Agarase OUC-Agai truncated mutant and application thereof

Abstract

The application discloses a gelase OUC-Aga1 truncated mutant, and an amino acid sequence of the gelase OUC-Aga1 truncated mutant is shown as SEQ ID NO. 3. A nucleotide sequence of a coding gene of the gelase OUC-Aga1 truncated mutant is shown as SEQ ID NO. 4. The application also discloses application of the gelase OUC-Aga1 truncated mutant in degradation of agar or / and preparation of neoagarooligosaccharide. The application obtains the gelase OUC-Aga1 truncated mutant by knocking out amino acid residues at positions 945-995 of the gelase OUC-Aga1 through truncated mutation, and the specific enzyme activity of the gelase OUC-Aga1 truncated mutant is 300.72 U / mg, which is greatly improved compared with the specific enzyme activity of the original enzyme gelase OUC-Aga1. The gelase OUC-Aga1 truncated mutant can degrade agar to obtain neoagarooligosaccharide, has high catalytic activity, good temperature stability and pH stability, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to a truncated mutant of agarase OUC-Aga1 and its application, belonging to the field of hydrolytic enzyme technology. Background Technology

[0002] Agar, also known as agar gum, is mainly composed of agarose and agar gum. Agarose is a linear polysaccharide formed by the alternating linkage of D-galactose and 3,6-endo-L-galactose via α-1,3-glycosidic bonds and β-1,4-glycosidic bonds. Agarase is an enzyme that degrades agarose into oligosaccharides. Based on the glycosidic bonds it cleaves, it is classified into α-agarase and β-agarase. α-Agarase hydrolyzes α-1,3-glycosidic bonds to generate agarooligosaccharides (AOSs); β-agarase specifically cleaves β-1,4-glycosidic bonds to degrade them into neo-agarooligosaccharides (NAOSs), such as neo-agarobiose, neo-agarotetraose, and neo-agarohexaose. Neo-Qiong oligosaccharides possess a variety of physiological activities, including antioxidant, anti-inflammatory, anti-fatigue, anti-aging, whitening, and gut microbiota regulation, and show broad prospects for industrial application in functional health foods, pharmaceuticals, and cosmetics.

[0003] β-Agarase (EC 3.2.1.81, GH50 family) belongs to the GH-A superfamily of glycosidic hydrolases. It specifically cleaves the β-1,4-glycosidic bonds within agarose, degrading large agarose molecules into new agaroligosaccharides. The enzymatic hydrolysis process follows a retention-type glycosidic hydrolysis mechanism. Most GH50 family β-agarases originate from marine microorganisms. Comparative analysis of the amino acid sequences of reported GH50 family β-agarases revealed low overall sequence similarity among GH50-type β-agarases from different strains. Conserved regions are concentrated within the catalytic domain, where two glutamate (Glu) residues constitute a catalytic binary, acting as a generalized acid-base catalyst and a nucleophilic group, respectively, to complete glycosidic bond cleavage. GH50 family β-agarases exhibit a modular structure, with the N-terminus mostly consisting of a carbohydrate-binding domain (CBM-like) and the C-terminus a classic (α / β) 8-barrel-shaped catalytic domain. Currently, there is a severe shortage of commercially available GH50-type β-agarase products. The few commercially available agarases generally suffer from defects such as poor heterologous soluble expression, low catalytic activity, insufficient stability under high temperature conditions, wide distribution of polymerization degree of enzymatic hydrolysis products, and difficulty in the targeted preparation of high-purity new agar oligosaccharides with specific polymerization degrees. Summary of the Invention

[0004] To address the aforementioned limitations of existing technologies, this invention provides a truncated mutant of agarase OUC-Aga1 and its applications. This invention modifies agarase OUC-Aga1 using protein engineering techniques to obtain a truncated mutant with improved catalytic performance, which is of significant engineering importance for expanding the agarase resource library and promoting the high-value utilization of marine polysaccharides.

[0005] This invention is achieved through the following technical solution: A truncated mutant of agarase OUC-Aga1, the amino acid sequence of which is shown in SEQ ID NO.3.

[0006] The encoding gene of the truncated mutant of agarase OUC-Aga1 has the nucleotide sequence shown in SEQ ID NO.4.

[0007] Application of the OUC-Aga1 truncated mutant agarase in the degradation of agar and / or the preparation of novel agar oligosaccharides.

[0008] This invention obtained the agarase OUC-Aga1 through mining, with a specific enzyme activity of 108.31 U / mg. Then, through truncation mutation modification, amino acid residues 945-995 of agarase OUC-Aga1 were knocked out, resulting in the truncated mutant agarase OUC-Aga1-Δ945-995, with a specific enzyme activity of 300.72 U / mg, a significant increase compared to the original agarase OUC-Aga1. The truncated mutant agarase OUC-Aga1 of this invention can degrade agar to obtain new agar oligosaccharides (new agarobiose, new agarotetraose), exhibiting high catalytic activity, an optimal temperature of 50℃, and an optimal pH of 7.0, demonstrating good temperature and pH stability, and showing promising prospects for industrial application.

[0009] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0010] Figure 1 SDS-PAGE electrophoresis results of wild-type enzymes and truncated mutants.

[0011] Figure 2 Results of relative enzyme activity assays for truncated mutants under different temperature conditions.

[0012] Figure 3 Results of relative enzyme activity determination of truncated mutants after incubation at different temperatures for different times.

[0013] Figure 4 Results of relative enzyme activity assays for truncated mutants under different pH conditions.

[0014] Figure 5The results of relative enzyme activity determination of truncated mutants after incubation for different times with different pH buffers.

[0015] Figure 6 The relative enzyme activity of truncated mutants was measured under the influence of different metal ions and chemical reagents.

[0016] Figure 7 HPLC detection results of substrates and products.

[0017] Figure 8 Results of molecular docking between agarase OUC-Aga1 and substrate.

[0018] Figure 9 Results of molecular docking between agarase OUC-Aga1-Δ945~995 and substrate. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0020] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0021] Example 1: Discovery of agarase OUC-Aga1 This invention discovered a source in the NCBI database. Vibrio sp.EJY3 (This bacterium currently has no universally accepted Chinese name; it can be temporarily translated as Vibrio strain EJY3.) A gene fragment (NCBI ID: AEX22316.1) shows that the AEX gene open reading frame is 3048 bp in length, encoding 1016 amino acids. Preliminary analysis suggests that the encoded protein may possess β-agarase activity. Based on the host *Escherichia coli* (…), E. coli The codons of the gene fragment were optimized based on the codon preference of the gene fragment, and the N-terminal signal peptide sequence was deleted. The optimized target gene fragment was obtained by whole-genome synthesis technology and inserted into the multiple cloning site of the pET-32a(+) plasmid to obtain the recombinant plasmid.

[0022] The gene fragment after deleting the N-terminal signal peptide and codon optimization is shown in SEQ ID NO.1. The protein after deleting the N-terminal signal peptide contains 995 amino acid residues and is named agarase OUC-Aga1, with an amino acid sequence shown in SEQ ID NO.2. The theoretical molecular weight of the protein is predicted to be 103.3 kDa using software. Due to the presence of tags such as thioredoxin co-solubilizing tags in the pET-32a(+) expression cassette, the theoretical molecular weight of the fusion protein is predicted to be 128.8 kDa.

[0023] Example 2 Heterologous expression of agarase OUC-Aga1 (1) Construction of recombinant engineered bacteria The target gene fragment (shown in SEQ ID NO.1) and the linearized vector were ligated using seamless cloning technology to construct the recombinant plasmid pET-32a-OUC-Aga1.

[0024] The recombinant plasmid was transformed into Escherichia coli DH5α competent cells using the heat shock method, spread on LB solid plates containing the corresponding antibiotics, and incubated upside down in a 37°C incubator for 12 h before observing the colonies.

[0025] After overnight incubation, single colonies were picked for PCR verification. Colonies with correct positive clones were selected for sequencing. Single colonies with correct sequencing results were activated and preserved at -80°C.

[0026] The activated DH5α bacterial culture was used to extract recombinant plasmids using a rapid plasmid mini-prep kit. The recombinant plasmids were then transformed into Escherichia coli BL21 strain according to the transformation steps described above. Colony verification, sequencing verification, and preservation operations were performed to obtain recombinant engineered bacteria. The preservation tubes were stored at -80°C for subsequent fermentation and enzyme production.

[0027] (2) Cultivation of recombinant engineered bacteria Recombinant engineered bacterial strains grown on ampicillin-resistant plates were selected and inoculated into 5 mL of LB liquid medium containing 25 μg / mL ampicillin, and cultured at 37°C and 220 rpm for 12 h. Then, 1% of the inoculum was added to LB liquid medium containing 25 μg / mL ampicillin and cultured at 20°C and 200 rpm until OD (dose retardation). 600 =0.6, add IPTG as an inducer at 1‰, and continue induction at 20℃ and 220 rpm for 16 hours to express agarase OUC-Aga1.

[0028] (3) Extraction and purification of agarase OUC-Aga1 Take the culture medium, centrifuge at 8000 rpm for 15 min at 4℃, collect the bacterial cells, resuspend them in Tirs-HCl buffer (50 mM Tris, 150 mM NaCl, pH 7.6), and sonicate (300 W, 3 s operation time, 3 s interval, 15 min operation time). After disruption, centrifuge completely (4℃, 10000 rpm, 20 min). The supernatant is the crude enzyme solution.

[0029] The crude enzyme solution was purified by affinity chromatography using a nickel column. The column was equilibrated with 10 mM imidazole solution (10 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl), followed by elution of weakly binding proteins with 40 mM imidazole solution (40 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). The target protein was then eluted with 80 mM imidazole solution (80 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). The eluent was collected as the purified enzyme solution (pure enzyme solution) and analyzed by SDS-PAGE. The results are shown below. Figure 1 As shown, the purified protein band is single, with a molecular weight of approximately 135 Kda, slightly larger than the predicted result. Enzyme activity verification confirmed that the purified protein has agar hydrolysis activity, confirming that the obtained protein is agarase OUC-Aga1.

[0030] Example 3: Determination of the activity of agarase OUC-Aga1 Take 190 μL of 0.3% low-melting-point agarose solution (low-melting-point agarose purchased from Solarbio) (prepared using 0.1 M phosphate buffer at pH 7.0), add 10 μL of diluted enzyme solution (the pure enzyme solution from Example 2 diluted to a concentration of 3.5 μg / μL), react at 60℃ for 20 min (Note: the optimal reaction temperature for agarase OUC-Aga1 is 60℃), inactivate the enzyme by boiling in a water bath for 5 min; add 300 μL of DNS solution, boil in a water bath for 5 min, stop the reaction by cooling in a water bath, add 1 mL of distilled water, and detect the content of galactose (D-Gal).

[0031] The method for detecting D-Gal content is as follows: First, a D-Gal standard curve is plotted: D-Gal (analytical grade) dried to constant weight at 60℃ is dissolved in a certain amount of distilled water to obtain gradient standard solutions with concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.8 mg / mL. 200 μL of each gradient standard solution is taken, and 300 μL of DNS colorimetric reagent is added to each solution. The mixture is incubated in a boiling water bath for 5 min, and the reaction is immediately terminated by cooling with cold water. The OD is then measured. 540 Plotting D-Gal concentration on the x-axis, OD... 540Plot a standard curve on the ordinate. Then, measure the OD of the test solution. 540 Substitute the values ​​into the standard curve to calculate the concentration of D-Gal in the test solution.

[0032] One β-agarase activity unit (U) is defined as the amount of enzyme required to hydrolyze agarose to produce 1 μmol of reducing sugar per minute.

[0033] The specific enzyme activity is calculated based on the mass of pure enzyme added. Specific enzyme activity (U / g) = A / M; where A represents the hydrolytic enzyme activity (U); M represents the mass of pure enzyme (mg), and the unit of specific enzyme activity is U / mg.

[0034] Each reaction was performed in triplicate, and the average value was taken. The same applies below.

[0035] The specific activity of agarase OUC-Aga1 was measured to be 108.31 U / mg.

[0036] Example 4: Truncated modification of agarase OUC-Aga1 The enzyme activity assay results in Example 3 showed that the enzyme activity of agarase OUC-Aga1 was low and not ideal. This invention attempts to modify agarase OUC-Aga1 using molecular truncation technology to improve its hydrolytic ability on agar substrates.

[0037] The three-dimensional structure of agarase OUC-Aga1 was modeled using the online analysis tools Swiss-Model and AlphaFold-3 to analyze the flexibility of amino acid residues in the C-terminal region, intermolecular forces, and functional division of domains. Based on the structural analysis results, the region containing amino acid residues from position 945 to 995 was selected as the deletion modification region. The truncated mutant agarase OUC-Aga1 contains 995 amino acid residues in its full length. By deleting 51 amino acid residues from positions 945 to 995, and retaining only amino acid residues from positions 1 to 944, the truncated mutant agarase OUC-Aga1-Δ945~995 was obtained.

[0038] The amino acid sequence of agarase OUC-Aga1-Δ945~995 is shown in SEQ ID NO.3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.4.

[0039] Example 5 Construction and expression of truncated mutants The coding gene sequence corresponding to amino acids 945-995 of agarase OUC-Aga1 was knocked out by whole-plasmid reverse PCR. The specific operation is as follows: Using the recombinant plasmid pET-32a-OUC-Aga1 carrying the full-length OUC-Aga1 gene as a template, primers were designed to add a stop codon after the coding sequence of amino acid 944. High-fidelity PCR amplification yielded a linear plasmid lacking the coding region 945–995. The methylated wild-type template plasmid was removed by DpnI digestion, followed by homologous recombination to achieve plasmid circularization. Transformation into *E. coli* DH5α followed by sequencing verification confirmed complete deletion of amino acid 945–995 and correct reading frame. Finally, the correct plasmid was introduced into *E. coli* BL21-DE3 competent cells to obtain genetically engineered bacteria expressing agarase OUC-Aga1-Δ945–995.

[0040] Expression and purification were performed according to the method in Example 2 to obtain crude enzyme solution and pure enzyme solution containing agarase OUC-Aga1-Δ945~995. SDS-PAGE analysis of the pure enzyme solution yielded the following results: Figure 1 As shown, the purified protein has a molecular weight of approximately 135 kDa, which is smaller than that of the agarase OUC-Aga1.

[0041] Example 6: Determination of the enzymatic properties of agarase OUC-Aga1-Δ945~995 (1) Enzyme activity assay Following the enzyme activity assay method in Example 3, the specific enzyme activity of agarase OUC-Aga1-Δ945~995 was measured at a reaction temperature of 50℃. The specific enzyme activity of agarase OUC-Aga1-Δ945~995 was found to be 300.72 U / mg, a significant increase compared to the 108.31 U / mg of wild-type agarase OUC-Aga1. This indicates that knocking out the flexible non-catalytic region at the C-terminus of agarase OUC-Aga1 significantly affected the catalytic efficiency of the mutant enzyme.

[0042] (2) Determination of the optimal temperature Following the enzyme activity assay method of Example 3, the truncated mutant was reacted for 20 min at different reaction temperatures (25℃, 35℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 80℃) to determine the specific enzyme activity at different reaction temperatures. The highest specific enzyme activity was taken as 100%, and the relative enzyme activity under each temperature condition was calculated.

[0043] Results: The relative enzyme activity of the truncated mutant under different temperature conditions is as follows: Figure 2 As shown, the optimal temperature for the truncated mutant is 50℃, and it exhibits high relative enzyme activity in the range of 45–60℃.

[0044] (3) Determination of temperature stability Pure enzyme solutions containing agarase OUC-Aga1-Δ945~995 were incubated at 50℃ for 0 min, 15 min, 30 min, 1 h, 2 h, 3 h, and 4 h, or at 55℃ for 0 min, 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 1 h, and 2 h, respectively.

[0045] Then, following the enzyme activity assay method of Example 3, at a reaction temperature of 50°C, the specific enzyme activity of the truncated mutant after incubation at different temperatures for different times was measured. The highest specific enzyme activity was taken as 100%, and the relative enzyme activity under each condition was calculated.

[0046] Results: The relative enzyme activity of the truncated mutant after incubation at different temperatures and for different times is as follows: Figure 3 As shown, the truncated mutant retains more than 80% of its activity after incubation at the optimal temperature of 50℃ for 1 h, and still retains more than 50% of its activity after incubation for 4 h, indicating that the truncated mutant has good temperature stability.

[0047] (4) Determination of optimal pH Agarose solutions were prepared using buffers with different pH values. The buffers used included: 0.1 M citrate-sodium citrate buffer with pH values ​​of 4.0, 5.0, and 6.0; 0.1 M phosphate buffer with pH values ​​of 6.0, 7.0, and 8.0; 0.1 M Tris-HCl buffer with pH values ​​of 8.0 and 9.0; 0.1 M Gly-NaOH buffer with pH values ​​of 9.0 and 10.0; and 0.1 M NaHCO3-Na2CO3 buffer with pH values ​​of 10.0 and 11.0.

[0048] Then, following the enzyme activity assay method of Example 3, at a reaction temperature of 50°C, the specific enzyme activity of the truncated mutant under different pH conditions was measured, and the highest specific enzyme activity was taken as 100%, and the relative enzyme activity under each condition was calculated.

[0049] Results: The relative enzyme activity of the truncated mutant under different pH conditions is as follows: Figure 4 As shown, the optimal pH for the truncated mutant is 7.0, and it exhibits high enzyme activity in the pH range of 6.0 to 8.0.

[0050] (5) Determination of pH stability Pure enzyme solutions containing agarase OUC-Aga1-Δ945–995 were diluted with buffers of different pH values ​​and incubated for different times (0, 24 h, 48 h, 72 h, 96 h). The buffers used included: 0.1 M citrate-sodium citrate buffer at pH 5.0; and 0.1 M phosphate buffer at pH 6.0, 7.0, and 8.0.

[0051] Then, following the enzyme activity assay method of Example 3, at a reaction temperature of 50°C, the specific enzyme activity of the truncated mutant after incubation in different pH buffers for different times was measured. The highest specific enzyme activity was taken as 100%, and the relative enzyme activity under each condition was calculated.

[0052] Results: The relative enzyme activity of the truncated mutants after incubation with different pH buffers for different times is as follows: Figure 5 As shown, after incubation in buffer solutions with pH 5.0–7.0 for 96 h, no significant decrease in activity was observed. After incubation in buffer solutions with pH 8.0 for 96 h, approximately 60% of the activity remained, indicating that the truncated mutant has excellent pH stability.

[0053] (6) Effects of metal ions on enzyme activity Following the enzyme activity assay method in Example 3, different metal ions and chemical reagents were added to the reaction system to achieve final concentrations of 1 mM and 10 mM, respectively, with distilled water as a control. The metal ions involved included: Na+. + K + Mg 2+ Ni 2+ Mn 2+ Ba 2+ Ca 2+ Zn 2+ Co 2+ Cu 2+ Fe 2+ (The corresponding anion is Cl) - The chemical reagents involved include: sodium dodecyl sulfate (SDS), disodium ethylenediaminetetraacetate (Na2EDTA), urea, and dithiothreitol (DTT). The reaction temperature was 50℃. The specific enzyme activity of the truncated mutant was measured under different metal ions and chemical reagents. The specific enzyme activity in distilled water was taken as 100%, and the relative enzyme activity under each condition was calculated.

[0054] Results: The relative enzyme activity of the truncated mutant under the influence of different metal ions and chemical reagents is as follows: Figure 6 As shown, it can be seen that 1 mM Mg 2+ Ba 2+ Co 2+ Zn has a promoting effect on enzyme activity. 2+ Cu2+ SDS and Na2EDTA have significant inhibitory effects on enzyme activity.

[0055] Example 7: Identification of the minimum hydrolytic substrate and products of the truncated mutant To investigate the minimum hydrolytic substrate for agarase OUC-Aga1-Δ945–995, 0.3% of even-numbered neo-agar oligosaccharides (NA2, NA4, NA6, NA8, NA10, NA12) with a degree of polymerization of 2–12 were mixed with pure enzyme solution containing agarase OUC-Aga1-Δ945–995. Take 190 μL of a 0.3% solution of even-numbered neo-agar oligosaccharides with a degree of polymerization of 2–12 (neo-agar disaccharide, neo-agar tetrasaccharide, neo-agar hexasaccharide, neo-agar octasaccharide, neo-agar decasaccharide, and neo-agar dodecasaccharide, abbreviated as NA2, NA4, NA6, NA8, NA10, and NA12, respectively) (all of which were purchased from Qingdao Bozhi Huili Biotechnology Co., Ltd.), add 10 μL of pure enzyme solution containing agarase OUC-Aga1-Δ945–995, react at 50℃ for 20 min, and then boil for 10 min to inactivate the enzyme and terminate the reaction. Filter the reaction solution through a 0.22 μm filter membrane and perform HPLC to detect the reaction substrate and products.

[0056] HPLC-RID detection conditions: Sugar-Pak I column (Waters, 300 mm × 6.5 mm, USA), analysis temperature 75℃, mobile phase 50 mg / mL EDTANa2Ca solution, flow rate 0.5 mL / min, injection volume 20 μL.

[0057] Results: The HPLC detection results of the substrate and product are as follows: Figure 7 As shown, the minimum hydrolytic substrate of the truncated mutant is neo-agarhexasaccharide, and the hydrolysis products are neo-agarhexasaccharide and neo-agartetrasaccharide. The cleavage unit is a tetrasaccharide, which is the same as the hydrolysis mode of the original enzyme.

[0058] Example 8 Molecular docking To elucidate the mechanism of the enhanced hydrolytic activity of the truncated mutant, the smallest cleavage unit, neo-agartetrasaccharide, was used as the substrate (Note: we attempted to use the smallest hydrolytic substrate, neo-agarhexasaccharide, but the neo-agarhexasaccharide molecule was too large, and the program reported an error during docking). Molecular docking was performed with agarase OUC-Aga1 and agarase OUC-Aga1-Δ945~995, respectively, and the structure with the lowest binding free energy was selected.

[0059] Molecular docking results as follows Figure 8 , 9As shown in the figure. Molecular docking analysis revealed that, compared to the original enzyme, the mutant exhibited significant structural optimization in both substrate binding mode and catalytic mechanism. The mutant not only fully preserved the key hydrogen bond contact between Glu639 (a generalized base) and the substrate hydroxyl group, but also enabled stable hydrogen bonding between the other core catalytic residue, Glu807 (a generalized acid), and the glycosidic bond region of the catalytic center, promoting efficient proton transfer during glycosidic bond hydrolysis. Furthermore, based on the Glu639 / Glu807 dual catalytic center, a multi-point hydrogen bond network formed by residues such as Arg319, Arg474, Arg864, Trp579, and Tyr779 significantly optimized the conformational localization of the substrate polysaccharide backbone, greatly enhanced the overall binding affinity of the complex, and provided stable electrostatic support for the catalytic transition state. In contrast, the active site of the original enzyme can only maintain a single effective hydrogen bond of Glu639, while Glu807, due to improper spatial orientation, can only contact the substrate through weak van der Waals forces, making it difficult to cooperate spatially with Glu639 to complete acid-base mediated proton transfer. Furthermore, the glycosidic catalytic region lacks polar anchoring sites and has a large number of missing hydrogen bond interactions with auxiliary stabilizing residues. Even with the limited hydrogen bond anchoring of Arg592 and Pro600 at both ends of the substrate, it is still difficult to compensate for the structural defects caused by the lack of two-residue synergistic catalysis.

[0060] In summary, the truncated mutation of this invention activates the generalized acid Glu807 and enables it to construct a highly efficient two-residue synergistic catalytic conformation with the generalized base Glu639, while being enhanced by a global hydrogen bond network, providing a direct structural mechanism basis for the significant enhancement of mutant enzyme activity at the molecular level.

[0061] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A truncated mutant of agarase OUC-Aga1, characterized in that: The amino acid sequence is shown in SEQ ID NO.

3.

2. The encoding gene of the agarase OUC-Aga1 truncated mutant according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

4.

3. The use of the OUC-Aga1 truncated mutant of agarase according to claim 1 in the degradation of agar and / or the preparation of new agar oligosaccharides.

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