Neutral heat-resistant xyloglucanase MtXG and application thereof

Abstract

The invention provides neutral heat-resistant xyloglucanase MtXG and an application of the neutral heat-resistant xyloglucanase MtXG. Belongs to the technical field of gene engineering. According to the invention, the neutral heat-resistant xyloglucanase MtXG and the gene of the neutral heat-resistant xyloglucanase MtXG are successfully cloned from a thermophilic fungus Mycothermophilus CGMCC (China General Microbiological Culture Collection Center) 3.4393, and the neutral heat-resistant xyloglucanase MtXG and the gene are subjected to heterologous expression in pichia pastoris. Research results show that the MtXG not only shows the optimal enzymatic activity at the neutral pH of 7.0 and the high temperature of 75 DEG C, but also keeps good stability under wide pH and temperature conditions, and has high specificity and good heavy metal ion tolerance to xyloglucan at the same time. The MtXG is integrated into a commercial cellulase system, so that the yield of reducing sugar in tobacco leaves is greatly increased, the capability of improving the efficiency of a biomass degradation process is shown, and the MtXG has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to a neutral thermostable xyloglucanase MtXG and its applications. Background Art

[0002] Xylglucan is a heteropolysaccharide primarily found in plant cell walls, especially in dicotyledons and some monocotyledons. It consists of a backbone of glucose units linked by β-1,4-glycosidic bonds, with xylose units in the side chains, which are linked to glucose residues via β-1,6-bonds. The structure of xylglucan varies among plant species; its degree of branching and the presence of other sugars such as galactose or fucose differ between plant types. In plant cell walls, xylglucan forms a network with cellulose fibers, helping to maintain the structural integrity of the cell wall and influencing its mechanical properties.

[0003] Xylglucanase (EC number 3.2.1.151) is a class of enzymes that catalyze the hydrolysis of xylglucan by breaking the β-1,4-glycosidic bonds in the glucan backbone. Based on their amino acid sequence and structural characteristics, these enzymes can be divided into several families, the most common being the glycosidase families 5, 12, 44, and 74. This reaction typically releases smaller oligosaccharides, which can be further processed by other enzymes. The unique properties of xylglucanase make it valuable in industrial applications. In the food industry, xylglucanase can be used to produce xylglucan oligosaccharides to improve yogurt quality; in lignocellulose-based biomanufacturing, xylglucanase helps break down plant cell walls, thereby promoting the release of fermentable sugars.

[0004] In industrial applications, enzymes active at neutral pH conditions offer numerous advantages. Using neutral enzymes reduces the need for additional pH-adjusting chemicals and improves the compatibility of enzymatic reactions with many industrial environments. Although many hydrolases derived from filamentous fungi have been used industrially, many of these enzymes are optimal at acidic pH. For example, three xyloglucanases from *Aspergillus japonicus*, *Chrysosporium lucknowense*, and *Trichoderma reesei* have been reported to have optimal pH values ​​of 5.0, 6.0, and 5.3, respectively. Similarly, two xyloglucanases from *Aspergillus niger* exhibit the highest activity at pH 4.5 and 5.0, respectively. To date, few fungal xyloglucanases with optimal activity at neutral pH have been reported.

[0005] Furthermore, the temperature tolerance, pH tolerance, and metal ion tolerance of xyloglucanase also determine its applicable range to a certain extent.

[0006] Therefore, finding a xyloglucanase and related genes that have the highest activity at neutral pH and are resistant to temperature, pH and metal ions is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a neutral thermostable xyloglucanase MtXG and its applications. The neutral thermostable xyloglucanase gene MtXeg74A was obtained from the thermophilic fungus *Mycothermus thermophilus*, and the gene was cloned, heterologously expressed, and its characteristics were analyzed. The MtXG enzyme expressed in *Pichia pastoris* exhibited optimal activity at pH 7.0 and 75°C, and showed significant stability over a wide pH range (2.5-7.5) and temperature range (50-85°C), with high temperature tolerance (not inactivated for up to 2 hours at 60°C). Furthermore, MtXG exhibits high specificity for xyloglucan and shows varying degrees of tolerance to various metal ions, especially Zn. 2+ NH4 + , K + Mn 2+ and Na + It exhibits significant resistance.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A neutral thermostable xyloglucanase MtXG, the amino acid sequence of which is shown in SEQ ID NO.8.

[0010] Another object of the present invention is to provide: a gene MtXeg74A encoding the above-mentioned neutral thermostable xyloglucanase, the nucleotide sequence of said MtXeg74A gene being shown in SEQ ID NO.3.

[0011] Another object of the present invention is to provide a primer set for amplifying the above-mentioned neutral thermostable xyloglucanase gene, said primer set comprising MtXG-F and MtXG-R, the specific sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0012] Another object of the present invention is to provide a biological material containing the above-mentioned neutral thermostable xyloglucanase gene MtXeg74A, said biological material being a recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria.

[0013] Another object of the present invention is to provide the application of the above-mentioned neutral thermostable xyloglucanase MtXG or the above-mentioned gene MtXeg74A or the above-mentioned biological material, wherein the application is in any of the following directions:

[0014] (1) Application in the preparation of xyloglucanase and / or products containing xyloglucanase;

[0015] (2) Application in the preparation of xyloglucanase mutants and / or products containing xyloglucanase mutants;

[0016] (3) Application in the preparation of recombinant xyloglucanase and / or related products containing recombinant xyloglucanase;

[0017] (4) Application in the degradation of xyloglucan;

[0018] (5) Application in promoting the enzymatic degradation of lignocellulose biomass.

[0019] Preferably, the reaction conditions for degradation in step (4) are as follows: the temperature of the reaction system is 60-80℃ and the pH value of the reaction system is 4.0-7.5.

[0020] Preferably, the degradation reaction conditions are as follows: the temperature of the reaction system is 75°C; the pH value of the reaction system is 7.0.

[0021] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention successfully cloned the gene for the neutral, thermostable xyloglucanase MtXG from the thermophilic fungus *Mycothermus thermophilus* CGMCC 3.4393 and expressed it heterologously in *Pichia pastoris*. The results showed that MtXG exhibited optimal enzyme activity not only at neutral pH 7.0 and high temperature 75°C, but also maintained good stability and tolerance to heavy metal ions under a wide range of pH and temperature conditions. This makes MtXG an attractive candidate for various industrial applications requiring a neutral and thermostable enzyme with heavy metal tolerance.

[0023] Furthermore, the high specificity of MtXG for xyloglucan (a key component of plant cell walls) enhances its potential for efficient degradation of lignocellulose biomass. Integrating MtXG into commercial cellulase systems significantly increased the yield of reducing sugars in tobacco leaves, demonstrating its ability to improve the efficiency of biomass degradation processes. These results indicate that this xyloglucanase shows great promise for industrial applications. Overall, this study highlights the unique value of MtXG and opens new avenues for its development in lignocellulose biotransformation biotechnology. Attached Figure Description

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0025] Figure 1 The results are as follows: structural information and SDS-PAGE analysis of MtXG; where A represents the structural domain information of MtXG protein; B represents the structure of MtXG predicted by AlphaFold3; and C represents the SDS-PAGE of the fermentation supernatant of MtXG.

[0026] Figure 2 The following are the biochemical properties of MtXG: A is the optimal pH value; B is the pH tolerance after incubation at 50℃ for 2 hours; C is the optimal temperature; D is the temperature tolerance after incubation at the optimal pH for 2 hours; the specific activity value is the average of three replicates.

[0027] Figure 3 The enzyme's tolerance to metal ions after incubation at 75°C for 30 minutes is defined as: the enzyme's tolerance to metal ions.

[0028] Figure 4 Here is the LC-MS spectrum of the products released from the enzymatic hydrolysis of xylglucan by MtXG.

[0029] Figure 5 The effect of adding MtXG to Cellic CTec2 on tobacco degradation. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0031] The materials, reagents, and strains used in the embodiments of this invention are as follows:

[0032] Tamarind beta-glucan was purchased from Megazyme (Wicklow, Ireland).

[0033] Sodium carboxymethyl cellulose, microcrystalline cellulose (Avicel), beech wood polysaccharide, barley β-glucan, and the commercial enzyme Cellic CTec2 were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

[0034] Glucose, citric acid, disodium hydrogen phosphate, and starch were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0035] The ultrafiltration tube with a molecular weight cutoff of 30 kDa was purchased from Millipore (USA).

[0036] YNB (yeast nitrogen-based, amino acid-free and ammonium sulfate-free) and yeast extract were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0037] Pichia pastoris GS115 and M. thermophilus CGMCC 3.4393 are preserved in the laboratory.

[0038] The plasmid pPIC9K was purchased from Invitrogen, USA.

[0039] Escherichia coli T1 was purchased from TransGen Biotech.

[0040] Example 1

[0041] Cloning of the neutral thermostable xyloglucanase gene MtXeg74A

[0042] (1) cDNA acquisition: Frozen thermophilic fungus M. thermophilus CGMCC 3.4393 was inoculated onto PDA solid medium slant and incubated statically at 45℃. Fresh spores were obtained by washing with physiological saline solution containing Tween-80. 500 μL of the spore suspension was inoculated into glucose medium and cultured at 45℃ with shaking at 200 rpm for 24 h. Then, 0.5% (w / v) mycelium was transferred to cellulose induction medium and cultured at 45℃ with shaking at 200 rpm for 24 h. Total RNA was extracted using Trizol reagent. The DNA was obtained using a TaKaRa reagent. RT reagent kit with gDNA Eraser (Perfect RealTime) for cDNA synthesis;

[0043] (2) Cloning of the target gene: Based on the transcriptome and secretome data of the thermophilic fungus M. 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). Restriction sites were analyzed using Primer5 software, and primers MtXG-F and MtXG-R were designed using this software. The target gene was amplified using MtXG cDNA as a template.

[0044] PCR amplification sequence:

[0045] MtXG-F: 5'-GCCACCTGGAAGAATGTCAA-3', SEQ ID NO.1;

[0046] MtXG-R: 5'-TCATGAGATACACTGGAAGT-3', SEQ ID NO. 2.

[0047] The PCR amplification reaction procedure is as follows: (1) Preheat at 95℃ for 3 min, (2) Denature at 95℃ for 15 s, (3) Anneal at 57℃ for 15 s, (4) Extend at 72℃ for the corresponding time (the extension rate of polymerase is 2 kb / min, and the time is calculated according to the length of the target gene fragment), (5) Extend completely at 72℃ for 10 min, and repeat steps (2-4) 30 times.

[0048] PCR reaction system: 50 μL, including 25 μL of 2×PhantaMax MasterMix, 1 μL of cDNA template, 2 μL of each of the two primers, and 20 μL of ddH2O.

[0049] The amplified gene fragment was identified by agarose gel electrophoresis and extracted using an OMEGA gel extraction kit to obtain the target gene fragment MtXeg74A, with the specific nucleotide sequence as follows:

[0050] MtXeg74A:

[0051]

[0052] Example 2

[0053] Construction of recombinant vectors and recombinant strains, and preparation of neutral thermostable xyloglucanase.

[0054] (1) Construction of recombinant vector

[0055] The target gene fragment was amplified by PCR using MtXG-oneF and MtXG-oneR to increase homologous arms. The pPIC9K plasmid was linearized with restriction endonucleases (EcoR I and Not I). The digested plasmid was recovered by gel excision and directionally recombinated with the purified target gene fragment containing overlapping regions (homologous arms) using the One Step Cloning Kit ClonExpress™ II (Nanjing Novozymes Vazyme) to obtain the recombinant plasmid.

[0056] MtXG-oneF: 5'-GAGAGGCTGAAGCTTACGTAGCCACCTGGAAGAAT GTCAA-3', SEQ IDNO.4;

[0057] MtXG-oneR: 5'-TCTAAGGCGAATTAATTCGCTCAATGGTGATGGTGATGATGTGAGATACACTGGAAGT-3', SEQ ID NO. 5.

[0058] (2) Screening of positive recombinant plasmids

[0059] The recombinant plasmid was then transformed into Trans-T1 *E. coli* competent cells, and 500 μL of LB liquid medium was added. The cells were incubated at 37°C and 1000 rpm for 60 min using a shaker. After incubation, 100 μL of the *E. coli* suspension was evenly spread onto LB agar plates containing 0.1% ampicillin and incubated at 37°C for 12 h. Single colonies grown on the LB agar plates were picked and placed in LB liquid medium containing 0.1% ampicillin, and incubated at 37°C and 1000 rpm for 8–12 h using a shaker. Using *E. coli* culture as a template and 5AOX1 and 3AOX1 as verification primers, colony PCR was performed to screen for positive transformants. The selected transformants were then sequenced for further verification. The pPIC9K recombinant plasmid was then extracted from the verified transformants and linearized using the restriction endonuclease BglII. The linearized plasmid was recovered by gel excision to obtain the linearized expression cassette.

[0060] 5AOX1: 5'-GACTGGTTCCAATTGACAAGC-3', SEQ ID NO.6;

[0061] 3AOX1: 5'-GCAAATGGCATTCTGACATCC-3', SEQ ID NO. 7.

[0062] (3) Pichia pastoris electroporation: Pichia pastoris GS115 was isolated by streaking on YPD solid plates. Single colonies were picked and cultured in 5 mL of YPD liquid medium at 30°C with shaking at 200 rpm for 24 h. 500 μL of bacterial culture was inoculated into 50 mL of YPD medium and cultured at 30°C with shaking at 200 rpm for 12 h. A horizontal rotor centrifuge was used and pre-cooled to 4°C. At the same time, sterile double-distilled water and 1 M sorbitol were placed on ice for pre-cooling. The electroporation cup (2 mm) was soaked in 75% ethanol and placed in a clean bench. After the ethanol evaporated, the cup was covered and placed on ice for pre-cooling. At the same time, 1.5 mL sterile centrifuge tubes were placed on ice for pre-cooling. Pour all activated bacterial suspension into a sterilized 50ml centrifuge tube, centrifuge at 1500g, 4℃ for 5min, and discard the supernatant. Resuspend the cells in 30mL of ice-cold double-distilled water, centrifuge at 1500g, 4℃ for 5min, and discard the supernatant. Resuspend the cells again in 30mL of ice-cold double-distilled water, centrifuge at 1500g, 4℃ for 5min, and discard the supernatant. Resuspend the cells in 2mL of ice-cold 1M sorbitol, centrifuge at 1500g, 4℃ for 5min, and discard the supernatant. Resuspend the cells in 50-100μL of ice-cold 1M sorbitol until the competent cells are viscous but still identifiable by aspiration. Add 80μL of the treated competent cells and 20μL of linearized plasmid to a 1.5mL pre-chilled centrifuge tube and mix well. Transfer the entire mixture into a pre-chilled transformation flask, gently tap it to allow the mixture to settle to the bottom, and place it on ice. Turn on the 30℃ water bath. Turn on the electrophoresis apparatus and set the parameters as follows: conversion cup size 2mm, voltage 1500V, resistance 400Ω. Remove the conversion cup from ice, wipe off surface moisture with absorbent paper, and place it in the electrophoresis tank. Start the pulsed cycle. After the pulsed cycle ends, quickly open the cap of the conversion cup near a lit alcohol lamp, add 1mL of 1M sorbitol, gently blow twice, and then close the cap. Under aseptic conditions, transfer the conversion solution from the conversion cup into a sterile 1.5mL EP tube and incubate at 30°C for 1.5 hours. Spread the conversion solution evenly on a pre-pollinated MD plate (containing 500× biotin) and incubate at 30°C until transformants appear. Draw a 1cm line on the back of the MD plate. 2The transformants were divided into small squares and numbered sequentially. Under aseptic conditions, using tweezers, a sterile 10 μL white pipette tip was used to spot all transformants sequentially in the center of each square on an MD plate. The white pipette tip was then placed into a sterile 1.5 mL centrifuge tube containing 1 mL of YPD liquid medium. A small opening was made at the top of the centrifuge tube with a red-hot needle. Both the MD plate and the 1.5 mL centrifuge tube were incubated at 30°C for 12 h. Subsequently, yeast genome was extracted from the bacterial culture in the 1.5 mL centrifuge tube. Using the extracted yeast genome as a template, colony PCR was performed using 5AOX1 and 3AOX1 as verification primers to verify the target gene. If the expression cassette successfully integrated into the GS115 strain, each transformant could amplify two bands, one approximately 2200 bp in size and the other approximately 500 bp larger than the target gene. In contrast, the control GS115 strain could only amplify one band approximately 2200 bp in size. Under aseptic conditions, colonies from MD plates that had been verified by PCR were picked using a sterile 10 μL pipette tip and transferred to 5 mL YPD shaker tubes. The tubes were incubated at 30°C and 200 rpm for 24 h with shaking. The bacterial culture was then aliquoted into sterile 1.5 mL centrifuge tubes, centrifuged at 12,000 rpm for 10 min, the supernatant was discarded, 1 mL of 30% glycerol was added, the tubes were capped, and the cell pellet was evenly dispersed using a vortex mixer. The tubes were then sealed with sealing film and stored at -20°C.

[0063] (4) Inducible expression of proteins in Pichia pastoris:

[0064] a: Inoculate 500 μL of the Pichia pastoris strain that has been activated in a shaker into BMGY medium and culture at 30°C and 200 rpm for 24 h.

[0065] b: Under aseptic conditions, pour the bacterial culture into a centrifuge tube, centrifuge at 5000 rpm and 4°C for 5 min, and discard the supernatant;

[0066] 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 BMMY medium, and incubate at 28℃ and 250 rpm for 5 days with shaking. During the incubation process, add 1% methanol to the bottle every 24 hours as a protein inducer. After induction, centrifuge at 5000 rpm and 4℃ for 10 min, and collect the supernatant.

[0067] d: The protein expression in the transformants was verified by SDS-PAGE. The strains with the correct target protein band were the correct recombinant Pichia pastoris strains.

[0068] The amino acid sequence of the neutral thermostable xyloglucanase MtXG was determined as follows:

[0069] ATWKNVKTDGGGGFVPGIIFHPKAKGVAYARTDIGGLYRLNEDDSW

[0070] TPVTDSLSTNERWGHWGIDAVALDPQDPDKVYAAVGMYTNSWDPNPGAI

[0071] IRSSDRGETWTSVDLPFKVGGNMPGRGMGERLAVDPANSNILYFGARSGN

[0072] GLWRSTDGGATWSKVTSFTNAGTYIPDPSDVGGYNGDIIGLAFVTFDSTSP

[0073] VINGTTSRIFVGTADTITASIYVSNDAGATWAPLAGQPGKYLPHKCKLQPD

[0074] EKALYITYSDGAGPYDGTNGAVYRYDLATETWKDITPVSGGDLYFGFGGL

[0075] ALDLQNPGTLVVAALNSWWPDTQLFRSTDSGETWSRLWEWAGYPDMNL

[0076] YYSINANNAPWIEAGFLSQDSKKLGWMIEALEIDPHNSDHFLYGTGLTLFG

[0077] GHDLTNWDTIHNITISSLAVGIEEMAVLGLASAPGGSELLAAVGDNCGFTY

[0078] PTANDLDTAPSKPWMNPQWATSTDVDYAGNDPSQVVRVGSGTGDQQVAI

[0079] STDGGKRWTRHPGADTATSGGAVAYSADGDTILWSSSNGGVLRSENQGP

[0080] FSPVASLPSGAVIAADRRNNSVFYAASSSSSGAKFYRSADAAATFSSISIPAF

[0081] TAAGAKSVRDIAPHPVVAGEVWVSTDKGLFRSVDFGETFTPVGQGVLTLT

[0082] EQVSLGKGKGDSWNVYAFGVGPSGAKLYASADGGQTWVDIQGERQGFG

[0083] AMGANRVVGSGNLEGVVYVGTNGRGVFYATIELPGTGGPTTTTTTTSSAS

[0084] VLPTTLVTSTVGVSSSAVTTTATTTTTTTAAEVSSTTESVTSSTSTTKSSAPT

[0085] PTAVAKQWAQCGGIGYTGPTQCEEPYRCHEWNPWYFQCIS*, SEQ ID NO.8.

[0086] Isolation and purification of neutral thermostable xyloglucanase: The recombinant protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% (w / v) separating gel to assess its molecular weight and purity. Its concentration was determined by the Bradford method.

[0087] The structural information and SDS-PAGE analysis results of the prepared neutral thermostable xyloglucanase MtXG are as follows: Figure 1 shown.

[0088] Results analysis: such as Figure 1 As shown in Figure A, structural analysis of the neutral thermostable xyloglucanase MtXG revealed that the enzyme consists of four main parts: a signal peptide (SP), a catalytic domain (CD) belonging to glycoside hydrolase family 74, a linker region, and a family 1 carbohydrate-binding module (CBM1). The structure of MtXG predicted by AlphaFold3 is shown in Figure A. Figure 1 As shown in B.

[0089] like Figure 1 C. SDS-PAGE results showed a distinct band in the fermentation supernatant of MtXG, corresponding to a protein with a molecular weight of approximately 120 kDa. Notably, the observed molecular weight was about 30 kDa higher than the theoretically predicted 89.8 kDa. Further analysis revealed that MtXG contains 2 N-glycosylation sites and 37 O-glycosylation sites. Therefore, the difference between the actual and theoretical molecular weight can be attributed to glycosylation modifications that occur during protein expression in the Pichia pastoris host. As a control, the fermentation supernatant of Pichia pastoris GS115 showed no distinct band, further confirming that the band detected by SDS-PAGE indeed originated from heterologously expressed xyloglucanase MtXG.

[0090] Example 3

[0091] Enzymatic property analysis of neutral thermostable xyloglucanase MtXG

[0092] (1) pH and temperature characteristics

[0093] Enzyme activity was measured in 0.2 mol / L citrate-disodium hydrogen phosphate buffer (pH 2.6–7.6). The enzyme activity at 0 h of incubation under optimal conditions (pH 7.0, 75 °C) was used as the control group (51.7 U / mL). The optimal pH was assessed by calculating the relative enzyme activity at different pH values. Simultaneously, the relative activity of the residual enzyme was measured after incubation at 50 °C for 2 hours in buffer (pH 2.6–7.6) to determine the enzyme's pH stability.

[0094] Enzyme activity was measured within a temperature range of 50-85℃. The enzyme activity at 0 h of incubation under optimal reaction conditions (pH 7.0, temperature 75℃) was used as the control group (enzyme activity 51.7 U / mL). Relative enzyme activities at different temperatures were calculated to assess the enzyme's optimal temperature. Simultaneously, the enzyme's thermostability was determined by evaluating the residual relative enzyme activity after 2 hours of incubation at different temperatures (40-80℃). The experimental results are as follows: Figure 2 shown.

[0095] Enzyme activity assay: The xyloglucanase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method.

[0096] The reaction mixture consisted of 75 μl of 1% xyloglucan substrate and 25 μl of purified xyloglucanase. The substrate and enzyme were prepared separately and diluted with citrate-disodium hydrogen phosphate buffer (100 mM, pH 7.0) to ensure optimal reaction conditions.

[0097] One unit of xyloglucanase activity (U) is defined as the amount of enzyme that releases 1 μmol of reducing sugar (equivalent to glucose) from xyloglucan per minute.

[0098] Results analysis: Figure 2This indicates that the optimal pH for MtXG is 7.0, and it exhibits significant stability within the pH range of 4.0–7.5. Furthermore, the optimal temperature for MtXG activity is as high as 75°C, demonstrating significant stability within the 60–80°C range. Simultaneously, under the optimal reaction conditions of pH 7.0 and 75°C, the highest enzyme activity reached 51.7 U / mL. In comparison, the optimal pH for TrXG from *T. reesei* is 5.3. Sequence analysis showed that MtXG contained a higher proportion of acidic amino acids (9.33% vs. 7.28%) and a slightly lower proportion of basic amino acids (7.12% vs. 7.16%) compared to TrXG (Grishutin, SG, Gusakov, AV, Markov, AV, Ustinov, BB, Semenova, MV, and Sinitsyn, AP (2004). Specific xyloglucanases as a new class of polysaccharide-degrading enzymes. Biochim Biophys Acta 1674, 268-81), (Lopes, DCB, Carraro, CB, Silva, RN, and de Paula, RG (2021). Molecular Characterization of Xyloglucanasecel74a from Trichodermareesei. Int J Mol Sci 22).

[0099] (2) Effects of different concentrations of metal ions on xyloglucanase activity

[0100] The metal ions studied include Zn 2+ NH4 + , K + Fe 3+ Co 2+ Mn 2+ Na + Ca 2+ and Mg 2+ Prepare stock solutions (0.1 mol / L) of these ions, with two concentration gradients (1 mmol / L and 10 mmol / L).

[0101] For a final reagent concentration of 1 mmol / L: Add 75 μL of 1% xyloglucan substrate and 25 μL of enzyme solution sequentially to a centrifuge tube. The enzyme solution needs appropriate dilution; add a metal ion solution during dilution. The enzyme solution needs to be diluted 5 times (citric acid-disodium hydrogen phosphate), so the reaction system is: 75 μL 1% xyloglucan substrate + 25 μL enzyme solution (5 μL enzyme solution + 19.8 μL buffer + 0.2 μL reagent solution). Incubate the mixture in a 75°C water bath for 30 min. After the reaction, add 150 μL of DNS reagent to terminate the reaction, boil in an induction cooker for 10 min, add 1 mL of pure water, and measure the absorbance at OD540 nm using a spectrophotometer. Set up three replicates for each group, and calculate the relative enzyme activity as 100% if the enzyme activity is directly subjected to the water bath reaction without adding any reagents. For a concentration of 10 mmol / L, the operating steps are the same except for adding more metal ion solution during dilution.

[0102] Incubate the test tubes at 75°C for 30 minutes. Then add DNS reagent to terminate the reaction. Boil the sample for 10 minutes before adding distilled water. Measure the OD using a microplate reader. 540 The absorbance at the specified location. Three replicates were set up for each group, including a blank control. The relative enzyme activity was calculated with the activity of the sample without added metal ions as 100%. The specific enzyme activity determination method and the definition of xylo-glucanase activity unit (U) are the same as in (1), and the experimental results are as follows. Figure 3 shown.

[0103] Results Analysis: In such Figure 3 As shown, at a concentration of 1 mM, Fe 3+ Co 2+ Ca 2+ and Mg 2+ It exhibited a significant inhibitory effect on MtXG, reducing its enzyme activity by more than 60%. This may be due to Fe 3+ Co 2+ Ca 2+ and Mg 2+ Heavy metal ions can chelate with the reaction system or react with enzymes, disrupting the enzyme's spatial structure and thus significantly inhibiting or even inactivating its activity. This result highlights the sensitivity of MtXG to specific metal ions and underscores the potential of these ions as regulators in biological processes involving MtXG. Conversely, Zn... 2+ NH4 + , K + Mn 2+ and Na +The effects on enzyme activity are minimal. This indicates that these ions do not significantly interfere with the enzyme's active site or its overall structure, allowing MtXG to maintain its catalytic efficiency. Furthermore, our analysis shows that the inhibitory effect of metal ions on MtXG is concentration-dependent. At higher concentrations of 10 mM, all tested metal ions exhibited strong inhibitory effects on MtXG, leading to a significant decrease in enzyme activity. This suggests that the enzyme's tolerance to these ions is concentration-limited. These findings provide valuable insights into the regulation of MtXG activity by metal ions and are significant for understanding the broader roles of metal ions in biological processes.

[0104] Example 4

[0105] Substrate specificity

[0106] The enzymatic activity of xyloglucanase MtXG was tested using various substrates at a concentration of 1%. Substrates included xyloglucan, starch, xylan, carboxymethyl cellulose (CMC), Avicel, and barley β-D-glucan. The relative activities of other substrates were calculated using the specific enzyme activity against xyloglucan as a baseline (set as 100%). Each experiment was repeated three times to collect reliable data, and the average value was then calculated. The results showed that MtXG exhibited primarily hydrolytic activity against xyloglucan, weakly hydrolytic activity against β-D-glucan, and no hydrolytic activity against the other substrates.

[0107] Example 5

[0108] To further understand the MtXG-catalyzed hydrolysis process, we used LC-MS to analyze the hydrolysis products of xyloglucan, selecting the products obtained by the hydrolysis of xyloglucan catalyzed by the xyloglucanase MtXG under optimal temperature and pH conditions as the analytical samples.

[0109] First, 1.5 ml of the liquid sample was centrifuged and then freeze-dried. Next, 1.5 ml of methanol was added, and the mixture was vortexed until the sample was completely dissolved. Then, 3 ml of dichloromethane was added, and the resulting system was incubated at room temperature for 1 hour. Subsequently, 1.25 ml of water was added, and the mixture was allowed to stand at room temperature for 10 minutes. After evaporating the solvent to dryness under a slow inert gas flow, 200 μl of a mixed solvent (composed of dichloromethane, methanol, and water in a volume ratio of 60:30:4.5) was added. Then, 250 μl of the sample was accurately pipetted and mixed with 750 μl of acetonitrile. The resulting mixture was thoroughly mixed and then centrifuged at 13,000 rpm for 10 minutes at 4 °C. Finally, the supernatant was collected for analysis by liquid chromatography-mass spectrometry (LC-MS) on a Thermo Scientific LTQ Orbitrap Velos system. The experimental results are as follows: Figure 4 shown.

[0110] Results analysis: The results showed that several oligosaccharides were released during the hydrolysis of xyloglucan by MtXG, namely XXXG, XLXG, XXLG and XLLG.

[0111] Each (1→4)-β-linked D-glucose residue in the main chain is assigned a single-letter code based on its substituent: G, β-D-Glc; X, α-D-Xyl-(1→6)-β-D-Glc; L, β-D-Gal-(1→2)-α-D-Xyl-(1→6)-β-D-Glc. This further confirms that MtXG can reduce the degree of polymerization of xyloglucan by directly cleaving the β-1,4-glycosidic bonds within its main chain. This unique catalytic property of MtXG highlights its potential importance in various biological processes, including cell wall degradation and plant cell wall remodeling.

[0112] Example 6

[0113] Enzymatic hydrolysis of tobacco leaves

[0114] MtXG was introduced into a cellulase hydrolysis system for tobacco leaves to investigate its effect on the degradation of tobacco leaves by Cellic CTec2. Fresh tobacco leaves were collected in July 2023 in Xuancheng City, Anhui Province, China (118.76°E, 30.95°N). The tobacco leaves were freeze-dried and then pulverized using an MF 10basic Microfine mill (IKA, Germany) through a 2.0 mm diameter sieve. Enzymatic hydrolysis of the tobacco leaves was carried out in 50 mL flasks with a reaction volume of 20 mL. The solids loading was 2% (w / v), and the pH was 5. Hydrolysis was conducted in a constant-temperature air bath shaker at 50 °C and 150 rpm. Xyloglucanase (1.2 mg protein per gram of substrate) and Cellic CTec2 (6.0 mg protein per gram of substrate) were added to the hydrolysis system to evaluate their effects on enzymatic hydrolysis. Samples were taken at 0, 4, 24, and 72 hours. The samples were boiled for 10 minutes to inactivate the enzyme, then centrifuged at 10,000 rpm for 10 minutes to obtain the supernatant, and the reducing sugar concentration was determined using the DNS method. A sample without crude enzyme and reacted for 0 hours served as a control. All experiments were repeated three times. The changes in reducing sugar production over time during the hydrolysis of tobacco leaves by different enzymes are shown below. Figure 5 shown.

[0115] Results analysis: From Figure 5It is evident that the addition of MtXG significantly increased reducing sugar production compared to the control group and the group treated with Cellic CTec2 alone. Particularly after 72 hours of enzymatic hydrolysis, the addition of MtXG increased reducing sugar production by 149% relative to the control group and 11% relative to the group treated with Cellic CTec2 alone. This enhancement can be attributed to the presence of xylglucan in tobacco leaves. Xylglucan can entangle or encapsulate polysaccharides such as cellulose, thereby hindering the enzymatic hydrolysis of cellulose and other polysaccharides. Conversely, in the presence of MtXG, xylglucan is broken down into smaller polymeric fragments. This effect not only alleviates the physical barrier caused by xylglucan but also improves the accessibility of Cellic CTec2 enzymes to cellulose and hemicellulose in tobacco leaves. By breaking down the xylglucan barrier, MtXG promotes a more efficient and thorough enzymatic hydrolysis process, ultimately leading to increased reducing sugar production.

[0116] The various embodiments in the specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0117] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

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

8.

2. A gene MtXeg74A encoding the neutral thermostable xyloglucanase according to claim 1, characterized in that: The nucleotide sequence of the gene MtXeg74A is shown in SEQ ID NO.

3.

3. A primer set for amplifying the neutral thermostable xyloglucanase gene MtXeg74A according to claim 2, characterized in that: The primer set includes MtXG-F and MtXG-R, and the specific sequences are shown in SEQ ID NO.1 and SEQ ID NO.

2.

4. A biomaterial containing the neutral thermostable xyloglucanase gene MtXeg74A according to claim 2, characterized in that: The biological material is a recombinant vector, an expression box, a transgenic cell line or a recombinant bacterium.

5. Use of the neutral thermostable xyloglucanase MtXG according to claim 1 or the gene MtXeg74A according to claim 2 or the biomaterial according to claim 4, characterized in that: The application is any of the following: (1) Application in the preparation of xyloglucanase and / or products containing xyloglucanase; (2) Application in the preparation of xyloglucanase mutants and / or products containing xyloglucanase mutants; (3) Application in the preparation of recombinant xyloglucanase and / or related products containing recombinant xyloglucanase; (4) Application in the degradation of xyloglucan; (5) Application in promoting enzymatic degradation of lignocellulosic biomass.

6. The use according to claim 5, characterized in that: The reaction conditions of the degradation in step (4) are as follows: the temperature range of the reaction system is 60-80° C., and the pH value of the reaction system is 4.0-7.

5.

7. The use according to claim 6, characterized in that: The degradation reaction conditions are as follows: the temperature of the reaction system is 75° C.; and the pH value of the reaction system is 7.0.

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