Antarctic krill chitosanase gene and application thereof
By screening out a new chitosan enzyme from Antarctic krill and applying the enzyme to efficiently degrade chitosan at 30°C, the problem of backward and high cost of enzymatic chitosan production in the prior art is solved, and an efficient and environmentally friendly chitosan preparation process is achieved.
Patent Information
- Application Number
- CN202510052264.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2024-11-22
- Filing Date
- 2025-01-14
- Publication Date
- 2025-03-18
AI Technical Summary
The prior art has problems such as high cost, serious resource waste, backward process and poor product quality when preparing marine chitosan derivatives. Especially when using enzymatic methods to produce chitosan, there is a lack of efficient enzyme preparations and processes.
A new type of chitosanase was screened from Antarctic krill, which has good catalytic activity at 30°C and can efficiently degrade chitosan and produce oligomeric chitosan. The optimal acting temperature of this enzyme is lower than that of other reported chitosanases and is expressed by recombinant techniques and applied to the process of preparing chitooligosaccharides.
It realizes efficient preparation of oligomeric chitin oligosaccharides under low temperature conditions, reduces production costs, improves product quality, and has a more environmentally friendly and controllable process.
Smart Images
Figure CN119639723A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of functional proteases, and in particular relates to an Antarctic krill chitosanase gene and an application thereof. Background Art
[0002] Marine chitosan is an important class of marine biological preparations, which has important physiological effects in the human body. As one of the degradation products of chitosan, oligosaccharide (DP2-20) almost covers all the advantages of chitosan. Scientific research shows that the functional effects and biological activities of chitosan oligosaccharide are dozens of times higher than those of chitosan. It has good water solubility and is easily absorbed by the human body (the absorption rate of the human body is nearly 100%, and the absorption rate of chitosan is 6.48%). It has multiple physiological effects such as anti-oxidation, antibacterial, anti-tumor, anti-inflammatory, regulating blood lipids and blood sugar, enhancing immunity, activating intestinal flora, etc.
[0003] At present, the main preparation methods of marine chitin derivatives are chemical method, physical method and bioenzyme method. Among them, the chemical method is the earliest preparation method used for industrial production. However, due to the particularity of the chemical properties of chitin, the single molecule of amino sugar molecules is easily destroyed when the acid hydrolysis process is applied, resulting in an increase in the impurity content in the product, and a large amount of wastewater and high treatment costs. Compared with the chemical method, although the physical method has good controllability and less pollution, the common problem of this method is that the degradation rate is fast in the early stage and slow in the later stage, which means that it takes a long time and high energy to degrade into smaller oligosaccharide molecules, and the equipment investment is large. The bioenzyme method has mild degradation conditions, purer products, controllable process conditions, easy to obtain oligosaccharides with the required degree of polymerization, and environmental protection. It is currently the most ideal preparation method. In the late 1990s, the enzymatic production of chitosan oligosaccharides was industrialized in Japan and South Korea, but there are very few companies in China that use enzymatic production.
[0004] The main constraints that have held back the development of this industry in my country are the following three aspects: First, the industrialization level of chitosanase preparations is not high. Limited by the difficulty in effectively developing expression genes for chitosan oligosaccharide enzyme preparations and excellent chitosanase production strains, it is also difficult to scale up the efficient fermentation and refining processes of chitosan oligosaccharide enzyme preparations. At present, there is no specialized production line for chitosan oligosaccharide enzyme preparations in my country. Second, the process of enzymatic production of chitosan oligosaccharides is backward. Limited by the source of enzyme preparations, domestic research on the process of enzymatic production of chitosan oligosaccharides is still mainly concentrated at the laboratory level. The process is generally backward, the enzymatic hydrolysis rate of the product is low, it is difficult to separate efficiently, and the quality is poor. Third, the production cost is high and resources are wasted seriously. Due to the backward production process, the waste of chitosan oligosaccharide production is high, and there is no comprehensive utilization product of chitosan oligosaccharide production waste in China, which reduces the added value of the product, resulting in high product costs and relatively low profits. Summary of the invention
[0005] The present invention aims to provide an Antarctic krill chitosanase gene and its application, which can use chitosan as a substrate to efficiently prepare low-molecular-weight chitosan oligosaccharides at a relatively low temperature of 30°C.
[0006] The present invention first provides an Antarctic krill chitosanase, which comprises:
[0007] 1) a protease having an amino acid sequence of SEQ ID NO: 2;
[0008] 2) A protease derived from 1) by replacing, deleting or adding one or more amino groups to 1).
[0009] A specific nucleotide sequence of the gene encoding the Antarctic krill chitosanase is SEQ ID NO: 1.
[0010] The present invention also provides a recombinant expression vector, into which a gene fragment encoding the Antarctic krill chitosanase is inserted;
[0011] The present invention further protects a recombinant engineered bacterium, which contains the above-mentioned recombinant expression vector.
[0012] The present invention also provides an application of the Antarctic krill chitosanase, which is the application of degrading chitosan to prepare oligochitosan oligosaccharides;
[0013] The present invention also provides a method for preparing low-molecular-weight chitosan by degrading chitosan, wherein the method comprises using the above-mentioned Antarctic krill chitosanase to degrade chitosan;
[0014] Furthermore, the reaction system of the method described contains 3% glacial acetic acid and 15% chitosan, and the preparation temperature is 30°C.
[0015] The present invention screened and obtained a new type of chitosanase from Antarctic krill. Compared with the reported chitosanase with an action temperature higher than 40°C, the chitosanase provided by the present invention has good catalytic activity at relatively low temperatures and can achieve efficient degradation of the substrate chitosan at 30°C. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 : Electrophoresis of the chitosanase gene of Antarctic krill, wherein M:D2000 marker, lane 1 is the chitosanase gene of Antarctic krill (1704 bp);
[0017] Figure 2 : Electrophoresis of Antarctic krill chitosanase protein, wherein M Marker (10-180Kda), lanes 1 and 2 are recombinant Antarctic krill chitosanase;
[0018] Figure 3: Antarctic krill optimal action temperature map;
[0019] Figure 4 : Mass spectrometry analysis of oligochitosan oligosaccharide DP2-20 product. DETAILED DESCRIPTION
[0020] The invention provides a chitosanase gene from Antarctic krill. The chitosanase can efficiently degrade chitosan and simultaneously cut off the glycosidic bonds GlcN-GlcNAc and GlcN-GlcN in chitosan molecules to generate low-molecular-weight chitosan oligosaccharides with a degree of polymerization of DP2-20. Moreover, the optimum action temperature (30°C) of the chitosanase is much lower than the optimum enzymolysis temperature (40°C) of other reported chitosanases.
[0021] The present invention is described in detail below in conjunction with embodiments and drawings.
[0022] Example 1: Cloning of Antarctic krill chitosanase gene and construction of expression vector
[0023] Using the Antarctic krill cDNA genome sequence as a template, CHF / CHR was used as primers to amplify the Antarctic krill chitosanase gene (the primers contain EcorⅠ and HindⅢ restriction sites). The agarose gel electrophoresis results of the PCR-amplified Antarctic krill chitosanase are shown in Figure 2. Figure 1 As shown, the primers are as follows:
[0024] KF 5 ′ -GAATTCAGGACTTTGGTGGTAATAGGGA-3 ′ ,
[0025] FR 5 ′ -AAGCTTTTAGTACATTGGAGCGGGTAG-3 ′ ;
[0026] The pCold-Ⅱ vector and the Antarctic krill chitosanase gene were double-digested with EcoI and HindIII, and the digestion products were recovered with a DNA product purification kit. The Antarctic krill chitosanase gene and the double-digested pCold-Ⅱ vector were recombined using a ligase and transformed into DH5a competent cells. A single bacterium was picked the next day, and the positive recombinant clone was verified by bacterial liquid PCR. The positive clone was then subjected to DNA sequencing to verify the correctness of the gene sequence.
[0027] The full length of the obtained Antarctic krill chitosanase gene is 1704bp, the nucleotide sequence is SEQ ID NO: 1, and the amino acid sequence of the encoded protein is SEQ ID NO: 2. Sequence alignment was performed in NCBI, and the chitosanase sequence from Palaemon carinicauda (NCBI accession number XM_068361049.1) with the highest homology was only 71%, indicating that the chitosanase provided by the present invention is a new type of polysaccharide hydrolase.
[0028] Example 2: Preparation of recombinant Antarctic krill chitosanase
[0029] The nucleotide fragment of the coding gene was inserted into the recombinant expression vector to construct a recombinant engineering strain. 100 μL of the positive recombinant bacteria BL21 (DE3) / pCold-Ⅱ chitosanase bacterial solution identified by sequencing was inoculated into 5.0 mL of LB liquid medium containing ampicillin and cultured at 37°C overnight. The next day, 50.0 μL was taken and inoculated into 50.0 mL of LB liquid medium containing ampicillin and continued to be cultured for 3-4 h (OD 600nm After about 0.4-0.6), IPTG was added for induction with a final concentration of 1.0 mM, cultured overnight at 20°C, and centrifuged at 8000 g for 5 min to collect the bacteria.
[0030] The cells were washed twice with 20 mmol / L Tris-HCL phosphate buffer (pH 7.2), and then with 20 mmol / L Tris-HCL phosphate buffer (pH 7.2). The cells were broken by ultrasound and centrifuged at 12000 g for 20 min. The supernatant was collected as the crude enzyme solution of recombinant Antarctic krill chitosanase ( Figure 2 ).
[0031] The supernatant is concentrated by ultrafiltration 25 times (membrane molecular weight cutoff 10Kda), and freeze-dried to obtain recombinant chitosanase.
[0032] Example 3: Activity determination of Antarctic krill chitosanase
[0033] Take clean test tubes, add 10 ml of 1% chitosan solution (pH 5.5-6) to each test tube, preheat in a constant temperature water bath at 30°C for 10 minutes, then add 1 ml of enzyme solution (10 mg / mL) to each test tube, immediately shake and mix, and start timing. Add 1 ml of 2% sodium hydroxide to a test tube every 10 minutes, mix immediately to terminate the enzymatic reaction, observe the condition of the remaining chitosan precipitation (precipitation amount and precipitation particle size), and record the enzymatic time (T) of the test tube with no precipitation at the earliest:
[0034] Enzyme activity calculation:
[0035] Enzyme activity = 0.1 × 1000 / T (min)
[0036] If the enzymatic hydrolysis reaction is terminated at 10 minutes and no chitosan precipitation is observed, the calculation results are as follows according to the above formula:
[0037] 0.1×1000 / 10=10 units / ml. (That is, within one minute, 1 ml of liquid enzyme preparation can convert 10 mg of chitosan into water-soluble components)
[0038] The activity of recombinant Antarctic krill chitosanase was determined to be 50,000 U / g.
[0039] Example 4: Preparation of oligomeric DP2-20 chitosan oligosaccharide
[0040] Add 800 kg of water and 150 kg of chitosan to the enzymolysis tank, heat it to 30 degrees, slowly add 30 kg of glacial acetic acid, stir while adding, and swell for 30 minutes to form gel after adding glacial acetic acid. Dissolve 1.2 kg of recombinant chitosanase in 20 kg of water, add it to the gelled chitosan, and enzymolyze for 3-5 hours. The content of oligomeric DP2-20 chitosan oligosaccharide in the product accounts for more than 90% ( Figure 3 ).
[0041] Example 5: Aquaculture effect of oligomeric DP2-20 chitosan oligosaccharide
[0042] 120 one-day-old AA healthy broiler chicks were selected, with an average initial body weight of 45.3±0.3g. They were completely randomly divided into three treatments, with 4 replicates in each treatment and 10 chickens in each replicate. The treatment groups were: Group 1 was the chlortetracycline group, with an initial addition of 80 mg / kg and a later addition of 50 mg / kg; Group 2 was the chitosan oligosaccharide 150 group, with an initial and later addition of 150 mg / kg; Group 3 was the chitosan oligosaccharide 100 group, with an initial and later addition of 100 mg / kg. The effect of chitosan oligosaccharide replacing chlortetracycline on the growth of broiler chickens was studied. The results are shown in Table 1.
[0043] Table 1: Effects of oligosaccharide DP2-20 on weight gain of chickens
[0044]
[0045] The results showed that the growth performance of broilers in the chitosan oligosaccharide 150 group and chitosan oligosaccharide 100 group was significantly improved compared with the chlortetracycline group. The daily weight gain of the two groups was 12.5% higher than that of the chlortetracycline group.
Claims
1. An Antarctic krill chitosanase, characterized in that: The Antarctic krill chitosanase comprises: 1) a protease having an amino acid sequence of SEQ ID NO: 2; 2) A protease derived from 1) by replacing, deleting or adding one or more amino groups to 1).
2. A gene, characterized in that The gene is used to encode the Antarctic krill chitosanase according to claim 1.
3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is SEQ ID NO:
1.
4. A recombinant expression vector, characterized in that: The gene fragment for encoding the Antarctic krill chitosanase according to claim 1 is inserted into the recombinant expression vector.
5. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria contain the recombinant expression vector described in claim 4.
6. Use of the Antarctic krill chitosanase according to claim 1 in degrading chitosan to prepare oligochitosan oligosaccharides.
7. A method for preparing oligochitosan by degrading chitosan, characterized in that: The method is to use the Antarctic krill chitosanase described in claim 1 to degrade chitosan.
8. The method according to claim 7, characterized in that In the method, the reaction system contains glacial acetic acid.
9. The method according to claim 8, characterized in that The concentration of the glacial acetic acid is 3%, and the content of chitosan is 15%.
10. The method according to claim 7, characterized in that The reaction temperature of the method described is 30°C.