A cellulase gene, cellulase, recombinant vector and application

Abstract

The application discloses a cellulase gene, a cellulase, a recombinant carrier and application, and the cellulase gene sequence is shown as SEQ ID NO.1 or SEQ ID NO.2, or the nucleotide sequence is obtained after site mutation of the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO.2, the substrate binding domain of the cellulase is PKD-Fn3, and the KD-Fn3 nucleotide sequence is shown as SEQ ID NO.3. The application uses metagenomics technology to mine and research novel and efficient cellulase genes in a soil metagenome library of Mount Qomolangma, heterologously expresses the screened cellulase genes, purifies cellulase proteins, and analyzes enzymatic characteristics and kinetic properties of the cellulase. The novel and efficient cellulase screened in the research can not only enrich an existing cellulase resource library, but also provide a reference basis for improving utilization efficiency of lignocellulose.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a cellulase gene, cellulase, recombinant vector, and their applications. Background Technology

[0002] Cellulose is the most abundant and widely distributed renewable energy source on Earth. Utilizing cellulases derived from microorganisms to convert cellulose into biofuels or produce high-value-added industrial products offers an effective way to alleviate the current global environmental pollution and energy shortages. However, existing cellulases suffer from low activity, limited enzymatic hydrolysis efficiency, poor stability, and low yield, which have consistently hindered their industrialization. Therefore, the discovery of novel and highly efficient cellulases is of great significance. Although traditional microbial culture methods have screened out a large number of highly efficient cellulase-producing strains, most microorganisms in the natural environment remain unculturable. Metagenomics, on the other hand, bypasses traditional microbial culture methods, directly studying the genomic information of microorganisms in all environmental samples, breaking down the operational barriers of unculturable microorganisms. Therefore, metagenomics holds the promise of maximizing the discovery of active enzyme genes in nature and obtaining novel cellulases.

[0003] Cellulose is a naturally abundant and inexpensive renewable biomass raw material with enormous potential for addressing the global energy crisis. However, its complex structure and difficulty in degradation result in low overall utilization rates. Currently, the main challenge in the industrial production of biomass energy lies in the scarcity of highly efficient cellulose-degrading enzymes. Although many excellent cellulase genes have been screened using in vitro microbial culture techniques, a large number of cellulase genes from unculturable microorganisms remain to be discovered. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a cellulase gene, cellulase, recombinant vector, and its applications. The cellulase ZFEG1907 has the nucleotide sequence shown in SEQ ID NO.1. The truncated form ZFEG1907t is the catalytic functional sequence remaining after removing the substrate-binding domain (PKD-Fn3) from the nucleotide sequence shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2. The substrate-binding domain gene sequence of the cellulase, PKD-Fn3, has the nucleotide sequence shown in SEQ ID NO.3. This experimental project avoids traditional direct microbial culture methods and utilizes metagenomics technology to mine and study novel and efficient cellulase genes in a metagenomic library of Mount Everest soil. After heterologous expression and protein purification of the screened cellulase genes to obtain cellulase, its enzymatic characteristics and kinetic properties are analyzed. The novel and efficient cellulase screened in this study not only enriches the existing cellulase resource library but also provides a reference for improving the utilization efficiency of lignocellulose. The enzyme protein corresponding to the novel cellulase SEQ ID NO.1 was discovered, which catalyzes the hydrolysis of cellulose degradation; in addition, SEQ ID NO.3 facilitates substrate binding. Therefore, in-depth exploration of cellulase-producing genes and achieving their efficient expression are of great practical significance.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The present invention provides a cellulase gene sequence, the nucleotide sequence of which is shown in SEQ ID NO.1, or the nucleotide sequence obtained by site mutation of the nucleotide sequence shown in SEQ ID NO.1, wherein the mutation includes substitution and / or deletion and / or addition and / or translocation of one or more nucleotides.

[0007] After screening for cellulase-positive clones, cellulase-positive subclones were constructed. Sequencing results of the positive subclones were analyzed using SnapGene Viewer 4.1.9.0 software, predicting a full-length ORF (nucleotide sequence shown in SEQ ID NO.1) of 2118 bp. This sequence has a GC content of 69%, encodes a protein of 705 amino acids, has a predicted molecular weight of 75.3 kDa, and a predicted isoelectric point (pI) of 6.02. The predicted ORF was uploaded to the NCBI database for BALSTX homology comparison. The results showed that the protein encoded by this gene belongs to the 44th family of glycosidases (GH44), and shares 76.38% similarity with bacteria from the Pyromonaceae family (MBA3515422.1), 73.17% similarity with bacteria from the Armorobacteria phylum (MCC2671073.1), and 63.65% similarity with bacteria from the Acidobacteria phylum (AHL27901.1). The alignment results showed that the protein encoded by this ORF is a potential cellulase, and the gene was named ZFEG1907 (nucleotide sequence shown in SEQ ID NO.1). Multiple sequence alignment of the cellulase ZFEG1907 encoded by this gene, along with some cellulases from the GH44 family and other GH families, was performed using ClustalW, and a phylogenetic tree was constructed using MEGA5.1. The phylogenetic tree further confirmed that ZFEG1907 belongs to the GH44 family. The ZFEG1907 sequence was submitted to the SWISS-PROT database for protein structure analysis, obtaining the template 3ii1.1 with the highest similarity, showing 76.25% identity. The protein corresponding to this template is the GH44 family cellulase celM2 (ABL11223.1).

[0008] Preferably, the nucleotide sequence is the catalytic functional sequence remaining after removing the substrate-binding domain from the nucleotide sequence shown in SEQ ID NO.1, the catalytic functional sequence being shown in SEQ ID NO.2, or obtained by site mutation of the nucleotide sequence shown in SEQ ID NO.2, the mutation including substitution and / or deletion and / or addition and / or shift of one or more nucleotides.

[0009] In any of the above embodiments, it is preferred that the nucleotide sequence shown in SEQ ID NO.2 is obtained by removing the last 561 base pairs of nucleic acid from the nucleotide sequence shown in SEQ ID NO.1.

[0010] This invention also discloses the amplification of the above-mentioned cellulase gene. Specific primers zfeg1907-F and zfeg1907-R (primer sequences P1907-F (5'-TTT AAG AAG GAG ATA TAC ATA TGC AGA ACC CCG CCGTCAACA TCA-3') and P1907-R (5'-GTG GTG GTG GTG GTG GTG CTC GAG TCT CTT AAG AGTTCT GGC GCC CGC-3') were designed to amplify the cellulase gene zfeg1907 by PCR. The plasmid expressing the protein ZFEG1907t was constructed, and the cellulase gene zfeg1907t was amplified by corrective PCR using specific primers zfeg1907t-F and zfeg1907t-R. The target band was recovered and purified.

[0011] This invention also discloses a method for inducing expression and purifying the above-mentioned cellulase gene, comprising:

[0012] (1) The recombinant plasmids pET-30a(+)-zfeg1907 and pET-30a(+)-zfeg1907t were induced to express at low temperature by adding the inducer IPTG to liquid LB medium;

[0013] (2) The recombinant plasmids pET-30a(+)-zfeg1907 and pET-30a(+)-zfeg1907t were induced to express in Escherichia coli BL21(DE3), and the target protein was purified.

[0014] Pure cellulases ZFEG1907 and ZFEG1907t were successfully obtained after purification by nickel column affinity chromatography.

[0015] This invention also discloses a study on the effects of the above-mentioned cellulase recombinase activity and stability, including the following methods:

[0016] (1) Effects of temperature on the activity and stability of cellulase recombinase;

[0017] (2) Effect of pH on the activity and stability of cellulase recombinase;

[0018] (3) Effects of metal ions and chemical reagents on the activity of cellulose recombinase.

[0019] The study on the effect of temperature on the activity and stability of recombinant cellulases revealed that the enzyme activities of cellulases ZFEG1907 and truncated ZFEG1907t gradually increased with increasing temperature, exhibiting high activity within the range of 40℃-50℃, with maximum activity at 50℃. Above 50℃, the activity of cellulases decreased with increasing temperature. At 70℃, the activity of cellulase ZFEG1907 decreased to 43% of its maximum activity, while that of cellulase ZFEG1907t decreased to 61% of its maximum activity. In conclusion, the optimal reaction temperature for both cellulases ZFEG1907 and truncated ZFEG1907t is 50℃, classifying them as thermophilic enzymes with broad industrial application prospects.

[0020] The study on the effect of pH on the activity and stability of recombinant cellulases revealed that the optimal reaction pH for both cellulase ZFEG1907 and its truncated form ZFEG1907t was 5.0. The pH stability of cellulase ZFEG1907 and its truncated form ZFEG1907t was generally similar. Both were relatively stable within a pH range of 4.0-8.0, maintaining over 50% of their activity. Cellulase ZFEG1907 maintained over 40% activity after treatment at pH 3.0 and pH 9.0, while the activity of truncated form ZFEG1907t was below 30% after treatment at both pH 3.0 and pH 9.0. This indicates that cellulase ZFEG1907 has stronger pH stability than its truncated form ZFEG1907t.

[0021] Results of the study on the effects of metal ions and chemical reagents on the activity of cellulosic recombinase: 1 mM metal ions, except for Cu... 2+ and Mn 2+ Both ZFEG1907 and the truncated ZFEG1907t enzyme showed some inhibitory activity. Mg 2+ and Ca 2+ Except for the promotion effect of all metal ions on the activity of ZFEG1907t enzyme, other metal ions have no significant effect on the activity of this enzyme. 1% organic solvents, except for acetonitrile, ethanol, acetone and methanol, all have a certain inhibitory effect on the activity of ZFEG1907t enzyme, while acetonitrile, acetone, methanol and ethyl acetate have a certain promoting effect on the activity of ZFEG1907t enzyme, other chemical reagents have no significant effect on the activity of this enzyme.

[0022] The present invention also discloses a substrate-binding domain gene sequence of a cellulase, wherein the substrate-binding domain is PKD-Fn3, the nucleotide sequence of PKD-Fn3 is shown in SEQ ID NO.3, or is obtained by site mutation of the nucleotide sequence shown in SEQ ID NO.3, wherein the mutation includes substitution and / or deletion and / or addition and / or translocation of one or more nucleotides.

[0023] The PFAM website was used to predict the potential catalytic and substrate-binding domains of ZFEG1907, and other cellulase domains of the GH44 family were also predicted and compared with ZFEG1907. The results showed that in addition to the catalytic domains of the GH44 family, the ZFEG1907 protein also contains PKD and Fn3 domains.

[0024] The present invention also discloses the application of the above-described substrate-binding domain gene sequence in promoting the binding of enzymes to macromolecular substrates, improving the affinity between enzymes and substrates, and improving the catalytic efficiency of enzymes.

[0025] Preferably, the substrate includes at least sodium carboxymethyl cellulose (CMC-Na), konjac gum (KGM), and lichenan. Experimental results show that the PKD-Fn3 domain can improve the catalytic efficiency of cellulase ZFEG1907 by enhancing its binding affinity to the three substrates CMC, KGM, and lichenan. Therefore, the PKD-Fn3 domain of cellulase ZFEG1907 helps promote the binding of the enzyme to macromolecular substrates and improves the enzyme-substrate affinity.

[0026] The present invention also discloses a cellulase whose amino acid sequence is encoded by the nucleotide sequence described in any one of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 above, or obtained by site mutation of the nucleotide sequence shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3, wherein the mutation includes substitution and / or deletion and / or addition and / or translocation of one or more nucleotides.

[0027] This invention also discloses a recombinant vector containing the nucleotide sequence shown in any one of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3. The vector is a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, or mammalian cell virus. In this invention, the nucleotide sequence encoding cellulase can be inserted into the vector to form a recombinant vector containing the polynucleotides described in this invention. "Vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.

[0028] Preferably, the expression vector for this recombinant vector is *Escherichia coli*. The recombinant plasmids pET-30a(+)-zfeg1907 and pET-30a(+)-zfeg1907t were induced to express in *Escherichia coli* BL21(DE3).

[0029] This invention also discloses the application of the aforementioned cellulase in the degradation of sodium carboxymethyl cellulose, konjac gum, lichen polysaccharides or microcrystalline cellulose, and sodium carboxymethyl cellulose. Under optimal conditions, the substrate specificity of cellulase ZFEG1907 and its truncated form ZFEG1907t was determined, showing similar substrate specificity. Both enzymes exhibited degradation activity only on four substrates: sodium carboxymethyl cellulose, konjac gum, lichen polysaccharides, and microcrystalline cellulose. The activity was highest for konjac gum, followed by lichen polysaccharides and sodium carboxymethyl cellulose, while no degradation activity was observed for locust bean gum, xylan, dextran, poria cocos polysaccharide, guar gum, chitin, or pNPG.

[0030] The present invention has the following beneficial effects:

[0031] This study focuses on the metagenomics of soil microorganisms in Tibet. Cellulase-positive clones were identified from a soil metagenomic library using functional screening methods. Bioinformatics analysis was applied to identify potential cellulase genes within these clones, and biochemical experiments were used to further investigate the enzymatic and domain functions of cellulase hydrolases. This provides a solid theoretical foundation for the industrial application of cellulases. The main research contents and results are described below:

[0032] (1) A novel cellulase gene, named zfeg1907, was obtained from a soil library using a functional metagenomics strategy. Its nucleotide sequence is shown in SEQ ID NO.1. The gene is 2118 bp in length, with a GC content of 69%, encoding a protein of 705 amino acids. Its predicted molecular weight is 75.3 kDa, and its predicted isoelectric point (pI) is 6.02. Homology analysis showed that the protein encoded by this gene belongs to the 44th family of glycosidases (GH44), and shares 76.38% homology with hydrolases derived from Porphyromonas bacteria. Protein structure prediction indicated that the ZFEG1907 protein contains not only the catalytic domain of the GH44 family but also PKD and Fn3 domains.

[0033] (2) To elucidate the function of the PKD-Fn3 domain in the ZFEG1907 protein within the cellulase structure, a PKD-Fn3 domain-deficient cellulase, ZFEG1907t, was constructed, with its nucleotide sequence shown in SEQ ID NO.2. The target gene fragment was linked to the expression vector pET-30a(+), and a 6× His tag was fused to the carboxyl terminus of the protein. E. coli BL21(DE3) was selected as the expression host bacterium for heterologous expression of the target gene. After nickel column affinity purification, the target proteins ZFEG1907 and ZFEG1907t were obtained, respectively. SDS-PAGE electrophoresis results showed that the molecular weights were consistent with the predicted values.

[0034] (3) The recombinant protease properties showed that the optimal temperature for both cellulase ZFEG1907 and its truncated form ZFEG1907t was 50℃, with good thermal stability below 40℃. Both had an optimal pH of 5.0, exhibiting high enzyme activity and good stability under neutral and slightly acidic conditions (pH 4.0-6.0), indicating they are acidophilic enzymes. 2+ and Mn 2+ (1 mM and 10 mM) showed significant inhibitory effects on both, Mg 2+ (1mM and 10mM) and Ca 2+ K+ (1mM) significantly promoted the activity of ZFEG1907t enzyme, while K+ (10mM) significantly promoted the activity of ZFEG1907. 1% acetonitrile and 1% methanol significantly inhibited the activity of cellulase ZFEG1907. 15% organic solvents inhibited both ZFEG1907 and ZFEG1907t enzymes, with a more significant inhibitory effect on ZFEG1907. SDS, as a strong denaturant, significantly inhibited the activity of both ZFEG1907 and ZFEG1907t enzymes regardless of concentration. 100mM imidazole significantly inhibited the activity of both ZFEG1907 and ZFEG1907t.

[0035] (4) Substrate specificity and kinetic parameters of the recombinant protease were analyzed. The results showed that ZFEG1907 and its truncated form ZFEG1907t exhibited similar substrate specificity. Both showed hydrolytic activity only on four substrates: sodium carboxymethyl cellulose, konjac gum, lichen polysaccharide, and microcrystalline cellulose, with the highest activity on konjac gum. Enzymatic kinetic parameters of sodium carboxymethyl cellulose, konjac gum, and lichen polysaccharide were determined. The results showed that when sodium carboxymethyl cellulose was used as the reaction substrate, the Kc of cellulase ZFEG1907 was significantly higher. m The value is 42.27% lower than ZFEG1907t, k cat / K m The K+ value was 35.85% higher than that of ZFEG1907t. When konjac gum was used as the reaction substrate, the K+ value of cellulase ZFEG1907 was... m The value is 33.79% lower than ZFEG1907t, k cat / K m The value was 70.08% higher than that of ZFEG1907t; when lichen polysaccharide was used as the reaction substrate, the K of cellulase ZFEG1907 was higher. m The value is 61.62% lower than ZFEG1907t, k cat / K mThe value was 92.73% higher than that of ZFEG1907t. These results indicate that the PKD-Fn3 domain can improve the catalytic efficiency of cellulase ZFEG1907 by enhancing its binding affinity to three substrates: sodium carboxymethyl cellulose, konjac gum, and lichen polysaccharide. The optimal reaction temperature for both cellulase ZFEG1907 (nucleotide sequence shown in SEQ ID NO.1) and its truncated form ZFEG1907t (nucleotide sequence shown in SEQ ID NO.2) is 50℃, classifying them as thermophilic enzymes with broad industrial application prospects.

[0036] (5) In summary, a novel cellulase gene, zfeg1907, was obtained from a Tibetan soil library using a functional metagenomics strategy. The protein ZFEG1907 contains both the catalytic domain of the GH44 family and the PKD-Fn3 domain. The target gene was ligated into the vector pET-30a(+), and heterologously expressed in E. coli BL21(DE3) to obtain proteins ZFEG1907 and the PKD-Fn3 domain-deficient protein ZFEG1907t. Recombinant proteomic characterization studies showed that the PKD-Fn3 domain had almost no effect on the conventional characteristics of cellulase (optimal temperature, optimal pH, tolerance to metal ions and chemical reagents, substrate specificity). However, the PKD-Fn3 domain could improve the catalytic efficiency of cellulase ZFEG1907 by enhancing its binding affinity to three substrates: sodium carboxymethyl cellulose, konjac gum, and lichen polysaccharide. Therefore, the PKD-Fn3 domain of cellulase ZFEG1907 helps promote the binding of the enzyme to macromolecular substrates, improving the enzyme-substrate affinity. The PKD-Fn3 domain (nucleotide sequence shown in SEQ ID NO.3) can improve the catalytic efficiency of cellulase ZFEG1907 by enhancing its binding affinity to three substrates: CMC, KGM, and Lichenan. Thus, the PKD-Fn3 domain of cellulase ZFEG1907 helps promote the binding of the enzyme to macromolecular substrates, improving the enzyme-substrate affinity. Attached Figure Description

[0037] Figure 1 Functional screening of cellulase-positive clones: In the figure, A represents positive clones screened from the metagenomic library, and B represents secondary screening of the first round of positive clones;

[0038] Figure 2 To construct positive subclones containing cellulase, A in the figure shows restriction endonuclease digestion of the positive clone with Sau3A I, B shows restriction endonuclease digestion of the positive subclones with BamHI, and C shows functional screening of positive subclones with 2000-3000bp DNA fragment insertion.

[0039] Figure 3 Phylogenetic analysis for ZFEG1907;

[0040] Figure 4 Multiple sequence ratio plot for ZFEG1907;

[0041] Figure 5 Comparison of ZFEG1907 with other cellulase domains in the GH44 family;

[0042] Figure 6 Agarose gel electrophoresis detection of zfeg1907 and zfeg1907t; 1: PCR amplification product of zfeg1907; 2: PCR amplification product of zfeg1907t; 3: Recombinant plasmid pET-30a(+)-zfeg1907 enzyme digestion verification; 4: Recombinant plasmid pET-30a(+)-zfeg1907t enzyme digestion verification; M: DNA Marker;

[0043] Figure 7 The diagram shows the construction scheme and SDS-PAGE analysis of the truncated ZFEG1907; A: SDS-PAGE analysis of purified ZFEG1907 and ZFEG1907t; M: protein molecular weight marker; Lane 1: purified recombinant ZFEG1907 protein, expected molecular weight 75.3 kDa; Lane 2: purified recombinant ZFEG1907 protein with truncated molecular weight, showing an expected molecular weight of 56.8 kDa; B shows the construction scheme of the truncated cellulase ZFEG1907t.

[0044] Figure 8 The effect of temperature on the enzyme activities of ZFEG1907 and ZFEG1907t (20-90℃);

[0045] Figure 9 The effect of temperature on the stability of ZFEG1907 and ZFEG1907t enzymes;

[0046] Figure 10 The effect of pH on the enzyme activities of ZFEG1907 and ZFEG1907t (pH 2.0-9.0);

[0047] Figure 11 The effect of pH on the stability of ZFEG1907 and ZFEG1907t enzymes;

[0048] Figure 12 The effects of different metal ions (1 mM) on the enzyme activities of cellulases ZFEG1907 and ZFEG1907t were investigated.

[0049] Figure 13The effects of different metal ions (10 mM) on the enzyme activities of cellulases ZFEG1907 and ZFEG1907t were investigated.

[0050] Figure 14 The effects of different chemical reagents (1%) on the enzyme activities of cellulase ZFEG1907 and ZFEG1907t were investigated.

[0051] Figure 15 The effects of different chemical reagents (15%) on the enzyme activities of ZFEG1907 and ZFEG1907t were investigated.

[0052] Figure 16 The effects of different organic compounds on the enzyme activities of ZFEG1907 and ZFEG1907t were investigated. Detailed Implementation

[0053] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The various terms and phrases used in this invention have their general meanings known to those skilled in the art. The present invention provides a general or specific description of the materials and experimental methods used in the experiments. Although many materials and operating methods used to achieve the objectives of the present invention are known in the art, the present invention will still be described in as much detail as possible herein. Unless otherwise specified, the instruments, reagents, materials, etc., involved in the following embodiments are all conventional instruments, reagents, materials, etc., already existing in the prior art; and unless otherwise specified, they are all conventional experimental methods, detection methods, etc., already existing in the prior art.

[0054] Example 1

[0055] 1.1 Screening of cellulase-positive clones

[0056] Tibet Archives: Soil samples were taken from the Mount Everest region of Tibet (28.21°N, 86.56°E, 4276 meters above sea level). This region has rugged terrain, unique environmental conditions, and is sensitive to climate change, resulting in a rich variety and abundance of microorganisms in the soil.

[0057] The Tibetan soil metagenomics contains approximately 1.04 × 10⁻⁶ cells. 7 The library clones, with an average inserted target fragment of 38,000 bp, encompassed a large number of microbial gene resources. This project used a functional metagenomic screening strategy with CMC-Na (sodium hydroxymethyl cellulose) as the screening substrate to mine cellulase genes from a Tibetan soil metagenomic library. After screening approximately 2 million library clones, a single positive clone with a hydrolysis zone around the colony was obtained. This positive clone was further screened and verified; by streaking the clone, a clear hydrolysis zone was still observed around it, confirming that this clone was a positive clone producing cellulase. Figure 1 As shown.

[0058] Screening Principle: The screening substrate selected for cellulase-producing microorganisms in this experiment is sodium carboxymethyl cellulose (CMC-Na). Cellulase-producing microorganisms can decompose CMC-Na in the culture medium, while other microorganisms cannot. After staining with acidic Congo red, Congo red binds to CMC-Na to form a red complex, but cannot bind to cellulase hydrolysis products such as cellobiose and glucose. Therefore, after staining and destaining, a transparent hydrolysis zone will appear around the colonies of cellulase-producing microorganisms.

[0059] Screening method: After determining the appropriate dilution factor of the library bacteria through preliminary experiments, the library bacteria were diluted and evenly spread on solid LB culture plates. After the bacteria grew, a layer of screening medium was poured on top. After staining and decolorization, positive clones producing cellulase were found by observing whether there was a transparent hydrolysis zone around the colonies.

[0060] The moderately diluted bacterial suspension was evenly spread on three LB agar plates (containing antibiotics Amp and Chl, with final concentrations of 100 μg / mL and 12.5 μg / mL, respectively. This ensures complete coverage of approximately 5000 clones in the numbered library). The plates were incubated at 37°C for 2-3 days. A layer of selection medium was then poured on top of the colonies, and the plates were incubated under the same conditions for another 1-2 days. After cultivation, stain with acidic Congo red for 40-60 minutes, then destain with sodium chloride solution for 40-60 minutes. Discard the destaining solution and observe in a well-lit environment (if no clear hydrolysis zone is observed after destaining, it may be due to weak cellulase activity; extend the destaining time and observe again). If a clear hydrolysis zone appears around a single colony on the culture plate, use a sterilized pipette tip to pick it up and streak it again onto an LB agar plate. After colony growth, select a single colony and incubate at 37°C. Rinse the corresponding single colony on the plate with tap water and repeat the staining and destaining method described above to observe whether a clear hydrolysis zone still exists around the selected single colony to rule out false positives. After confirming a positive clone, incubate overnight at 37°C with shaking at 225 rpm, then store in 20% glycerol and freeze at -80°C.

[0061] 1.2 Construction of cellulase subclones

[0062] Cellulase-positive clone plasmids were extracted and partially digested. DNA fragments between 2-3 kb were recovered. Electrophoresis results of the partial digestion are shown below. Figure 2 As shown in Figure A, the electrophoretic bands after enzyme digestion are diffuse. The enzyme-recovered DNA fragments were ligated with the enzyme-recovered pWEB-TNC using T4 ligase. TMSubclones were constructed using vector ligation. Using the same Congo red staining method, positive subclones with cellulase activity were selected, as shown in Figure 2C. These positive subclones underwent secondary screening; after confirming activity again by streaking, the subclone plasmids were extracted, digested, and verified by electrophoresis. The electrophoresis results for enzyme digestion verification are shown below. Figure 2 As shown in Figure B, lane 2 shows the plasmid electrophoresis results after BamHI digestion of the positive subcloning plasmid. The electrophoretic bands, from largest to smallest, correspond to the 5812 bp linear plasmid pWEB-TNC. TM The cellulase gene insertion fragment, approximately 2000 bp in size, was successfully constructed, with the inserted fragment band size matching expectations (2000-3000 bp). Enzyme digestion confirmed the successful construction of the cellulase-positive subclone. The plasmid of the successfully digested positive subclone was sent to Qingke Technology Co., Ltd. for sequencing.

[0063] 1.2.1 Preparation of subcloning vectors

[0064] pWEB-TNC TM The strain was streaked onto LB solid medium (containing antibiotics Amp and Chl, with final concentrations of 100 μg / mL and 12.5 μg / mL, respectively). Single colonies were picked and concentrated in LB liquid medium by shaking overnight at 37°C and 225 r / min. The pWEB-TNCTM vector plasmid was extracted according to the plasmid miniprep kit instructions. The vector plasmid, after detection by agarose gel electrophoresis, was digested with the restriction endonuclease BamHI to obtain the linearized pWEB-TNCTM plasmid. The digestion reaction temperature was 37°C, and the time was 60 min. The digestion system (10 μL) is shown in Table 1.

[0065] Table 1 Enzyme digestion of pWEB-TNCTM plasmid

[0066]

[0067] After the reaction, the enzyme was inactivated at 65℃ for 20 min. Electrophoresis was performed and the band containing the target gene was recovered and purified using a gel extraction kit.

[0068] 1.2.2 Partial restriction enzyme digestion of positive clone plasmids

[0069] Incomplete digestion of cellulase-positive clone plasmids was performed using the restriction endonuclease Sau3A I, which had been diluted 500-fold with FD buffer. The digestion reaction was carried out at 37°C for 40 min. The digestion system (10 μL) is shown in Table 2.

[0070] Table 2. Partial enzyme digestion of positive clones.

[0071]

[0072] After the reaction was complete, the enzyme was inactivated at 65°C for 20 minutes. Then, the enzyme digestion system was subjected to agarose gel electrophoresis, and the DNA fragments of 2000-3000 bp were recovered by gel digestion according to DNAMaker.

[0073] 1.2.3 Connection and transformation of target fragment and vector

[0074] The purified pWEB-TNCTM vector was ligated with the recovered 2000-3000 bp DNA fragment using T4 ligase. The ligation system (10 μL) is shown in Table 3.

[0075] Table 3. Connection system between target fragment and vector

[0076]

[0077] The ligation system was incubated at 16℃ for 18-20 h, then the reaction was terminated by incubation at 65℃ for 20 min. The ligation system was then desalted in a glucose-agarose gel for 40-60 min. The desalted ligation system was mixed with 50 μL of *E. coli* EPI100 competent cells on ice and transferred to a pre-chilled electroporation vessel for 1.5 kV electroporation. Immediately after electroporation, 1 mL of antibiotic-free LB was added, mixed, and then transferred to a sterile EP tube. The mixture was incubated at 37℃ for 225 rpm for 50-60 min. Finally, the entire incubated bacterial culture was spread onto LB agar plates and cultured at 37℃ for 2-3 days. Cellulase-positive subclones were then screened using the screening method described in section 1.2.1.

[0078] 1.2.4 Subcloning plasmid sequencing

[0079] Positive subclones were cultured in liquid LB at 37°C and 227 rpm for 10–12 h, followed by plasmid extraction. The subclones were digested with BamHI (see Table 4 for the digestion system). Digestion was performed at 37°C for 30 min, followed by inactivation at 65°C for 20 min. Electrophoresis was used to verify the correctness of the inserted target fragment. If the inserted fragment was correct, the obtained cellulase-positive clone plasmids were sent to Kexin Technology Co., Ltd. for sequencing using vector primers pWEBcexuUS (5'-AAATAGGGGTTCCGCGCA-3') and pWEBcexuUS (5'-ATCCAGGGTGACGGTGCC-3').

[0080] Table 4. BamH I digestion subcloning plasmids

[0081]

[0082] 1.3 Sequence analysis of potential cellulase genes

[0083] The positive subclone sequencing results were analyzed using SnapGene Viewer 4.1.9.0 software, predicting a full-length ORF of 2118 bp (sequence information shown in SEQ ID NO.1). This sequence has a GC content of 69%, encodes a protein of 705 amino acids, has a predicted molecular weight of 75.3 kDa, and a predicted isoelectric point (pI) of 6.02. The predicted ORF was uploaded to the NCBI database for BALSTX homology comparison. The results showed that the protein encoded by this gene belongs to the 44th family of glycosidases (GH44), and shares 76.38% similarity with bacteria from the Pyromonaceae family (MBA3515422.1), 73.17% similarity with bacteria from the Armorobacteria phylum (MCC2671073.1), and 63.65% similarity with bacteria from the Acidobacteria phylum (AHL27901.1). The alignment results showed that the protein encoded by this ORF is a potential cellulase, and the gene was named zfeg1907. Multiple sequence alignment of the cellulase ZFEG1907 encoded by this gene, along with some cellulases from the GH44 family and other GH families, was performed using ClustalW, and a phylogenetic tree was constructed using MEGA5.1. Figure 3 As shown, by constructing an evolutionary tree, it was further determined that ZFEG1907 belongs to the GH44 family.

[0084] Example 2

[0085] 2.1 ZFEG1907 domain prediction and multiple alignment analysis

[0086] The ZFEG1907 sequence, i.e., the sequence shown in SEQ ID NO.1, was submitted to the SWISS-PROT database for protein structure analysis. The template with the highest similarity, 3ii1.1, was obtained, showing 76.25% identity with this template. The protein corresponding to this template is the GH44 family cellulase celM2 (ABL11223.1). Based on this template structure as a reference structure, multiple alignments were performed on ZFEG1907 with several GH44 family cellulase proteins using Clustal X 2.0 and ESPript 3.0. The results are as follows... Figure 4 As shown in the figure, cellulase ZFEG1907 has 14 α-helices and 26 β-sheets. Its core catalytic domain (amino acids 58-304) shows high similarity to the GH44 family, while the sequence after amino acid 520 shows low similarity to other proteins in the GH44 family. It is speculated that ZFEG1907 may possess other domains besides the core catalytic domain, playing specific roles in the catalytic process.

[0087] Therefore, the PFAM website was used to predict the potential catalytic and substrate-binding domains of ZFEG1907, and other cellulase domains of the GH44 family were also predicted and compared with ZFEG1907. The results showed that in addition to the catalytic domains of the GH44 family, the ZFEG1907 protein also contains PKD and Fn3 domains, such as... Figure 5 As shown in Table 5, the only cellulase similar to this domain is Lc-celH (AHL27901.1), which also comes from unculturable microorganisms. However, research on Lc-celH has only been limited to the gene level, and the properties of this enzyme and the specific functions of the related PKD-Fn3 domain have not been studied in depth. The nucleotide sequence of the PKD-Fn3 domain is shown in SEQ ID NO.3.

[0088] Table 5 PFAM Predictions for ZFEG1907

[0089]

[0090] Example 3

[0091] 3.1 Amplification of the cellulase gene:

[0092] To construct the plasmid expressing protein ZFEG1907, specific primers zfeg1907-F and zfeg1907-R (with restriction sites Nde I and Xho I) were designed for PCR amplification of the cellulase gene zfeg1907. To construct the plasmid expressing protein ZFEG1907t, specific primers zfeg1907t-F and zfeg1907t-R (with restriction sites Nde I and Xho I) were used for corrected PCR amplification of the cellulase gene zfeg1907 (using zfeg1907 as a template to remove the last 561 base pairs of nucleic acid, i.e., the last 186 amino acids at the C-terminus of cellulase ZFEG1907, truncated as follows). Figure 7 (As shown in B). After the PCR amplification reaction, agarose gel electrophoresis was performed. The electrophoresis results of the PCR amplification products are shown below. Figure 6 Lanes 1 and 2 are shown. Lane 1 shows the electrophoresis results of the zfeg1907 gene PCR amplification product; the bright band near 2000 bp corresponds to the 2118 bp cellulase zfeg1907 gene, and its nucleotide sequence is shown in SEQ ID NO. 1. Lane 2 shows the electrophoresis results of the zfeg1907t gene PCR amplification product; the bright band near 1500 bp corresponds to the 1557 bp cellulase zfeg1907t gene, and its nucleotide sequence is shown in SEQ ID NO. 2. After confirming the PCR amplification results were correct, the target bands were recovered and purified.

[0093] Primers for PCR amplification of the zfeg1907 and zfeg1907t genes were designed using Primer software. Nde I and Xho I restriction sites were introduced at both ends of zfeg1907 and zfeg1907t, respectively, leaving the histidine tag at the C-terminus to facilitate co-expression of the target proteins. The specific primer sequences are shown in Table 6.

[0094] Table 6 Primers for amplifying genes zfeg1907 and zfeg1907t

[0095]

[0096] The bold and underlined parts in the sequence are restriction endonuclease recognition sites.

[0097] Using the successfully constructed positive subclone plasmid as a PCR template, and with zfeg1907-F, zfeg1907-R, zfeg1907t-F, and zfeg1907t-R listed in Table 7 as upstream and downstream primers, the target genes zfeg1907 and zfeg1907t were amplified, respectively. The PCR amplification system (50 μL) is as follows:

[0098] Table 7. Cellulase Gene PCR Amplification System

[0099]

[0100] The PCR amplification program for the target genes zfeg1907 and zfeg1907t is as follows:

[0101]

[0102] After the PCR amplification reaction is completed, the PCR amplification products are subjected to electrophoresis to detect the amplified target gene fragment and purify the PCR amplification products.

[0103] Example 4

[0104] 4.1 Construction of recombinant plasmids pET-30a(+)-zfeg1907 and pET-30a(+)-zfeg1907t

[0105] The constructed recombinant plasmids pET-30a(+)-zfeg1907 and pET-30a(+)-zfeg1907t were verified by double enzyme digestion and electrophoresis. The results are as follows: Figure 6Lanes 3 and 4 are shown. Lane 3 shows the electrophoresis results of the recombinant plasmid pET-30a(+)-zfeg1907 after double digestion with NdeI and XhoI. The bands, from largest to smallest, correspond to the 5422 bp linear plasmid pET-30a(+) fragment and the 2118 bp zfeg1907 insert fragment. Lane 4 shows the electrophoresis results of the recombinant plasmid pET-30a(+)-zfeg1907t after double digestion with NdeI and XhoI. The bands, from largest to smallest, correspond to the 5422 kb linear plasmid pET-30a(+) fragment and the 1559 bp zfeg1907t insert fragment. Enzyme digestion verifies the successful construction of the recombinant plasmids pET-30a(+)-zfeg1907 and pET-30a(+)-zfeg1907t.

[0106] The successfully digested and validated pET-30a(+)-zfeg1907 and pET-30a(+)-zfeg1907t plasmids were sent to Qingke Company for sequencing. The sequencing results were compared with the zfeg1907 and zfeg1907t gene sequences, and the results showed 100% match. Sequencing confirmed the successful construction of the recombinant plasmids pET-30a(+)-zfeg1907 and pET-30a(+)-zfeg1907t.

[0107] 4.1.1 Ligation and Transformation of Recombinant Vector and Target Fragment

[0108] After gel recovery, the vector and target DNA fragment were added to each component according to the ligation system in Tables 8 and 9, and the system was gently mixed several times with a pipette tip. After incubating the above ligation system at 16℃ for 18-20 h, remove it and inactivate it at 65℃ for 20 min. Then, transform the ligation system into E. coli EPI100. After single colonies grow, pick single colonies and culture them in liquid LB at 37℃ and 225 r / min for 10-12 h. Extract plasmids and verify them by digestion with restriction endonucleases NdeI and Xho I (Table 10). Electrophoresis detection was performed. Recombinant plasmids with correct insertion fragments were sent to Kexin Technology Co., Ltd. for sequencing. The recombinant plasmids with correct sequences were transformed into E. coli BL21(DE3) to construct recombinant expression strains E. coli BL21(DE3) / pET-30a(+)-zfeg1907 and E. coli BL21(DE3) / pET-30a(+)-zfeg1907t.

[0109] Table 8. Ligation of pET-30a(+) vector with zfeg1907 gene fragment

[0110]

[0111] Table 9. Ligation of pET-30a(+) vector with zfeg1907t gene fragment

[0112]

[0113] Table 10 Double-enzyme digested recombinant plasmid pET-30a(+)-zfeg1907 / ET-30a(+)-zfeg1907t

[0114]

[0115]

[0116] 4.2 Induction and purification of cellulase gene

[0117] Recombinant plasmids pET-30a(+)-zfeg1907 and pET-30a(+)-zfeg1907t were induced to express at low temperature in liquid LB medium with the addition of IPTG as the inducer, and crude enzyme solutions were prepared accordingly. Cellulase activity was determined using the DNS method with 0.4% CMC-Na (sodium carboxymethyl cellulose) as the substrate (cellulase inactivated at 99℃ for 15 min served as a control). The results showed that no cellulase activity was detected in the non-induced expression group, while cellulase activity was detected in all induced groups.

[0118] Recombinant plasmids pET-30a(+)-zfeg1907 and pET-30a(+)-zfeg1907t were induced to express in *E. coli* BL21(DE3). Since the recombinant plasmid vectors contain six His tags, according to Ni... 2+ The target protein was purified based on its affinity for recombinant proteins with a His tag. Purification was confirmed by SDS-PAGE electrophoresis. The protein SDS-PAGE electrophoresis results are shown below. Figure 7 As shown in Figure A, lane 1 displays the electrophoresis results of purified cellulase ZFEG1907. The band near 70.0 kDa corresponds to a 75.3 kDa cellulase ZFEG1907 protein, indicating successful expression of the recombinant cellulase ZFEG1907 in *E. coli*. Lane 2 displays the electrophoresis results of purified cellulase ZFBG1907t. The band near 55 kDa corresponds to a 56.8 kDa cellulase ZFEG1907 protein, indicating successful expression of the recombinant cellulase ZFEG1907t in *E. coli*. The results show that pure cellulases ZFEG1907 and ZFEG1907t were successfully obtained after purification by nickel column affinity chromatography. The nucleotide sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0119] 4.21 Induced expression of recombinant plasmids pET-30a(+)-zfeg1907 and pET-30a(+)-zfeg1907t

[0120] Recombinant Escherichia coli E. coli BL21 / pET-30a(+)-zfeg1907 and E. coli BL21 / pET-30a(+)-zfeg1907t were concentrated overnight in liquid LB medium containing Kanamycin resistance. They were then transferred to 400 mL of liquid LB medium (Kanamycin resistance) at an inoculation ratio of 1:100 and cultured at 37℃ and 225 r / min for 2.5-3 h until the OD600 reached 0.4-0.6. The culture was then placed in ice water and allowed to cool slightly before adding the inducer IPTG (final concentration 0.4 mM). Fermentation was carried out at 20℃ and 180 r / min for 20-24 h.

[0121] 4.2.2 Affinity chromatography purification of cellulase

[0122] The fermentation broth after induced expression was transferred to a pre-chilled clean centrifuge bottle and centrifuged at 4500 rpm for 15 min in a pre-chilled low-temperature high-speed centrifuge. The supernatant was gently discarded, and 10 ml of Lysisi buffer was added to resuspend the cells. The cells were then transferred to centrifuge tubes. The cells were lysed using an ultrasonic cell disruptor (6 mm probe diameter). The processing conditions were: 20 min total processing time, 2 s processing time on ice, 2.5 s rest time. Finally, the disrupted cells were centrifuged at 12000 rpm for 30 min at 4 °C to separate the supernatant from the precipitate. The supernatant was collected and stored at 4 °C. The supernatant is the crude enzyme obtained from induced expression. Since the cellulase zfeg1907 and zfeg1907t genes already carry His tags, the crude enzyme solution can be purified using affinity chromatography coupled with nickel ions. The specific steps are as follows.

[0123] (1) Rinse the chromatography column with 5-10 column volumes of ddH2O;

[0124] (2) Rinse the chromatography column with 5-10 column volumes of Lysisi buffer to equilibrate the nickel column;

[0125] (3) Add the crude enzyme solution to the chromatography column. When adding the sample, pay attention to controlling the flow rate of the enzyme solution in the chromatography column to ensure that His and Ni in the enzyme solution are in good condition. 2+ Fully integrate;

[0126] (4) Pass the column through 5-10 column volumes of washing buffer to wash away impurities in the crude enzyme;

[0127] (5) The column was passed through different concentrations of imidazole solution (50mM, 100mM, 200mM) and the components were collected.

[0128] (6) After elution, use 5-10 column volumes of ddH2O, then use 3-5 column volumes of 20% ethanol to pass through the column, and leave 3 mL of ethanol for sealing.

[0129] Example 5: Effect of Cellulose Recombinase Activity and Stability Test

[0130] 5.1 Effect of temperature on the activity and stability of cellulase recombinase

[0131] The results are as follows Figure 8 As shown, the enzyme activities of cellulase ZFEG1907 (nucleotide sequence as shown in SEQ ID NO.1) and its truncated form ZFEG1907t (nucleotide sequence as shown in SEQ ID NO.2) gradually increase with increasing temperature, exhibiting high activity in the range of 40℃-50℃, with maximum activity at 50℃. Above 50℃, the activity of cellulase decreases with increasing temperature. At 70℃, the enzyme activity of cellulase ZFEG1907 decreases to 43% of its maximum activity, and the enzyme activity of cellulase ZFEG1907t decreases to 61% of its maximum activity. In summary, the optimal reaction temperature for both cellulase ZFEG1907 and its truncated form ZFEG1907t is 50℃, classifying them as thermophilic enzymes with broad industrial application prospects.

[0132] The results of enzyme temperature stability are as follows: Figure 9 As shown, after incubation at 20-40℃ for 1 hour, the residual cellulase activity of both ZFEG1907 and the truncated ZFEG1907t can maintain more than 80% of the activity. When the temperature is higher than 50℃, the enzyme activity of both enzymes begins to show a significant downward trend. For example, after incubation at 60℃ for 1 hour, the residual enzyme activity of both enzymes is less than 10%, and they are basically inactive. This indicates that the two enzymes have good thermal stability at temperatures below 40℃.

[0133] This experiment employed the classic Miller method to determine the hydrolytic activity of cellulase. This method uses DNS (3,5-dinitrosalicylic acid) reagent to quantitatively detect the content of reducing sugars in the reaction system. By measuring the amount of reducing sugars released from the polysaccharide substrate, the hydrolytic activity of cellulase on the polysaccharide substrate is determined. The main experimental principle is as follows: cellulase catalyzes the hydrolysis of cellulose to produce reducing sugars such as glucose and cellobiose. Cellulase catalyzes the hydrolysis of sodium carboxymethyl cellulose (CMC-Na) polysaccharide substrate to produce reducing sugars such as glucose and cellobiose. The reducing sugars reduce the nitro group in 3,5-dinitrosalicylic acid (DNS) to an amino group, generating the product 3-amino-5-nitrosalicylic acid. This product appears brownish-red after boiling and shows a high OD value. 540The maximum absorption peak is observed at a certain concentration, and within a certain concentration range, the intensity of the brownish-red color is directly proportional to the reducing sugar content in the sample. The reducing sugar content is determined colorimetrically, indirectly revealing the cellulase activity.

[0134] Experimental methods

[0135] Based on the DNS colorimetric principle in section 4.2.1, the specific procedure for cellulase activity assay is as follows: Using a pipette, take 60 μL of 0.4% (w / v) CMC-Na solution corresponding to the corresponding pH and add 5 μL of diluted, pre-chilled enzyme solution. Mix thoroughly (all operations should be performed on ice). Use an inactivated enzyme solution (99℃ for 15 min) as a control. Incubate the PCR instrument at 50℃ for 30 min. After the reaction, add 60 μL of DNS solution to the ice-based reaction system and mix thoroughly. Incubate at 99℃ for 15 min, then immediately place the PCR tube on ice. Add 100 μL of the reaction solution to an ELISA plate, mix gently, and then measure the OD. 540 Value. OD of the experimental group. 540 Value minus control group OD 540 Each experiment was repeated three times. The average value was calculated. The cellulase activity was calculated based on the glucose standard curve.

[0136] Enzyme activity (U) is defined as the amount of enzyme required to break down a polysaccharide substrate to produce 1 μmol of glucose per unit time under specific conditions (25℃, other conditions being optimal).

[0137] Formula for calculating enzyme activity:

[0138]

[0139] Where: C - concentration of purified enzyme solution (μg / mL); V - volume of enzyme solution added to the reaction system (μL); M - molar mass of glucose; N - dilution factor of purified enzyme solution; m - reducing sugar content (mg) (from OD) 540 The value is obtained from the standard curve; T - test reaction time (min).

[0140] 5.2 Effect of pH on the activity and stability of cellulase recombinase

[0141] In this experiment, the buffer solution with pH 3.0-6.0 was prepared using a citrate-sodium citrate buffer system, the buffer solution with pH 6.0-8.0 was prepared using a phosphate buffer system, and the buffer solution with pH 8.0-9.0 was prepared using Tris-HCl solution. Cellulase is an amphoteric compound, and its structure mostly contains a large number of basic amino and acidic carboxyl groups. Therefore, proteases exhibit different dissociation states in different pH environments. The pH value of the solution in the most suitable dissociation state for catalytic reaction is the optimal reaction pH for the enzyme. Through a series of enzymatic reactions, the experimental results are as follows: Figure 10 The optimal reaction pH for both cellulase ZFEG1907 and its truncated form ZFEG1907t is 5.0. Above pH 5.0, enzyme activity decreases with increasing pH; below pH 5.0, enzyme activity decreases with decreasing pH. At pH 8.0-9.0, ZFEG1907t retains 30% of its activity, while ZFEG1907 retains only 10%.

[0142] The pH stability results of the cellulase recombinase are as follows: Figure 11 As shown, the pH stability of cellulase ZFEG1907 and its truncated form ZFEG1907t is generally similar. Both are relatively stable in the pH range of 4.0-8.0, maintaining more than 50% of their activity. Cellulase ZFEG1907 retains more than 40% of its activity after treatment at pH 3.0 and pH 9.0, while the activity of the truncated form ZFEG1907t is below 30% after treatment at both pH 3.0 and pH 9.0. This indicates that the pH stability of cellulase ZFEG1907 is stronger than that of its truncated form ZFEG1907t.

[0143] 5.3 Effects of temperature, pH, metal ions and chemical reagents on recombinant enzymes

[0144] The appropriately diluted cellulase sample, i.e., the purified cellulase solution, was mixed with 0.4% (w / v) CMC-Na substrate at pH 5.0 and incubated at 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ respectively. The effect of different temperatures on cellulase activity was studied by measuring the relative enzyme activity under different temperature conditions (the DNS method was used for enzyme activity determination). The highest enzyme activity was set as 100%, and the remaining values ​​were converted to percentages to plot the relative enzyme activity curves.

[0145] The appropriately diluted cellulase samples were mixed with 0.4% (w / v) CMC-Na solution and reacted in buffer solutions ranging from pH 2.0 to 9.0 (with a gradient of 1 pH unit) at 50°C. The effect of different pH values ​​on cellulase activity was studied by measuring relative enzyme activity. The highest enzyme activity was set as 100%, and the remaining values ​​were converted to percentages to plot relative enzyme activity curves.

[0146] The moderately diluted cellulase sample was incubated at 20-80℃ for 1 hour. The incubated enzyme solution was then mixed with 0.4% (w / v) CMC-Na substrate at pH 5.0 for enzymatic reaction. The biological temperature stability of cellulase was studied by measuring residual enzyme activity. Enzyme activity was measured, with enzyme activity at 4℃ considered 100%.

[0147] Appropriately diluted cellulase samples were incubated at 4°C for 2 hours in buffer solutions ranging from pH 2.0 to 9.0 (with a pH gradient of 1 unit). After incubation, the samples were mixed with 0.4% (w / v) CMC-Na solution and reacted at 50°C. pH stability was investigated by measuring residual enzyme activity. Enzyme activity was determined according to method 4.2.2, with the enzyme activity of the solution incubated at 4°C representing 100%.

[0148] Metal ions (including Li) were added to a reaction system of moderately diluted cellulase sample and 0.4% (w / v) CMC-Na at final concentrations of 1 mM and 10 mM. + Mg 2+ Ca 2+ Mn 2+ K + Na + Cu 2+ Fe 3+ Zn 2+ The effects of different metal ions on cellulase activity were studied by measuring residual enzyme activity under optimal enzymatic reaction conditions. The control group consisted of cells without any added metal ions, with enzyme activity set at 100%.

[0149] The effects of chemical reagents on cellulase activity were investigated by adding appropriately diluted cellulase samples to a 0.4% (w / v) CMC-Na reaction system at final concentrations of 1% and 15% (including DMSO, DMF, acetonitrile, ethanol, methanol, isopropanol, acetone, and ethyl acetate), and final concentrations of 1 mM and 10 mM (EDTA, SDS, urea, and imidazole), respectively. The residual enzyme activity was measured under optimal enzymatic reaction conditions. The control group consisted of samples without any added chemical reagents, with enzyme activity set at 100%.

[0150] 5.3 Effects of metal ions and chemical reagents on the activity of cellulosic recombinase

[0151] Different metal ions and chemical reagents can cause changes in the structure of cellulase, thereby promoting or inhibiting its activity. Various metal ions were added to the reaction system at final concentrations of 1 mM and 10 mM, and the residual enzyme activity in the enzymatic reaction was measured to reflect the effects of different metal ions on the activity of ZFEG1907 and the truncated ZFEG1907t enzyme. Figure 12 As shown, 1 mM metal ions except Cu 2+ and Mn 2+ Both ZFEG1907 and the truncated ZFEG1907t enzyme showed some inhibitory activity. Mg 2+ and Ca 2+ While all metal ions have a certain promoting effect on the activity of ZFEG1907t enzyme, other metal ions have no significant effect on the activity of this enzyme. Figure 13 The 10mM metal ions shown are excluding Cu 2+ Li + and Mn 2+ Both ZFEG1907 and the truncated ZFEG1907t enzyme have a certain inhibitory effect on enzyme activity. 2+ K + and Ca 2+ Both Mg and Mg have a certain promoting effect on the enzyme activity of ZFEG1907. 2+ and Ca 2+ While all metal ions have a certain promoting effect on the activity of ZFEG1907t enzyme, other metal ions have no significant effect on the enzyme's activity. Therefore, ZFEG1907 and its truncated form, ZFEG1907t, exhibit moderate tolerance to these metal ions. Metal ions can regulate enzyme activity by forming covalent bonds with nucleophilic residues of certain amino acids. If these amino acids bind to the enzyme's active site, the resulting metal-amino acid complex will inhibit enzyme activity. Some metal ions can enhance enzyme activity. For example, calcium interacts with electron-donating amino acid residues and nitrogen atoms in peptide bonds, even though these nitrogen atoms are not necessarily incorporated into the enzyme's active site.

[0152] Various organic reagents were added to the reaction system to final concentrations of 1% and 15%, respectively. The effects of different organic reagents on the enzyme activities of ZFEG1907 and truncated ZFEG1907t were assessed by measuring the residual enzyme activity in the enzymatic reaction. Figure 14 As shown, 1% organic solvents, except for acetonitrile, ethanol, acetone, and methanol, all had a certain inhibitory effect on the activity of ZFEG1907 enzyme, while acetonitrile, acetone, methanol, and ethyl acetate had a certain promoting effect on the activity of ZFEG1907 enzyme. Other chemical reagents had no significant effect on the activity of this enzyme. Figure 15As shown, 15% organic solvent significantly inhibited both cellulase ZFEG1907 and its truncated form ZFEG1907t, with ZFEG1907 exhibiting worse tolerance to 15% organic solvent. This indicates that ZFEG1907t enzyme is more tolerant to 15% organic solvent than ZFEG1907 enzyme. Figure 16 As shown, EDTA, as a metal chelating agent, did not significantly inhibit the enzyme activities of ZFEG1907 and ZFEG1907t regardless of concentration, indicating that this enzyme is not a metalloenzyme. SDS, as a strong denaturant, significantly inhibited the enzyme activities of both ZFEG1907 and ZFEG1907t regardless of concentration. Low concentrations of imidazole (1 mM, 10 mM) had little effect on the enzyme activities of ZFEG1907 and ZFEG1907t, but a concentration of 100 mM significantly inhibited their activities. Urea at both 10 mM and 100 mM had a certain promoting effect on the enzyme activities of both.

[0153] Example 6

[0154] 6.1 Detection of substrate specificity and kinetic parameters of cellulase recombinase

[0155] The enzyme activity was measured after reacting appropriately diluted cellulase samples with 0.4% (w / v) of substrates (including sodium carboxymethyl cellulose (CMC-Na), konjac gum (KGM), lichen polysaccharide (Lichenan), microcrystalline cellulose, locust bean gum (LBG), xylan, dextran, poria cocos polysaccharide, guar gum, chitin, and pNPG) under optimal conditions (pH 5.0, temperature 50℃), and the substrate specificity was analyzed.

[0156] Appropriately diluted cellulase samples were reacted with substrates (CMC-Na, KGM, Lichenan) at different concentrations (0.1% (w / v), 0.2% (w / v), 0.3% (w / v), 0.4% (w / v), 0.5% (w / v)) under optimal conditions (pH 5.0, temperature 50℃). Enzyme activity was then measured, and the Km and Vmax values ​​of the recombinant enzyme were determined using the Michaelis-Menten equation double reciprocal method. m Value, K mm / k cat value.

[0157] The substrate specificity of cellulase ZFEG1907 and its truncated form ZFEG1907t, measured under optimal conditions (pH 5.0, 50℃), is shown in Table 11. Cellulase ZFEG1907 and its truncated form ZFEG1907t exhibited similar substrate specificity. Both enzymes showed degradation activity only on four substrates: sodium carboxymethyl cellulose, konjac gum, lichen polysaccharide, and microcrystalline cellulose. The highest activity was observed for konjac gum, followed by lichen polysaccharide and sodium carboxymethyl cellulose. No degradation activity was observed for locust bean gum, xylan, dextran, poria cocos polysaccharide, guar gum, chitin, or pNPG. The specific activities of recombinant cellulase ZFEG1907 and its truncated form ZFEG1907t against 0.4% (w / v) konjac gum were 27.79±0.12 U / mg and 25.53±0.17 U / mg, respectively; against 0.4% (w / v) lichen polysaccharide, they were 13.17±0.07 U / mg and 15.03±0.32 U / mg, respectively; against 0.4% (w / v) sodium carboxymethyl cellulose, they were 10.24±0.15 U / mg and 11.82±0.20 U / mg, respectively; and against 0.4% (w / v) microcrystalline cellulose, they were 3.24±0.22 U / mg and 4.56±0.21 U / mg, respectively. Based on the specific activity data, the truncated form ZFEG1907t showed little difference in specificity to the four substrates compared to ZFEG1907. This indicates that the PKD-Fn3 domain has almost no effect on the substrate specificity of cellulase ZFEG1907.

[0158] Based on the recombinant enzyme substrate specificity detection results, three substrates (CMC-Na, KGM, and Lichenan) with high specific hydrolytic activity of ZFEG1907 and its truncated form ZFEG1907t were selected for enzyme kinetic determination. The Kk of cellulase for each substrate was obtained by hyperbolic fitting of gradient substrate concentrations and corresponding enzyme reaction rates using Origin software. m (substrateaffinity), Vmax (maximum reaction velocity), k cat (turmover rate) and k cat / K m The catalytic efficiency was measured, and the results are shown in Table 12. Enzyme kinetic parameters not only reflect the affinity between the enzyme and the substrate but also measure the catalytic efficiency of the enzyme on the substrate. When CMC-Na was used as the substrate, cellulase ZFEG1907t exhibited a faster catalytic reaction rate, with a Vmax value 74.11% higher than that of ZFEG1907. Meanwhile, ZFEG1907 had a smaller Kc. m Value, its K mThe value is 42.27% lower than that of ZFEG1907t. The kinetic constant K... m The value K represents the affinity between the enzyme and the substrate. m The smaller the value, the greater the affinity; therefore, ZFEG1907 has a stronger affinity for CMC-Na substrates than ZFEG1907t. cat The value refers to the number of substrate molecules converted into product molecules by an enzyme molecule per unit time. From the results, cellulase ZFEG1907 and its truncated form ZFEG1907t showed differences in k... cat The values ​​are not significantly different, with ZFEG1907t being slightly higher; k cat / K m This is usually used to measure the catalytic efficiency of an enzyme; different enzymes react with the same substrate k. cat / K m The values ​​can vary greatly depending on the enzyme's response to different substrates. cat / K m The differences in values ​​can also be very large. The results show that ZFEG1907 has a faster catalytic reaction efficiency, k... cat / K m The value is 35.85% higher than that of ZFEG1907t.

[0159] When KGM is used as the reaction substrate, cellulase ZFEG1907t also exhibits a faster catalytic reaction rate, with a Vmax value 19.48% higher than that of ZFEG1907, but ZFEG1907 has a smaller K value. m Value, its K m The value was 33.79% lower than that of ZFEG1907t, indicating that ZFEG1907 has a stronger affinity for KGM substrates; the two enzymes at k cat The values ​​are not significantly different, with ZFEG1907 being slightly higher; ZFEG1907 has a faster catalytic reaction efficiency, k cat / K m The value is 70.08% higher than that of ZFEG1907t. This indicates that ZFEG1907 exhibits higher catalytic efficiency when KGM is used as the reaction substrate.

[0160] When Lichenan is used as the reaction substrate, ZFEG1907t also exhibits a faster catalytic reaction rate, with a Vmax value 86.67% higher than that of ZFEG1907, but ZFEG1907 has a smaller K value. m Value, its K m The value is 61.62% lower than that of ZFEG1907t, indicating that ZFEG1907 has a stronger affinity for Lichenan substrates; ZFEG1907 has a faster catalytic reaction efficiency, k cat / K mThe value is 92.73% higher than that of ZFEG1907t, which shows that ZFEG1907 has higher catalytic efficiency when Lichenan is used as the reaction substrate.

[0161] The above experimental results strongly demonstrate that the PKD-Fn3 domain can enhance the catalytic efficiency of cellulase ZFEG1907 by improving its binding affinity to three substrates: CMC, KGM, and Lichenan. Therefore, the PKD-Fn3 domain of cellulase ZFEG1907 helps promote the binding of the enzyme to macromolecular substrates and improves the enzyme-substrate affinity.

[0162] Table 11. Substrate specificity detection of ZFEG1907 and truncated ZFEG1907t enzymes

[0163]

[0164] The above data represents the mean ± standard deviation; ND: Not detected.

[0165] The values ​​shown are mean±standard deviation; ND: not detected.

[0166] Table 12 Detection of enzyme kinetic parameters of ZFEG1907 and truncated ZFEG1907t

[0167]

[0168]

[0169] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

[0170]

[0171]

Claims

1. A cellulase gene, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.1, or the nucleotide sequence is obtained by removing the substrate-binding domain from the nucleotide sequence shown in SEQ ID NO.1, leaving the remaining catalytic functional sequence, the catalytic functional sequence is shown in SEQ ID NO.2, the nucleotide sequence shown in SEQ ID NO.2 is obtained by removing the last 561 base pairs of nucleic acid from the nucleotide sequence shown in SEQ ID NO.

1.

2. A substrate-binding domain gene for a cellulase, characterized in that: The substrate-binding domain is PKD-Fn3, and the nucleotide sequence of PKD-Fn3 is shown in SEQ ID NO.

3.

3. The application of the substrate-binding domain gene as described in claim 2 in promoting the binding of enzymes to macromolecular substrates, improving the affinity between enzymes and substrates, and improving the catalytic efficiency of enzymes.

4. The application according to claim 3, characterized in that: The substrates include at least sodium carboxymethyl cellulose, konjac gum, and lichen polysaccharide.

5. A cellulase, characterized in that: Its amino acid sequence is encoded by the nucleotide sequence described in claim 1.

6. A recombinant vector, characterized in that, It contains the nucleotide sequence shown in claim 1.

7. The recombinant vector according to claim 6, characterized in that, The expression vector for this recombinant vector is Escherichia coli.

8. The application of the cellulase according to claim 5 in the degradation of sodium carboxymethyl cellulose, konjac gum, lichen polysaccharide or microcrystalline cellulose, and sodium carboxymethyl cellulose.

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