Exo-chitinase Cq181 and application thereof in preparation of chitobiose

Abstract

The invention relates to the technical field of gene engineering, and particularly discloses exo-chitinase Cq181 and application thereof in preparation of chitobiose. The amino acid sequence of the chitinase is as shown in SEQ ID NO: 1, and the nucleotide sequence of a Cq181 gene for coding the chitinase is as shown in SEQ ID NO: 2. According to determination, the molecular mass of the chitinase is 82.53 kDa, the chitinase shows high activity and specific production of chitobiose under the conditions that the temperature is 40 DEG C and the pH value is 7.0, and the chitinase is suitable for a mild and low-energy-consumption biological manufacturing process and particularly has good application potential in the aspect of preparing chitosan oligosaccharide with a specific polymerization degree (the polymerization degree is 2).

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a chitinase Cq181 and application thereof in preparation of chitobiose. BACKGROUND

[0002] Chitin, as a polysaccharide formed by N-acetylglucosamine (GlcNAc) connected by β-1,4 glycosidic bonds, is widely present in the exoskeleton structure of arthropods, some mollusks, coelenterates and protozoa, is the second largest natural polysaccharide in nature, and is also a key component of most fungal cell walls. With the rapid development of the global aquaculture industry, the amount of crustacean waste has increased dramatically, and its harmless treatment has become an important problem in the field of environmental protection.

[0003] The molecular structure of chitin is dense and difficult to degrade, and the extraction process needs to be deproteinized and demineralized by complex biological or chemical methods, and the product after purification has high crystallinity and poor water solubility. The traditional degradation method has many limitations: the use of strong acid, strong alkali and oxidizing agent can easily cause environmental pollution, and the treatment cost of waste liquid is high; the degradation process is random, and it is difficult to accurately control the target degree of polymerization of the oligosaccharide product; high temperature and high pressure reaction conditions are often required, resulting in a substantial increase in energy consumption.

[0004] Enzymatic hydrolysis method degrades chitin into oligosaccharides (chitooligosaccharides) or monosaccharides (N-acetylglucosamine) through specific or non-specific action. Compared with chemical method, this method has the advantages of more green and environmentally friendly reaction system, mild and controllable conditions, strong product specificity, no harmful by-products, etc. However, free chitinase has application defects such as insufficient stability, difficulty in recovery, and complex product separation process.

[0005] Chitooligosaccharides (N-acetyl COSs) are proved to have various beneficial biological activities due to the amide group in the molecular structure, including antioxidant, antibacterial, immune regulation, intestinal flora balance, and human health improvement. Different degrees of polymerization (DP) of N-acetyl COSs show unique activity advantages in specific functional fields: (GlcNAc)2 has significant effects in preventing arthritis, diabetes, and reducing blood sugar, blood lipids, etc.; a mixture rich in (GlcNAc)2, (GlcNAc)3 and (GlcNAc)4 can be used as a plant growth biological stimulant.

[0006] The biological activity of chitinous oligosaccharides is closely related to the degree of polymerization, so the preparation of high-purity specific degree of polymerization oligosaccharides is a key link to realize the high-value application. Due to the high similarity in physical and chemical properties of chitinous oligosaccharides with different degrees of polymerization, the separation and purification by using conventional chromatography and other technologies has problems such as low efficiency, complex process, high cost and the like. This technical bottleneck seriously limits the large-scale production and practical application of chitinous oligosaccharides. Therefore, developing a new method for directed and efficient preparation of chitobiose has important significance for significantly reducing the production cost and promoting the application of functional chitinous oligosaccharides in the fields of medicine, food and agriculture. SUMMARY

[0007] The present application aims to provide a novel chitinase Cq181 and a construction method and application of a recombinant expression vector and an engineering bacterium, so as to overcome the deficiencies in the prior art.

[0008] According to the embodiments of the present application, a chitinase is provided, and the amino acid sequence of the chitinase is shown in SEQ ID NO: 1.

[0009] The upstream primer sequence for amplifying the chitinase Cq181 coding gene is shown in SEQ ID NO: 3, and the downstream primer sequence is shown in SEQ ID NO: 4.

[0010] A recombinant expression vector is provided, and a nucleic acid fragment encoding the amino acid sequence of the above-mentioned chitinase Cq181 is integrated in the genetic sequence of the recombinant expression vector.

[0011] An engineering bacterium is provided, and the recombinant expression vector is introduced into the engineering bacterium.

[0012] The present application also relates to the application of the chitinase Cq181 in the development and utilization of chitin resources and the preparation of chitinous oligosaccharides by enzymatic hydrolysis of chitin.

[0013] The expression method of the chitinase comprises the following steps:

[0014] S1, designing specific primers according to the nucleotide sequence of the chitinase Cq181, wherein the upstream primer Cq181F is SEQ ID NO: 3, and the downstream primer Cq181R is SEQ ID NO: 4;

[0015] S2, performing PCR amplification on the genomic DNA of the Bacillus thuringiensis as a template, and Aeromonas Xba I and Hin dIII double enzyme digestion, and then connecting with a vector treated by the same enzyme, to construct a recombinant plasmid, which is transformed into E. coli DH5α competent cells, and positive clones are screened on an LB plate containing 50 μg / mL kanamycin sulfate, to obtain a recombinant expression vector containing the Cq181 gene;​

[0016] S3, extract the above recombinant vector and transform into E. coli BL21(DE3) host bacteria, obtain a recombinant strain; inoculate the strain into LB medium containing 50 μg / mL kanamycin sulfate and culture to the stationary phase, add 0.1 mM IPTG to induce expression, centrifuge to collect the bacterial cells, resuspend with lysis buffer, add 1 mg / mL lysozyme and ultrasonically break the cells, centrifuge to obtain the supernatant to obtain the crude enzyme solution;

[0017] S4, load the crude enzyme solution into a pre-equilibrated His-tag purification column, wash 3 times with 10 mL of washing buffer, and elute 4 times with 5 mL of elution buffer, collect the elution fractions step by step, and detect the purity by SDS-PAGE to finally obtain the purified chitinase Cq181.

[0018] The lysis buffer is composed of 20 mM Tris-HCl, 50 mM NaCl, pH 7.5; the washing buffer is 50 mM NaH2PO4, 300 mM NaCl, 2 mM imidazole, pH 8.0; and the elution buffer is 50 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole, pH 8.0.

[0019] The chitinase Cq181 (molecular weight about 82 kDa) provided has high activity and stability under the condition of 40℃ and pH=7.0, specifically produces chitobiose, is suitable for a mild and low-energy biological manufacturing process, and has good application potential in preparing chitooligosaccharides with a specific degree of polymerization (degree of polymerization 2). BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is an SDS-PAGE electropherogram of the purified chitinase Cq181 (column M: protein marker; column 1: purified Cq181; column 2: Cq181 crude enzyme solution).

[0021] Figure 2 It is a SWISS-MODEL structure modeling model of chitinase Cq181.

[0022] Figure 3 It is a pull chart of SWISS-MODEL model quality evaluation of chitinase Cq181.

[0023] Figure 4 It is an AlphaFold3 structure modeling model of chitinase Cq181.

[0024] Figure 5Pulling plot for quality assessment of AlphaFold3 model of chitinase Cq181.

[0025] Figure 6 Optimum temperature determination plot for chitinase Cq181.

[0026] Figure 7 Temperature stability determination plot for chitinase Cq181.

[0027] Figure 8 Optimum pH determination plot for chitinase Cq181.

[0028] Figure 9 pH stability determination plot for chitinase Cq181.

[0029] Figure 10 Determination plot for the effect of different metal ions on chitinase Cq181 enzyme activity.

[0030] Figure 11 Determination plot for the effect of different chemical reagents on chitinase Cq181 enzyme activity.

[0031] Figure 12 Effect plot of chitinase Cq181 enzyme on the hydrolysis of different substrates.

[0032] Figure 13 TLC analysis plot of the degradation products of chitinase Cq181 enzyme.

[0033] Figure 14 Binding site plot of (GlcNAc)5 in chitinase Cq181, key residues of (GlcNAc)5 interacting with Cq181. (GlcNAc)5 is shown in purple, key residues are shown in green. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application.

[0035] Example 1, preparation of chitinase Cq181

[0036] 1. Screening of chitinase genes

[0037] The inventors obtained a strain of bacteria, RPC2, with strong chitin-degrading ability through screening. This strain was isolated from the waters off Putao Beach, Weihai City, Shandong Province (N: 37.5°; E: 122.1°). 16S rRNA gene sequencing analysis showed that this strain belongs to the genus Aeromonas (deposited in our laboratory).

[0038] 2. Analysis of chitinase Cq181

[0039] This invention is based on Aeromonas Analysis of the sp. genome sequence identified a potential chitinase-encoding gene Cq181, which was verified to be the gene encoding mature chitinase. Its nucleotide sequence is shown in SEQ ID NO:2, and the corresponding amino acid sequence is shown in SEQ ID NO:1.

[0040] Sequence alignment analysis using the NCBI online BLAST tool confirmed that chitinase Cq181 belongs to the GH18 family. To accurately determine its spatial conformation, this study integrated homology modeling technology from the SWISS-MODEL server with AlphaFold3, a third-generation protein structure prediction algorithm developed by the DeepMind team, to conduct conformational simulations. Experimental data (e.g.) Figure 2 , 3 As shown in the figure, during the SWISS-MODEL modeling process, the template A0A433G603.1, which has 81.42% identity with the Cq181 sequence, was selected to construct a 3D model, and its reliability was verified using the server's built-in GMQE and QMEAN dual-index system. It is generally considered that when the GMQE score is in the 0-1 range and the QMEAN value is in the -4-0 range, the model has effective predictive value. The homology model obtained in this experiment has a GMQE value of 0.93 and a QMEAN value of -0.83, both meeting the model validity criteria.

[0041] Further in-depth evaluation of model quality was conducted using Laplace chart analysis on the SAVES v6.0 platform (e.g.) Figure 4 , 5 (As shown). According to industry evaluation standards, a core region residue ratio exceeding 90% is considered a high-quality model, and exceeding 80% is considered a reliable model. In this study, the core region residue ratios of the homology modeling and AlphaFold3 prediction models were 84.85% and 86.23%, respectively, both meeting the usability requirements. Considering the completeness of the model, the structure predicted by AlphaFold3 was ultimately selected as the structural basis for the molecular docking study of Cq181 and pentameric N-acetylglucosamine (GlcNAc)5.

[0042] 3. Preparation of chitinase Cq181

[0043] The method for preparing chitinase Cq181 includes the following steps:

[0044] S1. Based on the nucleotide sequence of chitinase Cq181, design and synthesize primers that can specifically bind to the Cq181 nucleotide sequence.

[0045] Upstream primer sequence Cq181F: (SEQ ID NO:3)

[0046] 5'-CTAGTCTAGAGCCGCTGCCCCGGGCAAA -3'

[0047] Downstream primer sequence Cq181R: (SEQ ID NO:4)

[0048] 5'- CCCAAGCTTCTACCACACCAGTTGCTTGTGGCTCAC-3'

[0049] S2, PCR amplification and construction of recombinant plasmids;

[0050] Extraction using a rapid bacterial genomic DNA extraction kit (Shanghai Sangon Biotech Co., Ltd.) Aeromonas Genomic DNA of the sp. strain. Extracted Aeromonas Using the genomic DNA of the sp. strain as a template, amplification was performed using primers Cq181F and Cq181R. The amplification system (50 μL) is shown in Table 1:

[0051] Table 1 PCR amplification system

[0052] Taq enzyme (5 U / μL, TaKaRa) 0.25 μL 10X PCR Buffer (Mg 2+ plus) 5 μL dNTP Mixture (2.5 mM each) 4 μL sp. genomic DNA 2 μL Cq181F (10 μM) 1 μL Cq181R (10 μM) 1 μL ddH2O 36.25 μL

[0053] The amplification program was set as follows: initial denaturation at 95℃ for 3 min; followed by 30 cycles, each consisting of 95℃ for 20 s, 55℃ for 20 s, and 72℃ for 60 s. After amplification, the PCR products were subjected to agarose gel electrophoresis, and the target band was recovered. The recovered products were then subjected to... Xba I and Hin After double digestion with dIII (Shanghai Beyotime Biotechnology Co., Ltd.), the cells were ligated with pET-28a (+) digested with the same restriction enzyme using T4 ligase (TaKaRa). The ligation system was mixed with *E. coli* DH5α competent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 45 s, and then 1000 μL of LB liquid medium was added. The cells were cultured at 37℃ with shaking at 150 rpm for 2 h. After centrifugation, the bacterial culture was plated on LB agar plates containing 50 μg / mL kanamycin sulfate (Shanghai Sangon Biotech Co., Ltd.) and cultured overnight at 37℃. Positive clones were screened. The recombinant plasmid was extracted, and after double digestion and sequencing verification, the recombinant plasmid containing the chitinase gene Cq181 was successfully constructed.

[0054] The LB medium formula is: 10 g peptone, 5 g yeast extract, and 10 g NaCl per liter, with the pH adjusted to 7.0.

[0055] S3. Gene induction expression and preparation of crude enzyme solution:

[0056] Using the SanPrep column-based plasmid DNA mini-extraction kit (Shanghai Sangon Biotech Co., Ltd.), from... E. coli Recombinant plasmids were extracted from DH5α and transformed into... E. coli Recombinant engineered bacteria were obtained from BL21(DE3) competent cells. This strain was inoculated into 20 mL of LB broth containing 50 μg / mL kanamycin sulfate and cultured overnight. Then, it was transferred at a 1% inoculation rate to 500 mL of LB broth containing the same antibiotic and cultured at 37°C with shaking at 150 rpm until OD (dose retardation). 600 When the concentration reached approximately 0.6, IPTG was added to a final concentration of 0.1 mM, and expression was induced overnight at 16°C and 150 rpm. The cells were collected by centrifugation (8000 rpm, 5 min), resuspended in 15 mL of lysis buffer (20 mM Tris-HCl, pH 7.4), and lysozyme was added to a final concentration of 1 mg / mL. The mixture was then incubated on ice for 30 min. Following this, the cells were sonicated (120 W, 4 s on, 4 s off, total 15 min), centrifuged at 4°C and 6000 rpm for 5 min, and the supernatant was collected as the crude enzyme solution.

[0057] S4. Protein purification:

[0058] The crude enzyme solution was mixed with pre-equilibrated BeyoGold™ His-tag purification resin (Shanghai Beyotime Biotechnology Co., Ltd.) and incubated at 4°C for 2 h, then the eluent was discarded. The resin was washed three times with 10 mL of washing buffer (50 mM NaH2PO4, 300 mM NaCl, 2 mM imidazole, pH 8.0), followed by elution with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole, pH 8.0). Four tubes were collected, each containing 5 mL. The protein purity of each eluent was analyzed by SDS-PAGE, and the results are shown below. Figure 1 As shown, this indicates that the purified chitinase Cq181 was successfully obtained.

[0059] Example 2: Enzymatic properties of chitinase Cq181

[0060] 1. Method for determining the activity of chitinase Cq181

[0061] Take 20 g of colloidal chitin powder and slowly add it to 400 mL of ice-cold concentrated hydrochloric acid while stirring. After stirring for about 30 min, place the mixture in a 4°C refrigerator overnight. After 24 h, take out the mixture and add it to 2000 mL of ice-cold 50% ethanol while stirring. After the milky white precipitate is separated, place it in a 4°C centrifuge and centrifuge at 5000 rpm for 20 min to collect the precipitate. Add distilled water, stir, and measure the pH. Rinse the precipitate with sterile distilled water several times until the pH of the precipitate reaches 7.0. Then, add 1000 mL of water to the precipitate to prepare a 2% colloidal chitin solution. After preparation, store the solution at 4°C and adjust the concentration to 0.5% when needed.

[0062] Determine the reducing sugar content using the DNS method. Mix 100 μL of diluted enzyme solution and 400 μL of 0.5% colloidal chitin substrate and react at 40°C for 20 min. After the reaction is complete, add 500 μL of DNS reagent (3,5-dinitrosalicylic acid, Shanghai Genechem Co., Ltd.), boil for 5 min, immediately place in running water to cool, and measure the OD 540 value (using inactivated enzyme solution as a control). Calculate the amount of reducing sugar and enzyme activity according to the standard curve. The definition of enzyme activity unit: the amount of enzyme required to cleave 1 μg of reducing sugar from colloidal chitin per minute under the above determination conditions is defined as one enzyme activity unit (U).

[0063] 2. Optimum temperature determination of chitinase Cq181

[0064] Determine the enzyme activity of the enzyme solution at 15°C to 80°C using 0.5% colloidal chitin prepared in 50 mM Tris-HCl buffer (pH 7.0) as the substrate. Take the maximum enzyme activity as 100% and calculate the relative enzyme activity at different temperatures. The results are shown in Figure 6 The optimum reaction temperature of the enzyme is 40°C, and the enzyme activity is relatively high at 35°C to 45°C, with a relative enzyme activity of more than 80%.

[0065] 3. Temperature stability determination of chitinase Cq181

[0066] Incubate the enzyme solution at 35°C, 40°C, and 45°C for different times and determine the activity of the residual enzyme. Determine the enzyme activity using 0.5% colloidal chitin prepared in 50 mM Tris-HCl buffer (pH 7.0) as the substrate. Take the maximum enzyme activity at the same temperature as 100% and calculate the relative enzyme activity at different temperatures. The results are shown in Figure 7 The residual enzyme activity of the enzyme after being placed at 35°C for 8 h is maintained at more than 60%.

[0067] 3. Optimum pH determination of chitinase Cq181

[0068] The enzyme solution was incubated at 40°C with 0.5% colloidal chitin as substrate prepared in different buffers (50 mM citric acid buffer, pH 3.0-6.0; 50 mM phosphate buffer, pH 6.0-7.0; 50 mM Tris-HCl buffer, pH 7.0-9.0; 50 mM glycine buffer, pH 9.0-15.0) to determine the enzyme activity. The relative activity of the enzyme at different pH was calculated based on the maximum enzyme activity of 100% in 50 mM Tris-HCl buffer, pH 7.0. The results are shown in Figure 8 The results show that the optimum pH of the enzyme is 7.0, and the enzyme activity is high in the pH range of 6.0-9.0, maintaining more than 80%. Similarly, under the condition of pH 7.0, Tris-HCl buffer is better than phosphate buffer.

[0069] 4. pH stability determination of chitinase Cq181

[0070] The enzyme solution was incubated at different pH conditions for 2 h, and the residual enzyme activity was determined after incubation. The enzyme activity at 0 min incubation was taken as 100% to calculate the stability of chitinase Cq181 at different pH conditions. The results are shown in Figure 9 The results show that Cq181 still maintains more than 80% activity after 2 h storage in an environment with pH > 7.0, indicating that it is alkali-resistant and completely inactivated in an environment with pH 3.0 and 4.0.

[0071] 5. Effect of metal ions on the activity of chitinase Cq181

[0072] Different metal ions (K + , Ca 2+ , Mg 2+ , Fe 3+ , Sr + , Ba 2+ , Mn 2+ , Cu 2+ , Fe 2+ ) were added to the colloidal chitin substrate, and the blank control group did not add the above-mentioned metal ions. The reaction was carried out under the optimum conditions of Cq181 for 20 min, and the enzyme activity of the blank control group was taken as 100%. The metal ions added in the experiment were set at two levels of concentration, i.e. low concentration (1 mM / 0.1%) and high concentration (10 mM / 1%). The results are shown in Figure 10 The results show that high concentration (10 mM) of Ca 2+ , K + promotes the activity of Cq181, and 10 mM Ca 2+Cq181 activity was increased by 184%, only Mn 2+ , Cu 2+ showed strong inhibition effect. High concentration (10 mM) of metal ions Mn 2+ , Cu 2+ , Fe 3+ , Fe 2+ and Mg 2+ inhibited Cq181 activity, in which Fe 3+ and Fe 2+ completely inactivated it.

[0073] 6, Effect of chemical reagents on chitinase Cq181 activity

[0074] Different chemical reagents (formic acid, glycerol, Tween 60, Tween 80, ethyl acetate, methanol, ethanol, EDTA, SDS, triton X-100) were added to the colloidal chitin substrate, and the blank control group was not added with the above-mentioned chemical reagents. The enzyme activity of the blank control group was 100% under the optimal conditions of Cq181 for 20 min. The concentration of the chemical reagents added in the experiment was 1%. As shown in Figure 11 , methanol and SDS completely inactivated Cq181.

[0075] 7, Determination of substrate specificity of chitinase Cq181

[0076] Prepare 0.5% different substrate solutions (colloidal chitin, α-chitin, chitosan, microcrystalline cellulose, sodium carboxymethyl cellulose), and measure the enzyme activity after 20 min under the optimal conditions of Cq181. The enzyme activity measured when colloidal chitin is used as the substrate is 100%, and the substrate specificity of Cq181 is explored. As shown in Figure 12 , the relative activities of Cq181 to colloidal chitin, α-chitin, chitosan, microcrystalline cellulose and sodium carboxymethyl cellulose are 100%, 29%, 18%, 6% and 44%, respectively. Cq181 can degrade crystalline chitin, but the optimal substrate is colloidal chitin, and the degradation ability of crystalline chitin is weak. Cq181 also has the ability to degrade cellulose.

[0077] 8, Analysis of degradation products of chitinase Cq181

[0078] The purified enzyme was mixed with colloidal chitin at a mass ratio of 1 :4 and hydrolyzed for 3 h under the optimal reaction conditions. At the predetermined time point, the reaction mixture was heated in a boiling water bath for 20 min to terminate the enzyme reaction. The enzymatic product was purified by ethanol precipitation, and the obtained purified product powder was dissolved in deionized water and filtered through a 0.22 μm sterile filter. The composition distribution of the enzymatic product was analyzed by thin layer chromatography (TLC). The developing agent was n-butanol: anhydrous ethanol: water = 5:3:2 (V:V:V), the color developing agent was aniline: diphenylamine: acetone: 85% phosphoric acid = 1:1:50:5 (V:V:V:V), and the color developing condition was color development at 105°C for 5 min. It is worth noting that the enzymatic product of Cq181 is mainly COS with DP 2, indicating that it is an exo-enzyme.

[0079] 9. Molecular docking of Cq181

[0080] To identify the key sites recognized by Cq181 for substrates, the results with the lowest binding free energy were analyzed by molecular docking screening. As shown in Figure 14 (GlcNAc)5 can bind to the crack of the chitinase catalytic domain of Cq181, and this region is highly conserved. Further analysis of the binding interaction between (GlcNAc)5 and Cq181 found that Trp152, Arg157, Gln305, Lys354, Asp376, Phe377, Phe381, Arg431, Trp524, and Glu525 can form hydrogen bonds with the (GlcNAc)5 molecule in the Cq181-(GlcNAc)5 complex.

[0081] It is obvious to those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be regarded as exemplary and non-limiting in any respect.

Claims

1. A chitinase Cq181, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. The chitinase Cq181 according to claim 1, characterized in that, The nucleotide sequence of the Cq181 gene is shown in SEQ ID NO:2; the upstream primer sequence used to amplify the nucleotide sequence is shown in SEQ ID NO:3, and the downstream primer sequence is shown in SEQ ID NO:

4.

3. A recombinant expression vector, characterized in that, The vector contains an inserted nucleic acid fragment encoding the amino acid sequence of chitinase Cq181 as described in claim 1.

4. An engineered bacterium, characterized in that, The engineered bacteria, after transformation, contain the recombinant expression vector as described in claim 3.

5. A method for expressing chitinase Cq181 as described in claim 1, characterized in that, Includes the following steps: S1. Design and synthesize primers that specifically bind to the nucleotide sequence of chitinase Cq181, wherein the upstream primer Cq181F is shown in SEQ ID NO:3 and the downstream primer Cq181R is shown in SEQ ID NO:

4. S2, with Aeromonas sp. genomic DNA was used as a template for PCR amplification, and the amplification products were then subjected to... Xba I and Hin After digestion with dIII, the plasmid is ligated to a vector that has undergone the same digestion treatment to construct a recombinant plasmid; the recombinant plasmid is then transformed into... E. coli DH5α competent cells were screened on LB plates containing 50 μg / mL kanamycin sulfate to obtain a recombinant expression vector containing chitinase Cq181. S3. Extract the above-mentioned recombinant vector and transform it into... E. coli Recombinant strains were obtained from BL21(DE3); the recombinant strains were inoculated into LB medium containing 50 μg / mL kanamycin sulfate, and after stable culture, 0.1 mM IPTG was added to induce expression; the bacterial cells were collected by centrifugation, resuspended with lysis buffer, 1 mg / mL lysozyme was added and sonicated, and the supernatant was collected by centrifugation to obtain crude enzyme solution; S4. Load the crude enzyme solution onto a pre-equilibrated His-tagged purification column, wash three times with 10 mL of washing buffer, and then elute four times with 5 mL of elution buffer. Collect the elution fractions in batches, and detect the purity by SDS-PAGE to obtain purified chitinase Cq181.

6. The expression method according to claim 5, characterized in that: The lysis buffer was 20 mM Tris-HCl buffer (containing 50 mM NaCl, pH 7.5). The washing buffer is: 50 mM NaH2PO4 (containing 300 mM NaCl, 2 mM imidazole, pH 8.0); The elution buffer is: 50 mM NaH2PO4 (containing 300 mM NaCl, 50 mM imidazole, pH 8.0).

7. The use of the chitinase according to claim 1 in the preparation of chitin oligosaccharides.

8. The application according to claim 7, characterized in that, The chitosan oligosaccharide is mainly composed of chitobiose.

9. A method for preparing chitobiose, characterized in that, The method describes the preparation of chitobiose by enzymatically hydrolyzing chitin using the chitinase described in claim 1 or the host cell described in claim 5.

10. The method according to claim 9, characterized in that, The conditions for the enzymatic hydrolysis reaction are: temperature 40℃, pH 7.0.

Images