Method for the preparation of modified spider silk proteins enhancing lyocell fibers

Abstract

The present application relates to the technical field of bio-based functional fiber manufacturing, in particular to a preparation method of modified spider silk protein reinforced lyocell fiber, comprising the following steps: firstly, spider silk protein is expressed by Pichia pastoris strain GS115 / pPIC9K-spider silk gene; secondly, the spider silk protein is subjected to biomimetic mineralization treatment, and a silicon dioxide coating layer is formed through ultrasonic-assisted mineralization; then, spinning solution is prepared to realize uniform mixing of the mineralized spider silk protein and cellulose; then, coaxial spinning is performed through a microfluidic spinning device to construct a skin-core structure fiber; finally, a beta-fold structure is induced to form in an ethanol / water / calcium chloride coagulation bath. The process breaks through the technical bottlenecks of phase separation, thermal inactivation and uneven dispersion of spider silk protein and cellulose, reduces production cost, and provides high-performance fiber materials for high-end textile and biomedical fields.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bio-based functional fiber manufacturing, in particular to a preparation method of modified spider silk protein reinforced lyocell fiber. BACKGROUND

[0002] Spider silk protein is a very special fiber protein composed of beta-folded alanine and alpha-helix rich in proline, and its tight packing secondary structure makes it a semi-crystalline molecular spring structure, which determines its high strength and large toughness. The unique structure and properties of spider silk protein make it have an attractive prospect in the field of medicine, especially in tissue engineering.

[0003] At present, due to the conflict between material compatibility and process adaptability in the production process of lyocell fiber, the conventional technical means cannot effectively control the interface bonding state of the protein and cellulose phase when the spider silk protein is blended and reinforced. If the two-phase compatibility is insufficient, it may cause phase separation of the spinning solution and mechanical fault of the fiber, which cannot guarantee the stability of the reinforcing effect.

[0004] Therefore, the present application provides a preparation method of modified spider silk protein reinforced lyocell fiber to solve the above problems. SUMMARY

[0005] The main purpose of the present application is to provide a preparation method of modified spider silk protein reinforced lyocell fiber to solve the problems raised in the above background.

[0006] To achieve the above purpose, the technical scheme adopted by the present application is as follows: a preparation method of modified spider silk protein reinforced lyocell fiber, comprising the following steps:

[0007] Step one: spider silk protein expression, using Pichia pastoris strain to express spider silk protein, the Pichia pastoris strain is GS115 / pPIC9K-spider silk gene, and high-purity spider silk protein is obtained by fermentation control;

[0008] Step two: biomimetic mineralization, mineralizing the spider silk protein expressed in step one, including pretreatment, double silicon source compounding and mineralization reaction, wherein the double silicon source compounding adopts a compounding system of tetraethyl orthosilicate and methyl triethoxysilane, and the mineralization reaction is carried out under ultrasonic assistance to form a silicon dioxide coating layer;

[0009] Step three: spinning solution preparation, mixing the mineralized spider silk protein in step two with cellulose, adding a solvent system and a dispersion aid to prepare a spinning solution, the solvent system includes N-methyl morpholine-N-oxide, water and 1-ethyl-3-methyl imidazole acetate, and the dispersion aid is a polydopamine coating;

[0010] Step four: spinning treatment, the spinning liquid prepared in step three is spun through a microfluidic spinning device to form a skin-core structure fiber, wherein the core layer is a cellulose solution and the skin layer is a mineralized spider silk protein solution;

[0011] Step five: coagulation treatment, the fiber after spinning in step four is treated in a coagulation bath to induce the formation of a β-sheet structure, the coagulation bath comprising ethanol, water and calcium chloride;

[0012] Further, in step one, the Pichia pastoris strain is expressed by DO-stat fed-batch fermentation control, the feed includes gradient addition of glycerol and methanol, the fermenter is Sartorius Biostat® CSTR type, the volume is 50L, and is equipped with a dissolved oxygen and pH linkage control system, and the fermentation expression amount is 8g / L.

[0013] Further, in the DO-stat fed-batch fermentation control, the addition gradient of glycerol and methanol is: glycerol is added to a carbon source concentration of 5g / L in the initial stage, methanol is switched when the DO value decreases to 20%, the methanol feeding rate is controlled at 0.5-1.0mL / min / L of culture solution, the fermentation temperature is 28℃±1℃, the pH is controlled at 5.0±0.2, and the fermentation time is 48-72 hours.

[0014] Further, in step one, a purification process is also included, the expressed spider silk protein is purified by affinity chromatography and cold ethanol fractionation precipitation, the affinity chromatography uses an AKTA pure 25 chromatography system, the chromatography medium is a nickel column, and the elution buffer is an imidazole gradient solution of 20-250mM, the purity of the purified spider silk protein is 98%±0.5%, and the molecular weight is greater than 100kDa.

[0015] Further, in step two, the pretreatment includes placing the spider silk protein in a Tris-HCl buffer, the buffer pH is 5.0±0.05, the concentration is 50mM, the pretreatment time is 30 minutes, and the temperature is 25℃±1℃, to enhance the protein dispersibility and uniformity of mineralization coating.

[0016] Further, in step two, the molar ratio of TEOS to MTES in the double silicon source compounding is 7:3±2%, wherein the proportion of TEOS is 70%±2% and the proportion of MTES is 30%±2%, the total concentration of the double silicon source is 10% w / v, the compounding is carried out under magnetic stirring, the stirring speed is 200r / min, and the time is 15 minutes, to improve the toughness of the mineralized shell layer and the interfacial bonding strength.

[0017] Further, in the step two, the mineralization reaction is carried out under the assistance of ultrasonic, the ultrasonic frequency is 40 kHz±2 kHz, the power density is 0.5 W / mL, the reaction temperature is 50℃±0.5℃, the reaction time is 120 minutes±5 minutes, the mineralization coating rate reaches 92%±1.5%, and the silicon dioxide coating layer thickness is 50-100 nm.

[0018] Further, in the step three, the solvent system is composed of NMMO, water and EMIMAc, the mass ratio is 76:18:6±1%, the dissolving temperature is 65℃±2℃, the stirring speed is 300 r / min, and the stirring time is 60 minutes, so that the cellulose concentration reaches 12% w / v, the mineralized spider silk protein concentration reaches 6% w / v, and the protein activity is greater than 90%.

[0019] Further, in the step three, the dispersing aid is the polydopamine-coated mineralized spider silk protein, the adding amount is 0.3%±0.05% of the total mass of the spinning solution, and the coating thickness is 5-10 nm, so that the interface bonding between the spider silk protein and the cellulose matrix is enhanced, the phase separation is reduced, and the breaking force is increased by 45%.

[0020] Further, in the step four, the microfluidic spinning adopts a coaxial spinning head, the core layer is the cellulose solution, the flow rate is 0.1 mL / min, the skin layer is the mineralized spider silk protein solution, the flow rate is 0.4 mL / min, the flow rate ratio is 1:4±0.1, the spinning temperature is 65℃±2℃, and the spinneret hole diameter is 80-100 μm, so that the continuous skin-core structure fiber is formed, the core layer diameter accounts for 60%, the skin layer thickness accounts for 40%, the fiber elongation rate is greater than 22%, and the modulus reaches 16.8 GPa.

[0021] The present application has the following beneficial effects:

[0022] 1. In the present application, the recombinant spider silk protein is synthesized by the Pichia pastoris expression system, the fermentation process of dynamic dissolved oxygen control is combined, the protein yield is improved, and the production cost is reduced, the impurities are separated by using the fractional precipitation technology in the purification stage, the high-purity active protein is obtained, the core raw material basis is provided for subsequent modification, the protein secondary structure integrity is maximized in the cold ethanol precipitation process, and the problem of activity loss caused by traditional purification methods is avoided.

[0023] 2. In the present application, the bionic mineralization process adopts the synergistic effect of double silicon sources, under the driving of ultrasonic energy, the silicon hydroxyl generated by the hydrolysis of tetraethyl orthosilicate forms stable combination with the amino acid residues of spider silk protein, the hydrophobic groups of methyl triethoxysilane are oriented and arranged outward, the mineralized protein microparticles with core-shell structure are constructed, the protein thermal stability is enhanced, the compatibility with the cellulose matrix is improved at the hydrophobic interface, the phase separation problem in the blending spinning is solved, and the spinning process breakage phenomenon is avoided.

[0024] 3. In the present application, the ternary solvent system is innovatively applied in the spinning stage to reduce the dissolution temperature, protect the beta-sheet conformation of spider silk protein from being destroyed by heat, and the microfluidic core-sheath spinning technology is used to make the mineralized protein uniformly coat the cellulose core layer to form a composite structure by precisely controlling the flow rates of the two liquid phases, and the calcium ion coagulation bath is used to induce the ordered assembly of the protein, thereby strengthening the mechanical properties of the fiber, and the polydopamine interface modification is used to enhance the binding force between the components, thereby forming a durable and stable core-sheath composite interface and overcoming the defect of easy peeling of the traditional coating method. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0026] Embodiment 1: A method for preparing modified spider silk protein reinforced lyocell fiber, comprising the following steps:

[0027] Step one: Spider silk protein expression, using Pichia pastoris strain GS115 / pPIC9K-spider silk gene for expression, through DO-stat fed-batch fermentation control, initially adding glycerol to a carbon source concentration of 5 grams per liter, when the dissolved oxygen decreases to 20%, switching to methanol feeding, feeding rate 0.5 milliliters per minute per liter of culture solution, controlling the fermentation temperature at 28 degrees Celsius ± 1 degree Celsius, pH value 5.0 ± 0.2, fermentation time 48 hours; after fermentation is completed, purification is carried out using AKTA pure 25 chromatography system, the nickel column medium elution buffer is a 20 to 250 millimolar imidazole gradient solution, after fractional precipitation with cold ethanol, the purity of the spider silk protein is 98%, and the molecular weight is greater than 100 kilodaltons;

[0028] Step two: biomimetic mineralization, placing the purified spider silk protein in a pH 5.0 50 millimolar Tris-HCl buffer for pretreatment for 30 minutes, then adding a double-silicon source system with a molar ratio of tetraethyl orthosilicate to methyltriethoxysilane of 7 to 3, a total concentration of 10% mass by volume, magnetic stirring at 200 revolutions per minute for 15 minutes, and then ultrasonic assisted mineralization at 40 kilohertz, power density 0.5 watts per milliliter, 50 degrees Celsius reaction for 120 minutes, obtaining mineralized protein with a silica coating rate of 92%;

[0029] Step three: Spinning solution preparation, mixing the mineralized spider silk protein and cellulose in a solvent system, the solvent composition is NMMO 76 parts, water 18 parts, EMIMAc 6 parts mass ratio, dissolution temperature 65 degrees Celsius, stirring at 300 revolutions per minute for 60 minutes, adding 0.3% mass ratio of polydopamine coating as a dispersing agent;

[0030] Step four: spinning treatment, using coaxial microfluidic spinning equipment, the core layer is 12% mass volume ratio cellulose solution flow rate 0.1 milliliter per minute, the skin layer is 6% mass volume ratio mineralized protein liquid flow rate 0.4 milliliter per minute, the spinning temperature is 65 degrees Celsius, the spinneret aperture is 80 microns;

[0031] Step five: coagulation treatment, placing the chopped bamboo fiber in a coagulation bath with a volume ratio of ethanol to water of 6 to 4, adding 0.1 moles of calcium chloride, and treating for 5 minutes to obtain a skin-core structure fiber.

[0032] Example 2: a method for preparing modified spider silk protein reinforced lyocell fiber, comprising the following steps:

[0033] Step one: spider silk protein expression, using Pichia pastoris strain GS115 / pPIC9K-spider silk gene for expression, controlling by DO-stat fed-batch fermentation, initially adding glycerol to a carbon source concentration of 5 grams per liter, switching to methanol feeding when the dissolved oxygen decreases to 20%, feeding rate 0.5 milliliter per minute per liter of culture solution, controlling the fermentation temperature at 28 degrees Celsius ± 1 degree Celsius, pH value 5.0 ± 0.2, fermentation time 48 hours; after fermentation is completed, purifying using AKTA pure 25 chromatography system, nickel column medium elution buffer is 20 millimoles of imidazole gradient solution, cold ethanol fractionation precipitation to obtain spider silk protein with a purity of 98%, molecular weight greater than 100 kilodaltons;

[0034] Step two: biomimetic mineralization, placing the purified spider silk protein in a pH 5.0 50 millimoles Tris-HCl buffer for pretreatment for 30 minutes, then adding a double silicon source system with a mole ratio of tetraethyl orthosilicate to methyltriethoxysilane of 7 to 3, total concentration of 10% mass volume ratio, magnetic stirring 200 revolutions per minute for 15 minutes, then ultrasonic assisted mineralization at 40 kilohertz, power density 0.5 watts per milliliter, 50 degrees Celsius reaction for 120 minutes, obtaining mineralized protein with a silica coating rate of 92%;

[0035] Step three: spinning liquid preparation, mixing mineralized spider silk protein and cellulose in a solvent system, the solvent composition is NMMO 76 parts, water 18 parts, EMIMAc 6 parts mass ratio, dissolving temperature 65 degrees Celsius, stirring at 300 revolutions per minute for 60 minutes, adding 0.3% mass ratio of polydopamine coating as a dispersing agent;

[0036] Step four: spinning treatment, using coaxial microfluidic spinning equipment, the core layer is 12% mass volume ratio cellulose solution flow rate 0.1 milliliter per minute, the skin layer is 6% mass volume ratio mineralized protein liquid flow rate 0.4 milliliter per minute, the spinning temperature is 65 degrees Celsius, the spinneret aperture is 80 microns;

[0037] Step five: coagulation treatment, the cut bamboo fibers are placed in a coagulation bath with a volume ratio of ethanol to water of 6 to 4, 0.1 moles of calcium chloride are added, and the treatment time is 5 minutes, obtaining a skin-core structure fiber.

[0038] In step one, the Pichia pastoris strain is expressed by DO-stat fed-batch fermentation control, the feed includes gradient addition of glycerol and methanol, the fermenter is Sartorius Biostat® CSTR type, the volume is 50L, and it is equipped with a dissolved oxygen and pH linkage control system, and the fermentation expression amount is 8g / L.

[0039] In the DO-stat fed-batch fermentation control, the addition gradient of glycerol and methanol is: glycerol is added to a carbon source concentration of 5g / L in the initial stage, and when the DO value decreases to 20%, methanol feeding is switched, the methanol feeding rate is controlled at 0.5mL / min / L of culture solution, the fermentation temperature is 28℃±1℃, the pH is controlled at 5.0±0.2, and the fermentation time is 48 hours.

[0040] In step one, it also includes a purification process, the expressed spider silk protein is purified by affinity chromatography and cold ethanol fractionation, the affinity chromatography uses AKTA pure 25 chromatography system, the chromatography medium is a nickel column, and the elution buffer is an imidazole gradient solution of 20mM, after purification, the purity of the spider silk protein is 98%±0.5%, and the molecular weight is greater than 100kDa.

[0041] In step two, the pretreatment includes placing the spider silk protein in a Tris-HCl buffer, the buffer pH is 5.0±0.05, the concentration is 50mM, the pretreatment time is 30 minutes, and the temperature is 25℃±1℃, to enhance the protein dispersion and mineralization coating uniformity.

[0042] In step two, the molar ratio of TEOS to MTES in the double silicon source compounding is 7:3±2%, of which TEOS accounts for 70%±2% and MTES accounts for 30%±2%, and the total concentration of the double silicon source is 10% w / v, the compounding is carried out under magnetic stirring, the stirring speed is 200r / min, and the time is 15 minutes, to improve the toughness of the mineralized shell layer and the interface bonding strength.

[0043] In step two, the mineralization reaction is carried out under ultrasonic assistance, the ultrasonic frequency is 40kHz±2kHz, the power density is 0.5W / mL, the reaction temperature is 50℃±0.5℃, the reaction time is 120 minutes±5 minutes, the mineralization coating rate reaches 92%±1.5%, and the thickness of the silicon dioxide coating layer is 50nm.

[0044] In step three, the solvent system is composed of NMMO, water and EMIMAc with a mass ratio of 76:18:6±1%, the dissolution temperature is 65℃±2℃, the stirring speed is 300r / min, and the stirring time is 60 minutes, so that the concentration of cellulose reaches 12% w / v and the concentration of mineralized spider silk protein reaches 6% w / v, and the protein activity is greater than 90%.

[0045] In step three, the dispersion aid is polydopamine-coated mineralized spider silk protein, and the addition amount is 0.3%±0.05% of the total mass of the spinning solution, and the coating thickness is 5nm, so as to enhance the interface bonding between the spider silk protein and the cellulose matrix, reduce phase separation, and increase the breaking work by 45%.

[0046] In step four, the microfluidic spinning adopts a coaxial spinneret, the core layer is a cellulose solution with a flow rate of 0.1mL / min, and the skin layer is a mineralized spider silk protein solution with a flow rate of 0.4mL / min, the flow rate ratio is 1:4±0.1, the spinning temperature is 65℃±2℃, and the spinneret hole diameter is 80μm, so as to form a continuous skin-core structure fiber, the core layer diameter accounts for 60%, the skin layer thickness accounts for 40%, the fiber elongation rate is greater than 22%, and the modulus reaches 16.8GPa.

[0047] Example 3: Preparation method of modified spider silk protein enhanced lyocell fiber, comprising the following steps:

[0048] Step one: spider silk protein expression, using Pichia pastoris strain GS115 / pPIC9K-spider silk gene for expression, through DO-stat feeding fermentation control, initially adding glycerol to a carbon source concentration of 5 grams per liter, when the dissolved oxygen decreases to 20%, switching to methanol feeding, feeding rate 0.5 milliliters per minute per liter of culture solution, controlling the fermentation temperature at 28 degrees Celsius±1 degree Celsius, pH value 5.0±0.2, and the fermentation time is 48 hours; after fermentation, purification is carried out using AKTA pure 25 chromatography system, the nickel column medium elution buffer is 250 millimoles of imidazole gradient solution, and after fractional precipitation with cold ethanol, the purity of the spider silk protein is 98%, and the molecular weight is greater than 100 kilodaltons;

[0049] Step two: biomimetic mineralization, the purified spider silk protein is pretreated in a pH 5.0 50 millimolar Tris-HCl buffer for 30 minutes, then a double silicon source system with a molar ratio of tetraethyl orthosilicate to methyltriethoxysilane of 7 to 3 is added, the total concentration is 10% mass by volume, magnetic stirring at 200 revolutions per minute for 15 minutes, and then ultrasonic assisted mineralization at 40 kilohertz, power density 0.5 watts per milliliter, 50 degrees Celsius reaction for 120 minutes, to obtain mineralized protein with a silicon dioxide coating rate of 92%;

[0050] Step three: Spinning solution preparation, mixing mineralized spider silk protein and cellulose in solvent system, solvent composition is NMMO 76 parts, water 18 parts, EMIMAc 6 parts mass ratio, dissolution temperature 65 degrees Celsius, 300 revolutions per minute stirring for 60 minutes, adding 0.3% mass ratio of polydopamine coating as dispersing agent;

[0051] Step four: Spinning treatment, using coaxial microfluidic spinning equipment, core layer is 12% mass volume ratio cellulose solution flow rate 0.1 milliliter per minute, skin layer is 6% mass volume ratio mineralized protein solution flow rate 0.4 milliliter per minute, spinning temperature 65 degrees Celsius, spinneret aperture 80 microns;

[0052] Step five: coagulation treatment, placing the chopped bamboo fiber in a coagulation bath with a volume ratio of ethanol to water of 6 to 4, adding 0.1 mole of calcium chloride, and treating for 5 minutes to obtain a skin-core structure fiber.

[0053] In step one, the Pichia pastoris strain is expressed by DO-stat fed-batch fermentation control, which includes gradient addition of glycerol and methanol. The fermentation tank is a Sartorius Biostat® CSTR type with a volume of 50L and is equipped with a dissolved oxygen and pH linkage control system. The fermentation expression amount is 8g / L.

[0054] In the DO-stat fed-batch fermentation control, the addition gradient of glycerol and methanol is: glycerol is added to a carbon source concentration of 5g / L in the initial stage, and when the DO value decreases to 20%, methanol feeding is switched, the methanol feeding rate is controlled at 1.0mL / min / L culture solution, the fermentation temperature is 28℃±1℃, the pH is controlled at 5.0±0.2, and the fermentation time is 72 hours.

[0055] In step one, it also includes a purification process, which uses affinity chromatography and cold ethanol fractionation precipitation to purify the expressed spider silk protein. The affinity chromatography uses an AKTA pure 25 chromatography system, the chromatography medium is a nickel column, and the elution buffer is an imidazole gradient solution of 20-250mM. The purity of the spider silk protein after purification is 98%±0.5%, and the molecular weight is greater than 100kDa.

[0056] In step two, the pretreatment includes placing the spider silk protein in a Tris-HCl buffer, the buffer pH is 5.0±0.05, the concentration is 50mM, the pretreatment time is 30 minutes, and the temperature is 25℃±1℃, to enhance the dispersion of the protein and the uniformity of the mineralization coating.

[0057] In step two, the molar ratio of TEOS to MTES in the double silicon source complex is 7:3±2%, wherein the proportion of TEOS is 70%±2%, and the proportion of MTES is 30%±2%, the total concentration of the double silicon source is 10% w / v, the complex is prepared under magnetic stirring, the stirring speed is 200r / min, and the stirring time is 15 minutes, so as to improve the toughness and interface bonding strength of the mineralized shell layer.

[0058] In step two, the mineralization reaction is carried out under ultrasonic assistance, the ultrasonic frequency is 40kHz±2kHz, the power density is 0.5W / mL, the reaction temperature is 50℃±0.5℃, and the reaction time is 120 minutes±5 minutes, so that the mineralization coating rate reaches 92%±1.5%, and the thickness of the silicon dioxide coating layer is 100nm.

[0059] In step three, the solvent system is composed of NMMO, water and EMIMAc, and the mass ratio is 76:18:6±1%, the dissolution temperature is 65℃±2℃, the stirring speed is 300r / min, and the stirring time is 60 minutes, so that the concentration of cellulose reaches 12% w / v, the concentration of mineralized spider silk protein reaches 6% w / v, and the protein activity is greater than 90%.

[0060] In step three, the dispersion aid is mineralized spider silk protein coated with polydopamine, and the addition amount is 0.3%±0.05% of the total mass of the spinning solution, and the coating thickness is 10nm, so as to enhance the interface bonding between the spider silk protein and the cellulose matrix, reduce the phase separation, and improve the breaking work by 45%.

[0061] In step four, the microfluidic spinning adopts a coaxial spinneret, the core layer is a cellulose solution, the flow rate is 0.1mL / min, the skin layer is a mineralized spider silk protein solution, the flow rate is 0.4mL / min, the flow rate ratio is 1:4±0.1, the spinning temperature is 65℃±2℃, and the spinneret hole diameter is 100μm, so as to form a continuous skin-core structure fiber, the core layer accounts for 60%, the skin layer accounts for 40%, the fiber elongation rate is greater than 22%, and the modulus reaches 16.8GPa.

[0062] The application provides a technical scheme: a preparation method of modified spider silk protein reinforced lyocell fiber.

[0063] Comparative example 1

[0064] Without carrying out the biomimetic mineralization treatment, the recombinant spider silk protein is directly blended and spun with cellulose, and the other conditions are the same as those in example 1.

[0065] Comparative example 2

[0066] The conventional uniaxial spinning is adopted instead of the microfluidic spinning, the core layer and the skin layer solution are premixed before spinning, and the other conditions are the same as those in example 1.

[0067] Comparative example 3

[0068] The polydopamine coating dispersion aid was not added in the spinning solution, and the rest was the same as in Example 1.

[0069] Comparative Example 4

[0070] The double silicon source was compounded only with tetraethyl orthosilicate, and methyltriethoxysilane was not added, and the rest was the same as in Example 1.

[0071] The performance of the modified spider silk protein in Examples 1-3 and Comparative Examples 1-4 was tested, and the test items and test methods were as follows:

[0072] Test item Breaking strength (GPa) Breaking elongation (%) Modulus (GPa) Protein activity retention rate (%) Production cost (yuan / g) Example 1 1.3 22.5 16.0 92 300 Example 2 1.4 23.1 16.5 93 310 Example 3 1.5 24.0 16.8 94 320 Comparative Example 1 0.8 15.2 10.5 88 280 Comparative Example 2 0.9 16.8 11.2 85 290 Comparative Example 3 1.0 18.3 13.6 79 300 Comparative Example 4 1.1 19.7 14.3 83 305

[0073] Test method explanation:

[0074] Breaking strength and elongation: according to GB / T 14344-2008 "Chemical fiber filament tensile property test method";

[0075] Modulus: the initial modulus was measured by dynamic mechanical analyzer DMA;

[0076] Protein activity: the retention rate of beta-sheet structure was detected by circular dichroism spectrum;

[0077] Cost accounting: including strain culture, purification and spinning energy consumption.

[0078] By comparing the data of Examples 1-3 and Comparative Examples 1-4, it can be seen that:

[0079] Biomimetic mineralization treatment compared with direct blending improves breaking strength and modulus. The mineralized layer effectively inhibits the phase separation of the spinning solution, and the silica coating layer enhances the interfacial bonding force between protein and cellulose, avoiding the mechanical fault of the fiber.

[0080] Microfluidic core-sheath spinning improves elongation compared with conventional spinning. The core-sheath structure maintains the beta-sheet conformation activity through core support and sheath protection, and the calcium chloride in the coagulation bath directionally induces molecular chain rearrangement, realizing structure self-repairing.

[0081] The absence of polydopamine coating leads to a decrease in protein activity. The ortho-phenol group of polydopamine forms a hydrogen bond network with the silicon hydroxyl group on the surface of silica, realizing the nano-level positioning distribution of mineralized protein in the cellulose matrix and eliminating island defects.

[0082] The absence of methyltriethoxysilane in the double silicon source compound reduces the modulus. The hydrophobic group of MTES enhances the toughness of the mineralized layer and cooperates with TEOS to build a dense crosslinking network, improving the interface stress transfer efficiency.

[0083] The process breaks through the technical bottlenecks of low molecular weight, thermal inactivation and uneven dispersion of recombinant spider silk proteins, and provides new high-strength and high-functionality fiber materials for the field of biomedical textiles.

[0084] As a green and environmentally friendly fiber in the 21st century, lyocell is made of renewable pulp from plant sources, and its processing technology is environmentally friendly and its waste is biodegradable, so it is considered a sustainable fiber in the whole life cycle.

[0085] The production process of bamboo pulp lyocell fiber follows the process route of dry crushing, direct dissolution and dry spraying wet spinning. The production process takes bamboo pulp as the starting material, goes through bamboo pulp, crushed pulp, solvent and other links, and a total of 18-20 rigorous procedures. It is worth mentioning that almost no chemical reaction is involved in the entire production process, mainly physical reaction, thus ensuring the environmental performance of the product. The bamboo pulp used in the production process is a fast-growing plant and a renewable and recyclable material, and the solvent N-methyl morpholine-N-oxide used is non-toxic and non-polluting. After special process and equipment treatment, 99.7% of the solvent can be recycled and reused. Such a production process truly realizes zero emission, and the product is biodegradable and environmentally friendly.

[0086] Although embodiments of the present application have been shown and described, it is to be understood that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing modified spider silk protein-reinforced lyocell fibers, characterized in that, Includes the following steps: Step 1: Spider silk protein expression. Spider silk protein was expressed using a Pichia pastoris strain, GS115 / pPIC9K-spider silk gene. High-purity spider silk protein was obtained through fermentation control. Step 2: Biomimetic mineralization, which involves mineralizing the spider silk protein expressed in Step 1, including pretreatment, dual silicon source compounding and mineralization reaction. The dual silicon source compounding adopts a compounding system of tetraethyl orthosilicate and methyltriethoxysilane. The mineralization reaction is carried out under ultrasonic assistance to form a silica coating layer. Step 3: Preparation of spinning solution. The mineralized spider silk protein from Step 2 is mixed with cellulose, and a solvent system and dispersant are added to prepare the spinning solution. The solvent system includes N-methylmorpholine-N-oxide, water and 1-ethyl-3-methylimidazolium acetate, and the dispersant is a polydopamine coating. Step 4: Spinning treatment. The spinning solution prepared in step 3 is spun using a microfluidic spinning device to form a core-sheath structure fiber, wherein the core layer is a cellulose solution and the sheath layer is a mineralized spider silk protein solution. Step 5: Coagulation treatment. The fibers spun in Step 4 are placed in a coagulation bath to induce the formation of a β-sheet structure. The coagulation bath contains ethanol, water and calcium chloride. In step one, the Pichia pastoris strain is expressed using DO-stat fed-batch fermentation control. The feed consists of a gradient addition of glycerol and methanol. The fermenter is a Sartorius Biostat® CSTR type with a volume of 50L and is equipped with a dissolved oxygen and pH linkage control system. The fermentation expression level is 8g / L. In the DO-stat fed-batch fermentation control, the addition gradient of glycerol and methanol is as follows: in the initial stage, glycerol is added until the carbon source concentration is 5 g / L. When the DO value drops to 20%, methanol feeding is switched to methanol feeding. The methanol feeding rate is controlled at 0.5-1.0 mL / min / L of culture medium, the fermentation temperature is 28℃±1℃, the pH is controlled at 5.0±0.2, and the fermentation time is 48-72 hours. Step one also includes a purification process, in which the expressed spider silk protein is purified by affinity chromatography and cold ethanol fractionation. The affinity chromatography uses an AKTA pure 25 chromatography system with a nickel column as the chromatography medium and an imidazole gradient solution of 20-250 mM as the elution buffer. After purification, the spider silk protein has a purity of 98% ± 0.5% and a molecular weight greater than 100 kDa.

2. The method for preparing modified spider silk protein-reinforced lyocell fibers according to claim 1, characterized in that, In step two, the pretreatment includes placing spider silk protein in Tris-HCl buffer with a pH of 5.0 ± 0.05 and a concentration of 50 mM, for a pretreatment time of 30 minutes and a temperature of 25℃ ± 1℃.

3. The method for preparing modified spider silk protein-reinforced lyocell fibers according to claim 1, characterized in that, In step two, the dual silicon source system of tetraethyl orthosilicate and methyltriethoxysilane in a molar ratio of 7:3, with a total concentration of 10% w / v, is mixed under magnetic stirring at a speed of 200 r / min for 15 minutes.

4. The method for preparing modified spider silk protein-reinforced lyocell fibers according to claim 1, characterized in that, In step two, the mineralization reaction is carried out under ultrasonic assistance, with an ultrasonic frequency of 40kHz±2kHz, a power density of 0.5W / mL, a reaction temperature of 50℃±0.5℃, a reaction time of 120 minutes±5 minutes, a mineralization coating rate of 92%±1.5%, and a silica coating layer thickness of 50-100nm.

5. The method for preparing modified spider silk protein-reinforced lyocell fibers according to claim 1, characterized in that, In step three, the solvent system consists of N-methylmorpholine-N-oxide, water, and 1-ethyl-3-methylimidazolium acetate. The solvent composition is 76 parts N-methylmorpholine-N-oxide, 18 parts water, and 6 parts 1-ethyl-3-methylimidazolium acetate by mass ratio. The dissolution temperature is 65℃±2℃, the stirring speed is 300r / min, and the stirring time is 60 minutes, so that the cellulose concentration reaches 12% w / v, the mineralized spider silk protein concentration reaches 6% w / v, and the protein activity is retained to be greater than 90%.

6. The method for preparing modified spider silk protein-reinforced lyocell fibers according to claim 1, characterized in that, In step three, the dispersing agent is polydopamine-coated mineralized spider silk protein, and the amount added is 0.3% ± 0.05% of the total mass of the spinning solution, with a coating thickness of 5-10 nm.

7. The method for preparing modified spider silk protein-reinforced lyocell fibers according to claim 1, characterized in that, In step four, the microfluidic spinning uses a coaxial spinning head, with a core layer of cellulose solution at a flow rate of 0.1 mL / min and a sheath layer of mineralized spider silk protein solution at a flow rate of 0.4 mL / min. The flow rate ratio is 1:4±0.1, the spinning temperature is 65℃±2℃, and the spinneret orifice diameter is 80-100 μm to form a continuous core-sheath structure fiber. The core layer diameter accounts for 60%, the sheath thickness accounts for 40%, the fiber elongation is greater than 22%, and the modulus reaches 16.8 GPa.