Xyloglucanase mutant, coding gene thereof, application of xyloglucanase mutant and coding gene and method for degrading xyloglucan

Abstract

The invention relates to a genetic engineering technology, and discloses a xyloglucanase mutant, a coding gene thereof, application of the xyloglucanase mutant and the coding gene and a method for degrading xyloglucan. The amino acid sequence of the xyloglucanase mutant is as shown in SEQ ID NO. 1. The method for degrading the xyloglucan comprises the step of contacting the xyloglucan enzyme mutant and / or the gene with the xyloglucan. Compared with a wild type, the xyloglucanase mutant has lower optimal reaction temperature and pH, has stronger coordination with enzymolysis conditions of other enzymes, and has a wider application range.

Description

Technical Field

[0001] The present invention relates to genetic engineering technology, and particularly relates to a xyloglucanase mutant, its encoding gene, their applications, and a method for degrading xyloglucan. Background Art

[0002] Xyloglucan is a heteropolysaccharide mainly present in plant cell walls, especially in dicotyledonous plants and some monocotyledonous plants. It consists of a backbone of glucose units linked by β-1,4-glycosidic bonds, with side chains containing xylose units that are attached to glucose residues via β-1,6-bonds. The structure of xyloglucan varies among plant species, with differences in the degree of branching and the presence of other sugars such as galactose or fucose in different plant types. In the plant cell wall, xyloglucan forms a network with cellulose fibers, contributing to the maintenance of the structural integrity of the cell wall and affecting its mechanical properties.

[0003] Xyloglucanase is a class of enzymes that can catalyze the hydrolysis of xyloglucan, acting by breaking the β-1,4-glycosidic bonds in the glucan backbone. According to amino acid sequences and structural characteristics, these enzymes can be classified into multiple families, among which the most common are glycoside hydrolase families 5, 12, 44, and 74. This reaction usually releases smaller oligosaccharides, which can be further processed by other enzymes. The unique properties of xyloglucanase make it of great value in industrial applications. In the food industry, xyloglucanase can be used to produce xyloglucan oligosaccharides to improve the quality of yogurt; in lignocellulose-based biomanufacturing, xyloglucanase helps to decompose plant cell walls, thereby promoting the release of fermentable sugars.

[0004] However, when naturally isolated xyloglucanase exerts its enzymatic catalytic activity, it often requires a relatively high optimal reaction temperature and pH, which not only makes it difficult to meet the conditions of industrial yogurt production, but also cannot form coordinated enzymatic hydrolysis conditions when combined with other plant-degrading enzymes (such as the optimal action temperature of cellulase is 40 - 60°C and the optimal pH is 4 - 5), thus limiting the application of xyloglucanase. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problems existing in the prior art that the naturally isolated xyloglucanase has a relatively high optimal reaction temperature and pH and limited applications. The present invention provides a xyloglucanase mutant, its encoding gene, their applications, and a method for degrading xyloglucan. Compared with the wild type, this xyloglucanase mutant has a lower optimal reaction temperature and pH, stronger coordination of enzymatic hydrolysis conditions with other enzymes, and a wider application range.

[0006] To achieve the above purpose, the first aspect of the present invention provides a xyloglucanase mutant, and the amino acid sequence of the xyloglucanase mutant is as shown in SEQ ID NO.1.

[0007] In the second aspect of the present invention, there is provided a gene encoding a xyloglucanase mutant, which is a nucleotide sequence encoding the xyloglucanase mutant as described above.

[0008] Preferably, the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

[0009] In the third aspect of the present invention, there is provided the use of the xyloglucanase mutant as described above and / or the gene as described above in the preparation of xyloglucanase mutant and / or products related to xyloglucanase mutant.

[0010] In the fourth aspect of the present invention, there is provided the use of the xyloglucanase mutant as described above and / or the gene as described above in the degradation of xyloglucan.

[0011] In the fifth aspect of the present invention, there is provided the use of the xyloglucanase mutant as described above and / or the gene as described above in promoting the enzymatic degradation of lignocellulosic biomass.

[0012] In the sixth aspect of the present invention, there is provided a method for degrading xyloglucan, which includes: contacting the xyloglucanase mutant as described above and / or the gene as described above with xyloglucan.

[0013] Preferably, the conditions of the contact include: the temperature is 68 - 72 °C and the pH is 5 - 7.

[0014] Through the above technical solutions, the present invention uses genetic engineering to modify the xyloglucanase MtXG and its gene cloned from the thermophilic fungus Mycothermus thermophilus CGMCC 3.4393, and heterologously expresses them in Pichia pastoris to obtain a xyloglucanase mutant; the optimal pH for the enzymatic catalysis reaction of this xyloglucanase mutant is about 5.0, and the optimal temperature is about 70 °C. Its optimal reaction conditions, especially the optimal reaction pH, can better form coordinated enzymatic hydrolysis conditions with other plant degrading enzymes (such as cellulase), improve the efficiency of enzymatic hydrolysis with multiple enzymes in combination, and expand the application scenarios and scope of the xyloglucanase mutant. Further, the xyloglucanase mutant provided by the present invention has high pH stability and temperature stability.

[0015] Other features and advantages of the present invention will be described in detail in the subsequent specific implementation section. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1Structural information and SDS-PAGE analysis of MtXG and MtXG-ΔLC in Example 2; where A is the structure of MtXG predicted by AlphaFold3; B is the SDS-PAGE of the fermentation supernatants of MtXG and MtXG-ΔLC, with the fermentation supernatant of Pichia pastoris GS115 as the control;

[0017] Figure 2 Biochemical properties of MtXG and MtXG-ΔLC in Example 3 and Example 4; where A is the optimal pH value; B is the pH tolerance after incubation at 50 °C for 2 hours; C is the optimal temperature; D is the temperature tolerance after incubation at their respective optimal pH for 2 hours; the specific activity values are the average of three replicates. Detailed implementation manners

[0018] The endpoints and any values in the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0019] The first aspect of the present invention provides a xyloglucanase mutant, and the amino acid sequence of the xyloglucanase mutant is as shown in SEQ ID NO.1.

[0020] During the research process, the inventors of the present invention cloned the xyloglucanase MtXG and its gene from the thermophilic fungus Mycothermus thermophilus CGMCC3.4393 (the amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6). However, through verification, it was found that when the naturally isolated xyloglucanase exerted its enzymatic catalytic activity, its optimal temperature was as high as 75°C and the optimal pH was around 7.0. It not only difficult to meet the conditions of industrial production of yogurt, but also when combined with other plant-degrading enzymes (such as the optimal working temperature of cellulase is 40-60°C and the optimal pH is 4-5), it is impossible to form coordinated enzymatic hydrolysis conditions, reducing the combined enzymatic hydrolysis efficiency of multiple enzymes. Accordingly, the inventors creatively modified the naturally isolated xyloglucanase MtXG to obtain the xyloglucanase mutant MtXG-ΔLC with the amino acid sequence shown in SEQ ID NO.1. The optimal pH of this mutant MtXG-ΔLC is around 5.0 and the optimal temperature is around 70°C. Its optimal reaction conditions, especially the optimal reaction pH, can better form coordinated enzymatic hydrolysis conditions with other plant-degrading enzymes (such as cellulase), improve the combined enzymatic hydrolysis efficiency of multiple enzymes, and expand the application scenarios and scope of the xyloglucanase mutant. Further, the xyloglucanase mutant provided by the present invention also has high pH stability and temperature stability.

[0021] The xyloglucanase mutant provided by the present invention can be obtained by artificial synthesis, or its coding gene can be synthesized first and then obtained through biological expression.

[0022] The second aspect of the present invention provides a gene encoding a xyloglucanase mutant, which is the nucleotide sequence encoding the xyloglucanase mutant as described above, that is, the nucleotide sequence encoding the enzyme with the amino acid sequence shown in SEQ ID NO.1.

[0023] As is well known in the art, among the 20 different amino acids that make up proteins, except that Met (ATG) or Trp (TGG) are each encoded by a single codon, the other 18 amino acids are each encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, Second Edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic codons, there are usually more than one codon that determines an amino acid, and the substitution of the third nucleotide in the triplet codon often does not change the amino acid composition. Therefore, the nucleotide sequences of genes encoding the same protein can be different. Preferably, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0024] The nucleotide sequences provided by the present invention can generally be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis methods. Once the relevant nucleotide sequences are obtained, the relevant amino acid sequences can be obtained in large quantities by recombination methods. Usually, the obtained nucleotide sequences are cloned into a vector, then transferred into a genetically engineered bacterium, and then the relevant nucleotide sequences are isolated from the proliferated host cells by conventional methods.

[0025] In addition, the relevant nucleotide sequences can also be synthesized by well-known artificial chemical synthesis methods.

[0026] The present invention also provides a primer set, which is used to amplify the gene as described above.

[0027] According to the present invention, preferably, the nucleotide sequences of the primer set are as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0028] The present invention further provides a biological material, which contains the gene as described above, wherein the biological material is selected from at least one of recombinant vectors, expression cassettes, recombinant bacteria, and transgenic cell lines.

[0029] The "vector" used in the recombinant vector can be various vectors known in the art, such as various commercially available plasmids, cosmids, phages, and retroviruses. Preferably, the expression vector of the recombinant vector is the pPIC9K plasmid.

[0030] In the present invention, the recombinant vector can be constructed by ligating the expression vector and the gene fragment in a directional recombination manner to obtain a recombinant plasmid. Exemplarily, the pPIC9K plasmid can be linearized using restriction endonucleases (EcoR I and Not I), and the digested plasmid is recovered by gel cutting and then directionally recombined with the purified target gene fragment (MtXG-△LC) with overlapping regions (homologous arms) to obtain a recombinant plasmid pPIC9K-MtXG-△LC with homologous arms.

[0031] In the present invention, the recombinant vector can be transformed, transduced, or transfected into a host cell (strain) by conventional methods in the art, such as chemical transformation by the calcium chloride method or high-voltage electroporation transformation, to obtain a recombinant strain. The recombinant strain can be a prokaryotic cell or a eukaryotic cell, preferably selected from at least one of Pichia pastoris, Escherichia coli, and Saccharomyces cerevisiae. More preferably, the recombinant strain is Pichia pastoris. Further preferably, the host strain of the recombinant bacterium is Pichia pastoris GS115.

[0032] In the present invention, the xyloglucanase mutant can be used in the form of whole cells of recombinant bacteria, or in the form of a crude enzyme or a purified enzyme isolated from the cells of recombinant bacteria without purification. If necessary, the xyloglucanase mutant of the present invention can also be made into an immobilized enzyme or an immobilized cell using immobilization techniques known in the art.

[0033] As a method for preparing a xyloglucanase mutant, the preparation method includes: inoculating the aforementioned recombinant bacteria into a fermentation medium for fermentation to obtain a fermentation broth, and separating and purifying the fermentation broth to obtain the xyloglucanase mutant.

[0034] The method for preparing a xyloglucanase mutant provided by the present invention includes: culturing the recombinant bacteria provided by the present invention to induce the expression of the gene encoding the xyloglucanase mutant; separating and purifying the expressed xyloglucanase mutant. Among them, the culture conditions are conventional culture conditions, such as using BMMY medium (1% (v / v) methanol, 500× biotin), culturing at a temperature of 25-35°C and a rotation speed of 200-300 rpm, and using 0.5-1.5% methanol as a protein inducer.

[0035] Since the recombinant bacteria provided by the present invention contain the gene encoding the xyloglucanase mutant, it can efficiently express the xyloglucanase mutant. After culturing and separation and purification, a high-purity xyloglucanase mutant can be obtained. Specifically, methods well-known to those skilled in the art can be used for separation and purification, which will not be elaborated here.

[0036] Based on the xyloglucanase mutant provided by the present invention, an enzyme preparation containing the xyloglucanase mutant can be prepared. Specifically, it can exist in solid, semi-solid or liquid form. The enzyme preparation can contain excipients or additives for preparing the enzyme preparation, etc., which can be selected by those skilled in the art according to needs.

[0037] The third aspect of the present invention provides the application of the aforementioned xyloglucanase mutant and / or the aforementioned gene in the preparation of a xyloglucanase mutant and / or a product related to the xyloglucanase mutant.

[0038] The fourth aspect of the present invention provides the application of the aforementioned xyloglucanase mutant and / or the aforementioned gene in the degradation of xyloglucan.

[0039] The fifth aspect of the present invention provides the application of the aforementioned xyloglucanase mutant and / or the aforementioned gene in promoting the enzymatic degradation of lignocellulosic biomass.

[0040] The sixth aspect of the present invention provides a method for degrading xyloglucan, which includes: contacting the aforementioned xyloglucanase mutant and / or the aforementioned gene with xyloglucan.

[0041] According to the present invention, preferably, the conditions for the contact include: the temperature is 68 - 72°C, specifically it can be 68°C, 69°C, 70°C, 71°C, 72°C, or any value between the above two values, more preferably 69 - 71°C; the pH is 5 - 7, specifically it can be 5, 5.5, 6, 6.5, 7, or any value between the above two values, more preferably 5 - 6.

[0042] The present invention will be described in detail below through examples.

[0043] In the following examples, the materials, reagents and strains used are as follows:

[0044] Tamarind xyloglucan was purchased from Megazyme (Wicklow, Ireland) with the product number P - XYGLN.

[0045] Sodium carboxymethyl cellulose, microcrystalline cellulose (Avicel), beechwood xylan, barley - β - glucan and the commercial enzyme Cellic CTec2 were purchased from Sigma - Aldrich (St. Louis, Missouri, USA).

[0046] Glucose, citric acid, disodium hydrogen phosphate and starch were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0047] Ultrafiltration tubes with a molecular weight cut - off of 30 kDa were purchased from Millipore (USA).

[0048] YNB (Yeast Nitrogen Base without amino acids and ammonium sulfate) and yeast extract were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0049] Pichia pastoris GS115 and Mycothermus thermophilus CGMCC 3.4393 were stored in the laboratory.

[0050] The plasmid pPIC9K was purchased from Invitrogen (USA).

[0051] Escherichia coli T1 was purchased from TransGen Biotech.

[0052] The formula of PDA solid medium is: peeled potato 200 g / L, glucose 20 g / L, agar 20 g / L;

[0053] The formula of glucose medium is: glucose 20 g / L, Vogel's salt solution;

[0054] The formula of cellulose - induced medium is: cellulose 20 g / L, Vogel's salt solution;

[0055] The formula of LB liquid medium is: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L;

[0056] The formula of LB solid medium is: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 20 g / L;

[0057] The formula of YPD liquid medium is: tryptone 20 g / L, yeast extract 10 g / L, glucose 10 g / L;

[0058] The formula of YPD solid medium is: tryptone 20 g / L, yeast extract 10 g / L, glucose 10 g / L, agar 20 g / L.

[0059] Unless otherwise specified, the remaining reagents and raw materials are commercially available products.

[0060] Example 1 Cloning of xyloglucanase MtXG and mutant MtXG-ΔLC genes

[0061] 1.1 Cloning of xyloglucanase MtXG gene

[0062] 1. Obtaining cDNA: Inoculate the cryopreserved Mycothermus thermophilus CGMCC 3.4393 on the slant of PDA solid medium and place it in static culture at 45°C; wash with a physiological saline solution containing Tween-80 to obtain a fresh spore solution, take 500 μL of the spore suspension and inoculate it into the glucose medium, and culture it at 45°C and 200 rpm for 24 h. Then transfer 0.5% (w / v) of the mycelium to the cellulose induction medium and culture it at 45°C and 200 rpm for 24 h. Then extract the total RNA using Trizol reagent; use the RT reagent Kit with gDNA Eraser(Perfect Real Time) reverse transcription kit to synthesize cDNA.

[0063] 2. Cloning of the target gene: According to the transcriptome and secretome data of Mycothermus thermophilus CGMCC 3.4393, find the cDNA sequence of the target protein, and predict the signal peptide through SignalP6.0 (https: / / services.healthtech.dtu.dk / service.php?SignalP). Analyze the restriction enzyme sites using Primer5 software and design primers MtXG-F and MtXG-R with this software. Use MtXG cDNA as a template to amplify the target gene;

[0064] PCR amplified sequence:

[0065] MtXG-F: 5’-GCCACCTGGAAGAATGTCAA-3’ (SEQ ID NO.7);

[0066] MtXG-R: 5’-TCATGAGATACACTGGAAGT-3’ (SEQ ID NO.8).

[0067] The PCR amplification reaction procedure is as follows: (1) Preheat at 95°C for 3 min, (2) Denature at 95°C for 15 s, (3) Anneal at 57°C for 15 s, (4) Extend at 72°C for the corresponding time (the extension rate of the polymerase is 2 kb / min, and the time is calculated according to the length of the target gene fragment), (5) Thoroughly extend at 72°C for 10 min, and steps (2-4) are cycled 30 times.

[0068] The reaction system of PCR: 50 μL, including 25 μL of 2×Phanta Max Master Mix, 1 μL of cDNA template, 2 μL of each of the two primers, and 20 μL of ddH2O.

[0069] The amplified gene fragment was identified by agarose gel electrophoresis and subjected to gel extraction and recovery using the gel extraction kit of OMEGA to obtain the xyloglucanase MtXG gene. The specific amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6.

[0070] 1.2 Cloning of the xyloglucanase mutant MtXG-ΔLC gene

[0071] 1. Obtaining cDNA: Inoculate the cryopreserved Mycothermus thermophilus CGMCC 3.4393 on the inclined plane of PDA solid medium and place it in a static culture at 30°C; Wash with a physiological saline solution containing Tween-80 to obtain a fresh spore solution. Take 500 μL of the spore suspension and inoculate it into a glucose medium. After shaking culture at 30°C and 200 rpm for 24 h, transfer 0.5% (w / v) of the mycelium to a cellulose induction medium and culture at 30°C and 200 rpm for 24 h. Then extract the total RNA using Trizol reagent; Use the RT reagent Kit with gDNA Eraser (PerfectReal Time) reverse transcription kit to remove genomic DNA and synthesize cDNA;

[0072] 2. Cloning of the target gene: Based on the transcriptome and secretome data of the thermophilic fungus Mycothermus thermophilus CGMCC 3.4393, the cDNA sequence of the target protein was found, and the signal peptide was predicted using SignalP 6.0 (https: / / services.healthtech.dtu.dk / service.php?SignalP). The restriction enzyme cleavage sites were analyzed using Primer5 software, and primers MtXG-ΔLC-F and MtXG-ΔLC-R (nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4) were designed with this software; using MtXG cDNA as a template, the target gene (the gene of the xyloglucanase mutant MtXG-ΔLC) was amplified;

[0073] MtXG-ΔLC-F: 5’-GCCACCTGGAAGAATGTCAA-3’ (SEQ ID NO.3); MtXG-ΔLC-R: 5’-CGCATAAAACACTCCGCGCC-3’ (SEQ ID NO.4).

[0074] The PCR amplification reaction program and the PCR reaction system were the same as those in the cloning process of the xyloglucanase MtXG gene.

[0075] The amplified gene fragment was identified by agarose gel electrophoresis and gel extraction was performed using OMEGA's gel extraction kit to obtain the gene of the xyloglucanase mutant MtXG-ΔLC. The specific amino acid sequence is shown in SEQ ID NO.1 and the nucleotide sequence is shown in SEQ ID NO.2.

[0076] Example 2

[0077] Construction of recombinant vectors, recombinant strains, and preparation of xyloglucanase and enzyme mutants.

[0078] 1. The MtXG gene fragment was amplified by PCR using the primer pair MtXG-oneF and MtXG-oneR (nucleotide sequences are shown in SEQ ID NO.9 and SEQ ID NO.10) to increase the homologous arms.

[0079] The gene fragment of MtXG-ΔLC was amplified by PCR using the primer pair MtXG-ΔLC-oneF and MtXG-ΔLC-oneR (nucleotide sequences are shown in SEQ ID NO.11 and SEQ ID NO.12) to increase the homologous arms.

[0080] The pPIC9K plasmid was linearized with restriction endonucleases (EcoR I and Not I). After digestion, the plasmid was recovered by gel extraction and directionally recombined with the purified target gene fragments (MtXG or MtXG-ΔLC) with overlapping regions (homologous arms) using the OneStep Cloning Kit ClonExpressTMII (Vazyme, Nanjing) to obtain the recombinant plasmids pPIC9K-MtXG and pPIC9K-MtXG-ΔLC with homologous arms.

[0081] MtXG-oneF (SEQ ID NO.9):

[0082] 5’-GAGAGGCTGAAGCTTACGTAGCCACCTGGAAGAATGTCAA-3’;

[0083] MtXG-oneR (SEQ ID NO.10):

[0084] 5’-TCTAAGGCGAATTAATTCGCTCAATGGTGATGGTGATGATGTGAGATACACTGGAAGT-3’;

[0085] MtXG-ΔLC-oneF (SEQ ID NO.11):

[0086] 5’-GAGAGGCTGAAGCTTACGTAGCCACCTGGAAGAATGTCAA-3’;

[0087] MtXG-ΔLC-oneR (SEQ ID NO.12):

[0088] 5’-AAGGCGAATTAATTCGCGGCCGCATGGTGATGGTGATGATGCGCATAAAACACTCCGCGC-3’.

[0089] 2. Screening of positive recombinant plasmids: The recombinant plasmids pPIC9K-MtXG and pPIC9K-MtXG-ΔLC were separately transformed into Trans-T1 Escherichia coli competent cells. 500 μL of LB liquid medium was added, and the mixture was incubated at 37 °C and 1000 rpm for 60 min in a constant temperature shaking mixer. After incubation, 100 μL of the E. coli suspension was evenly spread on an LB solid plate containing 0.1% ampicillin using a spreader and cultured in an incubator at 37 °C for 12 h. Single colonies grown on the LB solid plate were picked and placed in LB liquid medium containing 0.1% ampicillin, and incubated at 37 °C and 1000 rpm for 8 - 12 h in a constant temperature shaking mixer.

[0090] Using the Escherichia coli bacterial solution as a template and the primer pair 5AOX1 and 3AOX1 (nucleotide sequences are shown in SEQ ID NO.13 and SEQ ID NO.14) as verification primers, colony PCR verification was carried out to screen for positive transformants, and the screened transformants were subjected to gene sequencing for further verification. Subsequently, the recombinant plasmid pPIC9K-MtXG and the recombinant plasmid pPIC9K-MtXG-△LC were extracted from the correctly verified transformants, and they were linearized using the restriction endonuclease Bgl II. The linearized plasmids were recovered by gel cutting to obtain the linearized expression cassette pPIC9K-MtXG containing the xyloglucanase gene and the linearized expression cassette pPIC9K-MtXG-△LC containing the xyloglucanase mutant gene respectively;

[0091] 5AOX1: 5’-GACTGGTTCCAATTGACAAGC-3’ (SEQ ID NO.13);

[0092] 3AOX1: 5’-GCAAATGGCATTCTGACATCC-3’ (SEQ ID NO.14).

[0093] 3. Electroporation of Pichia pastoris: Streak and isolate Pichia pastoris GS115 on a YPD solid plate, pick a single colony into 5 mL of YPD liquid medium, and shake culture at 30 °C and 200 rpm for 24 h; inoculate 500 μL of the bacterial solution into 50 mL of YPD liquid medium, and shake culture at 30 °C and 200 rpm for 12 h. Use a horizontal rotor and a pre-cooled centrifuge at 4 °C. At the same time, place sterile double-distilled water and 1 M sorbitol on ice for pre-cooling. Immerse the electroporation cuvette (2 mm) in 75% (v / v) ethanol and place it in a laminar flow hood. Turn on the ultraviolet lamp and wait for the ethanol to evaporate, then cover it and place it on ice for pre-cooling. At the same time, place 1.5 mL sterile centrifuge tubes on ice for pre-cooling. Pour all the activated bacterial solution into a sterilized 50 mL centrifuge tube, centrifuge at 1500 g and 4 °C for 5 min, and discard the supernatant; resuspend the cells with 30 mL of ice-bathed double-distilled water, centrifuge at 1500 g and 4 °C for 5 min, and discard the supernatant; resuspend the cells with 30 mL of ice-bathed double-distilled water again, centrifuge at 1500 g and 4 °C for 5 min, and discard the supernatant; resuspend the cells with 2 mL of ice-bathed 1 M sorbitol, centrifuge at 1500 g and 4 °C for 5 min, and discard the supernatant; resuspend the cells with 50 - 100 μL of ice-bathed 1 M sorbitol to make the competent cells viscous but aspirable.

[0094] Add 80 μL of the prepared competent cells and 20 μL of the linearized expression cassette (prepared in Step 2) to a 1.5 mL pre-cooled centrifuge tube and mix well. Transfer all of the mixture into a pre-cooled transformation cup, gently tap it to let the mixture fall to the bottom, and place it on ice; turn on the 30 °C water bath. Turn on the electrophoresis apparatus and set the parameters as follows: the transformation cup size is 2 mm, the voltage is 1500 V, and the resistance is 400 Ω. Take the electroporation cup out of the ice, dry the surface moisture with absorbent paper, and then place it in the electroporation chamber and start the pulse. After the pulse ends, quickly open the lid of the electroporation cup near the lit alcohol lamp, add 1 mL of 1 M sorbitol, gently pipette it twice, and then close the lid. Under sterile conditions, aspirate the transformation solution in the electroporation cup into a sterile 1.5 mL EP tube and incubate it in a 30 °C water bath for 1.5 h. Spread the transformation solution evenly on the previously poured MD plate (containing 500× biotin), and incubate it statically at 30 °C until transformants appear. Draw a 1 cm 2 small grid on the back of the MD plate and number it in sequence. Under sterile conditions, use sterile forceps to hold a sterile 10 μL white pipette tip and place all the transformants in the center of the MD plate grid one by one, and then put the white pipette tip into a sterile 1.5 mL centrifuge tube containing 1 mL of YPD liquid medium. Poke a hole in the centrifuge tube mouth with a red-hot needle tip. Both the MD plate and the 1.5 mL centrifuge tube are incubated statically at 30 °C for 12 h. Subsequently, extract the yeast genome using the bacterial liquid in the 1.5 mL centrifuge tube. Using the extracted yeast genome as a template and 5AOX1 and 3AOX1 as verification primers, perform colony PCR to verify the target gene.

[0095] If the expression cassette is successfully integrated into the GS115 strain, two bands can be amplified from each transformant, while only one band can be amplified from the control GS115 strain. Under sterile conditions, use a sterile 10 μL white pipette tip to pick the colonies on the MD plate with correct colony PCR verification into a 5 mL YPD shake tube and incubate it at 30 °C and 200 rpm for 24 h. Aliquot the above bacterial liquid into sterile 1.5 mL centrifuge tubes, centrifuge at 12000 rpm for 10 min, discard the supernatant, add 1 mL of 30% glycerol, close the lid, and use a vortex oscillator to evenly disperse the cell pellet. Seal it with a sealing film and store it at -20 °C.

[0096] 4. Induced expression of protein in Pichia pastoris:

[0097] a: Inoculate 500 μL of the Pichia pastoris strain activated in the shake tube into BMGY medium and incubate it at 30 °C and 200 rpm for 24 h.

[0098] b: Under sterile conditions, pour the bacterial liquid into a centrifuge tube, centrifuge at 5000 rpm and 4 °C for 5 min, and discard the supernatant.

[0099] c: Add an appropriate amount of BMMY medium (1% (v / v) methanol, 500× biotin) to the precipitate, gently pipette to suspend the precipitate, transfer the suspension to the BMMY medium, and culture with shaking at 28 °C and 250 rpm for 5 days. During the culture process, add 1% methanol to the flask every 24 h as a protein inducer; after the induction is completed, centrifuge at 5000 rpm and 4 °C for 10 min, and take the supernatant.

[0100] d: Use SDS-PAGE to verify the protein induction expression in the transformant. The transformant with the correct target protein band is the correct recombinant Pichia pastoris strain.

[0101] It was determined that the amino acid sequence of xyloglucanase MtXG is as shown in SEQ ID NO.5:

[0102] ATWKNVKTDGGGGFVPGIIFHPKAKGVAYARTDIGGLYRLNEDDSWTPVTDSLSTNERWGHWGIDAVALDPQDPDKVYAAVGMYTNSWDPNPGAIIRSSDRGETWTSVDLPFKVGGNMPGRGMGERLAVDPANSNILYFGARSGNGLWRSTDGGATWSKVTSFTNAGTYIPDPSDVGGYNGDIIGLAFVTFDSTSPVINGTTSRIFVGTADTITASIYVSNDAGATWAPLAGQPGKYLPHKCKLQPDEKALYITYSDGAGPYDGTNGAVYRYDLATETWKDITPVSGGDLYFGFGGLALDLQNPGTLVVAALNSWWPDTQLFRSTDSGETWSRLWEWAGYPDMNLYYSINANNAPWIEAGFLSQDSKKLGWMIEALEIDPHNSDHFLYGTGLTLFGGHDLTNWDTIHNITISSLAVGIEEMAVLGLASAPGGSELLAAVGDNCGFTYPTANDLDTAPSKPWMNPQWATSTDVDYAGNDPSQVVRVGSGTGDQQVAISTDGGKRWTRHPGADTATSGGAVAYSADGDTILWSSSNGGVLRSENQGPFSPVASLPSGAVIAADRRNNSVFYAASSSSSGAKFYRSADAAATFSSISIPAFTAAGAKSVRDIAPHPVVAGEVWVSTDKGLFRSVDFGETFTPVGQGVLTLTEQVSLGKGKGDSWNVYAFGVGPSGAKLYASADGGQTWVDIQGERQGFGAMGANRVVGSGNLEGVVYVGTNGRGVFYATIELPGTGGPTTTTTTTSSASVLPTTLVTSTVGVSSSAVTTTATTTTTTTAAEVSSTTESVTSSTSTTKSSAPTPTAVAKQWAQCGGIGYTGPTQCEEPYRCHEWNPWYFQCIS*(SEQ ID NO.5).

[0103] The amino acid sequence of the xyloglucanase mutant MtXG-ΔLC is shown in SEQ ID NO.1:

[0104] ATWKNVKTDGGGGFVPGIIFHPKAKGVAYARTDIGGLYRLNEDDSWTPVTDSLSTNERWGHWGIDAVALDPQDPDKVYAAVGMYTNSWDPNPGAIIRSSDRGETWTSVDLPFKVGGNMPGRGMGERLAVDPANSNILYFGARSGNGLWRSTDGGATWSKVTSFTNAGTYIPDPSDVGGYNGDIIGLAFVTFDSTSPVINGTTSRIFVGTADTITASIYVSNDAGATWAPLAGQPGKYLPHKCKLQPDEKALYITYSDGAGPYDGTNGAVYRYDLATETWKDITPVSGGDLYFGFGGLALDLQNPGTLVVAALNSWWPDTQLFRSTDSGETWSRLWEWAGYPDMNLYYSINANNAPWIEAGFLSQDSKKLGWMIEALEIDPHNSDHFLYGTGLTLFGGHDLTNWDTIHNITISSLAVGIEEMAVLGLASAPGGSELLAAVGDNCGFTYPTANDLDTAPSKPWMNPQWATSTDVDYAGNDPSQVVRVGSGTGDQQVAISTDGGKRWTRHPGADTATSGGAVAYSADGDTILWSSSNGGVLRSENQGPFSPVASLPSGAVIAADRRNNSVFYAASSSSSGAKFYRSADAAATFSSISIPAFTAAGAKSVRDIAPHPVVAGEVWVSTDKGLFRSVDFGETFTPVGQGVLTLTEQVSLGKGKGDSWNVYAFGVGPSGAKLYASADGGQTWVDIQGERQGFGAMGANRVVGSGNLEGVVYVGTNGRGVFYA(SEQ ID NO.1).

[0105] Isolation and purification of xyloglucanase MtXG and mutant MtXG-ΔLC: The recombinant proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% (w / v) separating gel to evaluate their molecular weights and purities, and their concentrations were determined by the Bradford method.

[0106] The structural information and SDS-PAGE analysis results of the prepared xyloglucanase MtXG and mutant MtXG-ΔLC are as Figure 1 shown.

[0107] Result analysis: The structure of xyloglucanase MtXG predicted by Alpha Fold3 is as shown in Figure 1 A; as shown in Figure 1 C, the SDS-PAGE results showed that there was an obvious band in the fermentation supernatants of both xyloglucanase MtXG and xyloglucanase mutant MtXG-ΔLC, corresponding to proteins with molecular weights of approximately 120 kDa and 90 kDa respectively. It is worth noting that these observed molecular weights are about 30 kDa and 10 kDa higher than the theoretically predicted 89.8 kDa and 78.2 kDa respectively. Further analysis revealed that MtXG contains 2 N-glycosylation sites and 37 O-glycosylation sites, while the mutant MtXG-ΔLC contains 2 N-glycosylation sites and 12 O-glycosylation sites. Therefore, the difference between the actual molecular weight and the theoretical molecular weight can be attributed to the glycosylation modification that occurs during the protein expression process in the Pichia pastoris host. As a control, there was no obvious band in the fermentation supernatant of Pichia pastoris GS115, further confirming that the bands detected by SDS-PAGE indeed originated from the heterologously expressed xyloglucanase MtXG and mutant MtXG-ΔLC.

[0108] Example 3

[0109] Determination of the optimal pH and temperature.

[0110] The enzyme activity was measured in 0.2 mol / L citric acid-disodium hydrogen phosphate buffer with a pH range of 2.6 - 7.6, and the enzyme activity at 0 h of incubation under the optimal reaction conditions was used as the control enzyme activity (the control enzyme activity of MtXG was 55.5 U / mL, and the control enzyme activity of MtXG-ΔLC was 56.8 U / mL) to calculate the relative enzyme activity at different pH values.

[0111] The enzyme activity was measured in the temperature range of 50 - 85 °C, and the enzyme activity at 0 h of incubation under the optimal reaction conditions was used as the control enzyme activity (the control enzyme activity of MtXG was 55.5 U / mL, and the control enzyme activity of MtXG-ΔLC was 56.8 U / mL) to calculate the relative enzyme activity at different temperatures to evaluate the optimal temperature of the enzyme.

[0112] Enzyme activity assay method: The 3,5-dinitrosalicylic acid (DNS) method was used to determine the activity of xyloglucanase and its mutants.

[0113] The reaction mixture consisted of 75 μL of 1% xyloglucan substrate and 25 μL of purified xyloglucanase MtXG or xyloglucanase mutant MtXG-ΔLC; the substrate and the enzyme were prepared separately and diluted with citric acid-disodium hydrogen phosphate buffer (100 mM, pH 7.0) to ensure the optimal reaction conditions.

[0114] One unit (U) of xyloglucanase activity is defined as the amount of enzyme that releases 1 μmol of reducing sugar (equivalent to glucose) from xyloglucan per minute.

[0115] The experimental results are as Figure 2 shown. The optimal pH of xyloglucanase MtXG is 7.0, and the optimal temperature is as high as 75 °C; while the optimal pH of xyloglucanase mutant MtXG-ΔLC is 5.0, and the optimal temperature is 70 °C (see Figure 2 A and C therein).

[0116] Example 4

[0117] Stability analysis

[0118] To determine the pH stability of the enzyme, the purified xyloglucanase MtXG or the xyloglucanase mutant MtXG-ΔLC was incubated at 50 °C for 2 hours in 0.2 mol / L citric acid-disodium hydrogen phosphate buffer with pH values of 2.6, 3, 4, 5, 6, 7, and 7.6 respectively. The enzyme activity at 0 h of incubation under the optimal reaction conditions was used as the control enzyme activity (the control enzyme activity of MtXG was 55.5 U / mL, and the control enzyme activity of MtXG-ΔLC was 56.8 U / mL), and the relative enzyme activity of the residual enzyme of MtXG and MtXG-ΔLC was measured.

[0119] To determine the temperature stability of the enzyme, the purified xyloglucanase MtXG or the xyloglucanase mutant MtXG-ΔLC was incubated at different temperatures (40 - 80 °C) for 2 hours in 0.2 mol / L citric acid-disodium hydrogen phosphate buffer with pH 5. The enzyme activity at 0 h of incubation under the optimal reaction conditions was used as the control enzyme activity (the control enzyme activity of MtXG was 55.5 U / mL, and the control enzyme activity of MtXG-ΔLC was 56.8 U / mL), and the relative enzyme activity of the residual enzyme of MtXG and MtXG-ΔLC was measured.

[0120] The experimental results are as Figure 2 shown. Compared with MtXG, MtXG-ΔLC retained higher residual enzyme activity after incubation at different pH values and different temperatures for 2 h (see Figure 2 B and D therein).

[0121] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A xyloglucanase mutant, characterized in that The amino acid sequence of the xyloglucanase mutant is shown in SEQ ID NO.

1.

2. A gene encoding a xyloglucanase mutant, characterized in that: The gene is a nucleotide sequence encoding the xyloglucanase mutant according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. Use of the xyloglucanase mutant according to claim 1 and / or the gene according to claim 2 or 3 in preparing the xyloglucanase mutant and / or products containing the xyloglucanase mutant.

5. Use of the xyloglucanase mutant according to claim 1 and / or the gene according to claim 2 or 3 in degrading xyloglucan.

6. Use of the xyloglucanase mutant according to claim 1 and / or the gene according to claim 2 or 3 in promoting enzymatic degradation of lignocellulosic biomass.

7. A method for degrading xyloglucan, characterized in that: The method comprises: contacting the xyloglucanase mutant according to claim 1 and / or the gene according to claim 2 or 3 with xyloglucan.

8. The method according to claim 7, characterized in that The contacting conditions include: temperature of 68-72° C. and pH of 5-7.

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