Alginate lyase YH7N1 and application thereof in enzymolysis of algin

Abstract

The invention discloses alginate lyase YH7N1 and an application of the alginate lyase YH7N1 in enzymolysis of alginate. The amino acid sequence of the alginate lyase YH7N1 is as shown in SEQ ID NO: 1, and the gene sequence for coding the amino acid is as shown in SEQ ID NO: 2. The invention also discloses construction of the YH7N1 gene on a vector, conversion of escherichia coli expression competent cells by the vector, extraction to obtain purified recombinant plasmids, conversion of BL21 protein competent cells, and purification to obtain the alginate lyase YH7N1. According to the alginate lyase YH7N1, sodium alginate is used as a substrate, under the optimal enzymolysis condition, Mn < 2 + > is used for assistance, the enzyme activity can reach 46.92 + / -0.54 U, all enzymolysis products are brown algae disaccharide, the purity is high, and the alginate lyase YH7N1 has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of alginate lyase preparation technology, specifically relating to an alginate lyase YH7N1 and its application in enzymatic hydrolysis of alginate. Background Technology

[0002] Alginate is an acidic linear polysaccharide composed of β-D-polymannuronic acid and α-L-polyguluronic acid linked by 1,4 glycosidic bonds. Due to its large molecular weight and high viscosity, alginate is difficult to hydrolyze, significantly limiting its activity. Studies have found that hydrolyzed alginate forms small-molecule alginate oligosaccharides with a degree of polymerization of 2-10, exhibiting low viscosity and easy hydrolysis. Therefore, how to efficiently enzymatically hydrolyze alginate to generate high-purity alginate oligosaccharides has become a research hotspot.

[0003] Fucoidan, a small molecule compound, possesses diverse biological activities and plays a vital role in medicine, cosmetics, and agriculture. In medicine, fucoidan dressings are a novel type of wound dressing. Using fucoidan as the main ingredient, the continuously released fucoidan effectively inhibits the growth of Staphylococcus aureus and Candida albicans, thereby promoting cell regeneration and accelerating wound healing. Sodium alginate / chitosan composite gel has a composition similar to human tissue matrix, making it an excellent injectable bone tissue engineering scaffold matrix material. Furthermore, tests have shown that the sodium alginate / chitosan composite gel exhibits zero cytotoxicity, making it suitable for clinical applications in treating diabetes and other conditions. Additionally, fucoidan can be used as an important component in oral liquids, playing a significant role in regulating intestinal absorption and alleviating obesity. Moreover, fucoidan plays a crucial role in the cosmetics industry. La Mer's The Renewal Oil, with fucoidan as one of its main ingredients, offers important benefits for skin repair and anti-oxidation. Algenist's Algenist Genius Liquid Collagen is an anti-aging serum whose main components are algae extract and biotechnology-based collagen. Clinically tested, it has been shown to slow down skin cell aging and keep skin firm and elastic. COSRX's Advanced Snail Peptide Eye Cream's main ingredients are brown algae oligosaccharides and snail mucus, and it has been tested to have anti-wrinkle and dark circle-reducing effects.

[0004] Fucoidan oligosaccharides, containing functional groups such as carboxyl and hydroxyl groups in their structure, also play an important role in agriculture. Using fucoidan oligosaccharides as fertilizer can bind to ammonium ions in fertilizers to form complexes, inhibiting NH4+ ions. 4+ NO 3-The conversion of these compounds can reduce nitrogen loss from fertilizers and increase nitrogen supply to plants. Furthermore, fucoidan can promote the efficiency of nutrient absorption by plant roots, such as the absorption of metal ions like B, Zn, Ca, and Mg. Organic fertilizers prepared using fucoidan as the main raw material are rich in trace organic elements, vitamins, proteins, and other nutrients, which can promote plant growth and enhance plant disease resistance. Oligomeric chitosan, a mixed feed additive prepared using oligosaccharides as the main raw material, is one of the most competitive antibiotic products. It is an intestinal regulator and growth promoter that can strengthen the immune system and leaves no residue in the animal body.

[0005] Fucoidan oligosaccharides have wide applications in various industries, making their preparation a focus of research. Currently, the main methods for preparing fucoidan oligosaccharides include physical, chemical, and enzymatic methods. Physical methods include ultraviolet (UV) irradiation, gamma-ray irradiation, and microwave irradiation. UV irradiation degrades alginate in an aqueous solution containing titanium dioxide, producing oligosaccharides, but the yield is low. Gamma-ray irradiation is unaffected by ambient temperature and additives, making it a relatively ideal method. Microwave irradiation directly acts on polar molecules, rapidly generating heat and creating localized high temperatures. The glycosidic bonds of polysaccharides break at these temperatures, forming oligosaccharides; this method is considered highly efficient and environmentally friendly. While physical methods for preparing oligosaccharides offer advantages such as environmental friendliness and efficiency, they are costly, produce numerous byproducts, and have unclear production mechanisms. In most cases, they require assistance from other methods, thus further optimization of physical methods is needed.

[0006] Compared to physical methods, chemical methods for preparing oligosaccharides are simpler to operate and lower in cost. Chemical methods for preparing oligosaccharides include acid-base degradation and oxidative degradation. Among these, acid-base degradation involves using H₂O… + Or OH - Unsaturated oligosaccharides are generated by breaking glycosidic bonds. Commonly used acids include oxalic acid, hydrochloric acid, and formic acid. Degrading alginate with 1 mol of oxalic acid at 100 °C yields 30% oligosaccharides. Hydrochloric acid degradation of alginate yields oligosaccharides with varying degrees of polymerization, and the oligosaccharides exhibit good free radical scavenging effects. Degrading alginate with 90% formic acid at 100 °C for 2 hours, followed by hydrolysis at 1.5 mol for 3 hours under the same conditions, completely degrades alginate, making it a relatively ideal chemical method for oligosaccharide preparation. Oxidative degradation utilizes H₂O₂ to degrade alginate, producing H₂O as the degradation product. This is a highly efficient and environmentally friendly method, but the degradation mechanism has not yet been clearly defined, requiring further clarification. Chemical methods are simple and low-cost, but the reaction process easily generates toxic and harmful chemicals, leading to environmental pollution. Therefore, chemical methods for oligosaccharide preparation need further optimization to reduce the production of toxic substances.

[0007] Enzymatic hydrolysis is a method for producing unsaturated oligosaccharides by breaking the β-1,4-glycosidic bonds in alginate under the action of alginate lyases, creating double bonds at the non-reducing ends. Alginate lyases are classified into three types: the first is a Poly-M specific lyase acting on the M segment; the second is a Poly-G specific lyase acting on the G segment, a bifunctional alginate lyase; and the third is an alginate lyase acting on both the G and M segments. Alginate lyases are widely sourced, mainly from marine bacteria, marine mollusks, terrestrial bacteria, and decaying seaweed, with bacteria being the most prevalent source. Compared with physical and chemical methods, enzymatic hydrolysis has advantages such as mild reaction conditions, high efficiency, high specificity, and fewer byproducts. Therefore, it is essential to explore new alginate lyases for the production of high-purity alginate oligosaccharides.

[0008] In conclusion, obtaining a novel alginate lyase for efficient enzymatic hydrolysis of alginate to produce high-purity alginate oligosaccharides can avoid the environmental harm caused by other methods of oligosaccharide production, thereby reducing preparation costs and environmental pollution. Summary of the Invention

[0009] The main objective of this invention is to provide an alginate lyase YH7N1 and its application in the enzymatic hydrolysis of alginate.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: An alginate lyase YH7N1, the amino acid sequence of which is shown in SEQ ID NO:1.

[0011] The gene encoding the aforementioned alginate lyase YH7N1 has the nucleotide sequence shown in SEQ ID NO:2.

[0012] A genetically engineered bacterium that produces the aforementioned alginate lyase YH7N1, wherein the aforementioned alginate lyase YH7N1 gene is introduced into the genetically engineered bacterium.

[0013] A method for preparing the above-mentioned alginate lyase YH7N1 specifically involves: synthesizing the gene of alginate lyase YH7N1 with the nucleotide sequence shown in SEQ ID NO:2, constructing it into an expression vector, introducing it into Escherichia coli, inducing culture, and obtaining secreted alginate lyase YH7N1.

[0014] Furthermore, the above method specifically includes the following steps: (1) The gene of alginate lyase YH7N1 shown in sequence SEQ ID NO:2 was fully synthesized and constructed into the expression vector pCold-1 to obtain the recombinant plasmid pCold-YH7N1; (2) The recombinant plasmid pCold-YH7N1 was transformed into Escherichia coli competent cells, and the purified recombinant plasmid was extracted. (3) The purified recombinant plasmid was transformed into BL21 protein competent cells and purified to obtain alginate lyase YH7N1.

[0015] Further, the specific purification steps in step (3) are as follows: collect the induced bacterial cells, wash them with ddH2O and 0.1 mM PBS buffer, sonicate and centrifuge to collect the crude protein solution; filter through a 0.45 μm filter membrane and load the sample onto a Ni-NTA affinity chromatography column, bind for 2 h and then wash with PBS, then elute in stages with a buffer containing 200 mM imidazole, collect the 200 mM imidazole eluent, which is the pure enzyme solution of alginate lyase YH7N1.

[0016] Application of alginate lyase YH7N1 in the preparation of alginate oligosaccharides.

[0017] Furthermore, the alginic oligosaccharide is a fucoidan.

[0018] Furthermore, the alginate lyase YH7N1 and Mn 2+ When used together, the substrate for enzymatic hydrolysis is sodium alginate.

[0019] The advantages of this invention are: (1) A new alginate lyase was obtained through metagenomic mining and identified as a new member of the PL7 family by bioinformatics analysis. Phylogenetic analysis showed that the enzyme originated from Clostridium grantii and had a sequence identity of 48.75% with the homologous protein WP_073337524.1 in the PL7 family. The similarity was low, indicating that it is a novel alginate lyase.

[0020] (2) Using sodium alginate as a substrate, enzymatic hydrolysis was performed under optimal conditions, with Mn 2+ With the aid of [unclear], under optimal enzymatic hydrolysis conditions, the enzyme activity reached 46.92±0.54 U, which was significantly higher than that of the control group under optimal enzymatic hydrolysis conditions and with Mn [unclear]. 2+ With the help of enzymes, enzyme activity increased by nearly 10 times.

[0021] (3) Under optimal enzymatic hydrolysis conditions, 1% sodium alginate was used as a substrate and YH7N1 alginate lyase was used for enzymatic hydrolysis. The product was entirely fucoidan.

[0022] (4) Enzymatic hydrolysis has mild reaction conditions, simple operation, low cost and green environmental protection. Attached Figure Description

[0023] Figure 1 :YH7N1 source sequence consistency.

[0024] Figure 2 Phylogenetic tree diagram of YH7N1 alginate digestase.

[0025] Figure 3 Diagram of recombinant plasmid construction.

[0026] Figure 4 Electrophoresis image of YH7N1 protein purified by heterologous expression (from left to right, imidazole concentrations are 10, 50, 100, 200, 300, and 400 mM, respectively. The protein concentration is highest when the elution buffer concentration is 200 mM, and no protein can be purified at 400 mM).

[0027] Figure 5 : The enzymatic hydrolysis product of alginate lyase identified by HPLC.

[0028] Figure 6 : The enzymatic hydrolysis product of alginate lyase identified by MS. Detailed Implementation

[0029] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative and not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.

[0030] Example 1 1. Metagenomics to discover novel alginate lyases A novel alginate lyase was obtained through metagenomic mining of kelp, and its amino acid sequence is shown in SEQ ID NO:1. Bioinformatics analysis identified it as a new member of the PL7 family and named it YH7N1 (National Microbial Science Data Center (NMDC) accession number: NMDCP0003426). Phylogenetic analysis showed that this enzyme has 48.75% sequence identity with Clostridium grantiiWP_073337524.1 (…). Figure 2 The gene was named YH7N1. The complete gene sequence of YH7N1 (SEQ ID NO:2) was obtained by optimizing according to the codon bias of E. coli and then sent to a biotechnology company for synthesis.

[0031] 2. Heterologous expression of alginate lyase synthesis gene (1) Using PCR amplification YH7N1,The expression vector pCold-1 was digested using restriction endonucleases. The expression vector and the PCR-amplified fragment were then recombined using recombinase in a metal bath at 37°C to achieve in vitro circularization of the two linearized DNAs, thus constructing the desired DNA structure. YH7N1 Recombinant expression plasmids of genes Figure 3 Take 1 μL of recombinant expression plasmid and gently mix it into 50 μL of competent E. coli expression cells. Incubate on ice for 30 min, then heat shock at 42 ℃ for 90 s, followed immediately by an ice incubation of 5 min. Add 500 μL of LB medium and incubate at 37 ℃ on a shaker for 30 min. After incubation, centrifuge at 12000 rpm for 1 min, pipette 400 μL of supernatant and discard it, leaving only 100 μL of bacterial cells. Gently mix the remaining bacterial cells and liquid with the supernatant, then spread the remaining bacterial cells and liquid onto LB agar plates (containing 100 μg / mL kanaciline) and incubate at 37 ℃ for 12 h. Pick a single clone of recombinant plasmid and inoculate it into 10 mL of LB liquid medium (containing 100 μg / mL kanaciline), and incubate at 37 ℃ and 180 rpm for 12 h. Take 1 mL of the medium, centrifuge at 12000 rpm for 1 min, aspirate the supernatant and retain the bacterial cells. Repeat five times to collect sufficient bacterial cells. Add 250 μL of PA lysin and lyse in a homogenizer for 1 min (60 Hz). Add 250 μL of PB lysin and gently mix. Let stand for 4 min (until the turbid bacterial solution becomes clear). Add 350 μL of PC lysin and mix. Centrifuge at 12000 rpm for 10 min. Transfer the supernatant to a silica gel centrifuge tube (pre-fill the tube with 200 μL of BL buffer, centrifuge at 12000 rpm for 30 s and discard the BL buffer). Centrifuge at 12000 rpm for 1 min and discard the waste liquid. Transfer 600 μL of PW wash buffer and centrifuge at 2000 rpm for 1 min. Discard the waste liquid. Repeat twice. Centrifuge at 12000 rpm for 2 min and discard the waste liquid. Add 65 μL of sterile ddH2O (preheated to 65°C) to a silica gel tube and centrifuge at 12000 rpm for 1 min. Repeat twice to obtain the recombinant plasmid pCold-YH7N1.

[0032] (2) Heterologous expression of recombinant plasmids 1 μL of recombinant plasmid pCold-YH7N1 was expressed in 50 μL of BL21(DE3) protein competent cells (transformation method was the same as for the E. coli colony competent cells). The transformants were inoculated into 10 mL of LB liquid medium (containing 100 μg / mL kanamycin) and cultured at 37 ℃ and 180 rpm for 12 h. 100 μL of bacterial culture was then inoculated into 100 mL of LB liquid medium (containing 100 μg / mL kanamycin) and cultured on a shaker at 37 ℃ and 180 rpm for 6 h. The culture was continued until the bacterial concentration reached OD500.540 When the value is 0.6, add 200 μL of IPTG (0.2 mM concentration) and induce at low temperature (16 °C) for 10 h.

[0033] (3) Purification of crude protein solution: The induced solution obtained in step (2) was first centrifuged at 9000 rpm for 10 min at 4 ℃ to collect the bacterial cells (repeated collection until all bacterial cells were collected). The bacterial cells were resuspended and washed with 20 mL of sterilized and pre-cooled ddH2O at 4 ℃, and centrifuged at 9000 rpm for 3 min, repeated 3 times. Then, 20 mL of PBS (0.1 mM) buffer was added for resuspending and washing, and centrifuged at 9000 rpm for 3 min. Finally, 20 mL of PBS buffer was added to resuspend the bacterial cells to prepare for cell disruption. Each sample was sonicated for 30 min, and finally centrifuged at 9000 rpm for 10 min to collect the bacterial solution, which is the crude protein solution. The crude protein solution was then purified using a NI-NTA affinity chromatography column. 10 mL of PBS (0.1 mM) buffer was used to equilibrate the Ni column. The crude protein solution was filtered through a 0.45 μm filter membrane to remove impurities. The filtered sample was loaded onto the Ni column and allowed to stand for 2 h (to allow the protein to fully bind with Ni ions). The column was washed again with 10 mL of PBS buffer (0.1 mM) to remove unbound impurity proteins. Finally, the column was eluted with buffers containing different concentrations of imidazole (10, 50, 100, 200, 300, 400 mM) (the eluted protein was the purified enzyme solution). The eluted protein was placed in 2 mL centrifuge tubes and stored at -20°C for protein electrophoresis verification.

[0034] (3) SDS-PAGE protein verification: 100 μL of each sample was added to 10 μL of protein buffer (10x protein loading buffer), and the protein was denatured at 100 °C for 5 min. 20 μL of sample was then spotted into each well of the protein gel, and the gel was run at 150 V for 1 h 30 min. After running, the protein gel was removed, and fixative was added to cover the gel. The gel was shaken for 5 min, and the fixative was discarded. Staining solution was added to cover the gel, and the gel was shaken for 10 min. Destaining solution was added to cover the gel, and the gel was shaken for 2-3 h. The destaining solution was discarded, and the process was repeated, with the gel being shaken overnight for destaining. Protein electrophoresis showed that the protein concentration was highest at a 200 mM imidazole concentration. Quantification of the 200 mM protein hydrolysate was performed using ImageJ software, revealing a protein concentration of 117.5 μg after hydrolysis. To investigate the optimal hydrolysis conditions, the protein with the highest 200 mM protein concentration was selected as the hydrolysate for subsequent validation of optimal enzyme activity. Figure 4 ).

[0035] 3. Enzymatic property analysis To investigate the optimal enzymatic hydrolysis temperature of YH7N1 (using purified protease solution eluted with 200 mM imidazole), the results are shown in Table 1. Starting from 20 °C, the enzyme activity continuously increased with increasing temperature, reaching its peak at 35 °C, after which the enzyme activity continuously decreased. The highest enzymatic hydrolysis efficiency at 35 °C was 16.17 ± 0.69 U. Therefore, the optimal temperature for H7N1 is 35 °C.

[0036] Table 1. Optimal reaction temperature of alginate dehydrogenase

[0037] The effect of pH on the activity of YH7N1 was investigated at the optimal reaction temperature. To determine the optimal pH value for this enzyme, measurements were performed between pH 4 and 10 (HAc-NaAc buffer for pH 4-6, Tris-HCl buffer for pH 6-8, and Gly-NaOH buffer for pH 8-10), as shown in Table 2. Enzyme activity increased with increasing pH, reaching a maximum of 20.01 ± 2.79 U at pH 6. After pH 6, enzyme activity began to decrease with further increases in pH. Therefore, the optimal pH value for this enzyme is 6.

[0038] Table 2 Optimal pH values ​​for YH7N1

[0039] The optimal enzyme hydrolysate concentration was verified under optimal temperature (35 ℃) and optimal pH (pH=6). Alginate lyase solution (i.e., purified protease solution eluted with 200 mM imidazole) with the highest protein concentration was used at concentrations of 50 μL, 100 μL, 150 μL, 200 μL, 250 μL, 300 μL, and 400 μL to investigate the optimal enzyme concentration. The results showed that starting from 50 μL, enzyme activity increased with increasing enzyme concentration, reaching a maximum of 26.43 ± 1.41 U at 150 μL. Furthermore, after 150 μL, enzyme activity decreased with further increases in enzyme concentration. Therefore, the optimal enzyme concentration is 150 μL.

[0040] Table 3 Optimal enzyme concentration for YH7N1

[0041] The optimal concentration of sodium alginate as a substrate was determined under the optimal reaction conditions of 35 °C, pH=6, and an enzyme addition of 150 μL. The results (Table 4) showed that enzyme activity increased with increasing sodium alginate concentration, reaching its highest level of 43.24 ± 0.26 U at a sodium alginate concentration of 1%. Thereafter, enzyme activity decreased with further increases in sodium alginate concentration. Therefore, the optimal reaction concentration of sodium alginate is 1%.

[0042] Table 4 Optimal Sodium Alginate Concentration for YH7N1

[0043] Under optimal conditions (35 °C), optimal pH (pH=6), enzyme addition of 150 μL, and substrate concentration of 1% sodium alginate, Mg was used. 2+ Cu 2+ Mn²⁺, Na + Fe 2+ K + Li + Ca 2+ An ionic solution (1% concentration, dissolved in ddH₂O) reacted with YH₇N₁. The results showed that only Mn... 2+ Ions can promote the reaction efficiency of YH7N1, and in Mn 2+ With the aid of ions, the enzyme activity can reach 46.92±0.54U.

[0044] The control group in the table represents the results of the reaction under the same conditions after the enzyme was inactivated by YH7N1a enzyme hydrolysate at 100℃ for 10 minutes, thus losing its enzyme activity.

[0045] Table 5. Effects of metal ions on the activity of YH7N1 enzyme

[0046] 4. Identification of catalytic products (1) High-performance liquid chromatography (HPLC) identification of catalytic products Take 150 μL of enzyme solution, 200 μL of HAc-NaAc buffer (pH=6), and 40 μL of 1% Mn. 2+The reaction was carried out at 35 °C for 1 h with an ionic solution (1% concentration, dissolved in ddH₂O). After the reaction was complete, the mixture was immediately placed on ice for 5 min to stop the reaction. The mixture was centrifuged at 12000 rpm for 2 min, and the reactants were filtered through a 0.22 μm water film to obtain the final enzymatic hydrolysis product, which was then analyzed by HPLC. The Shimadzu HPLC system was used with a SEC column (Superdex 30 Increase 10 / 300 GL), an oven temperature of 35 °C, a flow rate of 0.5 mL / min, an injection volume of 10 μl, and an elution time of 25 min. Isocratic elution was used, with mobile phase A being 100% methanol and mobile phase B being 0.1% aqueous acetic acid.

[0047] (2) Mass spectrometry (MS) identification of catalytic products Take 150 μL of enzyme solution (i.e., pure protease solution eluted with 200 mM imidazole), 200 μL of HAc-NaAc-buffer (pH=6), and 40 μL of Mn. 2+ The reaction was carried out at 35 °C for 1 h in a 1% solution dissolved in ddH₂O. After the reaction, the solution was immediately placed on ice for 5 min to stop the reaction. The mixture was centrifuged at 12000 rpm for 2 min, and the reactants were filtered through a 0.22 μm water membrane to obtain the final enzymatic hydrolysis product, which was then analyzed by MS. Data analysis was performed using a Thermo Scientific mass spectrometer. The analysis time was 60 min, the detection mode was negative ion mode, and the electrospray ionization (ESI) ion source was used. The capillary voltage was 3.0 kVolts, the cone voltage was 5 Volts, the ion source temperature was 150 °C, the desolvation gas temperature was 400 °C, the desolvation gas flow rate was 800 L / h (N₂, purity 99.9%), the cone gas flow rate was 50 L / h, the collision energy was 6 / 20 Volts, the Deterctor voltage was 1800 Volts, and the mass range was 50–1400 m / z. Analysis of alginate lyase hydrolysis products using MS ( Figure 6 ), Figure 6 The mass spectrum corresponding to the absorption peak of the enzymatic hydrolysis product is shown. The product corresponding to the mass-to-charge ratio (m / z) of 326.7705 is a disaccharide.

[0048] Based on the above experiments, the optimal reaction conditions for this alginate lyase were determined (substrate concentration of 1% alginate, reaction at 35 ℃ for 1 h, pH 6, enzyme addition of 150 μL, and Mn...). 2+ (Using a cofactor) The enzyme activity reached a maximum of 46.92 U. High-performance liquid chromatography and mass spectrometry confirmed that all the enzymatic hydrolysis products were fucoidan, with a molecular weight of 194 mAU, and the concentration of the disaccharide produced in the reaction system reached 1 ng / μl.

[0049] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. An alginate lyase YH7N1, characterized in that: The amino acid sequence of the alginate lyase YH7N1 is shown in SEQ ID NO:

1.

2. The gene encoding the alginate lyase YH7N1 of claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

2.

3. A genetically engineered bacterium for producing the alginate lyase YH7N1 as described in claim 1, characterized in that: The alginate lyase YH7N1 gene as described in claim 2 was introduced into the genetically engineered bacteria.

4. A method for preparing the alginate lyase YH7N1 as described in claim 1, characterized in that: After the gene of alginate lyase YH7N1 with nucleotide sequence as shown in SEQ ID NO:2 was fully synthesized, it was constructed into an expression vector, introduced into Escherichia coli, induced and cultured, and secreted alginate lyase YH7N1 was obtained.

5. The method according to claim 4, characterized in that: Specifically, the following steps are included: (1) The gene of alginate lyase YH7N1 shown in sequence SEQ ID NO:2 was fully synthesized and constructed into the expression vector pCold-1 to obtain the recombinant plasmid pCold-YH7N1; (2) The recombinant plasmid pCold-YH7N1 was transformed into Escherichia coli competent cells, and the purified recombinant plasmid was extracted. (3) The purified recombinant plasmid was transformed into BL21 protein competent cells and purified to obtain alginate lyase YH7N1.

6. The method according to claim 4, characterized in that: The specific purification steps in step (3) are as follows: Collect the induced bacterial cells, wash them with ddH2O and 0.1 mM PBS buffer, sonicate and centrifuge to collect the crude protein solution; filter through a 0.45 μm filter membrane and load the sample onto a Ni-NTA affinity chromatography column, bind for 2 h and then wash with PBS, then elute in stages with a buffer containing 200 mM imidazole, collect the 200 mM imidazole eluent, which is the pure enzyme solution of alginate lyase YH7N1.

7. The application of the alginate lyase YH7N1 as described in claim 1 in the preparation of alginate oligosaccharides.

8. The application according to claim 7, characterized in that: The alginic oligosaccharide is a fucoidan.

9. The application according to claim 7, characterized in that: alginate lyase YH7N1 and Mn 2+ When used together, the substrate for enzymatic hydrolysis is sodium alginate.

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