spinning could be normally carried out.

[0176] Example 10

[0177] In this example, ionic liquid 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) was used as a solvent for dissolving cellulose and recombinant spider silk protein to prepare recombinant spider silk regenerated fiber, and the specific process was as follows:

[0178] 1) The recombinant spider silk protein was obtained from Example 1, wherein the recombinant spider silk protein was F01 fusion protein (SEQ ID NO. 8)

[0179] 2) Preparation of recombinant spider silk protein / cellulose spinning solution: wood pulp board with 99wt% alpha cellulose content, 0.2wt% ash content, and an average degree of polymerization of 700 was used as the cellulose raw material. The wood pulp board was then immersed in a 300g / L sodium hydroxide solution at 40°C for 10h, followed by water washing, drying, and crushing to obtain the activated cellulose powder. Ionic liquid 1-butyl-3-methylimidazolium chloride 15g, cellulose powder 0.9g, and spider silk protein powder 0.018g were sequentially added to a mechanically stirred reaction kettle and mixed uniformly. The temperature in the kettle was controlled at 100°C and the stirring reaction was carried out for 1-2h. The spinning solution was observed under a polarizing microscope until there were no microfibers and solid particles, indicating that the cellulose and spider silk protein were completely dissolved. Subsequently, the dissolved slurry was vacuum degassed at 100°C for 1-2h and filtered to obtain a regenerated spider silk protein / cellulose spinning stock solution. The content of recombinant spider silk protein in the fiber was controlled at 2wt%.

[0180] 3) Preparation of recombinant spider silk regenerated fiber: the spinning stock solution was added to a syringe for spinning. The coagulation bath was a 5% ionic liquid solution, and the coagulation bath temperature was 50°C. The spinning speed was 10m / min, the spinning needle aperture was 260μm, and the air gap length was 5mm. The spinning line was vertically drawn into the coagulation bath after being stretched in the air to form a primary fiber. The obtained primary fiber was processed through water washing, stretching, oiling, drying, and other procedures to obtain the recombinant spider silk regenerated fiber.

[0181] The obtained recombinant spider silk regenerated fiber was subjected to mechanical testing, and the dry breaking strength and dry breaking elongation were tested according to GB / T14337-2022.

[0182] Comparative Example 7

[0183] The difference between the present comparative example and Example 10 is that no recombinant spider silk protein is added in the process of preparing the recombinant spider silk regenerated fiber, and other preparation processes and test methods are exactly the same as those of Example 10.

[0184] Comparative Example 8

[0185] The difference between the present comparative example and Example 10 is that the recombinant spider silk protein without CBM is used to prepare the recombinant spider silk regenerated fiber in the present comparative example, and the ionic liquid 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) is used as the solvent for dissolving the cellulose and the recombinant spider silk protein, and the following operation steps are included:

[0186] 1) The recombinant spider silk protein is obtained from Example 1, wherein the recombinant spider silk protein without CBM is F-04 recombinant protein (SEQ ID NO. 11).

[0187] 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 without CBM F-04, and the content of the recombinant spider silk protein in the fiber is controlled to be 2wt%.

[0188] 3) Preparation of recombinant spider silk regenerated fiber: the preparation process of the recombinant spider silk regenerated fiber is the same as that of Example 10.

[0189] The obtained recombinant spider silk regenerated fiber is subjected to mechanical test, and the properties such as dry breaking strength and dry breaking elongation are tested according to GB / T14337-2022.

[0190] The mechanical property test results are shown in FIG. 14 and Table 5. Compared with Comparative Examples 10 and 11, the recombinant spider silk protein fiber obtained from Example 10 shows more obvious performance improvement in the mechanical property test, the strength of the F-01 sample fiber is increased to 36.11 cN (increased by 43.98% compared with the control), and the elongation is increased by 185.71%. The strength of the F-04 sample fiber of Comparative Example 11 is 32.52 cN (only increased by 29.67%) compared with the protein-free control sample, and the elongation is 7.36% (only increased by 22.26%), which indicates that the double CBM structure is more conducive to the combination between the spider silk protein and the cellulose.

[0191] Table 5 Fiber mechanical data of Example 10 and Comparative Examples 7 and 8

[0192] Example 11

[0193] In this embodiment, 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, and the specific process is as follows:

[0194] 1) The recombinant spider silk protein is obtained from Example 1, wherein the recombinant spider silk protein is F02 fusion protein (SEQ ID NO. 9)

[0195] 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 content of recombinant spider silk protein in the fiber is controlled to be 2wt%.

[0196] 3) Preparation of recombinant spider silk regenerated fiber: the preparation process of the recombinant spider silk regenerated fiber is the same as that of Example 10.

[0197] The obtained recombinant spider silk regenerated fiber is subjected to mechanical test, and the dry breaking strength, dry breaking elongation and other properties are tested according to GB / T14337-2022.

[0198] The mechanical property test results are shown in FIG. 15 and Table 6. Compared with Comparative Examples 10 and 11, the fiber obtained in Example 11 shows more obvious performance improvement in the mechanical property test, and the strength of F-02 fiber is increased to 74.00 cN (increased by 195.06% compared with the control), and the elongation is increased by 147.84%. The strength of the F-04 fiber sample of Comparative Example 11 is 32.52 cN (only increased by 29.67%) compared with the protein-free control sample, and the elongation is 7.36% (only increased by 22.26%), which indicates that the double CBM structure is more conducive to the combination between spider silk protein and cellulose.

[0199] Table 6 Fiber mechanical data of Example 11 and Comparative Examples 7 and 8

[0200] Example 12

[0201] 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, and the specific process is as follows:

[0202] 1) The recombinant spider silk protein is obtained from Example 1, wherein the recombinant spider silk protein is F01 fusion protein (SEQ ID NO. 8)

[0203] 2) Recombinant spider silk protein / cellulose spinning solution preparation: Wood pulp board with 99wt% of alpha cellulose content, 0.2wt% of ash content, and an average degree of polymerization of 700 was used as the cellulose raw material. The wood pulp board was then immersed in a 300g / L sodium hydroxide solution at 40°C for 10 hours. After that, the wood pulp board was washed with water, dried, and crushed to obtain the activated cellulose powder. 30mL of DMAc was weighed, and 3g of LiCl was added to the DMAc solution. The mixture was heated to 100°C, and then 0.018g of recombinant spider silk protein was added. The mixture was then heated to 150°C and kept at this temperature for 10 minutes to ensure complete dissolution of the spider silk protein. Then, 0.9g of cellulose powder was added to the mixture and stirred for 20 minutes. The mixture was then cooled to 90°C and stirred for another 45 minutes. Finally, the mixture was cooled to 40°C, and the cellulose was completely dissolved during the cooling process. The spinning solution was observed under a polarizing microscope until there were no microfibers or solid particles, indicating that the cellulose and spider silk protein were completely dissolved.

[0204] 3) Preparation of recombinant spider silk regenerated fiber: The spinning solution was added to a syringe for spinning. Deionized water was used as the coagulation bath, and the coagulation bath temperature was 50°C. The spinning speed was 40m / min, the spinning needle aperture was 260μm, and the air gap length was 5mm. The spinning line was vertically drawn into the coagulation bath after being stretched in the air, and the obtained nascent fiber was processed through washing, stretching, oiling, and drying to obtain the recombinant spider silk regenerated fiber.

[0205] The obtained recombinant spider silk regenerated fiber was subjected to mechanical testing, and the dry breaking strength and dry breaking elongation were tested according to GB / T14337-2022.

[0206] Comparative Example 9

[0207] 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 process and testing method are exactly the same as those of Example 12.

[0208] Comparative Example 10

[0209] The difference between this comparative example and Example 12 is that the recombinant spider silk protein without CBM is used to prepare the recombinant spider silk regenerated fiber in this comparative example, and N,N-dimethylacetamide (DMAc) / lithium chloride (LiCl) system is used as the solvent for dissolving cellulose and recombinant spider silk protein, including the following operation steps:

[0210] 1) The recombinant spider silk protein was obtained from Example 1, and the recombinant spider silk protein without CBM was F04 recombinant protein (SEQ ID NO. 11).

[0211] 2) The preparation process and test method of recombinant spider silk protein fiber are the same as example 12. The content of spider silk protein in the fiber is controlled to be 2wt%.

[0212] The mechanical property test results are shown in Figure 16 and 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 F-02 fiber was increased to 62.13 cN (increased by 72.34% compared with the control), and the elongation was increased by 78.93%. The strength of the F-04 sample fiber of Comparative Example 13 was 39.27 cN (only increased by 8.93%) compared with the protein-free control sample, 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.

[0213] Table 7 Fiber mechanical data of Example 12 and Comparative Examples 12 and 13

[0214] Example 13

[0215] In this example, N,N-dimethylacetamide (DMAc) / lithium chloride (LiCl) system is used as the solvent for dissolving cellulose and spider silk protein to prepare recombinant spider silk regenerated fiber, and the specific process is as follows:

[0216] 1) The recombinant spider silk protein is obtained from Example 1, wherein the recombinant spider silk protein is F02 fusion protein (SEQ ID NO. 9)

[0217] 2) Preparation of recombinant spider silk protein / cellulose spinning solution: the spinning solution preparation process is the same as Example 12, except that the recombinant spider silk protein F01 fusion protein is replaced by F02 fusion protein, and the content of spider silk protein in the fiber is controlled to be 2wt%.

[0218] 3) Preparation of recombinant spider silk regenerated fiber: the preparation process of recombinant spider silk regenerated fiber is the same as Example 12.

[0219] The obtained recombinant spider silk regenerated fiber is subjected to mechanical test, and the properties such as dry breaking strength and dry breaking elongation are tested according to GB / T14337-2022.

[0220] The results of the mechanical property test are shown in Figure 17 and 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, the strength of F-02 fiber was increased to 65.41 cN (increased by 81.44% compared with the control), and the elongation was increased by 54.18%. The strength of 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%) compared with the control without protein, indicating that the double CBM structure is more conducive to the combination between the spider silk protein and cellulose.

[0221] Table 8 Fiber mechanical data of Example 13 and Comparative Examples 12 and 13

[0222] As shown in Figure 18, the fiber samples with and without added spider silk protein were prepared by industrial spinneret; Figures 19 and 20 are fiber samples with and without added spider silk protein prepared in the laboratory; there is no obvious difference in the appearance of the fiber samples with and without added protein, and the whiteness of the fiber is not affected.

[0223] Obviously, the above examples are only examples for the purpose of clarity, and are not intended to limit the embodiments. Based on the above description, other different forms of changes or variations can also be made by those of ordinary skill in the art. All embodiments do not need to be exhausted and cannot be exhausted. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for producing regenerated cellulose fibers bound to a recombinant protein, characterized by, The method comprises the following steps: The recombinant protein and the cellulose are dissolved in a solvent and uniformly mixed to obtain a spinning solution; and the spinning solution is used for spinning to obtain the regenerated cellulose fiber combined with the recombinant protein. The recombinant protein is obtained by fusion expression of at least one cellulose binding domain and at least one functional protein.

2. The method for producing regenerated cellulose fibers combined with recombinant proteins according to claim 1, characterized by, The amino acid sequence of the cellulose binding domain is shown in SEQ ID NO.

1.

3. The method for preparing regenerated cellulose fibers incorporating recombinant proteins 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 producing the regenerated cellulose fiber combined with the recombinant protein according to claim 3, characterized by, The spider silk protein is a MaSp type spider silk protein, and the amino acid sequence of the spider silk protein comprises at least one spider silk protein repeat sequence. The spider silk protein repeat sequence is shown in SEQ ID NO. 6 or SEQ ID NO.

7.

5. The method of producing regenerated cellulose fibers bound to recombinant proteins according to claim 4, characterized in that, The spider silk protein repeat sequence is connected with a spider silk protein N-terminal sequence at the N-terminal end and with a spider silk protein C-terminal sequence at the C-terminal end.

6. The method of claim 1, wherein the regenerated cellulose fiber is a combination of recombinant proteins. The N-terminal end and / or the C-terminal end of the functional protein in the recombinant protein are connected with at least one cellulose binding domain.

7. The method of claim 1, wherein the regenerated cellulose fiber is a combination of recombinant proteins. The recombinant protein comprises a plurality of functional proteins; and at least two functional proteins are connected by at least one cellulose binding domain.

8. The method of claim 1, wherein the regenerated cellulose fiber is bonded with the recombinant protein. The N-terminal end and the C-terminal end of the functional protein in the recombinant protein are both connected with at least one cellulose binding domain.

9. The method of claim 1, wherein the regenerated cellulose fiber is a combination of recombinant proteins. The recombinant protein is prepared by the following method: The nucleic acid sequence for expressing the recombinant protein is introduced into a bacterial strain to construct an engineered bacterium; the engineered bacterium is cultured, and the bacterial body is collected and the recombinant protein is extracted.

10. The method of producing regenerated cellulose fibers bound to recombinant proteins according to claim 9, characterized in that, 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.

11. The method for preparing regenerated cellulose fibers incorporating recombinant proteins according to claim 1, characterized in that, The cellulose is a powder-shaped cellulose after activation treatment.

12. The method of producing regenerated cellulose fibers bound to recombinant proteins according to claim 11, characterized in that, The powder-shaped cellulose after activation treatment is obtained by the following method: Cellulose raw materials with a content of alpha cellulose of not less than 95 wt% and an average degree of polymerization of 500-650 are subjected to immersion treatment, and then are subjected to water washing, drying, and crushing to obtain the powder-shaped cellulose after activation treatment; The immersion treatment is performed by using a concentrated sulfuric acid solution with a concentration of 9-36 g / L and a temperature of 60-90 DEG C or a sodium hydroxide solution with a concentration of 100-300 g / L and a temperature of 40-80 DEG C for immersion for 2-10 h.

13. The method for preparing regenerated cellulose fibers incorporating recombinant proteins according to claim 1, characterized in that, The solvent is an NMMO solvent or an ionic liquid solvent. Preferably, the ionic liquid solvent is 1-butyl-3-methylimidazolium chloride or N,N-dimethylacetamide / lithium chloride.

14. The method for preparing regenerated cellulose fibers incorporating recombinant proteins according to claim 1, characterized in that, The method of dissolving the recombinant protein and the cellulose raw materials in a solvent and uniformly mixing them is specifically as follows: Step one) The recombinant protein is dissolved in an NMMO solvent, the concentration of the NMMO solvent is 50-90 wt%, the dissolving temperature is 25-95 DEG C, and the dissolving time is 0.2-2 h to obtain an NMMO solution of the recombinant protein; Step two) The cellulose is added into the NMMO solution of the recombinant protein and is stirred and dissolved to obtain a spinning solution; and Preferably, the concentration of cellulose in step ii) is 4-13 wt% of the total mass of the recombinant spider silk protein / NMMO mixed spinning solution, the stirring and dissolving temperature is 80-110℃, and the dissolving time is 1-5h, until a transparent amber colored composite spinning solution is obtained.

15. The method for preparing regenerated cellulose fibers incorporating recombinant proteins according to claim 1, characterized in that, The mass ratio of the recombinant protein to cellulose is 1:0.5-100.

16. The method of producing regenerated cellulose fibers bonded with recombinant proteins according to claim 14, characterized in that, After the spinning solution is deaerated by high temperature standing, filtered, and then dry-jet wet spun through a spinneret, the spinning speed is 40-50m / min, the spinneret aperture is 100-300μm, the air gap length is 5-50mm, the spinning thread is vertically drawn into a coagulation bath through air and formed, the first coagulation bath uses a 5-15wt% NMMO solution at 25-70℃, the second coagulation bath uses an aqueous solution at 25-70℃; then the obtained nascent fiber is treated by cold-hot water exchange washing, cut, oiled, and dried to obtain recombinant spider silk protein lyocell fiber.

17. The method for preparing regenerated cellulose fibers incorporating recombinant proteins according to claim 1, characterized in that, The mixing and uniformity of the recombinant protein and cellulose raw materials in the solvent is achieved by: The ionic liquid, cellulose, and recombinant protein are mixed and stirred at 100℃ for 1-2h to obtain a composite spinning solution.

18. The method of producing regenerated cellulose fibers bound to recombinant proteins according to claim 17, characterized in that, After the composite spinning solution is deaerated by high temperature standing, filtered, and then spun by adding it into a syringe, the spinning speed is 40-50m / min, the spinning needle aperture is 100-300μm, the air gap length is 5-50mm; the spinning thread is vertically drawn into a coagulation bath through air and formed, the coagulation bath temperature is 25-70℃; then the obtained nascent fiber is washed, stretched, oiled, and dried to obtain recombinant spider silk regenerated fiber.

19. Recombinant protein-bound regenerated cellulose fiber prepared by the preparation method of any one of claims 1-18.

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