Chitosanase mutant as well as construction method and application thereof
By performing site-directed mutation of chitosanase CsnB and efficient expression in Pichia yeast, the problem of uneven distribution of the polymerization degree of enzymatic decomposition of enzymatic decomposition was solved, efficient production and activity of chitosan were achieved, and the needs of agricultural applications were met.
Patent Information
- Application Number
- CN202510704755.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-29
- Publication Date
- 2025-09-02
AI Technical Summary
When the existing endochitosan enzyme enzymatically dissolves chitosan, the polymerization degree distribution of the product is not concentrated, making it difficult to produce high-polymerization chitosan, resulting in insufficient anti-growth promotion activity in agriculture, and room for improvement in enzyme activity and thermal stability.
By performing site-directed mutations of Aspergillus fumigatus chitosanase CsnB, especially the transformation of Lys199Glu and Lys202Asp, combined with computer simulation and molecular dynamics analysis, the highly secreted and expressed chitosanase mutant CsnBV2 was constructed, and efficient expression was achieved in Pichia cerevisiae, optimizing its binding and catalytic activity with high polymerization chitosans.
The catalytic activity of the chitosanase mutant CsnBV2 was increased by 1.4 times, and the proportion of oligosaccharides with polymerization degree of 5 or above in the enzymatic solution products increased by 2.28 times, meeting the needs of efficient preparation of high-polymerization chitosaccharides.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of genetic engineering, and particularly relates to a chitosanase mutant and a construction method and application thereof. Background Art
[0002] Chitooligosaccharides (COS) are oligomers derived from chitin (chitin) or chitosan through chemical, enzymatic, or physical degradation. They are typically composed of 2 to 20 glucosamine or N-acetylglucosamine groups linked by β-1,4-glycosidic bonds. They have a low molecular weight, good water solubility, and biological activity, making them widely used in agriculture, medicine, food, cosmetics, and environmental protection.
[0003] The most important use of chitosan oligosaccharides in agriculture is as plant disease resistance inducers and plant growth promoters. They have the ability to activate plant systemic resistance (such as SAR), enhance defense against fungi, bacteria, and viruses, reduce the occurrence of diseases, promote plant growth, increase root development, and improve plant stress resistance (such as drought resistance, cold resistance, and disease resistance). Chitosan oligosaccharides with different degrees of polymerization (DP) have different inducing and promoting growth activities, among which chitosan oligosaccharides with a degree of polymerization (DP) of 6 to 8 (DP6-8) have the strongest inducing and promoting growth activities.
[0004] There are three main methods for producing chitosan oligosaccharides: chemical, physical, and enzymatic. Enzymatic degradation of chitosan to produce chitosan oligosaccharides offers significant advantages over the first two methods, including easier process control, milder reaction conditions, and a more complete product structure. This is currently the mainstream method for producing chitosan oligosaccharides.
[0005] Endochitosanase can hydrolyze the β-1,4 glycosidic bonds in chitosan from the inside of the sugar chain rather than from both ends, degrading it into chitosan oligosaccharides. It is currently the main enzyme used in the enzymatic production of chitosan oligosaccharides. The hydrolysis carried out by this endo-catalytic mechanism will result in a random distribution of the degree of polymerization (DP) of the product. After a long period of hydrolysis, the final product is mainly chitobiose, chitotriose, and chitotetraose. It is difficult to obtain chitosan oligosaccharides with a degree of polymerization (DP) greater than 6, resulting in low induction and promotion of the activity of oligosaccharide products. This is also the limiting factor in the current enzymatic production of agricultural chitosan oligosaccharides. In addition, there is room for improvement in the enzyme activity and thermal stability of endochitosanase.
[0006] In conclusion, appropriate modification of chitosanase, changing its binding properties with substrates, changing the degree of polymerization distribution of the product, and enhancing the activity and thermal stability of the enzyme itself are of great significance for the industrial preparation of high degree of polymerization (DP) chitosan oligosaccharides. Summary of the Invention
[0007] In order to improve the application value of chitosanase hydrolysis to produce chitosan oligosaccharides in agriculture, the present invention aims to obtain a chitosanase mutant with significantly increased oligosaccharide product degree of polymerization (DP) of 5 or above and improved activity when degrading chitosan.
[0008] The chitosanase's starting enzyme is chitosanase CsnB (NCBI ACCESSION: XP_746329), a member of the glycoside hydrolase family 75 from Aspergillus fumigatus Af293 (NCBI: txid330879). Using CsnB as the starting enzyme, computer simulations comprehensively analyzed the enzyme's preference for chitosan oligosaccharide substrates with varying degrees of polymerization (DP) from the perspectives of the enzymatic reaction energy barrier and enzyme-substrate binding free energy. Key sites influencing enzyme activity and the DP of the hydrolysis product were identified. Site-directed saturation mutagenesis of these amino acid sites was performed, resulting in a double-site mutant, CsnBV2, that exhibited simultaneous improvements in catalytic activity and the total DP of the hydrolysis product. This work resulted in the construction of an engineered Pichia pastoris strain that efficiently secretes and expresses CsnBV2, demonstrating promising industrial applications.
[0009] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:
[0010] One object of the present invention is to provide a chitosanase mutant CsnBV2, wherein the mutation positions are site-directed mutations of Lys199Glu and Lys202Asp in the amino acid sequence of Aspergillus fumigatus chitosanase. The amino acid sequence of the chitosanase mutant CsnBV2 is shown in SEQ ID NO.1.
[0011] Preferably, the nucleotide sequence encoding the amino acid sequence is shown as SEQ ID NO. 2, which is a coding sequence preferred by Pichia pastoris codons.
[0012] Another object of the present invention is to provide a recombinant expression vector pPIC9K-CsnBV2, comprising the sequence of the chitosanase mutant CsnBV2.
[0013] Another object of the present invention is to provide a recombinant bacterium, which is a host bacterium for transformation / transfection of the above-mentioned recombinant vector.
[0014] Preferably, the recombinant bacteria is a recombinant yeast, the recombinant expression vector pPIC9K-CsnBV2 is integrated into the chromosome of the yeast, and the recombinant bacteria can efficiently secrete and express the chitosanase mutant CsnBV2 after induction with methanol.
[0015] Preferably, the recombinant bacteria uses Pichia pastoris GS115 as a host bacteria.
[0016] Another object of the present invention is to provide a method for constructing the chitosanase mutant CsnBV2 as described above, the method comprising the following steps:
[0017] (1) Using AI protein modeling, molecular docking, and molecular dynamics simulation, we determined the key residues that affect the substrate preference and activity of Aspergillus fumigatus chitosanase CsnB.
[0018] (2) Combinatorial mutations were performed on the key residue sites obtained by calculation, and the corresponding single-copy Escherichia coli expression vector was constructed and transformed into the Escherichia coli strain; the enzyme activity of the transformed strain and the distribution of the degree of polymerization (DP) of the enzymatic hydrolysis products were tested; the best positive mutation was screened and its mutant sequence was obtained.
[0019] (3) The positive mutant sequence was codon-optimized for Pichia pastoris, and the resulting DNA sequence was chemically synthesized and inserted into the multiple cloning site of the pPIC9K plasmid to construct the Pichia pastoris secretory expression vector pPIC9K-CsnBV2. The vector was linearized and electroporated into the Pichia pastoris GS115 strain. The vector was integrated into the host chromosome, resulting in a genetically engineered Pichia pastoris strain that recombinantly expressed the target enzyme protein.
[0020] (4) Cultivating a genetically engineered strain of Pichia pastoris, during which methanol is induced, the culture fluid contains the chitosanase mutant CsnBV2 protein, which is filtered to obtain a crude enzyme solution that is directly used in the enzymatic hydrolysis process.
[0021] The last object of the present invention is to provide a use of the Pichia pastoris genetically engineered chitosanase mutant CsnBV2 in the preparation of chitosan oligosaccharides, wherein the chitosanase mutant is used to degrade chitosan, and the degradation products contain chitotriose, chitotetraose, chitopentaose, chitohexaose and chitoheptaose.
[0022] Compared with the prior art, the present invention has the following beneficial effects:
[0023] (1) The present invention provides a chitosanase mutant CsnBV2, whose activity is about 1.4 times higher than that of the original enzyme.
[0024] (2) The present invention provides a chitosanase mutant CsnBV2, in which the proportion of oligosaccharides with a degree of polymerization (DP) of 5 or above in the enzymatic hydrolysis product is 2.28 times that of the starting enzyme, reaching 48%. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.
[0026] Figure 1 A is the 3D structure of chitosanase CsnB combined with chitosan oligosaccharide (chitohexaose) with a degree of polymerization of 6, and B is the 3D structure of chitosanase CsnB combined with chitosan oligosaccharide (chitododecanose) with a degree of polymerization of 12;
[0027] Figure 2 A is the starting enzyme of chitosanase, and B is the HPLC spectrum of the enzymatic hydrolysis product of the mutant enzyme. DETAILED DESCRIPTION
[0028] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention is clearly and completely described below in conjunction with the examples of the present invention. Obviously, the examples described are only part of the examples of the present invention, rather than all the examples. Based on the examples in the present invention, those skilled in the art can make various modifications and improvements without departing from the principles and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.
[0029] Example 1 Construction of chitosanase mutant CsnBV2
[0030] A single-copy expression strain of wild-type chitosanase CsnB was constructed in Escherichia coli. The crude enzyme activity of the strain was only 12 U / mL. After 6 hours of chitosan degradation, the products were mainly chitosan tri-pentasaccharides, with low inducible and pro-inflammatory activity. Therefore, the present invention subjected the strain to mutation modification in order to obtain a chitosanase with higher activity and a higher product degree of polymerization (DP).
[0031] In order to change the mode of interaction between the enzyme and high-polymerization (DP) chitosan oligosaccharides and improve the enzymatic properties, the protein structure was first predicted online using AlphaFold2 (https: / / colab.research.google.com / github / sokrypton / ColabFold / blob / main / AlphaFo ld2.ipynb) to obtain a three-dimensional model of chitosanase CsnB. The active site was determined by referring to the homologous enzyme-substrate complex model and the position of the catalytic residues of the homologous enzyme. The chitosan oligosaccharides of DP12 and DP6 were docked into the active site of the CsnB three-dimensional model using Autodock software (see Figure 2). Figure 1 , Figure 1In the figure, A is the binding pattern diagram with chitosan oligosaccharide (chitohexaose) with a degree of polymerization of 6, and B is the binding pattern diagram with chitosan oligosaccharide (chitododecanose) with a degree of polymerization of 12; the chitosanase mutation sites are located at lysine (Lys) at positions 199 and 202. The molecular dynamics simulation software Amber12 was used to perform molecular dynamics simulations on the two complexes of CsnB-DP6 chitosan oligosaccharide and CsnB-DP12 chitosan oligosaccharide, calculate the binding free energy of the two systems and decompose them into each residue of the CsnB protein, and analyze the probability of catalytic close attack conformation; the differential residue region or site that affects the enzymatic preference for the substrate of DP6 / 12 chitosan oligosaccharide was determined to be R196~204. Alanine scanning calculation was performed on this region, and the residue sites most likely to affect the substrate preference were determined to be Lys199 and Lys202, see Figure 1 These two sites were mutated to Asp, Glu, Tyr, Gln, Thr, and Gly, respectively, in pairs, for a total of 36 mutations. The secretion signal peptide of the E. coli outer membrane protein was added to the N-terminus of the CsnB amino acid sequence. Based on this, the corresponding E. coli codon-preferred nucleic acid sequence was chemically synthesized (see SEQ ID NO.3). This was used as a PCR template to introduce mutations in the PCR primers, amplify the mutant DNA fragment, and insert it into the single-copy E. coli expression vector pETcoco-1 multiple cloning site. 36 corresponding E. coli expression vectors were constructed and transformed into E. coli strain Origami B to produce the corresponding mutant enzyme. After IPTG induction and expression for 48 hours, the bacterial solution was collected and the cells were broken and centrifuged. The supernatant was used as a crude enzyme sample to test the enzyme activity of each transformed strain and the distribution of the DP degree of the enzymatic hydrolysis product. Positive mutations were those with no decrease in enzyme activity and an increase in the average DP degree of the product. The experimental results showed that when the 199 position was mutated to glutamic acid (Glu) and the 202 position was mutated to aspartic acid (Asp), the activity of the mutant was 16.8 U / mL, which was 1.4 times higher than that of the starting enzyme. The proportion of product polymerization degree (DP>5) increased by more than 2 times, as shown in the following results. Figure 2 shown. Figure 2 After the chitosanase CsnB mutated, the product's degree of polymerization (DP>5) increased 2.28-fold from 21% to 48%. Figure A shows the HPLC spectrum of the hydrolysis product of the starting enzyme CsnB, and Figure B shows the HPLC spectrum of the hydrolysis product of the mutant enzyme CsnBV2. DP is the degree of polymerization, and the combination of DP and a number indicates chitosan N-saccharide, such as DP3 for chitotriose.
[0032] Enzyme activity detection method: ① Pipette 560 μL of 1%, pH 5.5 colloidal chitosan (80% deacetylation) solution (adjust pH with acetic acid) into a 2 mL centrifuge tube, add 20 μL of enzyme solution sample, vortex to thoroughly mix the substrate and enzyme solution, centrifuge briefly, place the centrifuge tube in a metal bath, 50°C, 800 rpm, and perform enzymatic reaction for 30 minutes; ② Add 580 μL of DNS reagent, mix by inversion, and centrifuge briefly; at the same time, take another 560 μL of 1%, pH 5 colloidal chitosan solution into a new 2 mL centrifuge tube, and add 580 μL of DNS reagent and 20 μL enzyme solution sample as a negative control; then, place the two centrifuge tubes in a 99°C water bath for 10 minutes for color development, cool them over water, and centrifuge at 10,000 rpm for 10 minutes. ③ 500 μL of supernatant from each tube is placed in a cuvette, and each cuvette is diluted with 1500 μL of ultrapure water. The spectrophotometer is zeroed using the negative control treatment, and colorimetry is performed at a wavelength of 540 nm to obtain the OD540 value of the sample. The same procedure is followed as above, except that the enzyme solution sample is replaced with glucose solutions of equal concentrations. The OD540 values of the reaction solutions at each concentration are measured. An OD540-reducing sugar concentration curve can be drawn, and the OD540 values of the sample reaction solutions can be converted into reducing sugar concentration values. Chitosanase activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of reducing sugar per minute under the above conditions, which is defined as one unit of enzyme activity (U).
[0033] Chitosan enzymatic hydrolysis product DP distribution test method:
[0034] Prepare 1 mL of a 5% chitosan solution (80% deacetylation, pH adjusted with acetic acid) at pH 5.5, place it in a 2 mL centrifuge tube, add 250 μL of crude enzyme solution, place the centrifuge tube in a metal bath, and incubate the reaction at 50°C, 800 rpm for 6 hours. Take a sample to detect the degree of polymerization (DP) of the product.
[0035] Using the ion chromatography method specified in the industry standard HG / T4926-2016, "Aminoligosaccharide Technical Product," the sample is diluted and injected directly without hydrolysis. This method can identify the chromatographic peaks and their corresponding areas of chitosan oligosaccharides of varying DP. The DP distribution is calculated as the percentage of the chromatographic peak area of chitosan oligosaccharides of varying DP relative to the total area.
[0036] Example 2 Construction of genetically engineered bacteria producing chitosanase mutant CsnBV2
[0037] While Escherichia coli can soluble and secrete chitosanase CsnB and its mutants, the enzyme activity in the fermentation broth is relatively low due to its obstructed secretory pathway. Most of the activity remains in the periplasm, requiring cell disruption to dissolve the target enzyme protein to achieve higher activity. Even so, the enzyme activity still fails to meet industrial needs. Therefore, the present invention uses yeast as a host bacteria for secretory expression of chitosanase CsnB and its mutants, as yeast has an unobstructed and efficient secretory pathway.
[0038] The coding sequence of the positive mutant sequence was codon-optimized for Pichia pastoris. The optimized sequence is shown in SEQ ID NO. 2. This DNA sequence was chemically synthesized with NotI / AvrII restriction sites at both ends and inserted into the multiple cloning site of the pPIC9K plasmid. Escherichia coli was transformed and the plasmid was extracted to obtain the Pichia pastoris secretory expression vector pPIC9K-CsnBV2. This vector was cleaved with Sac I and electroporated into Pichia pastoris GS115. The transformed bacterial solution was plated on standard MD plates and incubated at 30°C for 3-5 days. Single colonies that grew were identified as positive clones. The positive clones were then transferred and plated onto standard YPD plates containing G418 (3 mg / mL). The fastest-growing and largest colony was identified as the optimal positive clone, which was also the target product—the engineered Pichia pastoris strain expressing recombinant CsnBV2.
[0039] Example 3: Production of chitosanase using recombinantly expressed CsnBV2 Pichia pastoris and enzymatic hydrolysis of chitosan
[0040] The engineered strain was cultured in a shaking flask at 30°C and 250 rpm using BMGY culture medium. When the OD600 value reached 6, the bacteria were collected by centrifugation, resuspended with an equal amount of sterile BMMY culture medium, and poured into a new flask. Methanol was added to induce enzyme production. Methanol was added to a final concentration of 0.5% every 12 hours. After 7 days of induction culture, the bacterial liquid was harvested and filtered through 0.22 micron filter paper to remove the bacteria. The clear liquid was the crude enzyme liquid, and the activity test result was 2100 U / mL.
[0041] Prepare 7.5% chitosan (80% deacetylation) solution, place it in a reactor, add glacial acetic acid to make the pH reach about 5.5, at this time the chitosan solution is in colloidal state, add 5% crude enzyme solution, heat to 50°C, and stir. After 10 minutes, the colloidal system is fully liquefied, then add 7.5% chitosan, and continue stirring at 50°C for about 4 hours. Take 0.75ml of the reaction solution to a 2ml centrifuge tube, add an equal amount of ammonia water, mix well and let it stand. If it clumps, continue the reaction. If it does not clump, stop the reaction. Take samples to detect the degree of polymerization of the product. The degree of polymerization distribution of the enzymatic hydrolysis product using the wild enzyme CsnB is as follows Figure 2 A, the degree of polymerization distribution of the enzymatic hydrolysis products using the mutant enzyme is as follows Figure 2B.
[0042] SEQ ID NO.1:
[0043] YNLPNNLKQIYDKHKGKCSKVLAKGFTNGDASQGKSFSYCGDIPGAIFISSSKGYTNMDIDCDGANNSAGKCANDPSGQGETAFKSDVKKFGISDLDANIHPYVVFGNEDHSPKFKPQSHGMQPLSVMAVVCNGQLHYGIWGDTNGGVSTGEASISLADLCPNEHLDGNHGHDPNDVLFIGFTSKDAVPGATAKWKAENADEFEDSIKSIGDKLVAGLKA
[0044] SEQ ID NO.2:
[0045] TACAACTTACCAAACAATTTAAAGCAAATTTATGATAAACACAAAGGTAAGTGTAGTAAAGTTTTGGCCAAGGGTTTTACTAATGGTGATGCATCCCAAGGAAAGTCTTTTTCATATTGCGGTGACATTCCTGGTGCCATCTTTATCTCTTCTTCCAAAGGATACACCAACATGGATATTGACTGTGATGGTGCCAACAACAGTGCAGGAAAGTGTGCTAATGATCCATCCGGACAAGGTGAAACTGCTTTTAAGTCCGACGTGAAAAAGTTTGGAATTTCTGACTTGGACGCTAATATCCATCCATACGTCGTGTTCGGCAACGAAGATCAC TCACCTAAATTCAAACCTCAAAGTCATGGTATGCAGCCATTATCCGTGATGGCAGTGGTATGCAATGGACAGCTTCATTACGGTATTTGGGGAGATACAAACGGAGGAGTGTCTACCGGAGAAGCCTCCATTTCTTTGGCAGATTTGTGTTTCCCAAACGAGCACT TGGACGGTAATCACGGACACGATCCAAATGACGTTTTGTTCATCGGCTTTACCTCAAAGGATGCTGTCCCAGGTGCTACTGCTAAGTGGAAAGCTGAAAACGCTGATGAGTTCGAAGACTCTATTAAGTCCATTGGTGATAAACTGGTCGCCGGACTTAAAGCCTAA
[0046] SEQ ID NO.3:
[0047] TACAACCTCCCGAACAACCTGAAACAGATCTACGACAAACACAAAGGTAAATGCTCTAAAGTTCTGGCTAAAGGTTTCACCAACGGTGACGCTTCTCAGGGTAAATCTTTCTCTTACTGCGGTGACATCCCGGGTGCTATCTTCATCTCTTCTTCTAAAGGTTACACCAACATGGACATCGACTGCGACGGTGCTAACAACTCTGCTGGTAAATGCGCTAACGACCCGTCTGGTCAGGGTGAAACCGCTTTCAAATCTGACGTTAAAAAATTCGGTATCTCTGACCTGGACGCTAACATCCACCCGTACGTTGTTTTCGGTAACGAAGACCACTCTCCGAAATTCAAACCGCAGTCTCACGGTATGCAGCCGCTGTCTGTTATGGCTGTTGTTTGCAACGGTCAG CTGCACTACGGTATCTGGGGTGACACTAACGGCGGTGTAAGCACCGGTGAAGCTTCTATCTCTCTGGCTGACCTGTGCTTCCCGAACGAACACCTGGACGGTAACCACGGTCACGACCCGAACGACGTTCTGTTCATCGGTTTCACCTCTAAAGACGCTGTTCCGGGTGCTACCGCTAAATGGAAAGCTAAAAACGCTAAAGAATTCGAAGACTCTATCAAATCTATCGGTGACAAACTGGTTGCTGGTCTGAAAGCTTAA
Claims
1. A chitosanase mutant CsnBV2, characterized in that: The amino acid sequence of the mutant CsnBV2 is shown in SEQ ID NO.
1.
2. The chitosanase mutant CsnBV2 according to claim 1, characterized in that The nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO.
2.
3. A recombinant expression vector, characterized in that: The expression vector comprises the nucleotide sequence of the chitosanase mutant CsnBV2 according to claim 2.
4. A recombinant bacterium, characterized in that The recombinant bacteria is a host bacteria for transformation / transfection of the recombinant vector according to claim 3.
5. The recombinant bacterium according to claim 4, characterized in that The recombinant bacteria is a recombinant yeast.
6. The recombinant bacterium according to claim 5, characterized in that The base strain of the recombinant bacteria is Pichia pastoris GS115.
7. A method for constructing the chitosanase mutant CsnBV2 according to claim 1 or 2, comprising the following steps: (1) Identify the key residues that affect the substrate preference and activity of Aspergillus fumigatus chitosanase CsnB; (2) Combinatorial mutations were performed on the key residue sites obtained by calculation, and the corresponding single-copy Escherichia coli expression vector was constructed and transformed into the Escherichia coli strain; the enzyme activity of the transformed strain and the distribution of the degree of polymerization (DP) of the enzymatic hydrolysis products were tested; the best positive mutation was screened and its mutant sequence was obtained. (3) The positive mutant sequence was optimized for Pichia pastoris codons, the DNA sequence was chemically synthesized, and inserted into the multiple cloning site of the pPIC9K plasmid to construct the Pichia pastoris secretion expression vector pPIC9K-CsnBV2. The vector was linearized and then electroporated into the Pichia pastoris GS115 strain. The vector was integrated into the chromosome of the host strain to construct a Pichia pastoris genetic engineering strain that recombinantly expressed the target enzyme protein. (4) Cultivating a genetically engineered strain of Pichia pastoris, during which methanol is induced, the culture fluid contains the chitosanase mutant CsnBV2 protein, which is filtered to obtain a crude enzyme solution that is directly used in the enzymatic hydrolysis process.
8. Use of the chitosanase mutant CsnBV2 in the preparation of chitosan oligosaccharides, characterized in that: The chitosanase mutant is used for degrading chitosan, and the degradation products contain chitotriose, chitotetraose, chitopentaose, chitohexaose and chitoheptaose, and the proportion of chitopentaose, chitohexaose and chitoheptaose (DP>5) reaches 48%.