Beta-agarase mutant based on disulfide bond modification and application thereof

Abstract

The invention provides a beta-agarase mutant based on disulfide bond modification and application thereof, the mutant is obtained by performing amino acid modification and replacement on an amino acid sequence of beta-agarase Y3R1 as shown in SEQ ID NO: 1, and the amino acid modification and replacement position is S124C-K230C. Compared with wild beta-agarase Y3R1, the mutant enzyme activity is increased by 57.6%, and the thermal stability of the enzyme is improved by 15.0% after treatment at 80 DEG C for 1 h.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a beta-agarase mutant based on disulfide bond modification and application thereof. Background Art

[0002] β-Agarase plays an important role in the enzymatic degradation of agar and is used in the preparation of agar oligosaccharides, the preparation of seaweed protoplasts, and the extraction of bioactive substances from seaweed. However, the thermal stability of most natural agarases is not enough to meet the needs of industrial production, so it is necessary to modify their proteins. β-Agarase YM01-3 from Streptomyces agarophilus has high enzyme activity, but its thermal stability is poor.

[0003] The thermal stability of enzymes can be improved through protein engineering. Rational modification of enzyme structure is an effective way to improve enzyme properties. There is no improvement in the thermal stability of β-agarase by introducing additional disulfide bonds and some non-covalent bonds in the related art. Summary of the invention

[0004] The present invention aims to solve one of the technical problems in the above-mentioned technology at least to a certain extent. To this end, the present invention provides a β-agarase mutant based on disulfide bond modification and its application, and the thermal stability of the mutant is improved.

[0005] To this end, in the first aspect of the present invention, a disulfide bond-modified β-agarase mutant is provided, wherein the mutant is subjected to amino acid modification substitution on the amino acid sequence of β-agarase Y3R1 shown in SEQ ID NO: 1, and the amino acid modification substitution position is S124C-K230C.

[0006] According to the present invention, a β-agarase mutant based on disulfide bond modification is introduced into the β-agarase mutant by introducing an additional disulfide bond on the amino acid sequence of 313 amino acids as shown in SEQ ID NO: 1, and the amino acid modification substitution position is S124C-K230C; the thermal stability and enzyme activity of the mutant are improved; compared with the wild-type β-agarase Y3R1, the enzyme activity is increased by 57.6%. After being treated at 80°C for 1 h, the thermal stability of the enzyme is increased by 15.0%.

[0007] Optionally, the amino acid sequence of the mutant is shown in SEQ ID NO:2.

[0008] In the second aspect of the present invention, a gene encoding the above-mentioned β-agarase mutant is provided.

[0009] In the third aspect of the present invention, a construct is provided, which comprises a gene encoding the above-mentioned β-agarase mutant.

[0010] In a fourth aspect of the present invention, a recombinant cell expressing the above-mentioned β-agarase mutant, or containing the above-mentioned gene, or containing the above-mentioned construct is provided.

[0011] In a fifth aspect of the present invention, a method for constructing the above-mentioned β-agarase mutant is provided, which comprises the following steps: (1) The three-dimensional structure of β-agarase Y3R1 was submitted to the online server DBD 2.0. Under the condition of bond energy (χ3 angle) of +97 (± 20), the DBD 2.0 tool was used to predict candidate residue pairs suitable for additional disulfide bond formation, and mutants with larger B factors were selected as candidate results. Similarly, the Predict DisulfideBridges tool in Discovery Studio 2019 was used to predict the formation of additional disulfide bonds by cysteine ​​substitution, and residue pairs with lower energy after mutation were selected to screen out 4 disulfide bond mutants. (2) designing site-directed mutagenesis primers, and performing site-directed mutagenesis on the gene of β-agarase Y3R1 whose amino acid sequence is shown in SEQ ID NO: 1 by PCR to obtain a recombinant vector containing the gene sequence of the β-agarase mutant; (3) Transform the mutated recombinant vector into Escherichia coli E. coli In BL21(DE3), the expression was induced and purified to obtain four double-point mutation β-agarase mutants; (4) The enzymatic properties of four double-point mutation β-agarase mutants were analyzed, and the β-agarase mutant S124C-K230C with improved enzyme activity and thermal stability was screened out.

[0012] Optionally, the four disulfide bond mutants are K82C-K87C, E106C-G190C, S111C-A136C, and S124C-K230C.

[0013] Further, the amplification primer sequences for mutants K82C-K87C are SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8; The amplification primer sequences for mutant E106C-G190C are SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; The amplification primer sequences for mutant S111C-A136C are SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16; The amplification primer sequences of mutant S124C-K230C are SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

[0014] In the sixth aspect of the present invention, there is provided the use of the above-mentioned β-agarase mutant, or the above-mentioned encoding gene, or the above-mentioned construct, or the above-mentioned recombinant cell in degrading agar or preparing agar oligosaccharides.

[0015] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is an agarose gel electrophoresis analysis of the β-agarase gene according to an embodiment of the present invention, wherein M: Marker; 1-3: Y3R1 engineering strain; 4-6: YM01-3 engineering strain; Figure 2 The disulfide bond mutation site of β-agarase Y3R1 selected according to the embodiment of the present invention; Figure 3 SDS-PAGE analysis of β-agarase Y3R1 and its mutants according to the embodiments of the present invention; Figure 4 is the enzyme activity of β-agarase Y3R1 and its mutants according to the examples of the present invention; Figure 5 The thermal stability of β-agarase Y3R1 and its mutants according to the embodiments of the present invention; Figure 6 The tertiary structure analysis of β-agarase Y3R1 and its mutant S124C-K230 according to the embodiments of the present invention, wherein (A): fluorescence spectrum analysis of S124C-K230C; (B): surface hydrophobicity analysis of S124C-K230C; Figure 7 Molecular docking (AB) and hydrophobic interaction (C) between S124C-K230C and neoagarotetrose according to an embodiment of the present invention; Figure 8 is the Rg value of β-agarase Y3R1 and its mutant S124C-K230C at 338 K according to an embodiment of the present invention; Fig. 9 is the RMSD value of β-agarase Y3R1 and its mutant S124C-K230C at 338 K according to the embodiments of the present invention; Fig.10RMSF values ​​of β-agarase Y3R1 and its mutant S124C-K230C at 338 K according to an embodiment of the present invention, wherein (A): RMSF values ​​of S124C-K230C and WT at 338 K; (B): flexibility change of S124C-K230C relative to WT; wherein red and blue are regions with increased and decreased flexibility, respectively; Fig.11 SASA value of β-agarase Y3R1 and its mutant S124C-K230C at 338 K according to an embodiment of the present invention; Fig.12 LC-MS analysis of the S124C-K230C enzymatic hydrolysis product according to an embodiment of the present invention. DETAILED DESCRIPTION

[0017] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0018] The disclosure below provides many different embodiments or examples to realize different implementations of the present invention. In order to simplify the disclosure of the present invention, specific embodiments or examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the examples of various specific processes and materials provided by the present invention, those of ordinary skill in the art can be aware of the applicability of other processes and / or the use of other materials. Unless otherwise stated, the implementation of the present invention will adopt the traditional techniques in the fields of chemistry, molecular biology, etc. within the capabilities of those skilled in the art. In addition, unless otherwise stated, in this article, nucleic acids are written from left to right in the direction of 5' to 3', and amino acid sequences are written from left to right in the direction of amino terminal to carboxyl terminal.

[0019] In the following examples, the online servers and software used are shown in Table 1.

[0020] Table 1 Online servers and software

[0021] In the following examples, YM01-3 has a catalytic N-terminal domain (GH) and an R-terminal domain of unknown function (subdivided into R1 and R2 domains), and YM01-3 has 7 α-helices and 30 β-pleated sheets, numbered α1–α7 (Table 14) and β1–β30 (Table 15), respectively.

[0022] Table 14 Amino acid numbering of the α-helical region of agarase YM01-3

[0023] Table 15 Amino acid numbering of the β-folded region of agarase YM01-3

[0024] The present invention is described below by means of illustrative specific examples, which are not intended to limit the scope of the present invention in any way. It is particularly noted that the reagents used in the present invention are commercially available unless otherwise specified.

[0025] Example 1 Construction of β-agarase Y3R1 gene recombinant strain (1) Primer design. Using the recombinant plasmid pET-28a (+) / YM01-3 containing the wild-type enzyme gene fragment (GenBank accession number: No. KF413621.1) as the template, restriction sites were added at the 5' and 3' ends respectively. Bam HI and XOt I. Design primers on the primer design website (https: / / crm.vazyme.com / cetool / simple.html) and construct β-agarase Y3R1 according to the CD-Search analysis on the NCBI website. The primers for β-agarase Y3R1 are shown in Table 2.

[0026] Table 2 β-Agarase Y3R1 Primers

[0027] (2) PCR amplification of β-agarase Y3R1 gene. The recombinant plasmid pET-28a (+) / YM01-3 containing the wild-type enzyme gene fragment (GenBank accession number No. KF413621.1) was used as a template to amplify the target Y3R1 gene using PCR technology. The amplification reaction system and reaction conditions are shown below, where the specific upstream and downstream primers were purchased from Borui Biotechnology Co., Ltd. Plasmid extraction was performed according to the instructions of the bacterial plasmid mini-extraction kit of TianGen.

[0028] Table 3 Amplification reaction system

[0029] Table 4 Amplification reaction conditions

[0030] Refer to the instructions of TianGen's DNA purification kit to recover the target gene fragment and store it at -20 ºC.

[0031] (3) Double restriction enzyme digestion reaction of target gene and expression plasmid. The reaction conditions are as follows: mix the reagents, centrifuge briefly, and react at 37 ºC for 1 h. After the double restriction enzyme digestion reaction, use a DNA purification kit to recover the DNA fragments, and use an ELISA reader to measure the concentration of the recovered products. The reaction system is shown in Table 5.

[0032] Table 5 Double enzyme digestion reaction system

[0033] (4) Ligation reaction. The reaction conditions are as follows: after the reagents are mixed, centrifuge briefly and react at 16°C for 40 min; after the ligation reaction, use a DNA purification kit to recover the DNA fragments, then use 1% agarose gel electrophoresis to detect the target gene bands, and use an enzyme reader to determine the concentration of the recovered product. The reaction system is shown in Table 6.

[0034] Table 6 Ligation reaction system

[0035] (5) Conversion reaction. Thaw on ice E. coli DH5α or E. coli BL21 (DE3) competent cells. Add 20 μL of the ligation product to 100 μL of competent cells and place on ice for 30 min. Heat shock in a water bath at 42 °C for 90 sec, and immediately place on ice to cool for 2-3 min. Add 900 μL of LB liquid medium (without kanamycin) and shake at 37 °C for 60 min. Centrifuge at 3000-3500 rpm for 3 min. Remove 800 μL of supernatant and resuspend the cells with a pipette. Use a sterile spreader to spread the bacterial solution onto an LB solid medium plate (containing 50 μg / mL of kanamycin sulfate). Place the plate in a 37 °C incubator and invert for 12-16 h.

[0036] (6) Identification of positive transformants. Pick a single colony from the overnight culture and place it in 10 mL LB liquid medium (containing kanamycin sulfate) for expansion culture for 12-16 h. Perform PCR on the bacterial solution. The PCR reaction system and reaction conditions are shown in Tables 7 and 8. Use 1% agarose gel electrophoresis to detect the size of the PCR product. The results are as follows: Figure 1 As shown, the gene fragment size of β-agarase Y3R1 is between 1000 bp and 750 bp, which is consistent with the theoretical value of 939 bp. The bacterial solution was sent to Xiamen Borui Biotechnology Co., Ltd. for sequencing identification, and the sequencing results were analyzed using DNAMAN 9.0 software. The sequencing results showed that the target sequence of the inserted gene was consistent with the theoretical sequence.

[0037] Table 7 Amplification reaction system

[0038] Table 8 Amplification reaction conditions

[0039] Example 2 Selection of disulfide bond mutation sites in β-agarase The three-dimensional structure of β-agarase Y3R1 was submitted to the online server DBD 2.0. Under the condition of bond energy (χ3 angle) of +97 (± 20), the DBD 2.0 tool was used to predict candidate residue pairs suitable for the formation of additional disulfide bonds, and mutants with larger B factors were selected as candidate results. Similarly, the Predict DisulfideBridges tool in Discovery Studio 2019 was used to predict the formation of additional disulfide bonds by cysteine ​​substitution, and residue pairs with lower energy after mutation were selected. Based on the predictions of the two methods, four commonly predicted disulfide bond mutants (K82C-K87C, E106C-G190C, S111C-A136C, S124C-K230C) were screened out. The screening and analysis results are shown in Table 9. The selected disulfide bond mutation sites are as follows. Figure 2 shown.

[0040] Table 9 Analysis results of DBD 2.0 and DiscoveryStudio 2019

[0041] Example 3 Construction of mutant β-agarase recombinant strain The recombinant plasmid containing β-agarase Y3R1 gene (pET-28a-aga) E. coli After culturing the DH5α strain (obtained in Example 1) in LB liquid medium (containing 50 μg / mL kanamycin), the plasmid was extracted according to the instructions of the bacterial plasmid extraction kit of TianGen. Referring to the instructions of the Mut ExpressⅡ Fast Mutagenesis Kit V2 site-directed mutagenesis kit, the primer sequences designed according to the selected amino acid sites to be mutated (obtained in Example 2) are shown in Table 10.

[0042] Table 10 Mutation Primers

[0043] Note: Lowercase letters indicate mutation sites.

[0044] Using the β-agarase Y3R1 recombinant plasmid as a template, a PCR reaction was performed to amplify the mutant enzyme gene. The PCR reaction system is shown in Table 11, and the PCR reaction conditions are: pre-denaturation at 95°C for 30s; denaturation at 95°C for 15s; annealing at 62°C for 15s; extension at 72°C for 60s; denaturation, annealing, and extension as one cycle, 30 cycles; complete extension at 72°C for 5 min, and storage at 4°C.

[0045] Table 11 PCR reaction system

[0046] Digestion reaction: After the PCR reaction is completed, since the amplified product contains the original plasmid template, in order to prevent the formation of false positive transformants, 1 μL of Dpn Ⅰ digestion to remove the methylated plasmid template.

[0047] Recombination reaction: remove 4 μL of the final product, add 4 μL of 5×CE Ⅱ Buffer, 2 μL of Exnase Ⅱ, and 10 μL of sterile water, centrifuge briefly in a small centrifuge, mix well, react at 37°C for 30 min, and then store at 4°C.

[0048] Transformation reaction: Thaw on ice E. coli DH5α or E. coli BL21 (DE3) competent cells. Add 20 μL of the ligation product to 100 μL of competent cells and place on ice for 30 min. Heat shock in a water bath at 42 °C for 90 sec, and immediately place on ice to cool for 2-3 min. Add 900 μL of LB liquid medium (without kanamycin) and shake at 37 °C for 60 min. Centrifuge at 3000-3500 rpm for 3 min. Remove 800 μL of supernatant and resuspend the cells with a pipette. Use a sterile spreader to spread the bacterial solution onto an LB solid medium plate (containing 50 μg / mL of kanamycin sulfate). Place the plate in a 37 °C incubator and invert for 12-16 h.

[0049] Identification of positive transformants: Pick a single colony from overnight culture and place it in 10 mL LB liquid medium (containing kanamycin sulfate) for expansion culture for 12-16 hours. Perform PCR on the bacterial solution. The PCR reaction system and reaction conditions are shown in Tables 7 and 8 above.

[0050] Example 4 Inducible expression and purification of mutant β-agarase Inducible expression of mutant β-agarase: The recombinant plasmid containing mutant β-agarase (obtained in Example 3) was E. coliBL21 was inoculated into 50 mL LB liquid medium and cultured at 37 ºC and 180 rpm for 18 h. The bacterial solution was transferred to 300 mL LB liquid medium at a 1% inoculum volume and continued to be cultured at 37 ºC with shaking until the bacterial OD reached 0. 600 Between 1.0 and 1.2. Add 30 µL IPTG (0.5 mol / L) to the bacterial solution and culture at 16 ºC and 180 rpm for 24 h.

[0051] Purification and analysis of mutant β-agarase by affinity chromatography: Purify the protein by affinity chromatography according to the instruction manual of Ni Sepharose 6 FastFlow from GE Company. Add glycerol to the purified sample to 20%, mix well, and store at -20 ℃. Determine the protein concentration by Bradford method. Take an appropriate amount of sample for SDS-PAGE electrophoresis analysis. The results are as follows: Figure 3 As shown in the figure, M: Marker; 1: WT (β-agarase Y3R1); 2: K82C-K87C; 3: E106C-G190C; 4: S111C-A136C; 5: S124C-K230C. After purification, Y3R1 and mutants showed a single clear band in the gel image with a molecular weight of 34.1 kDa. The mutation did not change the molecular weight of agarase.

[0052] Example 5 Study on the enzymatic properties of mutant β-agarase Activity determination of β-agarase: The activity of β-agarase was determined using the 3,5-dinitrosalicylic acid method (DNS method). Take 5 µL of enzyme solution (enzyme content is 200 ng) and add 495 µL of 5 mg / mL agarose substrate solution (dissolved in 50 mmol / L sodium phosphate buffer, pH 7.0). After incubation at 60°C for 15 min, add 500 µL of DNS colorimetric agent, boil for 10 min, and use a microplate reader to measure the absorbance at 540 nm. The activity unit of agarase is defined as: under certain conditions, the amount of enzyme required to generate 1 µmoL galactose per minute is one enzyme activity unit (U). The experiment was repeated three times and the average value was taken. Figure 4 As shown in the figure, the S111C-A136C mutation inactivated the recombinant enzyme, and the enzyme activity of E106C-G190C was lost, with a relative activity of only 78.1%. In contrast, the activities of mutants K82C-K87C and S124C-K230C were increased by 25.8% and 57.6% respectively compared with WT (β-agarase Y3R1). Therefore, two disulfide bond mutants K82C-K87C and S124C-K230C were selected for further enzymatic property studies.

[0053] Analysis of enzyme thermal stability: β-agarase (enzyme content 400 ng) was incubated at different temperatures of 40-80℃ for 60 min, placed on ice for 5 min, and then added with 5 mg / mL agarose substrate (dissolved in 50 mmol / L sodium phosphate buffer, pH 7.0) for 15 min. The residual activity of β-agarase was measured with the enzyme activity without heat treatment as 100% to study its thermal stability. Y3R1 maintained 48.4% of residual activity when treated at 60℃ for 1h, while the residual activities of mutants K82C-K87C and S124C-K230C were 42.4% and 74.4%, respectively ( Figure 5 ). At a higher temperature of 80°C, Y3R1 only retained 9.4% of the initial activity, while the remaining activity of mutant S124C-K230C was 24.4%. The above results indicate that the thermal stability of mutant S124C-K230C is improved. By introducing an additional disulfide bond, the enzyme activity and thermal stability of mutant S124C-K230C are improved.

[0054] Determination of the optimal reaction temperature of the enzyme: Using 5 mg / mL agarose solution (dissolved in 50 mmol / L sodium phosphate buffer, pH 7.0) as the substrate, the catalytic activity of β-agarase (enzyme content of 200 ng) at different temperatures (50-75°C) was studied, and its optimal temperature was explored with the highest enzyme activity as 100%.

[0055] Determination of the optimal reaction pH of the enzyme: The catalytic activity of β-agarase (enzyme content 200 ng) on ​​agarose substrate was determined at 60°C in different pH buffers (pH 3.0-11.0). The optimal reaction pH of the enzyme was studied with the highest enzyme activity as 100%. The buffer used was 50 mmol / L citric acid-Na 2 HPO 4 buffer (pH 3.0-5.0), sodium phosphate buffer (pH 5.0-9.0), and Gly-NaOH buffer (pH 9.0-11.0).

[0056] Determination of enzyme kinetic parameters: 0.1%-0.3% agarose substrate solution was incubated with purified β-agarase at 60°C and pH 7.0 for 15 min. The Michaelis constant of β-agarase ( K m ) and the maximum enzyme reaction rate ( V max ).

[0057] The results are shown in Table 12. Consistent with WT (β-agarase Y3R1), the optimum temperature of mutant S124C-K230C was 65°C and the optimum pH was 7.0. K The m values ​​were 15.00 and 4.52 mg / mL, respectively, indicating that the substrate binding affinity was improved after mutation. V The maximum values ​​were 10000.7 and 14570.3 U / mg respectively, which indicated that the maximum reaction rate of the enzyme increased after mutation.

[0058] Table 12 Comparison of enzymatic properties of β-agarase Y3R1 and its mutant S124C-K230C

[0059] Example 6 Fluorescence spectral analysis of mutant β-agarase: The fluorescence spectra of Y3R1 and mutant β-agarase (in 50 mmol / L phosphate buffer, pH 8.0) were measured using a fluorescence spectrophotometer. The surface hydrophobicity of β-agarase was determined using the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS). Spectral analysis was performed using a fluorescence spectrophotometer. The excitation wavelength was 317 nm, and the emission spectrum from 330 to 500 nm was recorded. The optical diameter of the sample cell was 1 cm. The measurement temperature was 25°C, the excitation slit width and the emission slit width were both 5 nm, and the processed data were expressed as the average of three measurements. The fluorescence changes caused by mutations can reflect changes in the microenvironment of tryptophan residues. The fluorescence spectra of Y3R1 and its disulfide bond mutant S124C-K230C are shown in Figure 2. Figure 6 As shown in A, the highest absorption peaks are all at 392 nm, indicating that the introduction of additional disulfide bonds does not change the tertiary structure of β-agarase. The surface hydrophobicity of the protein was analyzed by ANS fluorescent probe, indicating that the surface hydrophobicity of S124C-K230C was significantly reduced ( Figure 6 B) is beneficial to the stability of protein structure, thereby improving the thermal stability of protein.

[0060] Residue interaction network analysis of mutant β-agarase: The Ring 4.0 online server was used to analyze the possible changes in residue interactions after mutation. The distance thresholds for interaction forces were set, including 3.9 Å for hydrogen bond donor-acceptor distance, 2.5 Å for hydrogen bond acceptor distance, 6.5 Å for π-π stacking center distance, 5 Å for π-cation distance, 2.5 Å for disulfide bond distance, and 4 Å for ionic bond distance. In addition, van der Waals forces (VDW) were identified by measuring the distance between atomic surfaces, and the intersection fraction of the van der Waals radii of two atoms was 0.01 Å. The residue interactions of β-agarase Y3R1 and its disulfide bond mutant S124C-K230C were analyzed by the Ring 4.0 online server, and the results are shown in Table 13. WT (β-agarase Y3R1) contains 192 hydrogen bonds, 25 π-π bonds, 3 π-cation bonds, 7 ionic bonds, 1 π-hydrogen bond, and 245 van der Waals forces. Compared with WT, the disulfide mutant S124C-K230C has 10 additional hydrogen bonds, 1 π-π bond, 5 ionic bonds, 1 disulfide bond and 11 van der Waals forces, of which the main increases are hydrogen bonds and van der Waals forces. Analysis of the interaction forces of amino acid residues in the R1 domain showed that WT had 22 hydrogen bonds and 6 van der Waals forces in the R1 domain, while S124C-K230C had 22 hydrogen bonds and 12 van der Waals forces. The additional hydrogen bonds and van der Waals forces may be the reason for the improved thermal stability of the mutant S124C-K230C.

[0061] Table 13 Residue interactions of β-agarase Y3R1 mutant S124C-K230C

[0062] Molecular docking of mutant β-agarase and neoagarotetrose: Molecular docking of β-agarase and neoagarotetrose was achieved using AutoDock Vina 1.2.0. β-agarase and neoagarotetrose were pre-processed using AutoDock Tools software, and the files were saved in .pdbqt format. The docking center and box size of the docking box were adjusted using AutoDock Tools, and the enzyme and substrate molecules were docked using AutoDock Vina, and the docking result with the lowest overall energy and the best structure was selected. Discovery Studio 2019 and LigPlot were used to analyze the docking results. + The program analyzes protein-ligand interactions in two-dimensional graphs. Three-dimensional modeling showed that S124C formed a disulfide bond with K230C ( Figure 7A). The binding energies of WT, S124C-K230C and neoagarotetrose are -8.51 kcal / mol and -8.58 kcal / mol, respectively. The molecular docking energy of mutant S124C-K230C is lower, making the conformation of the enzyme protein-ligand complex more stable. The number of hydrogen bonds between WT and S124C-K230C and the substrate did not change. Compared with WT, there is an additional Pi-Sigma bond between mutant S124C-K230C and neoagarotetrose ( Figure 7 B). Non-covalent interactions between proteins and ligands play a major role in determining the stability of the system. The increase in Pi-Sigma bonds provides additional stability between the enzyme and the substrate. This indicates that the mutant S124C-K230C binds more tightly to neoagarotetrose. During the binding of the mutant S124C-K230C to the neoagarotetrose substrate, 12 amino acid residues (Arg160, Trp55, Tyr51, Glu239, Pro58, Asp165, Gln241, His156, Trp243, Glu135, Trp174 and His152) formed hydrophobic interactions with neoagarotetrose ( Figure 7 C). Compared with WT, two hydrophobic interactions were added, which is beneficial to the catalytic reaction of the enzyme. The additional Pi-Sigma bonds and hydrophobic interactions promote the binding of the mutant S124C-K230C with the neoagarotetrose, which is beneficial for the enzyme to hydrolyze the substrate and improve the enzyme activity.

[0063] Molecular dynamics simulation of mutant β-agarase: Molecular dynamics simulation of β-agarase was performed using Gromacs 2022.3 software. The reference temperature of the simulation was set to 333 K and 338 K, the pressure was set to atmospheric pressure (1 Bar), the force field used was GROMOS96 54a7 force field, the solvent was water molecules (SPC water model) and by adding an appropriate amount of Na + ions to neutralize the total charge of the simulation system. The simulation system first uses the steepest descent method to minimize the energy, and then performs 100,000 steps of isothermal isochoric ensemble (NVT) equilibrium and isothermal isobaric ensemble (NPT) equilibrium, with a coupling constant of 0.1 ps and a duration of 100 ps. Finally, a free molecular dynamics simulation is run, which consists of 5,000,000 steps, a step size of 2 fs, and a total duration of 100 ns. After the simulation is completed, the software's built-in tools are used to analyze the trajectory, and the root mean square deviation (RMSD), root mean square fluctuation (RMSF), gyration radius (Rg), free energy morphology and other data of the motion trajectory of each amino acid are calculated. The results show that the Rg values ​​of WT and S124C-K230C are 2.06 and 2.03nm, respectively ( Figure 8). During the simulation, the Rg value of S124C-K230C was always slightly lower than that of WT, indicating that the introduction of a single additional disulfide bond can make the structure of the enzyme more compact. At 338 K, the average RMSD value of S124C-K230C was 0.59 nm, which is higher than the average RMSD value of WT (0.33 nm). Fig. 9 The RMSF value of the mutant S124C-K230C at 338K was studied by MD simulation ( Fig.10 A) and analyzed the RMSF changes between mutants S124C-K230C and Y3R1 to understand the stability of the amino acid residues in the enzyme protein. The flexibility of the mutants in most of the structure did not change significantly, and the highlighted red or blue indicated that the flexibility of the local area increased or decreased relative to the WT ( Fig.10 B). The regions with increased RMSF are α1, β12, β20 and some random coils, while the regions with decreased RMSF are concentrated in the random coils (Ala276–Leu288) and β21 of the R1 domain. The enhanced rigidity of the R1 domain may be the reason for the improved thermal stability of the mutant S124C-K230C. Fig.11 As shown, the SASA of S124C-K230C and WT are 144.48 and 156.72 nm / S2 / N, respectively. According to the 100 ns simulation, the decrease in Rg and SASA values ​​of S124C-K230C indicates that the enzyme structure is more compact.

[0064] Preparation of mutant β-agarase hydrolyzate: Take 15 mL, 0.5% (w / v) agar substrate solution (dissolved in 50mmol / L sodium phosphate buffer, pH 7.0), add 15 U purified agarase, and react in a constant temperature water bath at 60°C. Shake well every 15 minutes to allow full enzymolysis, and measure the reducing sugar content of the system until the reducing sugar content remains constant. After heating at 100°C for 10 minutes to terminate the reaction, add 3 times the volume of anhydrous ethanol and let it stand at 4°C overnight. Remove the undegraded agar in the system by suction filtration, remove anhydrous ethanol and excess water by rotary evaporator, and then freeze-dry to obtain the enzymolysis product.

[0065] LC-MS analysis of the enzymatic hydrolysis products of mutant β-agarase: The enzymatic hydrolysis products were analyzed by liquid chromatography-mass spectrometry (LC-MS). The liquid phase model was Waters I-Class, the mass spectrometer model was Waters XEVO-TQS micro, and the chromatographic column model was ACQUITY UPLCr BEH C18 1.7 μm (column length 100 mm). The separation conditions were: column temperature: 35°C; flow rate: 0.3mL / min; injection volume: 1 μL; mobile phase: 0.05% ammonia solution / acetonitrile. LC-MS was used to study the effect of introducing an additional disulfide bond on the enzymatic hydrolysis products of the mutant. The results showed that the mutant S124C-K230C showed absorption peaks at 0.57min and 0.75min. The mass spectrometry results showed that the enzymatic hydrolysis products were neoagarbiose, tetrasaccharide and hexasaccharide ( Fig.12 The degradation products of mutant S124C-K230C were the same as those of WT, indicating that the introduction of additional disulfide bonds would not change the composition of the enzymatic hydrolysis products.

[0066] In summary, according to an embodiment of the present invention, the potential disulfide bonds of Y3R1 were analyzed by DBD 2.0 and DS 2019 software, 4 disulfide bond mutants were constructed, and their enzymatic properties were analyzed. The results showed that the enzyme activity and thermal stability of the mutant S124C-K230C were improved. Compared with WT, the enzyme activity of the mutant increased by 57.6%, and the thermal stability at 60 ° C increased by 26.0%. The Pi-Sigma bond and hydrophobic interaction between the mutant S124C-K230C and the new agaronetetraose were increased, which was beneficial to the improvement of enzyme activity. The results of surface hydrophobicity analysis, residue interaction network analysis and kinetic simulation showed that the mutant increased multiple hydrogen bonds and van der Waals forces, and the surface hydrophobicity, the solvent accessible surface area and the gyration radius of the protein were reduced, making the structure more compact, and the rigidity of the R1 domain was enhanced, which was beneficial to the protein to ensure the stability of the structure at high temperatures.

[0067] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0068] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A disulfide-bond-modified β-agarase mutant, characterized in that: The mutant is obtained by performing amino acid modification and substitution on the amino acid sequence of β-agarase Y3R1 shown in SEQ ID NO: 1, and the amino acid modification and substitution position is S124C-K230C.

2. The β-agarase mutant according to claim 1, characterized in that The amino acid sequence of the mutant is shown in SEQ ID NO:

2.

3. A gene encoding the β-agarase mutant according to claim 1 or 2.

4. A construct, characterized in that Comprising the gene as claimed in claim 3.

5. A recombinant cell expressing the β-agarase mutant according to claim 1, or containing the gene according to claim 3, or containing the construct according to claim 4.

6. A method for constructing a β-agarase mutant according to claim 1 or 2, characterized in that: The following steps are involved: (1) The three-dimensional structure of β-agarase Y3R1 was submitted to the online server DBD 2.

0. Under the condition of bond energy (χ3 angle) of +97 (±20), the DBD 2.0 tool was used to predict candidate residue pairs suitable for additional disulfide bond formation, and mutants with larger B factors were selected as candidate results. Similarly, the Predict Disulfide Bridges tool in Discovery Studio 2019 was used to predict the formation of additional disulfide bonds by cysteine ​​substitution, and residue pairs with lower energy after mutation were selected to screen out 4 disulfide bond mutants. (2) designing site-directed mutagenesis primers, and performing site-directed mutagenesis on the gene of β-agarase Y3R1 whose amino acid sequence is shown in SEQ ID NO: 1 by PCR to obtain a recombinant vector containing the gene sequence of the β-agarase mutant; (3) Transform the mutated recombinant vector into Escherichia coli E. coli In BL21(DE3), the expression was induced and purified to obtain four double-point mutation β-agarase mutants; (4) The enzymatic properties of four double-point mutation β-agarase mutants were analyzed, and the β-agarase mutant S124C-K230C with improved enzyme activity and thermal stability was screened out.

7. The method for constructing a β-agarase mutant according to claim 6, characterized in that: The four disulfide bond mutants are K82C-K87C, E106C-G190C, S111C-A136C, and S124C-K230C.

8. The method for constructing a β-agarase mutant according to claim 7, characterized in that: The amplification primer sequences for mutants K82C-K87C are SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8; The amplification primer sequences for mutant E106C-G190C are SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; The amplification primer sequences for mutant S111C-A136C are SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16; The amplification primer sequences of mutant S124C-K230C are SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO:

20.

9. Use of the β-agarase mutant according to claim 1, or the encoding gene according to claim 3, or the construct according to claim 4, or the recombinant cell according to claim 5 in degrading agar or preparing agar-oligosaccharides.

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