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

Abstract

The present invention discloses the application of 3,6-anhydro-galactosidase OUC-ApdagB in the preparation of carrageenan pentasaccharide, belonging to the technical field of the preparation of carrageenan oligosaccharides. The amino acid sequence of the 3,6-anhydro-galactosidase OUC-ApdagB is shown as SEQ ID NO.1. When specifically applied, carrageenan pentasaccharide is prepared by using agar oligosaccharide as a substrate under the action of 3,6-anhydro-galactosidase OUC-ApdagB. The present invention can use agar as a substrate to prepare carrageenan pentasaccharide under the action of 3,6-anhydro-galactosidase OUC-ApdagB, filling the gap in the preparation of odd-numbered carrageenan oligosaccharides, and is of great significance for the industrial preparation of odd-numbered carrageenan oligosaccharides.

Description

Technical Field

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

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

[0003] 3,6-anhydro-galactosidase plays an important role in the generation of odd-numbered carrageenan oligosaccharides. It can excise even-numbered carrageenan oligosaccharides to generate corresponding odd-numbered carrageenan oligosaccharides and D-AHG. In the prior art, only 7 3,6-anhydro-galactosidases have been characterized and the study of their enzymatic properties is not perfect. Therefore, it is necessary to explore new and efficient 3,6-anhydro-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 prior art, the present invention provides the application of 3,6-anhydro-galactosidase OUC-ApdagB in the preparation of carrageenan pentasaccharide, belonging to the technical field of the preparation of carrageenan oligosaccharides.

[0005] The present invention is achieved by the following technical solutions:

[0006] The application of 3,6-anhydro-galactosidase OUC-ApdagB in the preparation of carrageenan pentasaccharide, wherein the amino acid sequence of the 3,6-anhydro-galactosidase OUC-ApdagB is as shown in SEQ ID NO.1.

[0007] Furthermore, in specific applications, carrageenan pentasaccharide is prepared under the action of 3,6-anhydro-galactosidase OUC-ApdagB using carrageenan oligosaccharide as a substrate.

[0008] Furthermore, the carrageenan oligosaccharide is prepared by degrading carrageenan with κ-carrageenase OUC-CgkA-Sn, and the amino acid sequence of the κ-carrageenase OUC-CgkA-Sn is as shown in SEQ ID NO.3.

[0009] Furthermore, the carrageenan 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 carrageenan solution with a concentration of 1 mg / ml, and the reaction is carried out at 35 °C for 3 h.

[0010] Furthermore, in specific applications, in combination with κ-carrageenase OUC-CgkA-Sn, the amino acid sequence of κ-carrageenase OUC-CgkA-Sn is as shown in SEQ ID NO.3. In specific applications, using carrageenan as a substrate, carrageenan pentasaccharide is prepared under the action of κ-carrageenase OUC-CgkA-Sn and 3,6-anhydro-galactosidase OUC-ApdagB.

[0011] Furthermore, in specific applications, in a 2 ml reaction system, 0.5 U of κ-carrageenase OUC-CgkA-Sn, 0.05 U of 3,6-anhydro-galactosidase OUC-ApdagB and water are added to 1 ml of a carrageenan solution with a concentration of 1 mg / ml, and the reaction is carried out at 35 °C for 3 h to prepare carrageenan pentasaccharide.

[0012] A method for preparing carrageenan pentasaccharide is as follows: using carrageenan as a substrate, carrageenan pentasaccharide is prepared under the action of κ-carrageenase OUC-CgkA-Sn and 3,6-anhydro-galactosidase OUC-ApdagB; the amino acid sequence of 3,6-anhydro-galactosidase OUC-ApdagB is as shown in SEQ ID NO.1, and the amino acid sequence of κ-carrageenase OUC-CgkA-Sn is as shown in SEQ ID NO.3.

[0013] 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-anhydro-galactosidase OUC-ApdagB and water are added to 1 ml of a carrageenan solution with a concentration of 1 mg / ml, and the reaction is carried out at 35 °C for 3 h to prepare carrageenan pentasaccharide.

[0014] The present invention has discovered a novel 3,6-anhydro-galactosidase, namely 3,6-anhydro-galactosidase OUC-ApdagB. Through research, it was surprisingly found that it can be used to prepare odd-numbered carrageenan oligosaccharides with a specific structure, namely carrageenan pentasaccharide. The specific structure is G-(DA-G4S)2, that is, a carrageenan pentasaccharide lacking a sulfate group in the non-reducing segment. Moreover, it has substrate specificity and can degrade κ-carrageenan oligosaccharides (i.e., agar oligosaccharides) without a sulfate group in the non-reducing segment, but cannot degrade κ-carrageenan oligosaccharides. When preparing carrageenan pentasaccharide, it has a low requirement for the concentration of the substrate and relatively high activity. In specific applications, κ-carrageenase OUC-CgkA-Sn and 3,6-anhydro-galactosidase OUC-ApdagB can be simultaneously added to the agar solution, and carrageenan pentasaccharide and D-AHG can be directly generated under the condition of 35 °C. Moreover, when the reaction time is more than 3 h, the oligosaccharide product is a single carrageenan pentasaccharide. The present invention can use agar as a substrate to prepare carrageenan pentasaccharide under the action of 3,6-anhydro-galactosidase OUC-ApdagB, filling the gap in the preparation of odd-numbered carrageenan oligosaccharides, and is of great significance for the industrial preparation of odd-numbered carrageenan oligosaccharides.

[0015] The various terms and phrases used in the present invention have the general meanings well-known to those skilled in the art. Brief Description of the Drawings

[0016] Figure 1 : Schematic diagram of the SDS-PAGE electrophoresis detection results. Among them, M is the standard protein Marker; 1 is the crude enzyme solution; 2 is the pure enzyme solution.

[0017] Figure 2 : Schematic diagram of the HPLC detection results of Example 4(1).

[0018] Figure 3 : Schematic diagram of the ESI-MS identification results of the degradation product P1.

[0019] Figure 4 : Schematic diagram of the HPLC detection results of Example 4(2).

[0020] Figure 5 : Schematic diagram of the ESI-MS identification results of the degradation product P2.

[0021] Figure 6 : Schematic diagram of the influence of reaction temperature on the relative yield of D-AHG.

[0022] Figure 7 : Schematic diagram of the influence of reaction pH on the relative yield of D-AHG.

[0023] Figure 8 : Schematic diagram of the temperature stability detection results.

[0024] Figure 9 : Schematic diagram of the pH stability test results.

[0025] Figure 10 : Schematic diagram of the HPLC test results of Example 8.

[0026] Note: Figure 2 、 Figure 4 、 Figure 6 、 Figure 7 When drawing the graph, for the convenience of intuitive comparison, the vertical positions of each curve were moved. Therefore, the ordinate in the graph has no actual meaning and does not need to be marked. What is compared is the peak area. Detailed implementation manners

[0027] The present invention will be further described below in conjunction with embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0028] The instruments, reagents, and materials involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, and materials existing in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods and detection methods existing in the prior art.

[0029] The Escherichia coli BL21(DE3) competent cells used in the present invention were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0030] The composition of the LB medium used in the present invention is as follows: sodium chloride 10 g / L; tryptone 10 g / L; yeast extract 5 g / L; agar powder 15 g / L (added when preparing solid medium); the balance is water; sterilization conditions: 115 °C, 30 min.

[0031] The gellan gum used in the present invention was purchased from Carbosynth Company, UK.

[0032] The κ-carrageenase OUC-CgkA-Sn used in the present invention is stored in the laboratory where the inventor is located. The relevant content of κ-carrageenase OUC-CgkA-Sn (amino acid sequence, preparation method, enzymatic properties, etc.) is recorded in Chinese Patent Application CN118374477 A.

[0033] Example 1 Mining of 3,6-anhydro-galactosidase OUC-ApdagB

[0034] The 3,6-anhydro-galactosidase OUC-ApdagB of the present invention was mined from the NCBI database and is derived from a marine microorganism Arenibacter palladensis strain DSM17539 that can survive using carrageenan as the sole carbon source, with the sequence number WP_072864264.1.

[0035] The gene of 3,6-anhydro-galactosidase contains 2016 bases, and the nucleotide sequence is shown in SEQ ID NO.2, encoding 672 amino acids, and the amino acid sequence is shown in SEQ ID NO.1. The phylogenetic tree analysis results show that it belongs to the glycoside hydrolase family 129 (GH129). To study the enzymatic properties of 3,6-anhydro-galactosidase OUC-ApdagB, the present invention expressed, purified the enzyme and conducted related experimental studies, as specifically shown in the following examples.

[0036] The amino acid sequence of 3,6-anhydro-galactosidase OUC-ApdagB is shown as follows, as shown in SEQ ID NO.1:

[0037] GNGNSTLAHVPEEPTVLENRAIKISVDGASGCFTVLEKKSGQLWGSDPWENAAGLLTLTDSKGKKQIVNLSKSKKIEVSKTDAGTVTIKFLDPVLEDGTVAKGVRIGTQLKLDQNAEHLNVLITEHQGGSFKLLDLRYPSRQFSLKTDEDKGAAVIPQKQGVICPSYIFPMNGGRFCKWDDATYNNKSVGSLELFNNGTGLTMPWWGTYNEKSAVVGILEEDSRPDMMYNVNNNGQYLFNPKGEMSPYQRIVFLDPVWKLDGDVGKMAINYHFIPGGDYVDMAKIYKKEAIKKGYFVTLKDKAARNPNVNKLPGAIYFGIYGGYPHYVNMPGMAFTFDELKEMIRSIREDLGVDKAFVHAWGTFSNFVPHNFPISEELGGPAKLKAAVDLAKSYGYLYSSYHAYSPMLENDPDFTTDLMQRDEEGKLMNTGSRWARVDPKFQKSLAQKNIENEISYLGLEADITDITFAAYRESGKEGRLELAKYIDSFNLVNGTEHGQEQWIPYFDMFEGMTYLEDRPLSVISHPAPLFNLVYHEAIANFGKIQDPDNEVTANGDFRIKALRSMLFGRGTTIFFAPYEFEGMRPMIKMAQKLVSPVHKETFFSELLKHEYLSADYKVQRSRFSNGTEVVANLGPVAQKIEGDIRIPGYGYRITMQDGSVKNGHFEVSLIAE。

[0038] The nucleotide sequence of the encoding gene of 3,6-epoxy-galactosidase OUC-ApdagB is shown below (direction 5'-3'), as shown in SEQ ID NO.2:

[0039]

[0040] Example 2 Preparation of 3,6-anhydro-galactosidase OUC-ApdagB

[0041] The steps are as follows:

[0042] (1) Entrust Sangon Biotech (Shanghai) Co., Ltd. to synthesize the coding gene of 3,6-anhydro-galactosidase OUC-ApdagB by total gene synthesis. Add 40 μl of pure water to the synthesized dry powder plasmid, and transform the plasmid into Escherichia coli BL21(DE3) competent cells. Coat the cells on a solid LB medium plate containing 50 μg / mL kanamycin sulfate, and culture them in an incubator at 37°C for 16 h. Pick a single colony into a liquid LB medium containing 50 μg / mL kanamycin sulfate, and culture it in a shaker at 37°C and 220 rpm for 12 h. The constructed engineering bacteria grow on the kanamycin sulfate resistance plate, and the recombinant expression strain is obtained.

[0043] (2) After activating the Escherichia coli recombinant strain in 5 ml of liquid LB medium containing 50 μg / mL kanamycin, inoculate it into 50 ml of liquid LB medium containing 50 μg / mL kanamycin sulfate at an inoculation amount of 1%, and culture it at 37°C and 200 rpm for 3 h. When the OD(600) value of the bacterial liquid is 0.6, add 1‰ isopropyl-β-D-thiogalactoside (IPTG) (100 mM / L), and induce it at 20°C for 16 h to express 3,6-anhydro-galactosidase.

[0044] (3) After the culture is completed, take the culture solution, centrifuge it at 4°C and 8000 rpm for 10 min to collect the bacteria. Resuspend the bacteria in ultrapure water, and ultrasonically disrupt them for 30 min (300 W, 3 s on, 3 s off). Centrifuge at 8000 rpm for 15 min, and the supernatant is the crude enzyme solution.

[0045] (4) Purify the crude enzyme solution using a Ni 2+ -NTA column for affinity chromatography: First, equilibrate the column with a 10 mM imidazole solution (10 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl, pH 8.0), add the crude enzyme solution to make it combine with Ni 2+-Bound to the NTA column, and then the weakly bound impurities were eluted with a 40 mM imidazole solution (40 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl). Then the target protein was eluted with a 200 mM imidazole solution (200 mM imidazole, 500 mM NaCl, 50 mM Tris-HCl), and the eluate was collected. Ultrafiltration concentration was carried out using an ultrafiltration centrifugal tube with a molecular weight cut-off of 30 kDa at 4 °C and 4000 rpm to obtain a pure enzyme solution containing 3,6-anhydro-galactosidase OUC-ApdagB. SDS-PAGE was performed to verify whether the band was single and the size was accurate. The results are shown in Figure 1. As Figure 1 can be seen, the molecular weight was approximately 78 kDa, which was consistent with the prediction.

[0046] Example 3 Substrate specificity analysis of 3,6-anhydro-galactosidase OUC-ApdagB

[0047] κ-carrageenase OUC-CgkA-Sn was used to prepare κ-carrageenan oligosaccharides and porphyran oligosaccharides respectively. The preparation method of κ-carrageenan oligosaccharides was as follows: in a 2 ml reaction system, 0.4 U of κ-carrageenase OUC-CgkA-Sn and ultrapure water were added to 1 ml of a κ-carrageenan solution with a concentration of 1 mg / ml, mixed, and reacted at 35 °C for 3 h. The product was mainly κ-carrageenan tetrasaccharide. The preparation method of porphyran oligosaccharides was as follows: in a 2 ml reaction system, 0.5 U of κ-carrageenase OUC-CgkA-Sn and ultrapure water were added to 1 ml of a porphyran solution with a concentration of 1 mg / ml, mixed, and reacted at 35 °C for 3 h. The product was mainly κ-carrageenan hexasaccharide lacking one sulfate group. The κ-carrageenan solution was prepared from κ-carrageenan and water; the porphyran solution was prepared from porphyran and water.

[0048] After the reaction, 3,6-anhydro-galactosidase OUC-ApdagB was added to the two substrates respectively. The addition amount was: 0.05 U of 3,6-anhydro-galactosidase OUC-ApdagB was added per 1 mg of the initial κ-carrageenan / porphyran, and the reaction was carried out at 35 °C for 3 h. After the reaction, 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, the supernatant was collected, and passed 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 boiled and inactivated 3,6-anhydro-galactosidase OUC-ApdagB was added.

[0049] The amino acid sequence of κ-carrageenase OUC-CgkA-Sn is shown in SEQ ID NO.3 as follows:

[0050] MVRRILFVSMVIVASLSVCNQTRLFAQDGKHNVPLSAASTDQWKIRWDRSDDFDGDDVDWRKWNKSPENFGAWVWDNESNVAVSNGILQITMRRLPTPVVKGRRPPTPYTSGMLKSHVTGTYGYYEARVKAAALFPGVCPSFWLYSKIDDSIVAVGETRYSEVDIVELTQRGDRVPGNERIADCNLHAILSNGKPGIGGRDWRRPNDQCYKDAQANELRLPFDPRDDFHTYGCEVTPETVTWFIDGKAIGQKPNRYWHREMNVALSLGLRPPYSTYTVKGFVPSDVEVDDEFPTTMEVDYVRVWERVK。

[0051] Example 4 Product Analysis of 3,6-anhydro-galactosidase OUC-ApdagB

[0052] (1)The sample prepared in Example 3 was detected by HPLC. The HPLC conditions were as follows: using a Superdex 30 10 / 300 gel filtration column, with 0.2 M NH4HCO3 solution as the mobile phase, a flow rate of 0.4 mL / min, a column temperature of room temperature, a sample loading volume of 100 μL, a refractive index detector (RID), and a detector temperature of 40 °C.

[0053] The HPLC detection results are as Figure 2 shown. 3,6-anhydro-galactosidase OUC-ApdagB cannot act on κ-carrageenan oligosaccharides, but can react with alginate oligosaccharides and produce a new degradation product P1. It can be seen that 3,6-anhydro-galactosidase OUC-ApdagB has substrate specificity.

[0054] The degradation product P1 was identified by ESI-MS. The results are as Figure 3 shown. The degradation product was detected at m / z of 475.0. Through analysis, this degradation product is a carrageenan pentasaccharide lacking one sulfate group.

[0055] (2)The sample prepared using alginate oligosaccharides as the substrate in Example 3 was detected by HPLC. The HPLC conditions were as follows: using a Sugar Pak I column (6.5×300 mm), with 50 mg / L EDTANa2Ca solution as the mobile phase, a flow rate of 0.5 mL / min, a column temperature of 75 °C, a sample loading volume of 30 μL, and differential detection of the product of 3,6-anhydro-galactosidase OUC-ApdagB and alginate oligosaccharides.

[0056] The HPLC detection results are as Figure 4 shown. It can be seen that compared with the control group, a new degradation product P2 was produced, and the retention time of the degradation product P2 was the same as that of the D-AHG standard.

[0057] The degradation product P2 was identified by ESI-MS, and the results are as Figure 5 shown. The degradation product was detected at m / z of 161.0. Through analysis, this degradation product was indeed D-AHG.

[0058] (3)Based on the above identification results, it was determined that when alginate oligosaccharide was used as the substrate, under the action of 3,6-anhydro-galactosidase OUC-ApdagB, the products were carrageenan pentasaccharide lacking one sulfate group and D-AHG.

[0059] Example 5 Determination of the Optimal Conditions of 3,6-Anhydro-Galactosidase OUC-ApdagB

[0060] A large amount of alginate oligosaccharide was prepared according to the preparation method of alginate 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 proteins in the system, the supernatant was collected, freeze-dried, and redissolved with pure water at 20% of the volume before freeze-drying to prepare freeze-dried alginate oligosaccharide as the subsequent substrate.

[0061] (1)Effect of reaction temperature on relative yield

[0062] Take 8 portions of substrate solutions (each 90 μL), add 10 μL of the pure enzyme solution in Example 2 to each, react at 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C for 10 min respectively, terminate the reaction by 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. The HPLC conditions are the same as in Example 4 (2). By detecting the yield of D-AHG, the effect of reaction temperature on the relative yield of D-AHG was evaluated.

[0063] The effect of reaction temperature on the relative yield of D-AHG is as Figure 6 shown. It can be seen that when the reaction temperature is 35 °C, the peak area is the largest. Therefore, the optimal reaction temperature of 3,6-anhydro-galactosidase OUC-ApdagB is 35 °C.

[0064] (2)Effect of reaction pH on relative yield

[0065] The different pH buffers used were: 50 mM citric acid-sodium citrate (CPBS) buffer (pH values were 3.0, 4.0, 5.0, 6.0 respectively), 50 mM phosphate (PBS) buffer (pH values were 6.0, 7.0, 8.0 respectively), 50 mM Tris-HCl buffer (pH values were 8.0, 9.0 respectively), and 50 mM glycine-sodium hydroxide buffer (pH values were 9.0, 10.0 respectively).

[0066] Dissolve freeze-dried alginate oligosaccharide with different pH buffers to obtain substrate solutions with different pH values. Take the substrate solutions with different pH values (90 μL each), add 10 μL of the pure enzyme solution in Example 2 to each, 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. The HPLC conditions are the same as in Example 4 (2). By detecting the yield of D-AHG, evaluate the effect of reaction pH on the relative yield of D-AHG.

[0067] The effect of reaction pH on the relative yield of D-AHG is as Figure 7 shown. It can be seen that when the reaction pH is 5.0 and 6.0, the peak areas are both relatively large, and when the pH is 6.0, the peak area is the largest. Therefore, the optimal pH of 3,6-anhydro-galactosidase OUC-ApdagB is 6.0.

[0068] Example 6 Determination of specific activity of 3,6-anhydro-galactosidase OUC-ApdagB

[0069] The determination method is as follows: Take 90 μL of alginate oligosaccharide solution, add 10 μL of the pure enzyme solution in 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 in Example 4 (2). According to the D-AHG concentration-peak area calibration curve drawn by the peak area and the liquid phase peak areas of D-AHG standard products with different concentrations, determine the yield of D-AHG. The alginate oligosaccharide solution is prepared from freeze-dried alginate oligosaccharide and citric acid-sodium citrate buffer (concentration 50 mM, pH 6.0), and the concentration of alginate oligosaccharide is 2.5 mg / ml (calculated based on the initial concentration of the alginate solution).

[0070] The definition of enzyme activity is: The amount of enzyme required to produce 1 mg of D-AHG per unit time is one enzyme activity unit, that is, 1 U.

[0071] The activity of 3,6-anhydro-galactosidase OUC-ApdagB in hydrolyzing alginate oligosaccharides was determined to be 0.471 U / mg.

[0072] Example 7 Determination of the Temperature and pH Stability of 3,6-anhydro-galactosidase OUC-ApdagB

[0073] Temperature Stability

[0074] The pure enzyme solution was incubated at 30 °C, 35 °C, and 40 °C for 8 h, and its residual enzyme activity after 8 h was measured under the optimal conditions (temperature 35 °C, pH 6.0) to obtain its temperature stability. The results of the temperature stability detection are as Figure 8 shown. When 3,6-anhydro-galactosidase OUC-ApdagB was placed at 35 °C for 8 h, more than 30% of the enzyme activity could still be retained, indicating good enzyme activity stability.

[0075] pH Stability

[0076] 80 μL of the 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. Its residual enzyme activity after 8 h was measured under the optimal conditions (temperature 35 °C, pH 6.0) to obtain its pH stability. The results of the pH stability detection are as Figure 9 shown. When 3,6-anhydro-galactosidase OUC-ApdagB was retained in the buffer at pH 5.0 and 6.0 for 8 h, more than 30% of the enzyme activity could still be retained, indicating good stability of the enzyme.

[0077] Example 8 Preparation of Carrageenan Pentasaccharide Using 3,6-anhydro-galactosidase OUC-ApdagB and κ-carrageenase OUC-CgkA-Sn

[0078] In a 2 ml reaction system, 0.5 U of κ-carrageenase OUC-CgkA-Sn and 0.05 U of 3,6-anhydro-galactosidase OUC-ApdagB were added to 1 ml of an alginate solution with a concentration of 1 mg / ml. Ultra-pure water was added to make up to 2 ml, and the reaction was carried out at 35 °C for 3 h; the reaction was terminated by boiling water bath for 10 min, centrifuged at 12000 rpm for 10 min, the supernatant was collected, filtered through a 0.22 μm filter membrane, and subjected to HPLC quantitative detection. The detection conditions were the same as in Example 4(1). The control group was the same reaction system, adding an equal amount of κ-carrageenase OUC-CgkA-Sn and an equal amount of boiled and inactivated 3,6-anhydro-galactosidase OUC-ApdagB.

[0079] The HPLC detection results are as Figure 10As shown, 3,6-anhydro-galactosidase OUC-ApdagB and κ-carrageenase OUC-CgkA-Sn can directly degrade carrageenan into carrageenan pentasaccharide, and there is basically no residual carrageenan hexasaccharide. It shows that 3,6-anhydro-galactosidase OUC-ApdagB is a tool enzyme that can efficiently prepare carrageenan pentasaccharide.

[0080] The above embodiments are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope disclosed herein. Modifications that are obvious to those skilled in the art will be 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: Carrageenan pentasaccharide was prepared by using carrageenan oligosaccharide as substrate under the action of 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; 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.

2. The use according to claim 1, 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.

3. 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.

4. The method for preparing carrageenan pentasaccharide according to claim 3, 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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