Chitinase truncated body and recombinant expression vector, engineering bacteria construction method and application thereof

Abstract

The present application relates to the technical field of genetic engineering, and particularly discloses a chitinase truncated body, a recombinant expression vector thereof, a construction method of an engineering bacterium and application. The chitinase truncated body is Cq181NC, the amino acid sequence of the Cq181NC is shown as SEQ ID NO:1, and the nucleotide sequence for encoding the enzyme is shown as SEQ ID NO:2. It is determined that the chitinase truncated body provided by the present application has a specific enzyme activity which is 1.8 times higher than that of the wild type, has strong tolerance to environmental changes, and has a thermal stability (incubation at 35°C for 8 hours) which is 1.43 times that of the wild type. More importantly, the spectrum of the enzymolysis product is changed: the wild type is mainly disaccharide, showing a typical exo-enzyme mode; and the truncated body Cq181NC produces various oligosaccharides including monosaccharide, disaccharide, trisaccharide and hexasaccharide, showing a double catalytic property of endo- and exo-enzyme, and having a better application prospect.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to chitinase truncated forms and their recombinant expression vectors, methods for constructing engineered bacteria, and their applications. Background Technology

[0002] Chitin is the second most abundant natural polysaccharide after cellulose. This polymer, formed by the polymerization of N-acetylglucosamine via β-(1,4) glycosidic bonds, is widely found in biological structures such as fungal cell walls, insect exoskeletons, and crustacean shells. However, chitin's dense molecular structure, high crystallinity, and poor water solubility, coupled with the complex deproteinization and demineralization steps involved in its extraction, make degradation difficult and limit its efficient utilization. Therefore, developing green and efficient chitin degradation processes to prepare chitin oligosaccharides is of great value.

[0003] Chitin, upon degradation, yields chitosan oligosaccharides and N-acetylglucosamine (GlcNAc). These degradation products possess a variety of biological activities, including antioxidant, antibacterial, immunomodulatory, and gut microbiota balancing functions, showing broad application potential in the food, agriculture, and pharmaceutical industries. As food additives, these products have a refreshing sweetness and good moisturizing ability, making them suitable for glucose substitution and food moisturizing modifications. They can also serve as effective plant growth regulators, enhancing disease resistance, promoting growth, and improving fruit quality in agriculture. As functional foods or biopharmaceuticals, chitosan oligosaccharides have the potential to be developed for the treatment of various diseases, such as cancer, gastritis, and Alzheimer's disease.

[0004] Chitinases are a class of glycoside hydrolases that specifically hydrolyze chitin and are widely used in the enzymatic preparation of chitin oligosaccharides. Most chitinases consist of multiple functional regions, including a catalytic domain (CD) and a carbohydrate-binding module (CBM). These domains, through functional complementarity and synergistic effects, jointly regulate the enzyme's substrate binding capacity, catalytic efficiency, thermal stability, and product polymerization degree distribution, making them widely used for chitin degradation and the preparation of chitin oligosaccharides.

[0005] However, naturally derived chitinases often suffer from limited catalytic activity, poor thermal stability, and high production costs, making it difficult to meet the demands of industrial-scale chitosan oligosaccharide production. Therefore, it is crucial to employ protein engineering strategies such as domain truncation to molecularly modify chitinases, thereby enhancing their catalytic performance and expanding product diversity. Summary of the Invention

[0006] This application aims to provide a novel chitinase truncated form Cq181NC and its recombinant expression vector, as well as a method for constructing engineered bacteria and its applications, in order to overcome the shortcomings of the existing technology.

[0007] According to an embodiment of this application, a chitinase is provided, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] The upstream primer sequence for amplifying the chitinase truncated form Cq181NC encoding gene is shown in SEQ ID NO:3, and the downstream primer sequence is shown in SEQ ID NO:4.

[0009] A recombinant expression vector is provided, the genetic sequence of which integrates a nucleic acid fragment encoding the amino acid sequence of the chitinase truncated form Cq181NC.

[0010] An engineered bacterium in which the recombinant expression vector is introduced.

[0011] This application also relates to the application of the chitinase truncated form Cq181NC in the field of chitin biotransformation, and particularly to its use in the enzymatic preparation of chitin oligosaccharides.

[0012] The expression method of the chitinase includes the following steps:

[0013] S1. Design specific primers based on the nucleotide sequence of the chitinase truncated form Cq181NC, wherein the upstream primer Cq181NCF is SEQ ID NO:3 and the downstream primer Cq181NCR is SEQ ID NO:4.

[0014] S2. The target fragment was amplified from the wild-type chitinase gene using PCR technology, and the resulting products were subjected to... Xba I and Hin Treatment with dIII restriction endonuclease. The amplified fragment after digestion was ligated to a vector that had undergone the same double digestion to construct a recombinant plasmid, which was then introduced into... E. coli DH5α competent cells were selected and positive transformants were obtained by screening on LB plates containing 50 μg / mL kanamycin sulfate, thereby constructing a recombinant expression vector containing the Cq181NC gene sequence.

[0015] S3. Extract the obtained recombinant plasmid and transform it into... E. coli A recombinant engineered strain was prepared from a BL21(DE3) expression host. This strain was inoculated into LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured at 37°C with shaking at 150 rpm until OD500 was reached. 600 When the concentration reaches 0.6, 0.1 mM IPTG is added to induce the expression of the target protein. The bacterial cells are collected by centrifugation, the bacterial pellet is resuspended in lysis buffer, 1 mg / mL lysozyme is added and the cells are sonicated to disrupt the cells, and the supernatant is collected after centrifugation to obtain the crude enzyme product.

[0016] S4. Load the above crude enzyme solution onto a pre-equilibrated nickel column affinity chromatography system, wash three times with 10 mL wash buffer, and then elute four times with 5 mL elution buffer, collecting each eluted fraction stepwise. Analyze the protein purity using SDS-PAGE electrophoresis to obtain a high-purity chitinase truncated form Cq181NC.

[0017] The lysis buffer consisted of 20 mM Tris-HCl, 50 mM NaCl, and pH 7.5; the washing buffer consisted of 50 mM NaH2PO4, 300 mM NaCl, 2 mM imidazole, and pH 8.0; and the elution buffer consisted of 50 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole, and pH 8.0.

[0018] The advantages of this invention are as follows: the chitinase truncated form Cq181NC (molecular weight approximately 58.4 kDa) provided exhibits a 1.8-fold increase in enzyme activity compared to the wild type, demonstrates strong tolerance to environmental changes, and its thermal stability (after incubation at 35°C for 8 hours) remains 1.43 times that of the wild type. More importantly, the enzymatic hydrolysis product profile undergoes a fundamental change: the wild type is predominantly disaccharides, exhibiting a typical exonuclease pattern; while the truncated form Cq181NC produces a variety of oligosaccharides, including monosaccharides, disaccharides, trisaccharides, and hexasaccharides, exhibiting dual catalytic characteristics of both endonucleases and exonucleases, thus showing better application prospects. Attached Figure Description

[0019] Figure 1 SDS-PAGE electrophoresis image of the purified chitinase truncated form Cq181NC (Column M: Protein Marker; Column 1: Crude Cq181NC enzyme solution; Column 2: Purified Cq181NC).

[0020] Figure 2 The optimal temperature curve for determining the chitinase truncated form Cq181NC.

[0021] Figure 3 The temperature stability curve of the chitinase truncated form Cq181NC is shown in the figure.

[0022] Figure 4 The optimal pH curve for determining the truncated chitinase Cq181NC.

[0023] Figure 5 The pH stability curve of the chitinase truncated form Cq181NC is shown.

[0024] Figure 6 The graph shows the effect of different metal ions on the activity of the chitinase truncated form Cq181NC.

[0025] Figure 7 The graph shows the effect of different chemical reagents on the activity of the chitinase truncated form Cq181NC.

[0026] Figure 8 This is a graph showing the effect of the truncated chitinase Cq181NC on the hydrolysis of different substrates.

[0027] Figure 9 TLC analysis of the degradation products of the chitinase truncated form Cq181NC.

[0028] Figure 10 The binding site of (GlcNAc)5 in the chitinase truncated form Cq181NC is shown, along with the key residues that interact between (GlcNAc)5 and Cq181NC. (GlcNAc)5 is shown in purple, and the key residues are shown in green. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1: Preparation of the chitinase truncated form Cq181NC

[0031] 1. Analysis of the chitinase truncated form Cq181NC

[0032] Domain analysis was performed using NCBI and InterPro servers. The predictions from both servers were consistent, indicating that the wild-type chitinase Cq181 contains an N-terminal chitin-binding domain (Chitinase A-N), a catalytic domain, and a C-terminal non-catalytic domain with unknown function. By removing the C-terminal non-catalytic domain, a truncated form, Cq181NC, was constructed. To further explore its three-dimensional conformational characteristics, this study employed the AlphaFold3 algorithm, recently developed by the DeepMind team, for protein structure modeling and analysis.

[0033] 2. Preparation of the chitinase truncated form Cq181NC

[0034] The method for preparing the chitinase truncated form Cq181NC includes the following steps:

[0035] S1. Based on the nucleotide sequence of the chitinase truncated form Cq181NC, primers that can specifically bind to the Cq181NC nucleotide sequence were designed and synthesized.

[0036] Upstream primer sequence Cq181NCF: (SEQ ID NO:3)

[0037] 5'-CCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAG-3'

[0038] Downstream primer sequence Cq181NCR: (SEQ ID NO:4)

[0039] 5'-CCCAAGCTTTTACGAGCCAGCGTTAGCCACAG-3'

[0040] S2, PCR amplification and construction of recombinant plasmids;

[0041] Plasmid templates were extracted from engineered bacteria containing the wild-type chitinase gene using a kit. PCR amplification was performed using the specific primers Cq181NCF / Cq181NCR. The amplification volume (50 μL) is shown in Table 1.

[0042] Table 1 PCR amplification system

[0043] Taq enzyme (5 U / μL, TaKaRa) 0.25μL <![CDATA[10X PCR Buffer(Mg 2+ plus)]]> 5μL dNTP Mixture (2.5mM each) 4μL plasmid 2μL Cq181NCF (10μM) 1μL Cq181NCR (10μM) 1μL <![CDATA[ddH2O]]> 36.25μL

[0044] The amplification program was set as follows: pre-denaturation at 95℃ for 3 min, followed by 30 cycles, each cycle consisting of amplification at 95℃ for 20 s, 55℃ for 20 s, and 72℃ for 60 s. The amplification products were separated by agarose gel electrophoresis, and the target fragment was recovered. Xba I and Hin After double digestion with dIII, the vector was ligated with the same digested pET-28a(+) vector under the catalysis of T4 ligase. The ligation product was then transformed into... E. coli DH5α competent cells were revived by heat shock and plated on LB selection plates containing 50 μg / mL kanamycin sulfate. Positive clones were obtained after incubation at 37°C. Double enzyme digestion and sequencing confirmed the successful construction of a recombinant plasmid containing the target gene.

[0045] S3. Gene induction expression and preparation of crude enzyme solution:

[0046] Transform the verified recombinant plasmid into E. coli BL21(DE3) expression host. The engineered bacteria were cultured at 37°C with shaking at 150 rpm until OD... 600When the concentration reached 0.6, 0.1 mM IPTG was added, and expression was induced at 16℃ and 150 rpm for 20 h. The bacterial cells were collected by centrifugation (8000 rpm, 5 min). After resuspending the bacterial cells in lysis buffer, they were treated with 1 mg / mL lysozyme and then sonicated (120 W, 3 s working time / 5 s intermittent time, total time 15 min). The cells were then centrifuged at 4℃ and 6000 rpm for 5 min, and the supernatant was collected as crude enzyme solution.

[0047] S4. Protein purification:

[0048] The crude enzyme solution was incubated with His-tag purification resin at 4°C for 2 h. After removing contaminating proteins with washing buffer, gradient elution was performed using elution buffer containing 50 mM imidazole. The collected eluted fractions were confirmed by SDS-PAGE analysis, and the results are as follows: Figure 1 As shown, the purified chitinase truncated form Cq181NC was successfully obtained.

[0049] Note: LB medium consists of 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl, pH 7.0; lysis buffer consists of 20 mM Tris-HCl, 50 mM NaCl, pH 7.5; wash buffer consists of 50 mM NaH2PO4, 300 mM NaCl, and 2 mM imidazole, pH 8.0; elution buffer consists of 50 mM NaH2PO4, 300 mM NaCl, and 50 mM imidazole, pH 8.0.

[0050] Example 2: Enzymatic properties of the chitinase truncated form Cq181NC

[0051] 1. Method for determining the activity of the chitinase truncated form Cq181NC

[0052] Colloidal chitin substrate was prepared by the following steps: 20 g of colloidal chitin powder was accurately weighed and slowly added to 400 mL of pre-cooled concentrated hydrochloric acid solution under continuous stirring. After stirring thoroughly for 30 min, the mixture was allowed to stand at 4°C for 24 h. The mixture was then slowly poured into 2000 mL of pre-cooled 50% ethanol solution. After a milky white precipitate formed, the precipitate was collected by centrifugation at 5000 rpm for 20 min at 4°C. The precipitate was repeatedly washed with sterile distilled water until neutral (pH 7.0), and finally, a 2% stock solution was prepared with an appropriate amount of distilled water and stored at 4°C for later use. It was diluted to 0.5% before use.

[0053] Enzyme activity was determined using the DNS method: 100 μL of appropriately diluted enzyme solution was mixed with 400 μL of 0.5% colloidal chitin substrate, reacted at 40℃ for 20 min, and then immediately 500 μL of DNS reagent was added to terminate the reaction. The mixture was heated in a boiling water bath for 5 min and then rapidly cooled. The absorbance at 540 nm was measured (using the heat-inactivated enzyme solution as a blank control). The amount of reducing sugar produced was calculated based on the standard curve. The enzyme activity unit was defined as the amount of enzyme required to catalyze the production of 1 μg of reducing sugar per minute under the above conditions.

[0054] 2. Determination of the optimal temperature for the chitinase truncated form Cq181NC

[0055] The catalytic activity of the enzyme solution was measured at different temperatures within the range of 15℃ to 80℃, using 0.5% colloidal chitin prepared with 50 mM Tris-HCl buffer (pH 7.0) as the reaction substrate. The highest enzyme activity was set as 100%, and the relative enzyme activity corresponding to each temperature point was calculated. The experimental results are as follows: Figure 2 As shown, the optimal reaction temperature of this enzyme is 40℃, and it exhibits significantly improved catalytic efficiency compared to the wild type at all test temperatures, demonstrating better thermal adaptability.

[0056] 3. Temperature stability determination of chitinase truncated form Cq181NC

[0057] The enzyme solution was incubated at 35℃, 40℃, and 45℃, and residual enzyme activity was measured at different time points. Using 0.5% colloidal chitin prepared in 50 mM Tris-HCl buffer (pH 7.0) as the substrate, and taking the maximum activity of the wild-type enzyme at the same temperature as 100%, the relative enzyme activity of the truncated form under each condition was calculated. The experimental results are as follows: Figure 3 The results showed that the truncated form Cq181NC retained over 80% of its catalytic activity after treatment at 35°C for 8 h, and retained 47% of its enzyme activity after treatment at 40°C for 1 h. Of particular note is that the truncated form exhibited 1.43 times the thermal stability of the wild type at 35°C for 8 h.

[0058] 3. Determination of the optimal pH for the chitinase truncated form Cq181NC

[0059] Enzyme activity was determined using 0.5% colloidal chitin prepared with different pH buffers at 40℃ as reaction substrates. The buffers included: 50 mM citrate 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), and 50 mM glycine buffer (pH 9.0–13.0). The highest enzyme activity measured in the pH 7.0 Tris-HCl buffer system was taken as 100%, and the relative enzyme activity under each pH condition was calculated. The results are as follows: Figure 4 The results showed that the optimal pH for this enzyme was 7.0, consistent with the wild type. In acidic environments (pH 3.0–6.0), its catalytic activity was enhanced compared to the wild type; however, under alkaline conditions (pH 9.0–15.0), the enzyme activity showed a decreasing trend.

[0060] 4. Determination of pH stability of chitinase truncated form Cq181NC

[0061] After incubating the enzyme solution in different pH buffer systems for 2 h, the residual enzyme activity was measured. Using the initial untreated enzyme activity as 100%, the stability of the chitinase truncated form Cq181NC under different pH conditions was evaluated. Results are as follows: Figure 5 The results showed that Cq181NC could still maintain more than 60% of its catalytic activity after 2 hours of incubation in an alkaline environment with pH > 7.0.

[0062] 5. Effects of metal ions on the activity of chitinase truncated form Cq181NC

[0063] Different types of metal ions (K) were added to the colloidal chitin reaction system. + Ca 2+ Mg 2+ Fe 3+ 、Sr + Ba 2 + Mn 2+ Cu 2+ Fe 2+ The system without added metal ions served as a blank control. After reacting for 20 min under the optimal reaction conditions of Cq181NC, the enzyme activity was compared with that of the control group (100%). Two concentration gradients of metal ions were set: low concentration (1 mM) and high concentration (10 mM). The results are as follows: Figure 6 As shown, under low concentration conditions, all tested metal ions exhibited varying degrees of inhibitory effects on the activity of Cq181NC. However, under high concentration conditions, Fe... 3+ Fe 2+ and Cu 2+Sr exhibits a strong inhibitory effect. + Ba 2+ and Ca 2+ This shows a significant enzyme activity promoting effect.

[0064] 6. Effects of chemical reagents on the activity of chitinase truncated form Cq181NC

[0065] Different chemical reagents (including formic acid, glycerol, Tween 60, Tween 80, ethyl acetate, methanol, ethanol, EDTA, SDS, and Triton X-100) were added at 1% concentration to the colloidal chitin reaction system, with the system without added reagents serving as a blank control. After catalysis for 20 min under the optimal reaction conditions of Cq181NC, the enzyme activity was compared with that of the control group, which had 100% enzyme activity. The results are as follows. Figure 7 As shown, SDS can completely inhibit the activity of Cq181NC, and this inactivation is consistent with the performance characteristics of the wild-type enzyme.

[0066] 7. Substrate specificity assay of chitinase truncated form Cq181NC

[0067] The substrate specificity of Cq181NC was evaluated using 0.5% of different polysaccharide substrates (colloidal chitin, α-chitin, β-chitin, chitosan, and microcrystalline cellulose). Enzyme activity was measured after catalysis for 20 min under optimal reaction conditions, with colloidal chitin used as a reference substrate (its relative activity was set at 100%). Results are as follows: Figure 8 As shown, the relative catalytic efficiencies of Cq181NC for the above substrates were 100% (colloidal chitin), 5% (α-chitin), 32% (β-chitin), 52% (chitosan), and 3% (microcrystalline cellulose). The experiments indicate that although this enzyme has the ability to degrade crystalline chitin, it exhibits the highest catalytic efficiency for colloidal chitin, while its degradation activity for crystalline substrates is relatively limited.

[0068] 8. Analysis of degradation products of chitinase truncated form Cq181NC

[0069] The purified enzyme was hydrolyzed with colloidal chitin at a mass ratio of 1:4 under optimal reaction conditions for 3 h. The reaction was terminated by boiling in a water bath for 20 min. The hydrolysate was purified by ethanol precipitation, and the resulting solid was reconstituted with deionized water and then filtered through a 0.22 μm filter for sterilization. The product composition was analyzed by thin-layer chromatography (TLC) with a developing system of n-butanol:anhydrous ethanol:water (5:3:2, V / V / V) and a colorimetric reagent of aniline:diphenylamine:acetone:85% phosphoric acid (1:1:50:5, V / V / V / V), and the reaction was carried out at 105 °C for 5 min. The results showed that the hydrolysate of Cq181NC contained chitosan oligosaccharides with various degrees of polymerization, ranging from monosaccharides to hexasaccharides. Compared with the wild type, which only has exonuclease activity, this truncated form exhibits dual catalytic characteristics of both endonuclease and exonuclease.

[0070] 9. Molecular docking of Cq181NC

[0071] To identify the key sites for Cq181NC to recognize substrates, this study employed molecular docking techniques to analyze the complex and screened for the conformational state with the optimal binding free energy for further analysis. The results are as follows: Figure 10 As shown, (GlcNAc)5 specifically binds to the cleft region of the Cq181NC catalytic domain. This binding site exhibits high sequence conservation within the chitinase family, suggesting its crucial role in maintaining catalytic function. Fine structural analysis of the Cq181NC-(GlcNAc)5 complex revealed that 11 residues—Arg150, Trp253, Thr254, Asp291, Gln298, Lys347, Tyr368, Asp369, Trp422, Arg424, and Glu451—form a multidimensional hydrogen bond network with the substrate molecule. Notably, compared to the wild-type enzyme, only three key residues—Lys347, Asp369, and Arg424—maintained the same hydrogen bond coordination pattern, while the remaining eight interaction sites underwent significant remodeling. This systematic reconstruction of the hydrogen bond network, especially the participation of newly emerging residues such as Gln298 and Tyr368, may affect the hydrolytic specificity of the enzyme by altering the spatial orientation and electron distribution of the substrate in the active site.

[0072] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and not restrictive.

Claims

1. A chitinase truncated form Cq181NC, characterized in that, It was obtained by removing the C-terminal non-catalytic domain after the 576th amino acid of wild-type chitinase Cq181, and its amino acid sequence is shown in SEQ ID NO:

1.

2. The chitinase truncated form Cq181NC according to claim 1, characterized in that, The nucleotide sequence of the Cq181NC 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 the chitinase truncated form Cq181NC 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 the chitinase truncated form Cq181NC as described in claim 1, characterized in that, The engineered bacteria of claim 4 were cultured under suitable culture conditions for protein expression, and the expression and purification were carried out to obtain the truncated form Cq181NC with chitinase activity.

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

7. The application according to claim 6, characterized in that, The chitin oligosaccharides are monosaccharides, disaccharides, trisaccharides, and hexasaccharides.

8. A method for preparing chitosan oligosaccharides with high degrees of polymerization, characterized in that, Under suitable reaction conditions for enzyme catalysis, the chitin substrate is reacted with the chitinase truncated form Cq181NC as described in claim 1 to generate chitin oligosaccharide products with multiple degrees of polymerization.

9. The method according to claim 8, characterized in that, The conditions for the enzymatic hydrolysis reaction are: temperature 40℃, pH 7.0.

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