Application of 3, 6-lactone-galactosidase OUC-ApdagB in preparation of carrageenan pentasaccharide

Abstract

The invention discloses application of 3, 6-lactone-galactosidase OUC-ApdagB in preparation of carrageenan pentasaccharide, and belongs to the technical field of preparation of carrageenan oligosaccharides. The amino acid sequence of the 3, 6-lactone-galactosidase OUC-ApdagB is as shown in SEQ ID NO. 1 (sequence identifier number 1). When the carrageenan pentasaccharide is specifically applied, the carrageenan pentasaccharide is prepared under the action of the 3, 6-lactone-galactosidase OUC-ApdagB by taking the red algae gum oligosaccharide as a substrate. The carrageenan pentasaccharide can be prepared under the action of 3, 6-lactone-galactosidase OUC-ApdagB by taking the red algae gum as a substrate, so that the vacancy of preparation of odd-numbered carrageenan oligosaccharides is filled, and the method has important significance on industrial preparation of odd-numbered carrageenan oligosaccharides.

Description

Technical Field

[0001] The invention relates to application of 3,6-endoether-galactosidase OUC-ApdagB in preparing carrageenan pentasaccharide, belonging to the technical field of preparation of carrageenan oligosaccharide. Background Art

[0002] Carrageenan is a linear macromolecular sulfated polysaccharide and one of the main components of the cell wall of red algae. It is composed of disaccharide units D-galactose (D-Gal) and 3,6-anhydro-D-galactose (D-AHG). Carrageenan oligosaccharides have strong functional activity and are widely used. There are large differences in the activity of carrageenan oligosaccharides with different structures. Compared with even-numbered carrageenan oligosaccharides, odd-numbered carrageenan oligosaccharides have better effects in anti-inflammatory and antioxidant aspects, so the preparation of odd-numbered carrageenan oligosaccharides is of great significance.

[0003] 3,6-Ether-galactosidase plays an important role in the production process of odd-numbered carrageenan oligosaccharides, which can exoclease even-numbered carrageenan oligosaccharides and generate corresponding odd-numbered carrageenan oligosaccharides and D-AHG. Only seven 3,6-Ether-galactosidases have been characterized in the prior art, and the study of enzymatic properties is incomplete. Therefore, it is necessary to explore new and efficient 3,6-Ether-galactosidases to provide tool enzymes for the preparation of odd-numbered carrageenan oligosaccharides with specific structures. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention provides the use of 3,6-endo-galactosidase OUC-ApdagB in the preparation of carrageenan pentasaccharide, belonging to the technical field of preparation of carrageenan oligosaccharides.

[0005] The present invention is achieved through the following technical solutions: Application of 3,6-endo-galactosidase OUC-ApdagB in the preparation of carrageenan pentasaccharide, wherein the amino acid sequence of the 3,6-endo-galactosidase OUC-ApdagB is shown in SEQ ID NO.1.

[0006] Furthermore, in a specific application, carrageenan pentasaccharide is prepared by using carrageenan oligosaccharide as a substrate under the action of 3,6-endo-galactosidase OUC-ApdagB.

[0007] Furthermore, the furcellaran oligosaccharide is prepared by degrading furcellaran with κ-carrageenase OUC-CgkA-Sn, and the amino acid sequence of the κ-carrageenase OUC-CgkA-Sn is shown in SEQ ID NO.3.

[0008] Furthermore, the furcellaran oligosaccharide is prepared by the following method: in a 2 ml reaction system, 0.5 U of κ-carrageenase OUC-CgkA-Sn and water are added to 1 ml of a 1 mg / ml furcellaran solution, and the mixture is reacted at 35° C. for 3 hours.

[0009] Furthermore, in specific applications, kappa-carrageenase OUC-CgkA-Sn is combined, and the amino acid sequence of the kappa-carrageenase OUC-CgkA-Sn is shown in SEQ ID NO. 3. In specific applications, carrageenan pentasaccharide is prepared by using furcellaran as a substrate under the action of kappa-carrageenase OUC-CgkA-Sn and 3,6-endo-galactosidase OUC-ApdagB.

[0010] Furthermore, in a specific application, in a 2 ml reaction system, 0.5 U of κ-carrageenase OUC-CgkA-Sn, 0.05 U of 3,6-endo-galactosidase OUC-ApdagB and water were added to 1 ml of 1 mg / ml red seaweed solution, and the reaction was carried out at 35°C for 3 h to prepare carrageenan pentasaccharide.

[0011] A method for preparing carrageenan pentasaccharide is as follows: taking red algae as a substrate, preparing carrageenan pentasaccharide under the action of kappa-carrageenanase OUC-CgkA-Sn and 3,6-endo-galactosidase OUC-ApdagB; the amino acid sequence of the 3,6-endo-galactosidase OUC-ApdagB is shown in SEQ ID NO.1, and the amino acid sequence of the kappa-carrageenanase OUC-CgkA-Sn is shown in SEQ ID NO.3.

[0012] Furthermore, the preparation method is as follows: in a 2 ml reaction system, 0.5 U of κ-carrageenase OUC-CgkA-Sn, 0.05 U of 3,6-endo-galactosidase OUC-ApdagB and water are added to 1 ml of 1 mg / ml red seaweed solution, and the reaction is carried out at 35°C for 3 h to prepare carrageenan pentasaccharide.

[0013] The present invention has discovered a new type of 3,6-endo-galactosidase, 3,6-endo-galactosidase OUC-ApdagB. After research, it was surprisingly found that it can be used to prepare carrageenan oligosaccharides with a specific structure, carrageenan pentasaccharide, with a specific structure of G-(DA-G4S) 2, that is, the carrageenan pentasaccharide lacking a sulfate group in the non-reducing section, and having substrate specificity, can degrade κ-carrageenan oligosaccharides (i.e., furcellaran oligosaccharides) without sulfate groups in the non-reducing section, but cannot degrade κ-carrageenan oligosaccharides. When preparing carrageenan pentasaccharides, the concentration of the substrate is low and the activity is high. In specific applications, κ-carrageenanase OUC-CgkA-Sn and 3,6-endo-galactosidase OUC-ApdagB can be added to the furcellaran solution at the same time, and carrageenan pentasaccharides and D-AHG can be directly generated at 35°C, and when the reaction time is more than 3 h, the oligosaccharide product is a single carrageenan pentasaccharide. The present invention can use furcellaran as a substrate, and prepare carrageenan pentasaccharides under the action of 3,6-endo-galactosidase OUC-ApdagB, which fills the gap in the preparation of odd-numbered carrageenan oligosaccharides and is of great significance for the industrial preparation of odd-numbered carrageenan oligosaccharides.

[0014] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Schematic diagram of SDS-PAGE electrophoresis detection results, where M is the standard protein Marker; 1 is the crude enzyme solution; 2 is the pure enzyme solution.

[0016] Figure 2 : Schematic diagram of the HPLC detection results of Example 4 (1).

[0017] Figure 3 : Schematic diagram of ESI-MS identification results of degradation product P1.

[0018] Figure 4 : Schematic diagram of the HPLC detection results of Example 4 (2).

[0019] Figure 5 : Schematic diagram of ESI-MS identification results of degradation product P2.

[0020] Figure 6 : Schematic diagram of the effect of reaction temperature on the relative yield of D-AHG.

[0021] Figure 7 : Schematic diagram of the effect of reaction pH on the relative yield of D-AHG.

[0022] Figure 8 : Schematic diagram of temperature stability test results.

[0023] Fig. 9 : Schematic diagram of pH stability test results.

[0024] Fig.10 : Schematic diagram of the HPLC detection results of Example 8.

[0025] Note: Figure 2 , Figure 4 , Figure 6 , Figure 7 When drawing the graph, in order to facilitate intuitive comparison, the upper and lower positions of each curve were moved. Therefore, the vertical axis in the figure has no practical meaning and does not need to be marked. What is compared is the peak area. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0027] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0028] The Escherichia coli BL21 (DE3) competent cells used in the present invention were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0029] The LB culture medium used in the present invention comprises the following components: 10 g / L sodium chloride; 10 g / L tryptone; 5 g / L yeast extract; 15 g / L agar powder (added when preparing the solid culture medium); the balance is water; and the sterilization conditions are: 115°C, 30 min.

[0030] The furcellaran used in the present invention is purchased from Carbosynth Company of the United Kingdom.

[0031] The κ-carrageenase OUC-CgkA-Sn used in the present invention is stored in the laboratory of the inventor. The relevant contents of κ-carrageenase OUC-CgkA-Sn (amino acid sequence, preparation method, enzymatic properties, etc.) are recorded in the Chinese invention patent application CN118374477 A.

[0032] Example 1 Mining of 3,6-galactosidase OUC-ApdagB The 3,6-endo-galactosidase OUC-ApdagB of the present invention is mined from the NCBI database, is derived from a marine microorganism Arenibacter palladensis strain DSM17539 that can survive with carrageenan as the only carbon source, and has a sequence number of WP_072864264.1.

[0033] The gene of 3,6-galactosidase contains 2016 bases, the nucleotide sequence is shown in SEQ ID NO.2, and it encodes 672 amino acids, the amino acid sequence is shown in SEQ ID NO.1. The results of phylogenetic tree analysis show that it belongs to the glycoside hydrolase 129 family (GH129). In order to study the enzymatic properties of 3,6-galactosidase OUC-ApdagB, the present invention expresses and purifies the enzyme and conducts relevant experimental studies, as shown in the following examples.

[0034] The amino acid sequence of 3,6-endo-galactosidase OUC-ApdagB is as follows, as shown in SEQ ID NO.1: .

[0035] The nucleotide sequence of the gene encoding 3,6-endo-galactosidase OUC-ApdagB is as follows (direction 5'-3'), as shown in SEQ ID NO.2:

[0036] Example 2 Preparation of 3,6-endo-galactosidase OUC-ApdagB Here are the steps: (1) We commissioned Sangon Biotech (Shanghai) Co., Ltd. to synthesize the coding gene of 3,6-endo-galactosidase OUC-ApdagB. We added 40 μl of pure water to the synthesized dry powder plasmid, and transformed the plasmid into competent Escherichia coli BL21 (DE3) cells. We spread the plasmid on a solid LB medium plate containing 50 μg / mL kanamycin sulfate, and cultured it in a 37°C incubator for 16 h. We picked a single clone and transferred it to a liquid LB medium containing 50 μg / mL kanamycin sulfate. We cultured it in a shaking incubator at 37°C and 220 rpm for 12 h. The engineered bacteria were grown on a kanamycin sulfate resistance plate to obtain a recombinant expression strain.

[0037] (2) After the recombinant E. coli strain was activated in 5 ml of liquid LB medium containing 50 μg / mL kanamycin, it was inoculated with 50 ml of liquid LB medium containing 50 μg / mL kanamycin sulfate at a 1% inoculum size and cultured at 37°C and 200 rpm for 3 h. When the OD (600) value of the bacterial liquid reached 0.6, 1‰ isopropyl-β-D-thiogalactoside (IPTG) (100 mM / L) was added and the culture was induced at 20°C for 16 h to express 3,6-tetramethyl-galactosidase.

[0038] (3) After the culture is completed, the culture medium was taken out and centrifuged at 8000 rpm for 10 min at 4°C. The bacteria were collected and resuspended in ultrapure water. Ultrasonic disruption was performed for 30 min (300 W, 3 s on, 3 s off). The supernatant was centrifuged at 8000 rpm for 15 min. The supernatant was the crude enzyme solution.

[0039] (4) Use Ni in crude enzyme solution 2+ -NTA column affinity chromatography purification: First, equilibrate the column with 10 mM imidazole solution (10 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl, pH 8.0), add the crude enzyme solution to allow it to bind to Ni 2+-NTA column binding, and then 40 mM imidazole solution (40 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl) was used to elute the weakly bound impurities, and then 200 mM imidazole solution (200 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl) was used to elute the target protein, and the eluate was collected. Ultrafiltration and concentration were performed using an ultrafiltration centrifuge tube with a molecular weight cutoff of 30 kDa at 4°C and 4000 rpm to obtain a pure enzyme solution containing 3,6-endo-galactosidase OUC-ApdagB. SDS-PAGE detection was performed to verify whether the bands were single and the size was accurate. The results are shown in Figure 1. Figure 1 It can be seen that the molecular weight is about 78 kDa, which is consistent with the prediction.

[0040] Example 3 Analysis of substrate specificity of 3,6-galactosidase OUC-ApdagB κ-carrageenan oligosaccharides and furcellan oligosaccharides were prepared using κ-carrageenanase OUC-CgkA-Sn, respectively. The preparation method of κ-carrageenan oligosaccharides is as follows: in a 2 ml reaction system, 0.4 U of κ-carrageenanase OUC-CgkA-Sn and ultrapure water were added to 1 ml of κ-carrageenan solution with a concentration of 1 mg / ml, mixed, and reacted at 35°C for 3 h, and the product was mainly κ-carrageenan tetrasaccharide. The preparation method of furcellan oligosaccharides is as follows: in a 2 ml reaction system, 0.5 U of κ-carrageenanase OUC-CgkA-Sn and ultrapure water were added to 1 ml of 1 mg / ml furcellan solution with a concentration of 1 mg / ml, mixed, and reacted at 35°C for 3 h, and the product was mainly κ-carrageenan hexasaccharide lacking a sulfate group. The κ-carrageenan solution is prepared from κ-carrageenan and water; the furcellan solution is prepared from furcellan and water.

[0041] After the reaction, 3,6-galactosidase OUC-ApdagB was added to the two substrates respectively. The amount of addition was: 0.05 U of 3,6-galactosidase OUC-ApdagB was added to each 1 mg of initial κ-carrageenan / furyl gum, and the reaction was carried out at 35°C for 3 h. After the reaction was completed, the reaction was terminated by boiling water bath for 10 min, centrifuged at 12000 rpm for 10 min to remove impurities such as proteins in the system, and the supernatant was collected and filtered through a 0.22 μm filter membrane as a sample for HPLC detection. The control group was the same reaction system, and an equal amount of boiling-inactivated 3,6-galactosidase OUC-ApdagB was added.

[0042] The amino acid sequence of κ-carrageenase OUC-CgkA-Sn is shown in SEQ ID NO.3, as shown below: MVRRILFVSMVIVASLSVCNQTRLFAQDGKHNVPLSAASTDQWKIRWDRSDDFDGDDVDWRKWNKSPENFGAWVWDNESNVAVSNGILQITMRRLPTPVVKGRRPPTPYTSGMLKSHVTGTYGYYEARVKAAALFPGVCPSFWLYSKIDDSIVA VGETRYSEVDIVELTQRGDRVPGNERIADCNLHAILSNGKPGIGGRDWRRPNDQCYKDAQANELRLPFDPRDDFHTYGCEVTPETVTWFIDGKAIGQKPNRYWHREMNVALSLGLRRPYSTYTVKGFVPSDVEVDDEFPTTMEVDYVRVWERVK.

[0043] Example 4 Product Analysis of 3,6-Ether-Galactosidase OUC-ApdagB (1) The sample prepared in Example 3 was subjected to HPLC detection. The HPLC conditions were: using a Superdex 30 10 / 300 gel filtration column with 0.2 M NH 4 HCO 3 The solution was used as the mobile phase, the flow rate was 0.4 mL / min, the column temperature was room temperature, the sample volume was 100 μL, the differential refractive index detector (RID) was used, and the detector temperature was 40°C.

[0044] HPLC test results are as follows Figure 2 As shown, 3,6-galactosidase OUC-ApdagB cannot act on κ-carrageenan oligosaccharides, but can react with furcellan oligosaccharides to produce a new degradation product P1. It can be seen that 3,6-galactosidase OUC-ApdagB has substrate specificity.

[0045] The degradation product P1 was identified by ESI-MS. Figure 3 As shown, the degradation product was detected at m / z 475.0. Through analysis, the degradation product was found to be carrageenan pentasaccharide lacking a sulfate group.

[0046] (2) The sample prepared using furcellaran oligosaccharide as substrate in Example 3 was subjected to HPLC detection. The HPLC conditions were as follows: using a Sugar Pak I column (6.5×300 mm) and 50 mg / L EDTANa 2Ca solution was used as the mobile phase, the flow rate was 0.5 mL / min, the column temperature was 75°C, the sample volume was 30 μL, and the products of 3,6-endo-galactosidase OUC-ApdagB and furcellaran oligosaccharides were detected by differential detection.

[0047] HPLC test results are as follows Figure 4 As shown, it can be seen that compared with the control group, a new degradation product P2 was generated, and the retention time of the degradation product P2 was the same as that of the D-AHG standard.

[0048] The degradation product P2 was identified by ESI-MS. Figure 5 As shown, the degradation product was detected at m / z 161.0. Through analysis, the degradation product was indeed D-AHG.

[0049] (3) Based on the above identification results, it was determined that when carrageenan oligosaccharides were used as substrates, under the action of 3,6-endo-galactosidase OUC-ApdagB, the products were carrageenan pentasaccharide lacking a sulfate group and D-AHG.

[0050] Example 5 Determination of the Optimal Conditions for 3,6-Ether-Galactosidase OUC-ApdagB A large amount of furcellaran oligosaccharide was prepared according to the preparation method of furcellaran oligosaccharide in Example 3. After the reaction was completed, the reaction was terminated by boiling water bath for 10 min, centrifuged at 8000 rpm for 10 min to remove impurities such as protein in the system, and the supernatant was collected and freeze-dried. Pure water was added to 20% of the volume before freeze-drying to prepare freeze-dried furcellaran oligosaccharide as a subsequent substrate.

[0051] (1) Effect of reaction temperature on relative yield Take 8 portions of substrate solution (90 μL each), add 10 μL of pure enzyme solution of Example 2 to each, react at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C for 10 min, terminate the reaction in boiling water bath for 10 min, centrifuge at 12000 rpm for 10 min, collect the supernatant, filter through 0.22 μm filter membrane, and perform HPLC quantitative detection. The HPLC conditions are the same as those of Example 4 (2). By detecting the yield of D-AHG, the effect of reaction temperature on the relative yield of D-AHG was evaluated.

[0052] Effect of reaction temperature on the relative yield of D-AHG Figure 6 As shown, it can be seen that when the reaction temperature is 35°C, the peak area is the largest, so the optimal reaction temperature of 3,6-endo-galactosidase OUC-ApdagB is 35°C.

[0053] (2) Effect of reaction pH on relative yield The buffers with different pH values ​​used were: 50 mM citric acid-sodium citrate (CPBS) buffer (pH 3.0, 4.0, 5.0, and 6.0, respectively), 50 mM phosphate (PBS) buffer (pH 6.0, 7.0, and 8.0, respectively), 50 mM Tris-HCl buffer (pH 8.0 and 9.0, respectively), and 50 mM glycine-sodium hydroxide buffer (pH 9.0 and 10.0, respectively).

[0054] Different pH buffers were used to dissolve freeze-dried algae oligosaccharides to obtain substrate solutions of different pH. Substrate solutions of different pH (90 μL each) were taken, and 10 μL of the pure enzyme solution of Example 2 was added to each solution. The solution was reacted at 35°C for 10 min, and the reaction was terminated by boiling water bath for 10 min. The solution was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane for quantitative HPLC detection. The HPLC conditions were the same as those of Example 4 (2). The effect of reaction pH on the relative yield of D-AHG was evaluated by detecting the yield of D-AHG.

[0055] Effect of reaction pH on relative yield of D-AHG Figure 7 As shown, it can be seen that when the reaction pH is 5.0 and 6.0, the peak area is relatively large, and the peak area is the largest when the pH is 6.0. Therefore, the optimal pH of 3,6-endo-galactosidase OUC-ApdagB is 6.0.

[0056] Example 6 Determination of the specific activity of 3,6-galactosidase OUC-ApdagB The determination method is as follows: take 90 μL of the furcellaran oligosaccharide solution, add 10 μL of the pure enzyme solution of Example 2, react at 35°C for 10 min, terminate the reaction in a boiling water bath for 10 min, centrifuge at 12000 rpm for 10 min, collect the supernatant, filter through a 0.22 μm filter membrane, and perform HPLC quantitative detection to detect the yield of D-AHG. The detection conditions are the same as those of Example 4 (2). The yield of D-AHG is determined by plotting the D-AHG concentration-peak area curve based on the peak area and the liquid phase peak area of ​​D-AHG standards of different concentrations. The furcellaran oligosaccharide solution is prepared from freeze-dried furcellaran oligosaccharide and citric acid-sodium citrate buffer (concentration 50 mM, pH 6.0), and the concentration of furcellaran oligosaccharide is 2.5 mg / ml (based on the initial furcellaran solution concentration).

[0057] Enzyme activity is defined as: the amount of enzyme required to produce 1 mg of D-AHG per unit time is one enzyme activity unit, i.e. 1 U.

[0058] It was determined that the activity of 3,6-endo-galactosidase OUC-ApdagB in hydrolyzing algae oligosaccharides was 0.471 U / mg.

[0059] Example 7 Determination of Temperature and pH Stability of 3,6-Ether-Galactosidase OUC-ApdagB Temperature stability The pure enzyme solution was incubated at 30℃, 35℃, and 40℃ for 8 h, and its residual 8 h enzyme activity was measured under the optimal conditions (temperature 35℃, pH 6.0) to obtain its temperature stability. The temperature stability test results are as follows: Figure 8 As shown in the figure, the enzyme activity of 3,6-galactosidase OUC-ApdagB can still be retained by more than 30% after being placed at 35℃ for 8 h, and the enzyme activity stability is good.

[0060] pH stability 80 μL of pure enzyme solution was mixed with 120 μL of citric acid-sodium citrate buffer at pH 5.0, pH 6.0, and pH 7.0, respectively, and incubated at 4°C. The residual enzyme activity after 8 h was measured under the optimal conditions (temperature of 35°C and pH 6.0) to obtain its pH stability. The results of pH stability test are shown in Fig. 9 As shown in the figure, 3,6-endo-galactosidase OUC-ApdagB was retained in the buffer of pH 5.0 and 6.0 for 8 h, and the enzyme activity was still retained by more than 30%, indicating that the enzyme has good stability.

[0061] Example 8 Preparation of carrageenan pentasaccharide using 3,6-endo-galactosidase OUC-ApdagB and kappa-carrageenase OUC-CgkA-Sn In a 2 ml reaction system, 0.5 U of κ-carrageenase OUC-CgkA-Sn and 0.05 U of 3,6-galactosidase OUC-ApdagB were added to 1 ml of 1 mg / ml red seaweed solution, ultrapure water was added to 2 ml, and the mixture was reacted at 35°C for 3 h. The reaction was terminated by boiling in a water bath for 10 min, centrifuged at 12,000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane for HPLC quantitative detection. The detection conditions were the same as those in Example 4 (1). The control group was the same reaction system, and an equal amount of κ-carrageenase OUC-CgkA-Sn and an equal amount of boiling-inactivated 3,6-galactosidase OUC-ApdagB were added.

[0062] HPLC test results are as follows Fig.10 As shown, 3,6-galactosidase OUC-ApdagB and κ-carrageenase OUC-CgkA-Sn can directly degrade furcellan into carrageenan pentasaccharide, and there is basically no carrageenan hexasaccharide left, indicating that 3,6-galactosidase OUC-ApdagB is a tool enzyme that can efficiently prepare carrageenan pentasaccharide.

[0063] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. The use of 3,6-endo-galactosidase OUC-ApdagB in the preparation of carrageenan pentasaccharide, characterized in that: The amino acid sequence of the 3,6-endo-galactosidase OUC-ApdagB is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Carrageenan pentasaccharide was prepared by using carrageenan oligosaccharide as substrate under the action of 3,6-endo-galactosidase OUC-ApdagB.

3. The use according to claim 2, characterized in that: The furcellaran oligosaccharide is prepared by degrading furcellaran by κ-carrageenase OUC-CgkA-Sn, and the amino acid sequence of the κ-carrageenase OUC-CgkA-Sn is shown in SEQ ID NO.

3.

4. The use according to claim 3, characterized in that: The furcellaran oligosaccharide is prepared by the following method: in a 2 ml reaction system, 0.5 U of κ-carrageenase OUC-CgkA-Sn and water are added to 1 ml of a 1 mg / ml furcellaran solution, and the mixture is reacted at 35° C. for 3 h.

5. The use according to claim 1, characterized in that: Combined with κ-carrageenase OUC-CgkA-Sn, the amino acid sequence of the κ-carrageenase OUC-CgkA-Sn is shown in SEQ ID NO.

3.

6. The use according to claim 5, characterized in that: Carrageenan pentasaccharide was prepared by using furcellaran as substrate under the action of kappa-carrageenase OUC-CgkA-Sn and 3,6-endo-galactosidase OUC-ApdagB.

7. The use according to claim 6, characterized in that: In specific application, in a 2 ml reaction system, 0.5 U of κ-carrageenase OUC-CgkA-Sn, 0.05 U of 3,6-endo-galactosidase OUC-ApdagB and water were added to 1 ml of 1 mg / ml red seaweed solution, and the mixture was reacted at 35°C for 3 h to prepare carrageenan pentasaccharide.

8. A method for preparing carrageenan pentasaccharide, characterized in that: Carrageenan pentasaccharide is prepared by taking red algae as a substrate under the action of kappa-carrageenase OUC-CgkA-Sn and 3,6-endo-galactosidase OUC-ApdagB; the amino acid sequence of the 3,6-endo-galactosidase OUC-ApdagB is shown in SEQ ID NO.1, and the amino acid sequence of the kappa-carrageenase OUC-CgkA-Sn is shown in SEQ ID NO.

3.

9. The method for preparing carrageenan pentasaccharide according to claim 8, characterized in that: In a 2 ml reaction system, 0.5 U of κ-carrageenase OUC-CgkA-Sn, 0.05 U of 3,6-endo-galactosidase OUC-ApdagB and water were added to 1 ml of 1 mg / ml red seaweed solution, and the mixture was reacted at 35°C for 3 h to prepare carrageenan pentasaccharide.

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