Coding gene and enzyme of kappa-carrageenase CgkA as well as preparation and application of kappa-carrageenase CgkA

Abstract

The invention discloses a kappa-carrageenan hydrolase CgkA gene derived from Pedobacter hainanensis Q-13 as well as a preparation method and application of the kappa-carrageenan hydrolase CgkA gene, namely the kappa-carrageenan hydrolase CgkA gene is cloned to an escherichia coli expression vector by utilizing a genetic engineering technical method to obtain an escherichia coli recombinant strain capable of heterologously expressing the kappa-carrageenan hydrolase CgkA gene, and the kappa-carrageenan hydrolase CgkA gene can be used for preparing the kappa-carrageenan hydrolase CgkA gene. The kappa-carrageenan hydrolase prepared by heterologous expression of the strain can efficiently degrade kappa-carrageenan polysaccharide. The kappa-carrageenan hydrolase provided by the invention can be widely applied to the fields of agriculture, food, chemical industry, medicine and the like.

Description

Technical Field

[0001] This invention relates to the gene sequence of a κ-carrageenase CgkA, its preparation method, and its applications. The invention provides a recombinant plasmid of CgkA and a recombinant genetically engineered strain, as well as their applications in polysaccharide degradation. The κ-carrageenase provided by this invention can be widely used in agriculture, food, chemical, and pharmaceutical fields. Background Technology

[0002] Carrageenan is a natural linear sulfated polysaccharide, typically derived from marine red algae such as *Gynostemma pentaphyllum*, *Euphorbia milii*, and *Geochelone*. It is a major component of their cell walls, accounting for approximately 30%–75% of the algae's dry weight. The structure of carrageenan consists of repeating disaccharide units composed of D-galactose (D-Gal, G) and 3,6-anhydro-D-galactose (D-AHG, DA / D) linked by alternating β-1,4- and α-1,3-glycosidic bonds. Based on the number and type of sulfate groups, carrageenan polysaccharides are generally classified into kappa(κ)-, iota(ι)-, and lambda(λ)-carrageenan. The disaccharide units of κ-, ι-, and -λ carrageenan contain 1, 2, and 3 sulfate groups, respectively, with disaccharide monomer structures of G4S-DA, G4S-DA2S, and G2S-D2,6-2S, respectively. In addition, other carrageenan polysaccharides with mixed structures exist in nature.

[0003] Carrageenan is widely used due to its excellent physical properties, such as as a thickener, coagulant, and stabilizer in the food industry and other sectors. However, the high molecular weight and poor tissue penetration of this sulfated polysaccharide significantly limit its further applications. Low-molecular-weight carrageenan oligosaccharides, on the other hand, exhibit good physiological activity, are easily absorbed by the human body, and have significantly enhanced biological activity, including antioxidant, antitumor, anti-inflammatory, antibacterial, and antiviral activities. They have broad application prospects in the food, pharmaceutical, and chemical industries. Therefore, developing efficient preparation technologies for carrageenan oligosaccharides is key to realizing the high-value utilization of carrageenan.

[0004] Currently, commonly used methods for degrading carrageenan include chemical degradation, physical degradation, and enzymatic degradation, with acid hydrolysis being the most common. Acid hydrolysis offers advantages such as rapid reaction and the ability to process high substrate concentrations. However, it causes non-specific breakage of polysaccharide chains, resulting in complex products that cannot yield carrageenan oligosaccharides with a single degree of polymerization. Furthermore, it suffers from side reactions such as oxidation, leading to increased byproducts and sulfate loss, ultimately resulting in loss of biological activity. The most commonly used physical degradation method is ultrasonic degradation, which also suffers from uneven product polymerization, with products often exhibiting a high degree of polymerization (DP) of 15-30, making separation difficult. Enzymatic hydrolysis offers advantages such as mild reaction, uniform products, and easy separation. Therefore, enzymatic methods are a green and sustainable approach, and the development of carrageenases for the preparation of carrageenan oligosaccharides is of great significance.

[0005] Carrageenase is an enzyme that cleaves the internal β-1,4 glycosidic bonds in carrageenan polysaccharides, releasing carrageenan oligosaccharides composed of repeating disaccharide units with varying degrees of polymerization. Based on substrate specificity, carrageenases can be classified into κ-carrageenase (EC 3.2.1.83), ι-carrageenase (EC 3.2.1.157), and λ-carrageenase (EC 3.2.1.162), belonging to the galactosidase class. κ-carrageenase specifically hydrolyzes κ-carrageenan, producing κ-neocarrageenan oligosaccharides, which belong to the glycoside hydrolase family 16.

[0006] Currently, carrageenases mainly originate from two categories: marine animals, namely marine mollusks that primarily feed on marine red algae, and marine microorganisms. Among these, marine microorganisms are the primary source and focus of research for carrageenases. Bacteria reported to produce carrageenase include *Pseudoalteromonas*, *Pseudomonas*, *Cellulophaga*, *Cytophaga*, *Microbulbifer*, *Alteromonas*, *Pedobacter*, *Shewanella*, *Vibrio*, *Tamlana*, and *Zobellia*. However, only one κ-carrageenase (CgkB) from *Pedobacter* has been reported, and its amino acid sequence similarity to the κ-carrageenase reported in this patent is less than 60%. Currently reported carrageenases have not yet achieved large-scale industrial applications due to insufficient catalytic activity and stability. Therefore, finding a carrageenase with high catalytic activity, strong temperature stability, and the ability to efficiently degrade carrageenan polysaccharides is an effective way to reduce the production cost of carrageenan oligosaccharides. Furthermore, since carrageenases are present in very small amounts in organisms, using genetic engineering techniques to achieve overexpression of carrageenases in engineered strains or cells is an effective measure to increase carrageenase yield. Summary of the Invention

[0007] The first objective of this invention is to provide a novel κ-carrageenase CgkA derived from Pedobacter hainanensis Q-13 and its encoding gene.

[0008] A second objective of this invention is to provide a method for preparing a novel κ-carrageenase CgkA.

[0009] A third objective of this invention is to provide a recombinant expression plasmid containing the κ-carrageenase CgkA gene and a recombinant genetically engineered strain.

[0010] The fourth objective of this invention is to provide an application of a novel κ-carrageenase CgkA in the degradation of κ-carrageenan polysaccharides.

[0011] The κ-carrageenase CgkA provided by this invention is derived from *Agrobacterium hainanense* Q-13. The κ-carrageenase encoding gene (named cgka) amplified from it has one or more of the following nucleotide sequence characteristics:

[0012] 1) The deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing;

[0013] 2) The deoxyribonucleic acid (DNA) sequence encoding the amino acid sequence of SEQ ID NO.2 in the sequence listing;

[0014] 3) A deoxyribonucleic acid (DNA) sequence that has 80% or more homology with the deoxyribonucleic acid (DNA) sequence defined in SEQ ID NO.1 and that encodes a protein that degrades κ-carrageenan;

[0015] 4) A nucleotide sequence encoding κ-carrageenase activity obtained by substituting, deleting, or adding one or more nucleotides to the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing.

[0016] This invention also provides the amino acid sequence of κ-carrageenase CgkA, which has one or more of the following characteristics:

[0017] 1) The sequence of amino acid residues 1-262 from the amino terminus of SEQ ID NO.2 in the sequence listing, wherein positions 1-262 are amino acid sequences with κ-carrageenase CgkA activity.

[0018] 2) Substitute, delete, or add one or more amino acid residues from amino terminus 1 to 262 of SEQ ID NO.2 in the sequence listing to form an amino acid sequence that does not change the κ-carrageenase activity.

[0019] The amino acid sequence and its nucleotide coding sequence of the κ-carrageenase CgkA of the present invention can also be artificially synthesized based on the predicted amino acid sequence and its nucleotide coding sequence of the κ-carrageenase CgkA.

[0020] The method for preparing recombinant enzyme CgkA involves cloning the κ-carrageenase cgka gene into a recombinant expression vector, introducing it into host cells, and obtaining recombinantly expressed κ-carrageenase.

[0021] The gene encoding the aforementioned κ-carrageenase CgkA has one or more of the following characteristics in its nucleotide sequence:

[0022] 1) It has the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing;

[0023] 2) The deoxyribonucleic acid (DNA) sequence encoding the amino acid sequence of SEQ ID NO.2;

[0024] 3) A nucleotide sequence encoding κ-carrageenase activity obtained by substituting, deleting, or adding one or more nucleotides to the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing;

[0025] 4) A deoxyribonucleic acid (DNA) sequence that has 80% or more homology with the deoxyribonucleic acid (DNA) sequence defined in SEQ ID NO.1 and that encodes a deoxyribonucleic acid (DNA) sequence that degrades κ-carrageenan protein.

[0026] The recombinant expression vector for κ-carrageenase CgkA can be an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, a Streptomyces expression vector, a bacteriophage vector, a filamentous fungal expression vector, a plant expression vector, an insect expression vector, or a mammalian cell expression vector, etc.

[0027] Recombinant bacteria or transgenic cell lines used for recombinant expression of κ-carrageenase CgkA can be Escherichia coli host cells (such as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5α, etc.), yeast host cells (such as Saccharomyces cerevisiae, Pichiapastoris, Kluyveromyceslactis, etc.), Bacillus subtilis host cells (such as Bacillus subtilis R25, Bacillus subtilis9920, etc.), lactic acid bacteria host cells (such as Lactic acidbacteria COCC101, etc.), actinomycete host cells (such as Streptomyces spp., etc.), filamentous fungal host cells (such as Trichodermaviride, Trichodermareesei, Aspergillusniger, Aspergillusnidulans, etc.), and insect cells (such as Bombyxmori, Antharaea). eucalypti, etc.) or mammalian cells (such as Chinese hamster ovary cells CHO, young hamster kidney cells BHK, Chinese hamster lung cells CHL, etc.).

[0028] The gene sequence of the κ-carrageenanase CgkA of the present invention was cloned from Hainan Agrobacterium QCMCC Q-13 by PCR technology, and the coding region of the gene is 786 bp long.

[0029] The application of κ-carrageenase CgkA provided by this invention in the degradation of carrageenan polysaccharides includes one or two of the following applications:

[0030] 1) Application in obtaining carrageenan oligosaccharides by breaking the glycosidic bonds of carrageenan polysaccharides;

[0031] 2) Application in the synergistic cleavage of carrageenan glycosidic bonds when mixed with other carrageenases.

[0032] This invention utilizes the κ-carrageenase CgkA obtained through recombinant expression in *E. coli*, which can efficiently degrade κ-carrageenan polysaccharides to produce κ-carrageenan oligosaccharides with relatively concentrated degrees of polymerization (DP = 2, 4, 6). The optimal reaction temperature and pH of this enzyme are 40℃ and 7.0, respectively. CgkA exhibits a wide pH tolerance range; after treatment with buffers at pH 6.0 to 10.0 for 1 hour, the residual enzyme activity remains above 50%. Under optimal reaction conditions, the specific enzyme activity is 132.9 U / mg, which is relatively high among reported κ-carrageenases.

[0033] The κ-carrageenase CgkA of this invention can be widely used in the food, chemical, and pharmaceutical industries. Attached Figure Description

[0034] Figure 1 : Comparison of the amino acid sequences of CgkA with those of typical κ-carrageenases reported so far.

[0035] Figure 2 Map of the pET21a-cgka recombinant plasmid.

[0036] Figure 3 SDS-PAGE images of κ-carrageenase CgkA expression and purification. The samples added to each lane are: M protein marker; 1. Sample purified by anion exchange chromatography; 2. Sample purified by nickel affinity chromatography.

[0037] Figure 4 The optimal reaction conditions for κ-carrageenase CgkA: (a) shows the effect of temperature on the hydrolytic activity of CgkA; (b) shows the effect of pH on the hydrolytic activity of CgkA.

[0038] Figure 5 Liquid chromatography chromatograms of κ-carrageenan degradation products by κ-carrageenan enzyme CgkA.

[0039] Figure 6 MALDI-TOF-MS analysis of κ-carrageenan degradation products by κ-carrageenan enzyme CgkA. Detailed Implementation

[0040] sequence list

[0041] Information of SEQ ID No. 1 (a) Sequence characteristics

[0042] Length: 786 nucleotides

[0043] Type: Nucleotide

[0044] Chain type: Single strand (b) Molecular type: DNA

[0045] Sequence description: SEQ ID NO.1

[0046] ATGCTGTTTGATGAACTGAGCGATGAATTTGCGACCCGCGATATGGATAAATGGAACTTTAAAGGGGAAAGCTTTGGCGTGTGGTCCTGGGATGATAAAAACGCGGTGGTGGAAGATGGCATTCTGAAACTGAGCGCGGTGCATGAAGAACATACCCGCAAATTTTGGGATGGCTGCAACAAAAAACCGGTGGATGATTTTCCGCTGTATTTTAAAAGCGGCATGGCGAAAAGCAAAGCGACCGGCGTGTATGGCTATTATGAAGCGAAAATGAAAGGCGCGGATCTGCATCCGGGCGTGAGCCCGGCGTTTTGGCTGTATAGCGCGTTTGATCGCACCCTGAAAGAAGATGGCGATGTGCAGTATAGCGAAATTGA TGTGGTGGAACTGCAGCAAGAAAGCGATGATGTGTTTCATAGCGATCATAACCTGCATAACGTGATTGTGGAAAACGGCAAACCGAAATGGATGCGCCCGAAACCGTTTGCGGAAACCAATCAGAACATTCATAAACTGGATTTTGATCCGCGCGAAGAATTTGCGATTTATGCGGTGAACGTGACCCCGGAAGATATTACCTGGTATGTGAACGGCGAACAAGTGGGCTATAAAAAAAACCTGTATTGGCATCGCGATATGAACGTGGCGCTGAGCCTGGGCATGCGCGGCGATCAGTTTACCCGCTGGGATTGCAATCAGTTTTATCCGGTGGATCTGCAAGGCGAAACCGGCCTGCCGACCACCATGGAAGTGGAATATATTCGCGCGTGGAAACTGGCGGATAAASEQ ID No.2 information (a) Sequence characteristics

[0047] Length: 262 amino acids

[0048] Type: Amino acid

[0049] Chain type: Single-stranded (b) Molecular type: Protein

[0050] Sequence description: SEQ ID NO.2

[0051] MLFDELSDEFATRDMDKWNFKGESFGVWSWDDKNAVVEDGILKLSAVHEEHTRKFWDGCNKKPVDDFPLYFKSGMAKSKATGVYGYYEAKMKGADLHPGVSPAFWLYSAFDRTLKEDGDVQYSEIDVVELQ QESDDVFHSDHNLHNVIVENGKPKWMRPKPFAETNQNIHKLDFDPREEFAIYAVNVTPEDITWYVNGEQVGYKKNLYWHRDMNVALSLGMRGDQFTRWDCNQFYPVDLQGETGLPTTMEVEYIRAWKLADK

[0052] Example 1

[0053] Cloning of the full-length κ-carrageenase gene

[0054] Based on preliminary laboratory research, Pedobacter hainanensis Q-13 (China Brewing, 2013, 32:20. China Center for Type Culture Collection, accession number CCTCAB 2012076) was obtained from the laboratory. T The κ-carrageenase gene sequence was cloned from the genome of [organism name] using PCR technology, as shown in SEQ ID NO1. The PCR reaction system was as follows: 1 μL template DNA, 50 μL 2X Mix, 4 μL CgkA-F primer, 4 μL CgkA-R primer, and ddH2O added to a final volume of 100 μL.

[0055] The PCR reaction conditions were: 94℃ for 5 min, followed by denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min, repeated 30 times, and finally sequence completion at 72℃ for 10 min.

[0056] Example 2

[0057] κ-carrageenase gene sequence analysis

[0058] Gene sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) in the GenBank database, and multiple sequence alignment was performed using ClustalW and ESPript 3.0.

[0059] The obtained κ-carrageenase gene (named cgka) has a coding region of 786 bp, and its nucleotide sequence is shown in SEQ ID NO1. cgka encodes 262 amino acid residues, and its amino acid sequence is shown in SEQ ID NO2. The theoretical molecular weight of the protein is 30.6 kDa, and the predicted isoelectric point is 4.89. Amino acid sequence alignment analysis showed that CgkA has low sequence similarity to several reported typical κ-carrageenases, all below 60% (e.g., ...). Figure 1 (As shown).

[0060] Example 3

[0061] Recombinant expression and purification of CgkA in Escherichia coli

[0062] To facilitate gene recombination expression, NdeI and XhoI restriction sites were introduced into the designed upstream and downstream primers (see Table 1), respectively. The PCR-amplified cgka and empty expression vector pET21a were double-digested with NdeI and XhoI, respectively. The double digestion system (50 μL) is as follows:

[0063] PCR product / empty expression vector 43 μL

[0064] 10×FD Buffer 5μL

[0065] FD NdeI1μL

[0066] FD XhoI1μL

[0067] The reaction was carried out at 37℃ for 3 hours. The double-digested products were then recovered using a DNA cleaning and recovery kit and ligated using T4 DNA ligase (T4 DNA Ligase 0.5 μL, 10T4 DNA Ligase Buffer 0.5 μL, pET21a 2 μL, PCR product 2 μL, incubated overnight at room temperature). 5 μL of the ligation product was transformed into 100 μL of L.coli TOP10 competent cells (Novagen Biosciences), plated on solid Luria-Bertani medium containing 100 μg / mL ampicillin, and cultured at 37℃ for 12–16 hours. Single clones were picked for colony PCR verification. Correctly amplified single clones were inoculated into liquid Luria-Bertani medium containing 100 μg / mL ampicillin and the plasmid was extracted. The extracted plasmid was double-digested with restriction enzymes NdeI and XhoI. Correctly amplified recombinant plasmids were sent to BGI Genomics for sequencing. Sequencing results showed that the cgka gene shown in SEQ ID NO 1 was inserted between the NdeI and XhoI restriction sites of pET21a, and the insertion direction was correct, proving that the recombinant plasmid was successfully constructed. This recombinant plasmid was named pET21a-cgka. Figure 2 As shown.

[0068] pET21a-cgka was transformed into E. coli BL21(DE3) competent cells (Beijing Zhuangmeng International Biotechnology Co., Ltd.). Single colonies were picked and inoculated into 10 mL of LB medium. After overnight growth at 37°C and 180 rpm, 1% colonies were inoculated into 1 L of LB medium and cultured at 37°C and 180 rpm until OD600 = 0.6. IPTG was added to a final concentration of 0.1 mmol / L, and expression was induced for 24 h at 16°C and 180 rpm. The cells were collected at 8000 rpm, resuspended in 100 mL of Binding Buffer, sonicated, and centrifuged at 12000 rpm for 30 min. The supernatant was collected. Then, the cells were purified by nickel affinity chromatography and anion exchange chromatography. The expression and purification of κ-carrageenanase CgkA were detected by polyacrylamide gel electrophoresis. The results are shown below. Figure 3 As shown, the purified κ-carrageenase CgkA appears as a single band on the electrophoresis gel, and its position matches the predicted molecular weight.

[0069] Table 1. Primers used for cloning the CgkA encoding gene

[0070]

[0071] Example 4

[0072] Optimal reaction conditions for κ-carrageenase CgkA

[0073] CgkA activity was determined using the 3,5-dinitrosalicylic acid (DNS) method. Specifically, 50 μL of recombinant enzyme CgkA was added to 450 μL of 0.5% (w / v) κ-carrageenan polysaccharide solution. After reacting for 15 min, the reaction was terminated. 100 μL of the reaction solution was mixed with 200 μL of DNS and boiled for 2 min. After cooling to room temperature, water was added to bring the volume to 1.5 mL, and the absorbance was measured at 540 nm. To prepare the standard curve, 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of galactose standard solution (1 mg / mL) were placed in 15 mL test tubes, and distilled water was added to bring the volume to 1.0 mL. 2 mL of DNS reagent was accurately added to each tube, and the mixture was heated in a boiling water bath for 2 min. After cooling, water was added to bring the volume to 15 mL, and the absorbance was measured at 540 nm. The amount of reducing sugar produced was then calculated using this standard curve.

[0074] Enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute. Specific enzyme activity is defined as the ratio of enzyme activity to the corresponding protein mass. Enzyme-protein concentration is determined using the Beyotime BCA protein concentration assay kit.

[0075] Based on the method described above for determining CgkA activity, the CgkA hydrolytic κ-carrageenase activity was measured at different temperatures (25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃) under pH 7.0 conditions. The optimal reaction temperature was determined to be 40℃. The results are as follows: Figure 4 As shown in (a). Following the method described above for determining CgkA activity, the CgkA hydrolytic κ-carrageenase activity was measured at different pH values ​​(pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 10.0, pH 11.0) at 40℃. The optimal reaction pH was determined to be 7.0. The results are as follows. Figure 4 As shown in (b), the specific enzyme activity of CgkA was measured to be 132.9 U / mg under reaction conditions of 40 °C and pH 7.0.

[0076] Example 5

[0077] Analysis of products from the degradation of carrageenan polysaccharides by recombinant enzyme CgkA

[0078] 0.5% (w / v) carrageenan polysaccharide and recombinant enzyme CgkA were mixed at a ratio of 9:1 (volume ratio) and reacted at 37°C for 24 h. After removing the protein by the Savage method, the product was analyzed by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0079] The specific procedure was as follows: 200 μL of a 0.2 mol / L methanol solution of 3-amino-9-ethylcarbazole (AEC), 25 μL of a 0.5 mol / L sodium cyanoborohydride (NaBH3CN) aqueous solution, and 50 μL of acetic acid were added sequentially to 500 μL of a 1 mg / mL κ-carrageenan oligosaccharide solution. The mixture was incubated at 70 °C for 1 h. The labeling reaction was then terminated by neutralization with 100 μL of 1.0 mol / L NaOH. The aqueous phase was extracted with 500 μL of dichloromethane to remove excess AEC, followed by centrifugation at 8000 rpm for 3 min. The aqueous phase was then subjected to chromatographic analysis. HPLC separation was performed on a Shimadzu LC-2010AHT (Tokyo, Japan). The column used was a 250 mm × 4.6 mm id, 5 μm Sinochrom ODS-BP (Elite Analytical Instruments Co., Ltd., Dalian, China). Data were collected and processed using Kromstation software. Labeled oligosaccharides were analyzed by UV detection at 254 nm. The injection volume was 10 μL. Separation was performed at 40 °C in a linear gradient mode from 20% to 40% acetonitrile in a 10 mmol / L ammonium acetate aqueous solution (pH 4.5) over 60 min at a flow rate of 0.5 mL / min. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis was performed in negative ion mode with an injection volume of 10 μL. The analytical results are shown below. Figure 5 , 6 As shown, CgkA degrades carrageenan polysaccharides to generate oligosaccharides with even degrees of polymerization and relatively concentrated distribution, DP = 2, 4, and 6. Therefore, CgkA can be used for the preparation of carrageenan oligosaccharides and research related to carrageenan degradation, including in the food, chemical, and pharmaceutical fields.

Claims

1. A gene encoding a κ-carrageenase CgkA, the nucleotide sequence of which has one or more of the following characteristics: 1) It has the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing; 2) The deoxyribonucleic acid (DNA) sequence encoding the amino acid sequence of SEQ ID NO.2; 3) A nucleotide sequence encoding κ-carrageenase activity obtained by substituting, deleting, or adding one or more nucleotides to the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing; 4) A deoxyribonucleic acid (DNA) sequence that has 80% or more homology with the deoxyribonucleic acid (DNA) sequence defined in SEQ ID NO.1 and that encodes a deoxyribonucleic acid (DNA) sequence that degrades κ-carrageenan protein.

2. A κ-carrageenase encoded by the cgka gene of claim 1, characterized in that: Its amino acid sequence has one or two of the following characteristics: 1) The sequence of amino acid residues 1-262 from the amino terminus of SEQ ID NO.2 in the sequence listing; 2) An amino acid sequence with κ-carrageenase activity formed by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2 in the sequence listing.

3. A method for preparing κ-carrageenase according to claim 2, characterized in that: The gene encoding κ-carrageenase CgkA was cloned into a recombinant expression vector and introduced into host cells to obtain recombinant κ-carrageenase. The expression vector for recombinant expression of κ-carrageenase refers to one or more of the following: Escherichia coli expression vector, yeast expression vector, Bacillus subtilis expression vector, lactic acid bacteria expression vector, Streptomyces expression vector, bacteriophage vector, filamentous fungal expression vector, plant expression vector, insect expression vector, or mammalian cell expression vector.

4. The preparation method according to claim 3, characterized in that: Recombinant bacteria or transgenic cell lines used for recombinant expression of κ-carrageenase refer to Escherichia coli host cells (such as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5α, etc.), yeast host cells (such as Saccharomyces cerevisiae, Pichiapastoris, Kluyveromyceslactis, etc.), Bacillus subtilis host cells (such as Bacillus subtilis R25, Bacillus subtilis 9920, etc.), lactic acid bacteria host cells (such as Lactic acid bacteria COCC101, etc.), actinomycete host cells (such as Streptomyces spp., etc.), filamentous fungal host cells (such as Trichodermaviride, Trichodermareesei, Aspergillusniger, Aspergillusnidulans, etc.), and insect cells (such as Bombyxmori, Antharaea). Eucalypti, etc., is a type of mammalian cell (such as Chinese hamster ovary cells CHO, young hamster kidney cells BHK, Chinese hamster lung cells CHL, etc.).

5. The use of the κ-carrageenase CgkA according to claim 2 in the degradation of κ-carrageenan polysaccharides.

6. The application according to claim 5, characterized in that: Includes one or two of the following applications: 1) Application in breaking the glycosidic bonds of κ-carrageenan to obtain κ-neocarrageenan oligosaccharides; 2) Application in the synergistic cleavage of κ-carrageenan glycosidic bonds when mixed with other carrageenases.

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