Fuco-oligosaccharide, preparation method therefor, and use thereof
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- RENHE GLOBAL (SHANGHAI) GRAND HEALTH RESEARCH INSTITUTE CO LTD
- Filing Date
- 2025-08-15
- Publication Date
- 2026-07-02
Smart Images
Figure CN2025115159_02072026_PF_FP_ABST
Abstract
Description
A fucoidan, its preparation method and application
[0001] Cross-references to related applications
[0002] This application claims priority to Chinese Patent Application No. 202411913001.5, filed on December 24, 2024, entitled "A Fucoidan and its Preparation Method and Application", the entire contents of which are incorporated herein by reference. Technical Field
[0003] This disclosure relates to the field of bioenzyme technology, specifically to a fucoidan oligosaccharide, its preparation method, and its application. Background Technology
[0004] Fucoidan is a polysaccharide extracted from seaweed, belonging to the oligosaccharide family. It is mainly obtained from fucoidan molecules through enzymatic or acid hydrolysis and possesses certain biological activities and health benefits. Fucoidan is believed to have benefits such as regulating immune function, anti-oxidation, and lowering blood lipids, and is widely used in food, health products, and pharmaceuticals. However, although high molecular weight fucoidan possesses certain biological activities, its application is limited due to its large molecular weight, poor solubility, difficulty in absorption, and low oral bioavailability.
[0005] Fucoidosaccharides are generally prepared through chemical hydrolysis, physical degradation, and enzymatic hydrolysis. Acid hydrolysis can yield a large amount of product in a short time, but due to its mechanism of action, it can sometimes destroy the activity of fucoidan (sulfate groups, branched structures, etc.). In recent years, physical methods such as ultrasonic degradation have been used to prepare fucoidan. Although these methods are highly effective, have a short preparation cycle, do not destroy the bioactivity of the substance, and do not require the addition of other substances, their drawback is that the molecular weight of fucoidan cannot be controlled. Since bioactivity is closely related to molecular weight, this increases the uncertainty of the product.
[0006] Therefore, there is an urgent need to develop a method for preparing fucoidan oligosaccharides that is mild, produces products with a concentrated molecular weight distribution, and does not damage the bioactivity of the substances.
[0007] Application content
[0008] The purpose of this disclosure is to overcome the shortcomings of the prior art and provide a fucoidan, its preparation method, and its application.
[0009] To achieve the above objectives, the technical solution adopted in this disclosure is as follows:
[0010] In a first aspect, this disclosure provides an α-1,4-endo-fucoidanase, the amino acid sequence of which is shown in SEQ ID No: 3.
[0011] Secondly, this disclosure provides a nucleic acid encoding α-1,4-endofucoidase, the nucleotide sequence of which is shown in SEQ ID No: 4.
[0012] Thirdly, this disclosure provides a method for preparing fucoidan oligosaccharides, comprising the following steps:
[0013] (1) Add the α-1,4-endofucosaccharase to the fucoidan solution and hydrolyze to obtain the enzymatic hydrolysate; the conditions for the enzymatic hydrolysis are: pH 7-8, temperature 45℃-55℃, time 12h-24h;
[0014] (2) Inactivate the enzyme in the enzymatic hydrolysate, cool to room temperature, centrifuge and filter to obtain supernatant 1;
[0015] (3) Add ethanol to the supernatant 1, and centrifuge after precipitation is complete to obtain supernatant 2;
[0016] (4) The ethanol in the supernatant 2 is completely evaporated to obtain a solid; the solid is redissolved with deionized water to obtain supernatant 3;
[0017] (5) The supernatant 3 is dialyzed to remove impurities and then freeze-dried to obtain fucoidan oligosaccharide.
[0018] The preparation method disclosed herein is mild, environmentally friendly, does not damage the structure and bioactivity of the product, and allows for control of the product's molecular weight. Furthermore, the fucoidan product exhibits a high degree of molecular weight distribution concentration and uniformity, good antioxidant and hygroscopic properties, making it valuable for application, and eliminating the need for subsequent separation processes to obtain products with different molecular weights.
[0019] As a specific embodiment of the method for preparing fucoidan oligosaccharides as disclosed herein, in step (1): the concentration of the fucoidan solution is 10 g / L-15 g / L; the solvent of the solution is Tris-HCl buffer; and the amount of α-1,4-endo-fucoidan hydrolase added is 50-100 U / g fucoidan.
[0020] As a specific embodiment of the method for preparing fucoidan oligosaccharides as disclosed herein, in step (1): the method for preparing α-1,4-endofucoidase is as follows: recombinant Escherichia coli carrying the endofucoidase gene is inoculated into a liquid culture medium to activate and obtain seed liquid, and then the seed liquid is inoculated into a fermentation culture medium for fermentation to obtain crude enzyme solution.
[0021] The seed culture conditions are: temperature 30℃-37℃, time 10h-16h; the fermentation culture conditions are: temperature 25℃-30℃, time 12h-24h.
[0022] As a specific embodiment of the method for preparing fucoidan oligosaccharides as disclosed herein, in step (2): the enzyme inactivation conditions are: 90℃-105℃, 10min-20min; the centrifugation conditions are: temperature 20℃-25℃, rotation speed 8000rpm-10000rpm, time 15min-20min.
[0023] As a specific embodiment of the method for preparing fucoidan oligosaccharides as disclosed herein, in step (3): the volume ratio of the supernatant 1 to the ethanol is 1:3-5; the precipitation temperature is 2℃-8℃; the centrifugation conditions are: temperature 4℃-10℃, rotation speed 8000rpm-10000rpm, and time 10min-20min.
[0024] As a specific embodiment of the method for preparing fucoidan oligosaccharides as disclosed herein, in step (3): the concentration of ethanol is 50%-100%.
[0025] As a specific embodiment of the method for preparing fucoidan oligosaccharides as disclosed herein, in step (4): the volatilization temperature is 60℃-80℃, and the time is 1h-3h.
[0026] As a specific embodiment of the method for preparing fucoidan oligosaccharides as disclosed herein, in step (5): the dialysis uses a 100Da-400Da dialysis bag; the dialysis conditions are: temperature 2℃-8℃, time 24h-36h.
[0027] Fourthly, this disclosure provides a fucoidan oligosaccharide prepared by the above-described preparation method.
[0028] The fucoidan prepared in this disclosure has a small molecular weight (e.g., number average molecular weight of 497 Da and weight average molecular weight of 1356 Da), and significant antioxidant, hygroscopic and moisturizing properties.
[0029] Fifthly, this disclosure applies the α-1,4-endofucoidase, the nucleic acid, the preparation method, and the fucoidan to food, cosmetics, and pharmaceuticals. Attached Figure Description
[0030] Figure 1 shows the determination of the ABTS scavenging ability of fucoidan in this disclosure;
[0031] Figure 2 shows the hygroscopic capacity (RH-81%) of the fucoidan sample disclosed in this invention.
[0032] Figure 3 shows the hygroscopic capacity (RH-43%) of the fucoidan sample disclosed in this invention. Detailed Implementation
[0033] To better illustrate the purpose, technical solutions, and advantages of this disclosure, the following will further describe this disclosure in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of this disclosure and are not intended to limit this disclosure.
[0034] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified. The fucoidan raw material used in the examples is brown algae, purchased from Qingdao Mingyue Hailin Fucoidan Biotechnology Co., Ltd., product batch number: 572212002; all other chemical reagents are analytical grade.
[0035] Example 1: Construction of recombinant bacteria expressing mutants
[0036] 1. Constructing recombinant Escherichia coli
[0037] The gene of fucoidan hydrolase (GeneBank No: WP_132216013) was used to synthesize the cloning vector Pcola Duet by Tianlin Biotechnology (Shanghai) Co., Ltd. The vector was resistant to kanamycin and contained a gene with a base sequence of 6726pb. The plasmid was then introduced into competent cells of Escherichia coli to obtain recombinant bacteria BL21(DE3) / pCOLA-Duet-FcnA.
[0038] 2. Site-directed mutagenesis of α-1,4-endofucoidase
[0039] Primers were designed based on the gene encoding FcnA from Mariniflexile fucanivorans as shown in SEQ ID NO.1 to determine the mutation site. The specific primers are as follows:
[0040] T368S-F: TGTCGAGTGGagcTCTACCGGTATTGTTAATGGCGG;
[0041] T368S-R: TAGAgctCCACTCGACAAACTGCTCGTCAGTC;
[0042] P418A-F: TTCCAGTCCgcgGGTAAGCCGAACTGGTCGCG;
[0043] P418A-R:TTACCcgcGGACTGGAACTCCTTCAGGTAGGT.
[0044] The underlined portion represents the codons corresponding to the mutation of threonine to serine at position 368 and proline to alanine at position 418 in the protein encoded by the mutant gene.
[0045] The PCR amplification system is as follows:
[0046] Table 1 Amplification System
[0047] After PCR amplification, 2 μL of DpnⅠ restriction endonuclease (10 U / μL) was added to the reaction solution, and the sample was incubated at 37°C for 2 h to eliminate the template. The PCR product was transformed into E. coil DH5α cells, plated on LB agar plates, and incubated overnight at 37°C. Single colonies were picked from the plates and transferred to LB liquid medium, plasmids were extracted, and sequencing yielded the correct mutant plasmid pCOLA-Duet-FcnA. T368S / P418A The successfully constructed mutant plasmid was transformed into E. coil BL21(DE3) to obtain recombinant cells BL21(DE3) / pCOLA-Duet-FcnA that could express the mutant. T368S / P418A .
[0048] Example 2: Expression and purification of the original enzyme and the mutant enzyme
[0049] The original BL21(DE3) / pCOLA-Duet-FcnA and the mutant BL21(DE3) / pCOLA-Duet-FcnA prepared in Example 2 were picked separately. T368S / P418A Single colonies were cultured in LB liquid medium containing 50 μg / mL kanamycin at 37°C and 200 rpm for 12 h, then transferred to LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 rpm until OD reached. 600 At 0.7, 1 mmol / L IPTG was added and the mixture was induced overnight at 20 °C and 200 r / min. The fermentation broth was then collected.
[0050] Centrifuge the collected fermentation broth at 8000 rpm and 4°C for 10 min, discard the supernatant, collect the bacterial cells, resuspend the cells in 10 mL of Tris-HCl buffer, and sonicate for 20 min (30% power, 1 s disruption, 2 s pause). Centrifuge again at 8000 rpm and 4°C for 10 min, collect the supernatant, which is the crude enzyme solution. Use Binding Buffer to prepare the Ni... 2+ The chelated agar resin column was pre-equilibrated; crude enzyme solution was added and equilibrated with Binding Buffer and Washing Buffer respectively; the enzyme solution was eluted with Elution Buffer and recovered; the recovered enzyme solution was dialyzed in dialysis buffer at 4°C to finally obtain pure enzyme solution of α-1,4-endofucoidase.
[0051] The specific enzyme activity of the purified enzyme solution α-1,4-endofucoidase was measured separately:
[0052] Protein content determination: The protein content in the pure enzyme solution was determined using a BCA kit (Yamei Biotechnology).
[0053] Enzyme activity assay conditions: The reaction system included 200 μL of fermentation broth or supernatant diluted 100 times and purified, 200 μL of fucoidan solution with a concentration of 15 g / L (pH = 7.5), reacted at 50℃ for 30 min, boiled for 15 min to inactivate the enzyme, cooled and centrifuged, and the reducing sugar content was determined by the DNS method and calculated using the fucoidan standard curve.
[0054] Enzyme activity unit (U): The amount of enzyme required to produce 1 μmol of fucose per minute at 50°C is defined as one enzyme activity unit.
[0055] The formula for calculating the specific enzyme activity of α-fucopolysaccharide enzyme is as follows:
[0056] Fucoidan specific enzyme activity (U·mg) -1 ) = 1000AN / Mt / m
[0057] 1000 is the coefficient for conversion from mmol to μmol; A is the reducing sugar content (mg); N is the dilution factor of the enzyme solution; M is the relative molecular mass of fucose (164.16); T is the reaction time (min); m is the protein content (mg).
[0058] The results showed that the enzyme activity of the original BL21(DE3) / pCOLA-Duet-FcnA α-fucopolysaccharide enzyme was 55.0 U·mg. -1 The mutant BL21(DE3) / pCOLA-Duet-FcnA T368S / P418A The enzyme activity of α-fucopolysaccharide enzyme was 133.65 U·mg. -1 The mutant's enzyme activity was 2.43 times that of the mutant.
[0059] Example 3: Preparation of Fucoidan
[0060] The experimental groups and control groups 1-8 were set up as follows:
[0061] 1. The preparation method of fucoidan in the experimental group is as follows:
[0062] (1) Dissolve solid fucoidan in Tris-HCl buffer (pH=7.5) to obtain a 15 g / L fucoidan solution;
[0063] (2) Add the pure enzyme solution of α-1,4-endofucoidase prepared in Example 2 (mutant BL21(DE3) / pCOLA-Duet-FcnA) T368S / P418AEnzymatic hydrolysis was performed with an enzyme dosage of 100 U / g fucoidan, a hydrolysis pH of 7.5, a hydrolysis temperature of 50℃, and a hydrolysis time of 12 h to obtain the hydrolysate.
[0064] (3) Enzyme inactivation: Inactivate the enzyme in the enzyme hydrolysate by boiling water bath, and after cooling to room temperature, centrifuge at 8000 rpm for 15 min and filter to obtain supernatant 1.
[0065] (4) Alcohol precipitation: Add three times the volume of anhydrous ethanol to the supernatant 1, place at 4℃, and centrifuge at 8000 rpm for 15 min to obtain supernatant 2 after the precipitation is complete.
[0066] (5) Evaporation: The ethanol in supernatant 2 is completely evaporated at 65°C;
[0067] (6) Redissolution: The substance obtained in step (5) is redissolved with deionized water to obtain supernatant 3;
[0068] (7) Removal of impurities: The supernatant was dialyzed with a 300 Da dialysis bag to remove salts. The retentate was freeze-dried at -80℃ to obtain fucoidan.
[0069] 2. The preparation method of fucoidan in control group 1 is as follows:
[0070] Compared with the preparation method of the experimental group, the only difference in the preparation method of fucoidan in control group 1 is:
[0071] In step (2), the enzymatic hydrolysis pH is 6.5.
[0072] 3. The preparation method of fucoidan in control group 2 is as follows:
[0073] Compared with the preparation method of the experimental group, the only difference in the preparation method of fucoidan in control group 2 is:
[0074] In step (2), the enzymatic hydrolysis pH is 8.5.
[0075] 4. The preparation method of fucoidan in control group 3 is as follows:
[0076] Compared with the preparation method of the experimental group, the only difference in the preparation method of fucoidan in control group 3 is:
[0077] In step (2), the enzymatic hydrolysis temperature is 30℃.
[0078] 5. The preparation method of fucoidan in control group 4 is as follows:
[0079] Compared with the preparation method of the experimental group, the only difference in the preparation method of fucoidan in control group 4 is:
[0080] In step (2), the enzymatic hydrolysis temperature is 70℃.
[0081] 6. The preparation method of fucoidan in control group 5 is as follows:
[0082] Compared with the preparation method of the experimental group, the only difference in the preparation method of fucoidan in control group 5 is:
[0083] In step (2), the amount of enzyme added is 20 U / g fucoidan.
[0084] 7. The preparation method of fucoidan in control group 6 is as follows:
[0085] Compared with the preparation method of the experimental group, the only difference in the preparation method of fucoidan in control group 6 is:
[0086] In step (2), the amount of enzyme added is 5 U / g fucoidan.
[0087] 8. The preparation method of fucoidan in control group 7 is as follows:
[0088] Compared with the preparation method of the experimental group, the only difference in the preparation method of fucoidan in control group 7 is:
[0089] In step (4), three times the volume of 75% ethanol is added to the supernatant 1.
[0090] 9. The preparation method of fucoidan in control group 8 is as follows:
[0091] Compared with the preparation method of the experimental group, the only difference in the preparation method of fucoidan in control group 8 is:
[0092] In step (4), three times the volume of 50% ethanol is added to the supernatant 1.
[0093] Test Example 1: Determination of total sugar yield, number-average molecular weight, weight-average molecular weight, and area of fucoidan oligosaccharides.
[0094] The fucoidan prepared in the experimental group and control groups 1-8 of Example 3 above were used to detect the total sugar yield, number-average molecular weight, weight-average molecular weight and fucoidan area.
[0095] (1) Total sugar yield of fucoidan oligosaccharides
[0096] The yield of fucoidan oligosaccharides is expressed as a percentage, which is the ratio of the total sugar content in the product to the total sugar content in the reaction mixture (fucoidan solution and pure enzyme solution of fucoidanase).
[0097] Fucoidan total sugar yield (%) = Total sugar content of product / Total sugar content of mixture × 100%.
[0098] Total sugar determination method: phenol-sulfuric acid method. The specific method is as follows: Pipette 1 mL of sample solution diluted to an appropriate concentration into a stoppered test tube, add 1 mL of 6% phenol solution, mix thoroughly, then quickly add 5 mL of concentrated sulfuric acid and shake well. Incubate at room temperature for 30 min, and measure the absorbance at 490 nm using a microplate reader.
[0099] Standard curve determination method: Pipette fucose standard solutions diluted to different concentrations into test tubes and perform the determination according to the method described above.
[0100] The results are shown in Table 2:
[0101] Table 2. Total sugar yield of fucoidan for each group
[0102] (2) Number-average molecular weight, weight-average molecular weight and fucoidan area
[0103] The freeze-dried fucoidan product was dissolved in pure water to prepare an aqueous solution with a concentration of 5 mg / mL. The solution was filtered through a 0.22 μm membrane, and the molecular weight distribution of each group of fucoidan was determined by gel permeation chromatography (GPC). The chromatographic conditions were as follows: column TSKgel G3000SWXL (7.8 mm × 300 mm), mobile phase 0.1 M Na2SO4-0.1 M Na3PO4 (pH 6.7), flow rate 0.5 mL / min, column temperature 30 ℃, and 2487 UV detector at UV 220 nm.
[0104] The number-average molecular weight (Mn) and weight-average molecular weight (Mw) of each component were determined using high-performance gel permeation chromatography (HPGPC). The area percentage of fucoidan oligosaccharides was obtained by area normalization method according to high-performance liquid chromatography. Chromatographic column: Ultrahydrogel™ Linear (300 mm × 7.8 mm id × 2), mobile phase: 0.1 mol·L⁻¹ NaNO₃, flow rate: 0.9 mL·min⁻¹, column temperature: 45 °C, detector: differential detector.
[0105] The results are shown in Table 3:
[0106] Table 3. Number-average molecular weight, weight-average molecular weight, and fucoidan area for each group
[0107] The higher the percentage of fucoidan area, the greater the degree of removal of high molecular weight fucoidan. Table 3 shows that after alcohol precipitation of supernatant 1 with 100% ethanol, the number-average molecular weight of fucoidan was relatively small, and the substrate area percentage was large; this indicates that the removal of high molecular weight substances was more thorough and effective compared to the other two treatment methods. The results from the 75% and 50% ethanol precipitation groups showed little difference in number-average and weight-average molecular weights.
[0108] Test Example 2: Detection of Antioxidant Activity
[0109] The antioxidant activity of the fucoidan prepared in the experimental group and control groups 1-8 of Example 3 was tested.
[0110] The method for determining antioxidant activity is as follows:
[0111] Prepare 7 mmol / L ABTS and 2.45 mmol / L K2S2O8 solutions for later use. Mix 2 mL of ABTS and 2 mL of K2S2O8 solution in an EP tube, wrap with aluminum foil, and place in a dark environment at room temperature for 14 h to prepare ABTS free radical stock solution. Dilute the ABTS free radical stock solution with deionized water to make its absorbance at 734 nm 0.70 ± 0.02.
[0112] Take 2.9 mL of ABTS free radical assay solution, and add 0.1 mL of each group of fucoidan oligosaccharide sample or fucoidan dilution solution. React in the dark for 10 min and measure the absorbance at 734 nm (A1). Use K2S2O8 solution instead of ABTS free radical assay solution to measure the absorbance (A2). Then use deionized water instead of the sample and measure the absorbance (A3) using the same procedure. Use antioxidant vitamin C as a positive control. Perform the experiment in triplicate for each sample.
[0113] The free radical scavenging ability of ABTS is expressed as scavenging rate R%: R% = ((A3-A1+A2) / A3) × 100%.
[0114] The test results are shown in Figure 1. The results indicate that the fucoidan has a significant ability to scavenge ABTS free radicals, and this ability is positively correlated with the concentration of fucoidan. When the fucoidan concentration is 1.5 mg / mL, the free radical scavenging ability is 100%. However, the antioxidant ascorbic acid has a higher ability to scavenge ABTS free radicals, achieving 100% ABTS scavenging rate at a concentration of 0.2 mg / mL. However, compared to the original high molecular weight fucoidan, the ABTS free radical scavenging ability of fucoidan is only 9.2% at a concentration of 3 mg / mL. This demonstrates that the molecular weight of the polysaccharide plays an important role in its ability to scavenge ABTS free radicals, and that lower molecular weight polysaccharides enhance their antioxidant activity.
[0115] Test Example 3: Testing Hygroscopic Activity
[0116] Fucoidosaccharides prepared in the experimental group and control groups 1-8 of Example 3 were tested for hygroscopic activity.
[0117] The method for determining hygroscopic activity is as follows:
[0118] Weigh 0.1000 g of fucoidan or each group of fucoidan oligosaccharide samples and glycerol (positive control) into weighing bottles. Record the initial weight of the sample as Wo. Place the bottles in a desiccator, using saturated (NH₄)₂SO₄ solution and saturated K₂CO₃ or sodium carbonate solution instead of silica gel to achieve humidity levels of 81% and 43% respectively. Control the temperature at 25℃. Measure the sample weight at 2h, 5h, 8h, 12h, 24h, 36h, 48h, and 60h, and record this weight as Wn. Perform three parallel experiments for each sample. The formula for calculating the moisture absorption rate is as follows:
[0119] Moisture absorption rate % = (Wn - Wo) / Wo × 100%.
[0120] The test results are shown in Figures 2 and 3. The results show that under high humidity conditions (81%), the hygroscopicity of fucoidan did not change significantly, while the hygroscopicity of fucoidan oligosaccharide increased rapidly from 0 to 12 hours, reaching saturation at 24 hours. In the first 5 hours, the hygroscopicity of the sample was similar to that of glycerol, but the difference gradually increased with time after 5 hours. Under low humidity conditions (43%), the hygroscopicity of fucoidan oligosaccharide increased from 0 to 10 hours, then gradually stabilized; while the hygroscopicity of fucoidan showed almost no upward trend from 2 to 60 hours. Under high and low humidity conditions, the highest hygroscopicity of fucoidan was 22.1% and 3.4%, respectively; the hygroscopicity of fucoidan oligosaccharide at saturation was 59.5% and 10.15%, respectively, reaching 62.32% and 42.29% of the hygroscopicity of glycerol at saturation, indicating that fucoidan oligosaccharide has good hygroscopicity.
[0121] Compared with the prior art, the beneficial effects of this disclosure are as follows:
[0122] The method for preparing fucoidan disclosed herein is mild, does not damage the activity of the product, and allows for directional control of the product's molecular weight, resulting in high product uniformity. Furthermore, the fucoidan prepared exhibits significant antioxidant and hygroscopic activities, making it a promising candidate for applications in food, cosmetics, and pharmaceuticals.
[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this disclosure and are not intended to limit the scope of protection of this disclosure. Although this disclosure has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this disclosure without departing from the substance and scope of the technical solutions of this disclosure. Industrial applicability
[0124] The method for preparing fucoidan disclosed herein is as follows: Fucoidan is enzymatically hydrolyzed using α-1,4-endofuctanase expressed in *Escherichia coli*. The process involves enzyme inactivation, centrifugation, alcohol precipitation, volatilization, resolventization, impurity removal, and drying to obtain fucoidan. This method for preparing fucoidan is mild, does not damage the activity of the product, and allows for targeted control of the product's molecular weight, resulting in high product uniformity. Furthermore, the obtained fucoidan exhibits significant antioxidant and hygroscopic activities, showing broad application prospects in food, cosmetics, and pharmaceuticals.
[0125] Sequence information:
[0126] SEQ ID No: 1 (Amino acid sequence of FcnA)
[0127] SEQ ID No: 2 (Gene sequence of FcnA)
[0128] SEQ ID No: 3 (Amino acid sequence of FcnA mutant)
[0129] SEQ ID No: 4 (Mutant gene sequence of FcnA)
Claims
1. An alpha- 1,4-endofucanase, characterized in that, The amino acid sequence of which is shown as SEQ ID No:
3.
2. A nucleic acid encoding an alpha- 1,4-endofucanase, characterized in that, The nucleotide sequence of which is shown as SEQ ID No:
4.
3. A method for producing fucoidan, characterized by, The method comprises the following steps: (1) adding the alpha-1, 4-endofucanase of claim 1 to a fucoidan solution to obtain an enzymatic hydrolysate; the enzymatic hydrolysis is carried out under the conditions of pH 7-8, temperature 45-55℃, and time 12-24h; (2) inactivating the enzyme of the enzymatic hydrolysate, cooling to room temperature, centrifuging and filtering to obtain supernatant 1; (3) adding ethanol to the supernatant 1, and centrifuging after the precipitation is completed to obtain supernatant 2; (4) evaporating the ethanol in the supernatant 2 completely to obtain a solid; redissolving the solid in deionized water to obtain supernatant 3; (5) removing impurities by dialysis of the supernatant 3, and freeze-drying to obtain fucoidan oligosaccharide.
4. The method for producing fuco-oligosaccharides according to claim 3, characterized by, In step (1), the concentration of the fucoidan solution is 10-15g / L; the solvent of the solution is Tris-HCl buffer; and the addition amount of the alpha-1, 4-endofucanase is 50-100U / g fucoidan.
5. The method for producing fuco-oligosaccharides according to claim 3, characterized by, In step (2), the inactivation conditions are 90-105℃ for 10-20min; and the centrifugation conditions are temperature 20-25℃, rotation speed 8000-10000rpm, and time 15-20min.
6. The method for producing fuco-oligosaccharides according to claim 3, characterized by, In step (3), the volume ratio of the supernatant 1 to the ethanol is 1:3-5; the precipitation temperature is 2-8℃; and the centrifugation conditions are temperature 4-10℃, rotation speed 8000-10000rpm, and time 10-20min.
7. The method for producing fuco-oligosaccharides according to claim 3, characterized by, In step (4), the evaporation temperature is 60-80℃, and the evaporation time is 1-3h.
8. The method for producing fuco-oligosaccharides according to claim 3, characterized by, In step (5), the dialysis bag is 100-400Da; and the dialysis conditions are temperature 2-8℃ and time 24-36h.
9. A fucoid oligosaccharide, characterized by, The fucoidan oligosaccharide is prepared by the method of any one of claims 3-8.
10. The alpha-1, 4-endofucanase of claim 1, the nucleic acid of claim 2, the preparation method of any one of claims 3-8, or the fucoidan oligosaccharide of claim 9 is used in food, cosmetics, and medicine.