A fusion protein, its modified cellulose and preparation method

Abstract

A fusion protein and modified cellulose, as well as a preparation method, contain a protein that can bind to cellulose. This fusion protein can significantly improve the properties of cellulose. Compared to the original cellulose membrane, the fused spider silk membrane has significantly improved softness and maximum tensile strength, and its tear resistance is greatly enhanced.

Description

[0001] Priority information

[0002] This application claims priority and benefits from patent application number CN20211122 3656.6 filed with the State Intellectual Property Office of China on October 20, 2021. Technical Field

[0003] The present invention relates to the technical field of genetic engineering, and in particular to a fusion protein, modified cellulose and a preparation method thereof. Background Art

[0004] In recent years, young consumers have shown increasing concern for environmental issues. The traditional leather industry, with its high energy consumption and high pollution levels, has been severely tested, leading to the rise of the concept of vegan leather. Vegan leather refers to leather that does not contain any animal materials, including various polymer artificial leathers and natural leather materials made from natural plant materials. However, most polymer artificial leathers are made from polyethylene or other plastic materials. Their production and processing still contains heavy metals and toxic organic residues, and they easily generate large amounts of microplastic waste. Plastic leather materials are also criticized for their inability to biodegrade naturally. Natural plant materials are made from fiber materials such as palm fiber and pineapple fiber, and their production process is more complex. However, plants grow more slowly, and their toughness and softness do not yet meet the requirements of natural leather. Summary of the Invention

[0005] According to the first aspect, in one embodiment, a fusion protein is provided, wherein the fusion protein comprises a protein that can bind to cellulose.

[0006] According to the second aspect, in one embodiment, a modified cellulose is provided, wherein the modified cellulose comprises cohesin and cellulose.

[0007] According to the third aspect, in one embodiment, an isolated polynucleotide is provided, wherein the polynucleotide encodes the fusion protein of the first aspect.

[0008] According to the fourth aspect, in one embodiment, a construct is provided, wherein the construct contains the polynucleotide according to the third aspect.

[0009] According to the fifth aspect, in one embodiment, an expression system is provided, wherein the expression system comprises the construct as described in the fourth aspect or the exogenous polynucleotide as described in the third aspect integrated into the genome.

[0010] According to the sixth aspect, in one embodiment, a method for preparing the modified cellulose according to the second aspect is provided, comprising: mixing coagulant protein with cellulose to obtain the modified cellulose.

[0011] According to the seventh aspect, in one embodiment, a microorganism is provided, wherein the microorganism can express cellulose.

[0012] According to the fusion protein and modified cellulose and preparation method thereof of the above embodiment, the fusion protein can significantly improve the performance of cellulose.

[0013] In one embodiment, the fused spider silk membrane or honeycomb silk membrane has significantly improved softness and maximum tensile strength compared to the original cellulose membrane, and its tear resistance is greatly enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a screening diagram for BCM-producing strains in one embodiment.

[0015] Figure 2 1 is a diagram of the production process and output results of BCM in one embodiment.

[0016] Figure 3 A diagram of the scaled-up production and preparation process of BCM according to an embodiment.

[0017] Figure 4 This is a schematic diagram of the structural background and design of a recombinant spider silk protein according to an embodiment.

[0018] Figure 5 This is a diagram showing the expression results of the recombinant spider silk protein according to an embodiment.

[0019] Figure 6 This is a graph showing the expression level of recombinant spider silk protein in an embodiment.

[0020] Figure 7 This is a diagram showing the maximum tensile force test results of an embodiment.

[0021] Figure 8 This is a diagram showing the softness test results of an embodiment.

[0022] Figure 9 A schematic diagram of leather production according to an embodiment.

[0023] Figure 10 The graph shows the tensile strength results of different fusion protein treatment groups and the control group (protein-free BCM).

[0024] Figure 11 The graph shows the elongation at break of the different fusion protein treatment groups and the control group (protein-free BCM). DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below by means of specific embodiments in conjunction with the accompanying drawings. Similar elements in different embodiments are numbered with associated similar elements. In the following embodiments, many detailed descriptions are provided to enable the present application to be better understood. However, those skilled in the art will readily appreciate that some of the features may be omitted in different circumstances, or may be replaced by other elements, materials, or methods. In some cases, some operations related to the present application are not shown or described in the specification. This is to avoid the core portion of the present application being overwhelmed by excessive descriptions, and for those skilled in the art, it is not necessary to describe these related operations in detail. They will fully understand the related operations based on the description in the specification and the general technical knowledge in the art.

[0026] In addition, the features, operations, or characteristics described in the specification may be combined in any appropriate manner to form various embodiments. Furthermore, the steps or actions in the method description may be reordered or adjusted in a manner readily apparent to those skilled in the art. Therefore, the various sequences in the specification and drawings are provided solely for the purpose of clearly describing a particular embodiment and are not intended to be mandatory, unless otherwise specified.

[0027] The serial numbers assigned to components herein, such as "first," "second," etc., are used solely to distinguish the objects being described and do not convey any sequential or technical meaning. References to "connection" and "coupling" herein, unless otherwise specified, include both direct and indirect connections (couplings).

[0028] According to the first aspect, in one embodiment, a fusion protein is provided, wherein the fusion protein comprises a protein that can bind to cellulose.

[0029] In one embodiment, the protein capable of binding to cellulose comprises a CBM family protein.

[0030] In one embodiment, the CBM family protein includes but is not limited to at least one of CBM2 and CBM3 proteins. CBM2 and CBM3 are cellulose binding proteins, and their natural function is to be the domain of microbial cellulase for binding to cellulose.

[0031] In one embodiment, the protein capable of binding to cellulose is selected from CBM3 protein.

[0032] In one embodiment, the fusion protein further comprises at least one of a NT (N-terminal: hydrophilic N-terminal) region, a CT (C-terminal: hydrophilic C-terminal) region, and a repeat region.

[0033] In one embodiment, the fusion protein comprises a first CBM family protein, an NT region, a repeat region, a CT region, and a second CBM family protein.

[0034] In one embodiment, from N-terminus to C-terminus, the fusion protein comprises, in sequence, a first CBM family protein, an NT region, a repeat region, a CT region, and a second CBM family protein.

[0035] In one embodiment, the repeat region includes, but is not limited to, at least one of the spider silk MaSp1 repeat region (2Rep), the spider silk protein MaSp4 repeat region (3DB), the spider silk protein AcSp repeat region (Ac), the spider silk protein PySp repeat region (Py), and the honey bee silk protein structural region (AF). The repeat regions listed here are merely exemplary, and other repeat regions that can achieve similar functions are also within the scope of protection of the present invention.

[0036] In one embodiment, the first CBM family protein and the second CBM family protein are the same.

[0037] In one embodiment, the CBM2 has the amino acid sequence shown in SEQ ID No. 17 or a sequence having at least 90% similarity thereto.

[0038] In one embodiment, CBM3 comprises the amino acid sequence shown in SEQ ID No. 3 or a sequence having at least 90% similarity thereto.

[0039] In one embodiment, the spider silk MaSp1 repeat region comprises the amino acid sequence shown in SEQ ID No. 6 or a sequence having at least 90% similarity thereto.

[0040] In one embodiment, the spider silk protein MaSp4 repeat region comprises the amino acid sequence shown in SEQ ID No. 8 or a sequence having at least 90% similarity thereto.

[0041] In one embodiment, the spider silk protein AcSp repeat region comprises the amino acid sequence shown in SEQ ID No. 9 or a sequence having at least 90% similarity thereto.

[0042] In one embodiment, the spider silk protein PySp repeat region comprises the amino acid sequence shown in SEQ ID No. 10 or a sequence having at least 90% similarity thereto.

[0043] In one embodiment, the honeybee silk protein structural region comprises the amino acid sequence shown in SEQ ID No. 11 or a sequence having at least 90% similarity thereto.

[0044] In one embodiment, NT comprises the amino acid sequence shown in SEQ ID No. 4 or a sequence having at least 90% similarity thereto.

[0045] In one embodiment, CT comprises the amino acid sequence shown in SEQ ID No. 7 or a sequence having at least 90% similarity thereto.

[0046] In one embodiment, the method further comprises a connector;

[0047] In one embodiment, the linker comprises at least one of the following amino acid sequences:

[0048] GSGGGS;

[0049] GNS;

[0050] GGGGS;

[0051] GKL.

[0052] In one embodiment, there are linkers between the first CBM family protein and the NT region, between the NT region and the repeat region, between the repeat region and the CT region, and between the CT region and the second CBM family protein.

[0053] In one embodiment, the cellulose comprises at least one of cellulose produced by microorganisms and cellulose derived from plants. Cellulose produced by microorganisms and cellulose derived from various plants are all suitable for the present invention.

[0054] In one embodiment, the cellulose produced by the microorganism includes but is not limited to bacterial cellulose, and specifically can be bacterial cellulose membrane (BCM).

[0055] According to the second aspect, in one embodiment, a modified cellulose is provided, wherein the modified cellulose comprises cohesin and cellulose.

[0056] In one embodiment, the cohesive protein comprises at least one of spider silk protein, bee silk protein, collagen, muscle fiber protein, mussel mucin, anther mucin, snail mucin, and octopus mucin.

[0057] In one embodiment, the cohesive protein comprises the fusion protein of any one of the first aspects.

[0058] In one embodiment, the cellulose comprises bacterial cellulose, specifically a bacterial cellulose film.

[0059] In one embodiment, the cellulose can be produced using microbial strains, and the specific strains are not limited, including but not limited to Kombuchaea, Cellulobacter, Gluconobacter, etc. All microbial species that produce cellulose fall within the scope of protection of the present invention.

[0060] In one embodiment, the nucleotide sequence encoding the CBM3 protein comprises the following nucleotide sequence:

[0061] ATGGTTAGCGGCAACCTGAAAGTTGAATTTTATAGCAACCCGAGCGACACCAC CAATAGCATTAATCCGCAGTTTAAAGTGACCAACACCGGTAGCAGCGCAATTGATCTGAGCAAACTGACCCTGCGTTATTATTATACCGTTGACGGTCAGAAAGACCAGACCTTTTGGT GTGATCATGCAGCAATTATTGGCAGCAATGGCAGCTATAATGGCGTGACCAGCAATGTGA AAGGTACCTTTGTTAAAATGAGCAGCAGCACCAATAATGCCGATACCTATCTGGAAATTAGCTTCACCGGTGGTACCCTGGAACCGGGTGCACATGTTCAGATTCAGGGTCGTTTTGCA AAAAATGACTGGAGCAATTATACCCAGAGCAACGATTATAGCTTTAAGAGCGCAAGCCAGTTTGTGGAGTGGGATCAGGTTACCGCATATCTGAATGGTGTTCTGGTTTGGGGTAAAGA ACCG.

[0062] According to the third aspect, in one embodiment, an isolated polynucleotide is provided, wherein the polynucleotide encodes the fusion protein of the first aspect.

[0063] According to the fourth aspect, in one embodiment, a construct is provided, the construct containing the polynucleotide described in the third aspect. The construct can usually be obtained by inserting the isolated polynucleotide into a suitable vector. Those skilled in the art can select a suitable vector, and the vector can be a phage, a plasmid, a viral vector, such as a bacterium. In other words, the vector of the embodiment of the present invention comprises a polynucleotide of interest that can be expressed in a host cell or its separated fraction. The vector is also generally suitable as a cloning vector, i.e., replicable in a microbial system; the cloning vector can be designed for replication in a host, and the construct is designed for expression in different hosts. The vector comprising the polypeptide and protein of the embodiment of the present invention can also include a selection marker for propagation or selection in a host cell. The vector can be introduced into a prokaryotic or eukaryotic cell by conventional transformation or transfection technology.

[0064] According to the fifth aspect, in one embodiment, an expression system is provided, comprising the construct described in the fourth aspect or the exogenous polynucleotide described in the third aspect integrated into the genome. The expression system can be a host cell, and the host cell can express the fusion protein described in the first aspect. In another specific embodiment of the present invention, the host cell can be a eukaryotic cell and / or a prokaryotic cell.

[0065] In one embodiment, fusion proteins such as spider silk proteins or beeswax proteins can be produced using other strains of E. coli, such as the C41(DE3) strain, which is suitable for the pT7 expression system. Alternatively, genetic engineering vectors can be modified to express spider silk proteins using the pTac or pAra promoters, and proteins can be expressed in strains independent of the T7 system, such as DH5a. Spider silk proteins expressed in various strains fall within the scope of this invention.

[0066] According to the sixth aspect, in one embodiment, a method for preparing the modified cellulose according to the second aspect is provided, comprising: mixing coagulant protein with cellulose to obtain the modified cellulose.

[0067] In one embodiment, the mass ratio of the coagulant protein to the cellulose is (0.05-0.06) g: (0.5-10 mg).

[0068] In one embodiment, the mass ratio of the coagulant protein to the cellulose is (0.05-0.06) g: (0.5-5 mg).

[0069] In one embodiment, after the coacervate protein and cellulose are mixed, the standing time is ≥ 20 min, preferably 30 min.

[0070] In one embodiment, after the coacervate protein and cellulose are mixed and allowed to stand, a solvent is added and the mixture is soaked at least once.

[0071] In one embodiment, the method further includes a soaking step, comprising mixing coagulant protein and cellulose to obtain a mixture, and soaking the obtained mixture in a first soaking liquid to obtain a first soaked product.

[0072] In one embodiment, the first soaking liquid includes but is not limited to at least one of isopropyl alcohol, ethanol, methanol, n-butanol, formic acid, acetic acid, etc. The first soaking liquid is a coagulant.

[0073] In one embodiment, the soaking time is ≥20 min, preferably 20 min.

[0074] In one embodiment, a secondary soaking step is further included, comprising soaking the primary soaked object in a second soaking liquid to obtain a second soaked object.

[0075] In one embodiment, the second soaking liquid includes but is not limited to a glycerol (glycerol, CAS registration number: 56-81-5) aqueous solution.

[0076] In one embodiment, the mass percentage of glycerol in the glycerol aqueous solution is 1-10%.

[0077] In one embodiment, the secondary soaking time is ≥12 hours, preferably 20 to 24 hours.

[0078] In one embodiment, the method further includes three soaking steps, including soaking the second soaked material in a third soaking liquid to obtain a third soaked material, namely modified cellulose, which is then washed and dried to obtain the final product.

[0079] In one embodiment, the third soaking liquid includes but is not limited to a detergent containing a surfactant or an aqueous solution thereof.

[0080] In one embodiment, the surfactant-containing cleaning agent includes but is not limited to at least one of soap, hand soap, dishwashing liquid, and the like.

[0081] In one embodiment, the time for the three soakings is ≥30 min, preferably 30 min.

[0082] According to the seventh aspect, in one embodiment, a microorganism is provided, wherein the microorganism can express cellulose.

[0083] In one embodiment, the microorganism comprises at least one of Komagataeibacter intermedius and Komagataeibacter rhaeticus.

[0084] In one embodiment, the microorganism comprises at least one of strains B2, 12, 25, and 40.

[0085] In one embodiment, the present invention provides a new type of "pure vegan" leather that combines the advantages of animal leather while meeting the requirements of carbon reduction and environmental protection.

[0086] In one embodiment, the present invention proposes a new vegan leather production method, which uses microbial cellulose and genetically engineered microbial spider silk or beeswax to prepare a dual network system, which is mixed through a special process to form a new leather material.

[0087] In one embodiment, the present invention screened several high-cellulose-producing strains from commercial kombucha beverages, verified that these strains can be cultured with brewer's yeast, and used sucrose and tea as raw materials to produce a thick layer of cellulose hydrogel material.

[0088] Cellulose materials alone are brittle and thin, but their performance can be greatly improved if they are combined with other cross-linkable molecules to form a stable dual-network system. In one embodiment, the spider silk protein of the present invention is a recombinant spider silk protein produced by genetic engineering. Spider silk protein or bee silk protein has high water solubility during storage. When its concentration reaches a certain level and is subjected to a specific coagulant and tensile force, it can cross-link into a network through hydrogen bonds. The present invention designs a recombinant spider silk or recombinant bee silk that has high water solubility at high expression levels, and fused cellulose binding domains at both ends of the spider silk protein. These cellulose binding domains can further connect the spider silk network and the microbial cellulose network, forming a more compact dual-network structure. We have developed a production process that firmly fixes spider silk protein or bee silk protein in microbial cellulose and greatly improves the properties of the cellulose membrane. Through testing, we found that the fused spider silk membrane or bee silk membrane has significantly improved softness and maximum tensile strength compared to the original cellulose membrane, and its tear resistance is greatly enhanced.

[0089] In one embodiment, the present invention successfully combines plant-based cellulose and animal-based spider silk through microbial genetic engineering, creating a vegan leather that stands out from other materials. We believe this approach could create a convenient, rapid, and cost-effective alternative to traditional leather, with comparable performance and the ability to be 100% bioproduced and 100% biodegradable.

[0090] In one embodiment, the present invention verifies the production and function of four different types of fusion spider silk proteins and one bee silk protein, which show high water solubility and all have the activity of binding to cellulose. In the future, more types of artificial spider silk proteins may be fused, and different spider silk proteins all fall within the scope of protection of the present invention. Instead of using spider silk protein, other cohesive proteins may be used, such as bee silk protein, silk protein, collagen, muscle fiber protein, mussel mucin, anther mucin, snail mucin, octopus mucin and other proteins to replace spider silk protein. Since these proteins all have the function of physical structural support and adhesion in natural biological materials, or serve as natural leather filling materials, they can be applied to fusion proteins and provide physical properties of materials. Different adhesive proteins are still protected by the present invention when used as fusion protein domains.

[0091] In one embodiment, regarding the process of mixing pigmented proteins and other proteins into BCM, spider silk proteins form hydrogen-bonded networks through their repeating domains. These repeating domains allow different types of spider silk proteins to bind to other proteins carrying these domains and cross-link to the BCM structure. This technology could potentially be used to add pigmented proteins to BCM, thereby creating colored vegan leather. Other functional proteins, such as antimicrobial peptides and drug molecules, could also be added and bound to the BCM through the spider silk network, achieving antibacterial, anti-inflammatory, and drug delivery properties. Enhancing the efficacy of spider silk BCM by doping with other proteins also falls within the scope of this invention.

[0092] In one embodiment, the fusion protein of the present invention can also increase the shear force of cellulose.

[0093] Example 1

[0094] This embodiment includes the following steps:

[0095] 1. Screening of Kombucha microorganisms:

[0096] In this example, four strains of cellulose membrane (BCM) were screened from commercially available Kombucha beverages: B2, 12, 25, and 40. 16s RNA sequencing and phylogenetic tree analysis revealed that the strains were BCM-producing. Figure 1 ), strains B2, 12, and 25 belong to the genus Komagataeibacter intermedius, and strain 40 belongs to Komagataeibacter rhaeticus. All kombucha strains (red tea fungi) can grow in YPD glucose medium and can grow in shake flasks in 100 mL of medium. They were adjusted to an OD600 of 2.5 and inoculated into the target medium at a ratio of 1:50. Saccharomyces cerevisiae was then adjusted to an OD600 of 0.05 and inoculated into the target medium at a ratio of 1:100 for co-cultivation with the red tea fungi.

[0097] After co-cultivation with Saccharomyces cerevisiae, the dry weight was measured after 10 days. Figure 2 Figures show the production process and yield of BCM. Figure A represents the pilot production process, Figure B represents the dry weight of the strain after 10 days, and Figure C represents the growth curve of the B2 strain biofilm after 10 days. Figure 3 The results show that the BCM produced by strain B2 has the largest dry weight (per 7cm 2The B2 strain produced approximately 0.06g of BCM. After 10 rounds of passage, the B2 strain maintained its stability in producing BCM. It can stably grow and produce BCM in YPD and YPS media, as well as in black tea broth supplemented with sucrose. We tested it in 5mL, 15mL, 500mL, and 5L media, consistently producing BCM membranes and reaching maximum yield in approximately 10 days.

[0098] 2. Design and purification of spider silk protein:

[0099] Figure 4 Figures 1 and 2 illustrate the structural background and design ideas for the recombinant spider silk protein used in this example. Figure A is a schematic diagram of the spider silk protein network. Figure B illustrates the design of the recombinant spider silk protein. Figures C and D are schematic diagrams of the protein structure of the cellulose-binding region (CBM).

[0100] In this example, a pair of cellulose binding proteins CBM2 and CBM3 were designed, and a flexible linker (also called connector) NT2RepCT was used to connect them to form a recombinant spider silk protein.

[0101] The nucleotide sequence of CBM2 is as follows:

[0102] GGTGGTCCGGCAGGTTGTCAGGTTCTGTGGGGTGTTAATCAGTGGAATACCGGTTTT TACCGCAAATGTTACCGTTAAAAATACCAGCAGCGCACCGGTTGATGGTTGGACCTTAA CATTTAGCTTTCCGAGCGGTCAGCAGGTTACCCAGGCATGGAGTAGCACCGTTACCCAGAGCGGTAGCGCAGTTACCGTTCGTAATGCACCGTGGAATGGTAGCATTCCGGCAGGTGG TACCGCACAGTTTGGTTTTTAATGGTAGCCATACCGGTACCAATGCAGCACCGACCGCATTTAGCCTGAATGGTACCCCGTGTACCGTTGGTGGTAGCACCGGT (SEQ ID No. 16).

[0103] The amino acid sequence of CBM2 is as follows:

[0104] GGPAGCQVLWGVNQWNTGFTANVTVKNTSSAPVDGWTLTFSFPSGQQVTQAWSSTV TQSGSAVTVRNAPWNNGSIPAGGTAQFGFNGSHTGTNAAPTAFSLNGTPCTVGGSTG (SEQ ID No. 17).

[0105] The amino acid sequence of 2rep is as follows:

[0106] The complete amino acid sequence of CBM2-NT2RepCT-CBM2 is as follows:

[0107] The amino acid marked with a double underlined line is the starting amino acid, the sequence marked with a double underlined line is CBM3, the sequence marked with a dotted underline is the NT region, the sequence marked with a dot-dashed underline is the 2Rep region, the sequence marked with a single underlined line is the CT region, the sequence marked with a single underlined wavy line is also CBM3, and the sequence marked with an underlined bold line is the linker.

[0108] The amino acid sequence of CBM3-NT2RepCT-CBM3 is shown in SEQ ID No. 1.

[0109] CBM3 binds to cellulose, tightly connecting the two networks (the silk network and the cellulose network) through intermolecular forces. The natural functions of NT and CT are to sense coagulants (such as alcohols and pH changes) and stretching forces, facilitating the solidification of spider silk proteins. When a coagulant is added, the rep sequences can form beta-sheets with each other, linking the spider silk network through hydrogen bonds.

[0110] Figure 5 Schematic diagram of the expression of recombinant spider silk protein. Figures B and D verify the high water solubility of CBM3-bound spider silk protein.

[0111] Figure 6 Figure 4 shows the expression results of recombinant spider silk protein. Figure D shows that the fusion protein yield is 133.08 mg / L.

[0112] In this example, recombinant spidroin proteins were expressed using fully gold-rich BL21(DE3) competent cells and the pT7 system. We found that CBM3-bound spidroin proteins exhibited high yields and high water solubility, while CBM2-bound spidroin proteins exhibited poor water solubility. We fused a histidine tag to the CBM3 C-terminus and purified the spidroin proteins using a native nickel column. The concentration of the spidroin proteins was measured using a BCA assay, demonstrating that the fermentation yield of the fused spidroin protein reached 133 mg / L and that it remained water-soluble even at a high concentration of 100 mg / mL.

[0113] The nucleotide sequence of the CBM3-BsaI-CBM3 expression vector used in this example is shown in SEQ ID No.5.

[0114] 3. Process flow of cellulose mixed with spider silk protein:

[0115] We selected 10-day-old BCM cultured in 50 mL centrifuge tubes, soaked them in 20 mL of water for 30 minutes, rinsed, dried, and measured their dry weight. Forty samples of biofilm-infused spider silk were collected, weighing 0.054–0.058 g. The concentrations of the biofilm-infused spider silk samples were adjusted to 0 mg / L, 2 mg / L, 6 mg / L, 13.6 mg / L, and 20 mg / mL (other suitable concentrations were possible) using 20 mM Tris-HCl (pH 8.0). 250 μL of the spider silk solution was then added to the BCM to swell them. The BCM were allowed to stand for 30 minutes to allow the spider silk proteins to fully swell the BCM. The BCM were then soaked in isopropanol for one hour to fully denature the spider silk. Alcohols such as ethanol and methanol can also be used as an alternative to isopropanol. Afterwards, the BCM were rinsed three times with water to remove the alcohol solution, and then soaked in a 10% (by weight) glycerol solution for at least 12 hours. Rinse the BCM three times with clean water to remove the glycerin, then soak it in a 2% by volume detergent solution for at least 30 minutes. Finally, rinse the BCM with clean water, dry it at 50°C for 1 hour, and air dry it overnight to produce the new vegan leather.

[0116] If the size and thickness of the cultured cellulose membrane change, the amount of each reagent can be increased proportionally.

[0117] 4. Performance test of new leather:

[0118] The properties of the new vegan leather were tested using a tensile testing machine (Dongri Instruments, model STC-50KG) and a leather softness tester (Hebei Tianjian Engineering Instrument Co., Ltd., model YK3002). BCM was cut into 1cm x 2cm strips and clamped at both ends of the sample with a tensile testing machine to measure the maximum tensile force. Figure 7Figures show tensile force experiments. Panel A is a schematic diagram of the tensile experiment. Panel B shows the tensile force effect of 5 mg of protein binding. Panels C and D show the maximum tensile force results at different concentrations for spider silk proteins with and without CBM3 fusion, respectively.

[0119] Figure 7 There are three sample groups (samples 1 to 9) in Figure B. Three samples were tested in each sample group and the average value was taken.

[0120] Figure 7 There are 5 sample groups (samples 10 to 25) in C. Three samples were tested in each sample group and the average value was taken.

[0121] Figure 7 D shows 5 sample groups (samples 26 to 40), 3 samples were tested in each sample group, and the average value was taken.

[0122] We found that 3.4 mg and 5 mg of spider silk protein (CBM3-NT2RepCT-CBM3, i.e. Figure 7 The presence of CBM-2re p-CBM in the BCM significantly impacted the maximum tensile force, with the addition of 5 mg of spidroin increasing the maximum tensile force by approximately 1-2 times. We also tested spidroin with and without CBM fusions and found that the CBM-fused group exhibited significantly greater maximum tensile force.

[0123] We used a leather softness tester to test the softness of spider silk BCM. The experimental results are as follows: Figure 8 As shown in the results, it was found that the softness of BCM increased by 35% after the incorporation of 3.4mg of spider silk protein. We can also qualitatively observe from the touch that the addition of spider silk protein can make BCM more leathery and soft, while the unincorporated BCM is harder and brittle.

[0124] We also tested the tear strength of spider silk BCM leather. When a 2mm-long cut was made in the BCM and a slight stress was applied, the BCM immediately broke. However, after incorporating the spider silk protein CBM3-NT2RepCT-CBM3, the BCM could withstand even stronger stress and tear very slowly under stress, significantly improving its tear resistance.

[0125] The amino acid sequence of CBM3-NT2RepCT-CBM3 is as follows:

[0126]

[0127] The amino acid marked with a double underlined line is the starting amino acid, the sequence marked with a double underlined line is CBM3, the sequence marked with a dotted underline is the NT region, the sequence marked with a dotted-dashed underline is the 2Rep region, the sequence marked with a single underlined line is the CT region, the sequence marked with a single underlined wavy line is also CBM3, the sequence marked with a bold underlined line is the linker, and "*" indicates a stop codon (since the codon corresponding to this position does not encode any amino acid, it is no longer shown in the subsequent amino acid sequence).

[0128] The amino acid sequence of CBM3 is as follows:

[0129]

[0130] The NT region sequence is as follows (the NT region sequence in other amino acid sequences below is the same as this):

[0131]

[0132] The sequence of the 2Rep region (belonging to the spider silk MaSp1 repeat region) is as follows:

[0133]

[0134] The specific sequence of the CT region is as follows (the CT region sequence in other amino acid sequences below is the same as this):

[0135] VTSGGYGYGTSAAAGAGVAAGSYAGAVNRLSSAEAASRVSSNIAAIASGGASALPSVISNIYSGVVAS GVSSNEALIQALLELLSALVHVLSSASIGNVSSVGVDSTLNVVQDSVGQYV (SEQ ID No. 7).

[0136] The nucleotide sequence of CBM3-NT2RepCT-CBM3 is as follows:

[0137] ATGGTTAGCGGCAACCTGAAAGTTGAATTTTATAATAGCAACCCGAGCGACACCAC CAATAGCATTAATCCGCAGTTTAAAGTGACCAACACCGGTAGCAGCGCAATTGATCTGAGCAAACTGACCCTGCGTTATTATTATACCGTTGACGGTCAGAAAGACCAGACCTTTTGGT GTGATCATGCAGCAATTATTGGCAGCAATGGCAGCTATAATGGCGTGACCAGCAATGTGAAAGGTACCTTTGTTAAAATGAGCAGCAGCACCAATAATGCCGATACCTATCTGGAAATTAGCTTCACCGGTGGTACCCTGGAACCGGGTGCACATGTTCAGATTCAGGGTCGTTTTGCAAAAAATGACTGGAGCAATTATACCCAGAGCAACGATTATAGCTTTAAGAGCGCAAGCCA GTTTGTGGAGTGGGATCAGGTTACCGCATATCTGAATGGTGTTCTGGTTTGGGGTAAAGA ACCGGGCTCCGGCGGTGGTAGTATGAGCCATACCACCCCGTGGACCAATCCTGGTCTGGCGGAAAACTTTATGAACAGCTTTATGCAGGGCCTGAGCAGCATGCCTGGTTTTACCGCG AGCCAGCTGGATGATATGAGCACCATTGCGCAGAGCATGGTGCAGAGCATTCAGAGCTTAGCGGCGCAAGGTCGCACCAGCCCGAATAAATTACAGGCGCTGAACATGGCATTTGCGA GCAGCATGGCGGAAATTGCGGCGTCAGAAGAAGGTGGTGGTAGCCTGAGCACCAAAAC CAGCAGCATTGCGAGCGCGATGAGCAATGCGTTTCTGCAAACCACCGGCGTTGTTAACCAGCCGTTCATCAACGAAATTACCCAGCTGGTGAGCATGTTTGCACAGGCGGGCATGAATGATGTGAGCGCGGGCAATAGCGGTCGTGGTCAAGGTGGTTATGGCCAAGGCAGCGGTGGTAATGCAGCAGCAGCAGCGGCAGCAGCGGCAGCAGCGGCGGCGGCAGCGGGTCAAG GTGGTCAAGGTGGCTATGGTCGTCAAAGCCAAGGTGCAGGTTCAGCGGCGGCAGCAGCAGCGGCGGCAGCGGCAGCGGCGGCAGCGGGCTCAGGCCAAGGTGGTTATGGCGGCCA AGGTCAAGGTGGTTATGGCCAGAGCGGTAACAGCGTTACCAGCGGCGGCTATGGTTATGGTACCAGCGCAGCAGCAGGTGCAGGCGTTGCAGCAGGTTCATATGCAGGTGCGGTTAA CCGTTTAAGCAGCGCGGAAGCAGCGTCACGTGTGAGCAGCAACATTGCGGCGATTGCA AGCGGTGGTGCGTCAGCATTACCGAGCGTGATTAGCAACATTTATAGCGGCGTGGTGGCGTCAGGTGTTAGCTCAAACGAAGCGCTGATTCAGGCGTTACTGGAATTACTGAGCGCGC TGGTGCATGTTTTAAGCAGCGCGAGCATTGGCAATGTGAGCAGCGTGGGTGTTGATAGC ACCCTGAACGTGGTGCAGGATAGCGTGGGTCAGTATGTGGGCGGAGGTGGATCCGTTAGCGGTAATCTGAAAGTTGAATTTTACAACAGCAACCCGAGCGACACCACCAATAGCATTA ATCCGCAGTTTAAAGTGACCAACACCGGCAGCAGCGCAATTGATCTGAGCAAACTGACCCTGCGTTATTATTATACCGTTGACGGTCAGAAAGACCAGACCTTTTGGTGTGATCATGC AGCAATTATTGGCAGCAATGGCAGCTATAATGGCGTTACCAGCAATGTTAAAGGCACCTTTGTTAAGATGAGCAGCAGCACCAATAATGCCGATACCTATCTGGAAATTAGCTTCACCGGTGGTACCCTGGAACCGGGTGCACATGTTCAGATTCAGGGTCGTTTTGCAAAAAATGACT GGAGCAATTATACCCAGAGCAACGATTATAGCTTCAAGAGCGCAAGCCAGTTTGTGGAATGGGATCAGGTTACCGCATATCTGAATGGTGTTCTGGTTTGGGGTAAAGAACCGGGTAAACTGCACCACCACCACCACCACTGA (SEQ ID No. 2).

[0138] The nucleotide sequence of the CBM3-BsaI-CBM3 expression vector used in this example is as follows:

[0139] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTA CGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTT TAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCA CGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGAT TTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGT ATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAA ACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGA AATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCG CAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACG CTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCAT CCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTT AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCA GCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACT TCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGAT AAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCG CACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCC ACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATG TTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGG AAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTA TCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGA GCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAG CTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCC ACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCT CAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGC ACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAA ACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAA TGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGG CTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGA ATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCG CACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGA AAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGA GTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCG TACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGA TCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGG AGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACG TGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGC ACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGC CCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGC CGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGTTAGCGGCAACCTGAAAGTTGAATTTTATAATAGCAACCCGAGCGACACCACCAATAGCATTAATCCGCAGTTTAAAGTGACCAACACC GGTAGCAGCGCAATTGATCTGAGCAAACTGACCCTGCGTTATTATTATACCGTTGACGGTCAGAAAGACCAGACCTTTTGGTGTGATCATGCAGCAATTATTGGCAGCAATGGCAGCTAT AATGGCGTGACCAGCAATGTGAAAGGTACCTTTGTTAAAATGAGCAGCAGCACCAATAATGCCGATACCTATCTGGAAATTAGCTTCACCGGTGGTACCCTGGAACCGGGTGCACATGT TCAGATTCAGGGTCGTTTTGCAAAAAATGACTGGAGCAATTATACCCAGAGCAACGATTATAGCTTTAAGAGCGCAAGCCAGTTTGTGGAGTGGGATCAGGTTACCGCATATCTGAATGGTGTTCTGGTTTGGGGTAAAGAACCGGGCTCCAGAGACCGAGTCACTGCCAAGGTCTCAGGATCCGTTAGCGGTAATCTGAAAGTTGAATTTTACAGCAACCCGAGCGACACCA CCAATAGCATTAATCCGCAGTTTAAAGTGACCAACACCGGCAGCAGCGCAATTGATCTGAGCAAACTGACCCTGCGTTATTATTATACCGTTGACGGTCAGAAAGACCAGACCTTTTGG TGTGATCATGCAGCAATTATTGGCAGCAATGGCAGCTATAATGGCGTTACCAGCAATGTTAAAGGCACCTTTGTTAAGATGAGCAGCAGCACCAATAATGCCGATACCTATCTGGAAATT AGCTTCACCGGTGGTACCCTGGAACCGGGTGCACATGTTCAGATTCAGGGTCGTTTTGC AAAAAATGACTGGAGCAATTATACCCAGAGCAACGATTATAGCTTCAAGAGCGCAAGCCAGTTTGTGGAATGGGATCAGGTTACCGCATATCTGAATGGTGTTCTGGTTTGGGGTAAAG AACCGGGTAAACTGCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGG CCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT (SEQ ID No. 5).

[0140] like Figure 9 As shown, selected kombucha fungi and yeast are co-cultured to produce microbial cellulose membranes. Genetically engineered E. coli produce recombinant spider silk proteins. By mixing the two and applying a specific process, a new cellulose material can be produced. This material can be used to make leather, an alternative material for everyday items.

[0141] Figure 10 The graph shows the tensile strength results of different fusion protein treatment groups and the control group (protein-free BCM).

[0142] Figure 11The graph shows the elongation at break of the different fusion protein treatment groups and the control group (protein-free BCM).

[0143] It can be seen that after adding a certain amount of fusion protein, the tensile strength and ductility of the fusion protein treatment group were significantly increased compared with the control group.

[0144] Repeat region amino acid sequence:

[0145] 3DB: spider silk protein MaSp4 repeat region

[0146] PQSPYGPGPQGPGPQGPGPQGSGPGPQRPQGPGPQGPYGPGGVSVVSATVSGPGPQG PSGPGPQGPYGPGPQGPGPQGPGPQLPGPQGSGPGPQGPYGPGPQGPGPQGPGPQGPSGPGPQRPQGPGPQGPYGPGGVSVVSATVS (SEQ ID No. 8).

[0147] Ac: spider silk protein AcSp repeat region

[0148] STASASGASYATSTSSAVSSSQATGYSTAAGYGSAPAPAPRPRPLPAPIQAPRPAPAPQ PAPVYAPAPVVSQVQATSSSQASAQQSAFAQSQQSSVVQSQQSSNAYSAASTAGSSVSQSQAIVSSAPVYFNTQTLSSSLSSSLQSLSALNSLASGQLSSWNAASIIASAVAPSLGVSQASVQNSI SQQLRSVGPGSSTSSVAQAIANGVANAVGASGTGVAGQEQSISQSIYTSVSTALSQLAAPRSVAAYGGAGGVATSSSSATASGSRI (SEQ ID No. 9).

[0149] Py: spider silk protein PySp repeat region

[0150] GSGAQGGFGPTTGAQSALISRIANALANTSTLRSVLRTGVSQQTASSVVQRTIQTLASN LGIDGNNLSRIALQAISQVPAGSDTSAYAQAFSTALVTGGVLNANNVDTLGSQVLSAVLNGVSSAAQGLGINVDTGSVQSDIRSSSSSLSTSSSSASFSQTSGAASTTGFTGAGGYPGGAGPLGG GVGSLTGQTSFGQTSGFTRS (SEQ ID No. 10).

[0151] AF: honey bee silk protein structural region

[0152] GSMKIPVLLATCLYLCGFASAGLEGPGNSLPELVKGSASATASTAVTARSGLRAGQVAL ASQKDAVLQAQAAASAASEARAAADLTAKLSQESASVQSQAAAKGKETEEAAVGQARAGLESVSMAASATSAAKEASTAAKAAASALSTAVVQAKIAERAAKAEAVASDEAKAKAIAAA NLAAEASVAAEAALKAEKVAEEAIARAASAKAAARAAAAALASSKEAATASARNAAESEARNEVAVLIAEIDKKSREIDAASSLNARAAAKASSRNVETATIGANINSSKQVVSIPVEIKKFSE PEVSTSWREDEEVTKEKKEHINLNDFDLKSNVFSG (SEQ ID No. 11).

[0153] Complete protein sequence:

[0154] CBM3-NT-3DB-CT-CBM3:

[0155]

[0156] The amino acid marked with a double underlined line is the starting amino acid, the sequence marked with a double underlined line is CBM3, the sequence marked with a dotted underline is the NT region, the sequence marked with a dotted-dashed underline is the 3DB region, the sequence marked with a single underlined line is the CT region, the sequence marked with a single underlined wavy line is also CBM3, and the sequence marked with an underlined bold line is the linker.

[0157] CBM3-NT-Ac-CT-CBM3:

[0158]

[0159] The amino acid marked with a double underlined line is the starting amino acid, the sequence marked with a double underlined line is CBM3, the sequence marked with a dotted underline is the NT region, the sequence marked with a dot-dash underline is the Ac region, the sequence marked with a single underlined line is the CT region, the sequence marked with a single underlined wavy line is also CBM3, and the sequence marked with an underlined bold line is the linker.

[0160] CBM3-NT-Py-CT-CBM3:

[0161]

[0162] The amino acid marked with a double underlined line is the starting amino acid, the sequence marked with a double underlined line is CBM3, the sequence marked with a dotted underline is the NT region, the sequence marked with a dotted-dashed underline is the Py region, the sequence marked with a single underlined line is the CT region, the sequence marked with a single underlined wavy line is also CBM3, and the sequence marked with an underlined bold line is the linker.

[0163]

[0164] The amino acid marked with a double underlined line is the starting amino acid, the sequence marked with a double underlined line is CBM3, the sequence marked with a dotted underline is the NT region, the sequence marked with a dot-dash underline is the AF region, the sequence marked with a single underlined line is the CT region, the sequence marked with a single underlined wavy line is also CBM3, and the sequence marked with an underlined bold line is the linker.

[0165] The above examples are used to illustrate the present invention, which are only used to help understand the present invention and are not intended to limit the present invention. Those skilled in the art can make several simple deductions, modifications or substitutions based on the concept of the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein comprises a protein that can bind to cellulose; from the N-terminus to the C-terminus, the fusion protein comprises a first CBM family protein, an NT region, a repeat region, a CT region, and a second CBM family protein in sequence; NT comprises the amino acid sequence shown in SEQ ID No. 4; CT comprises the amino acid sequence shown in SEQ ID No. 7; The amino acid sequence of the fusion protein is shown in SEQ ID NO.

1.

2. A modified cellulose, characterized in that The modified cellulose comprises cohesive protein and cellulose; the cohesive protein comprises the fusion protein according to claim 1.

3. An isolated polynucleotide, characterized in that The polynucleotide encodes the fusion protein according to claim 1.

4. A construct, characterized in that The construct comprises the polynucleotide of claim 3.

5. An expression system, characterized in that The expression system comprises the construct according to claim 4 or the exogenous polynucleotide according to claim 3 integrated into the genome.

6. The method for preparing modified cellulose according to claim 2, wherein: include: The modified cellulose is prepared by mixing coacervate with cellulose.

7. The preparation method according to claim 6, wherein The mass ratio of the coagulant protein to the cellulose is (0.05-0.06) g: (0.5-10) mg; and / or, the mass ratio of the coagulant protein to the cellulose is (0.05-0.06) g: (0.5-5 mg); and / or, after the coacervate and cellulose are mixed, the mixture is allowed to stand for ≥20 min; And / or, further comprising a soaking step, comprising mixing cohesive protein with cellulose to obtain a mixture, and soaking the obtained mixture in a first soaking liquid to obtain a first soaked product; and / or, the first soaking liquid comprises at least one of isopropyl alcohol, ethanol, methanol, n-butanol, formic acid, and acetic acid; and / or, the soaking time is ≥20 min; And / or, further comprising a secondary soaking step, comprising soaking the primary soaked material in a second soaking liquid to obtain a second soaked material; and / or, the second soaking liquid comprises an aqueous glycerol solution; and / or, the secondary immersion time is ≥12h; And / or, further comprising a third soaking step, comprising soaking the second soaked material in a third soaking liquid to obtain a third soaked material; And / or, the third soaking liquid includes a detergent containing a surfactant or an aqueous solution thereof.

8. The preparation method according to claim 7, wherein After the cohesive protein and cellulose are mixed, the standing time is 20 minutes; And / or, the mass percentage of glycerol in the glycerol aqueous solution is 1 to 10%; and / or, the secondary soaking time is 20-24 hours; and / or, the three soaking times are ≥30 min; And / or, the surfactant-containing cleaning agent includes at least one of soap, hand soap, and dishwashing liquid.

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