A chitinase derived from tea trees, its preparation method and application

Abstract

This invention discloses a chitinase derived from tea plants, its preparation method, and its applications in the field of bioengineering technology. The invention clones the chitinase gene fragment CsChit1B from tea plants, ligates it into an expression vector, constructs a recombinant expression plasmid, transforms the recombinant expression plasmid into host cells to obtain a recombinant engineered strain, induces expression in the recombinant engineered strain, obtains the expression product, and isolates and purifies the expression product to obtain the chitinase derived from tea plants, achieving efficient heterologous expression of chitinase CsChit1B. The recombinant chitinase CsChit1B exhibits excellent catalytic performance: it maintains good stability within the range of 10-60 °C and pH range of 5.0-10.0, retaining a relative activity of over 80%. This enzyme can efficiently degrade the chitin in the cell walls of *Anthracis chinensis* and *Polytrichum pseudobulb*, while also showing efficient degradation ability for highly deacetylated chitosan, demonstrating great application potential in agricultural disease control and crustacean waste resource utilization.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a chitinase derived from tea trees, its preparation method, and its application. Background Technology

[0002] Chitin is the second most abundant natural polymer in nature, and it is widely found in the exoskeletons of arthropods, the organs of mollusks, and the cell walls of fungi. Chitosan, as a deacetylated derivative of chitin, has broad application prospects in biomedicine, food industry, agriculture and other fields.

[0003] The degradation of chitin and chitosan mainly involves chemical methods, physical methods, and bio-enzymatic methods. Among them, the bio-enzymatic method has become a key technology for promoting the industrial utilization of chitin and chitosan due to its advantages such as mild reaction conditions, environmental friendliness, and high product selectivity.

[0004] Currently, industrial chitinases mainly utilize microbial sources. However, these generally suffer from limited bioavailability, strict substrate specificity requirements, and poor adaptability to various enzyme properties, such as a wide optimal pH range and insufficient heat resistance, which restricts their effectiveness in complex applications. Plant-derived chitinases, particularly those from tea (Camellia sinensis), have been shown to be important defense factors against fungal diseases such as Camellia anthracnose, theoretically possessing excellent enzymatic properties. However, research on the heterologous expression, enzymatic properties, and applications of the tea chitinase gene CsChit1B remains lacking.

[0005] Based on the above, this invention targets the heterologous expression of the chitinase gene CsChit1B from tea trees, develops an efficient preparation method for chitinase from tea trees, and clarifies its application direction. This is of great significance for breaking through the technical bottlenecks in the application of existing chitinases and promoting the industrialization of chitin resource utilization. Summary of the Invention

[0006] The purpose of this invention is to provide a chitinase derived from tea trees, its preparation method, and its application, thereby solving the following technical problems: (1) Existing microbial chitinases have unstable enzymatic properties, such as narrow optimal pH range, poor heat resistance, and strict substrate requirements. In practical applications, their activity is easily inhibited and their application range is narrow. (2) There is a lack of systematic development of chitinases from tea trees, the potential of chitinases from tea trees has not been fully explored, and there are no relevant efficient preparation and application schemes in the existing technology.

[0007] The present invention achieves the above objectives through the following technical solutions: In a first aspect, the present invention provides a chitinase derived from tea plants, wherein the tea plant-derived chitinase CsChit1B is obtained by recombinant expression and purification of the tea plant chitinase gene fragment CsChit1B.

[0008] As a further optimization of the present invention, the nucleotide sequence of the chitinase gene fragment CsChit1B is shown in SEQ ID NO.1.

[0009] As a further optimization of the present invention, the protein sequence of the chitinase CsChit1B derived from tea trees is shown in SEQ ID NO.4.

[0010] A second aspect of the present invention provides the use of a tea-derived chitinase as described in any one of the foregoing claims in at least one of the following: (1) Inhibits the activity of Colletotrichum camelliae or Pseudopestalotiopsis spp. strains; (2) Degrading shrimp and crab shells or crustacean waste; (3) Degradation of chitosan with different degrees of deacetylation.

[0011] A third aspect of the present invention provides a method for preparing chitinase derived from tea trees, comprising the following steps: Step 1: Clone the chitinase gene fragment CsChit1B from the tea plant, with the nucleotide sequence shown in SEQ ID NO.1; Step 2: Ligate the chitinase gene fragment CsChit1B to the expression vector to construct a recombinant expression plasmid; Step 3: Transform the recombinant expression plasmid into host cells, clone and express it to obtain a recombinant engineered strain; Step 4: Induce the expression of the recombinant engineered strain to obtain the expression product; Step 5: Separate and purify the expressed product to obtain the chitinase derived from the tea plant.

[0012] As a further optimization of the present invention, the sequence of the forward primer for cloning the chitinase gene fragment CsChit1B is shown in SEQ ID NO. 2, and the sequence of the reverse primer is shown in SEQ ID NO. 3.

[0013] As a further optimization of the present invention, the recombinant expression vector is pMAL-c2x-CsChit1B.

[0014] As a further optimization of the present invention, the host cell for cloning is Escherichia coli DH5α; the host cell for expression is Escherichia coli Rosetta (DE3).

[0015] As a further optimization of the present invention, IPTG-induced expression is used, and the induction conditions are: IPTG final concentration 0.5 mM, 28 ℃, 120 rpm for 15 h.

[0016] As a further optimization of the present invention, the separation and purification adopts linear starch column affinity chromatography. Specifically, after the expression is induced, the bacterial cells are collected, sonicated, and the supernatant is taken. The supernatant is then adsorbed by linear starch column affinity chromatography, and eluted with TrisHCl buffer containing maltose. The eluent is collected to complete the separation and purification.

[0017] The beneficial effects of this invention are as follows: (1) This invention is the first to achieve heterologous high-efficiency expression and systematic development of the chitinase gene CsChit1B from tea tree, enriching the source library of chitinase and providing a new direction for the screening and development of new high-yield and high-quality chitinases; (2) The present invention has obtained a novel chitinase with excellent performance. The recombinant chitinase CsChit1B has a wide pH adaptability and can maintain high activity in the pH range of 5.0-10.0. It has good heat resistance and can stably play a role in the range of 20-60℃. It has a wide substrate range and solves the problems of existing chitinases being easily inhibited in complex application scenarios and the limited application range of single enzyme preparations. (3) Chitinase derived from tea trees has a significant inhibitory effect on pathogenic fungi such as Camellia anthracnose, and performs well in the degradation of shrimp and crab shells and the decomposition of chitosan with different degrees of deacetylation. It has dual application potential for agricultural disease control and shell waste resource utilization. Attached Figure Description

[0018] Figure 1 Agarose gel electrophoresis image of the PCR reaction product provided by this invention; Figure 2 This is a plasmid map and a schematic diagram of multiple cloning sites for the pMAL-c2x vector provided by the present invention. Figure 3 SDS-PAGE image of chitinase CsChit1B after separation and purification provided by the present invention; Figure 4 A standard curve of GlcNAc concentration of chitinase CsChit1B provided for this invention. Figure 5 The graph shows the results of the relative activity determination of chitinase CsChit1B at different temperatures provided by the present invention. Figure 6 The graph shows the results of the relative activity determination of chitinase CsChit1B at different pH values ​​provided by the present invention. Figure 7 The relative decomposition activity of chitinase CsChit1B against different substrates provided by the present invention; Figure 8 The graph shows the results of the relative activity determination of chitinase CsChit1B under different metal ions provided by the present invention. Detailed Implementation

[0019] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0020] I. Materials and Reagents The tea tree variety is Longjing 43, purchased from Nanjing Yarun Tea Co., Ltd. The strain was cultured using LB medium purchased from Shanghai Sangon Biotech Co., Ltd. After the medium was prepared, it was dispensed and autoclaved at 121 °C for 20 min. The colloidal chitin concentration was 1% (w / v) and it was purchased from Nanjing Ruiden Biotechnology Co., Ltd.

[0021] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0022] method Unless otherwise specified, the methods used in the following embodiments are conventional methods known to those skilled in the art.

[0023] 1. Cloning the chitinase gene fragment CsChit1B from tea plants 1.1 After grinding the tea leaves that had been frozen in liquid nitrogen using a ball mill, total RNA was extracted using a total RNA extraction kit.

[0024] 1.2. Total cDNA from tea plants was synthesized using extracted total RNA as a template; The components of the reverse transcription reaction system are: (a) Oligo dT Primer (50 μM) 1 μL, dNTP Mixture (10 mM each) 1 μL, template RNA 5 μL, RNase Free ddH2O 3 μL, incubated at 65 ℃ for 5 min and then rapidly cooled on ice; (b) Take 10 μL of the denatured reaction solution from (a), 4 μL of 5×PrimeScript II Buffer, 0.5 μL of RNase Inhibitor, 1 μL of PrimeScript II RTase, and 4.5 μL of RNase Free dH2O, mix them, and incubate at 42℃ for 45 min, then incubate at 95℃ for 5 min.

[0025] 1.3. PCR reaction was performed using 2×primer STAR max premix high-fidelity enzyme. The PCR reaction product was verified and purified to obtain the chitinase gene fragment CsChit1B. The PCR reaction system consisted of: 0.5 μL tea cDNA, 0.5 μL forward primer (with pMAL-c2X homologous recombination arm), 0.5 μL reverse primer (with pMAL-c2X homologous recombination arm), 12.5 μL 2×primer STAR max premix, and 12 μL ddH2O. The PCR reaction procedure is as follows: 98℃ for 10s, 52℃ for 15s, 72℃ for 15s, for a total of 30 cycles; hold at 16℃ for 3 min.

[0026] The chitinase gene fragment CsChit1B (NCBI: LOC114301399), with its nucleotide sequence shown in SEQ ID NO.1: ATGAGATTTTGCATACTAGTTTTGTTTTCTATCATCTCCCTACTAGGAGCCACAGCACAAGATATTAGCTCCCTTATCAGCAGAGATCTCTTCGATCAGATGTTGAAGCACCGGAACGATGCTAGTTGCCCCGGAAATGGGTTCTACACTTATGATGCTTTTGTAGCTGCTGCCAAGTCTTTTGGGGGTTTTGGAACCACCGGAGATACTGACACTCGTAAGAGAGAGATTGCGGCCTTTCTAGCTCAAACTTCGCATGAAACTACTGGTGGGTGGCCAAGTGCGCCAGATGGACCATATGCATGGGGATATTGCCATGTACGGGAACAAAACCCTGCTGGGGACTATTGTAGTCCAAGTCAAGAATGGCCCTGTGCTCCTGGTAAACAATACTATGGTCGTGGTCCAATCCAAATTTCACACAACTACAACTACGGTCCAGCAGGGAAAGCTATAGGGTCTGATCTGTTGGGCAACCCTGACCTAGTCGCAACTGATCCAACCATATCTTTCAAGACAGCATTCTGGTTCTGGATGACACCGCAATCCCCAAAACCCTCGTGTCACGATGTCATCACGGGCAGTTGGACACCATCTAGTGCGGACACCTCAGCGGGTCGGGTCCCTGGCTACGGTGTGATTACAAACATCATCAATGGCGGACTTGAATGTGGCAAAGGCTCTAATGCACAGGCAGAGGACCGGATCGGATTCTATAAAAGATACTGTGACTTGTTCGGAGTGGGATATGGCAACAATCTTGATTGCAATAACCAACAGCCTTTCGCATAA (SEQ ID NO.1) The forward primer (with pMAL-c2X homologous recombination arm) and reverse primer (with pMAL-c2X homologous recombination arm) of the chitinase gene fragment CsChit1B are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively: 5’-ATTTCAGAATTCGGATCCATGAGATTTTGCATACTAGTT-3’ (SEQ ID NO.2); 5'-GCTTGCCTGCAGGTCGACTGCGAAAGGCTGTTGGTTAT-3' (SEQ ID NO.3); The obtained PCR reaction products were subjected to agarose gel electrophoresis to verify fragment size. The electrophoresis image is shown below. Figure 1 As shown, the chitinase gene fragment CsChit1B is 792 bp in size, composed of... Figure 1 It can be seen that the strip size is correct; The gel was then cut, and the chitinase gene fragment CsChit1B was purified using a gel recovery kit.

[0027] 2. The chitinase gene fragment CsChit1B, purified by gel extraction, was ligated into an expression vector to construct a recombinant expression plasmid. 2.1 The pMAL-c2x empty plasmid (purchased from UBO Biotechnology, VT1247) was double-digested with restriction endonucleases to obtain the linearized pMAL-c2x vector; The plasmid map and multiple cloning site diagram of the pMAL-c2x vector are shown below. Figure 2 As shown; Preparation of the double enzyme digestion system for the pMAL-c2x vector: 6.0 μL of pMAL-c2x vector, 1.5 μL of restriction endonuclease BamHI, 1.5 μL of restriction endonuclease SalI, 5.0 μL of rCutSmart™ Buffer, and 36.0 μL of ddH2O were incubated at 37 °C for 2.5 h, followed by incubation at 65 °C for 20 min.

[0028] 2.2 Using Novizan's seamless cloning ClonExpress II One Step Cloning Kit enzyme, the chitinase gene fragment CsChit1B obtained in step 1 above was ligated with the linearized pMAL-c2x vector obtained in step 2.1 through seamless cloning to construct the recombinant plasmid pMAL-c2x-CsChit1B.

[0029] The components of the reaction system for seamless clonal linkage are: The following ingredients were used for seamless cloning: 1 μL Exnase II, 3 μL CsChit1B, 4 μL linearized pMAL-c2x vector, and 2 μL 5×Cell Buffer. The cloning was performed at 37 °C for 30 min.

[0030] 3. Transform the recombinant expression plasmid into host cells to obtain recombinant engineered strains. 3.1 Take 5 μL of the recombinant plasmid pMAL-c2x-CsChit1B obtained in step 2 and add it to 40 μL of E. coli DH5α competent cells. Incubate on ice for 30 min, heat shock in a 42 ℃ water bath for 70 s, then incubate on ice for 2 min. Add 200 μL of antibiotic-free LB medium and activate at 37 ℃ and 200 rpm for 1 h. Then plate on an LB plate containing ampicillin (50 μg / mL). The next day, perform colony PCR verification on the single clones that grow on the plate. Select positive clones for transfer, extract plasmids and send them for sequencing verification.

[0031] 3.2. Transform the validated plasmid into E. coli Rosetta (DE3) competent cells. The transformation process includes: 5 μL of the sequenced plasmid was added to 40 μL of Rosetta(DE3) competent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, then 200 μL of LB medium was added, and the mixture was incubated on a shaker at 37℃ and 200 rpm for 1 h. The mixture was then plated on ampicillin-resistant LB plates. Positive clones were selected for preservation, thus successfully constructing the engineered strain Rosetta(DE3) / pMAL-c2x-CsChit1B-MBP, which heterologously expresses recombinant chitinase. The engineered strain Rosetta(DE3) / pMAL-c2x-CsChit1B-MBP was heterologously expressed with recombinant chitinase.

[0032] 4. Induce the expression of the recombinant engineered strain to obtain the expression product. 4.1 Take 100 μL of the engineered strain Rosetta(DE3) / pMAL-c2x-CsChit1B-MBP, which heterologously expresses recombinant chitinase, from the preservation tube, inoculate it into a 5 mL LB test tube, add ampicillin to a final concentration of 50 μg / mL, and incubate at 37 ℃ and 200 rpm for 15 h. 4.2. Transfer the cultured bacterial suspension at a volume of 5% to a 250 mL Erlenmeyer flask containing 100 mL LB, add ampicillin to a final concentration of 50 μg / mL and glucose to a final concentration of 0.2%, and incubate at 37 ℃ and 200 rpm until OD reaches 0.5%. 600 =0.7; 4.3 Add 0.5 mM IPTG, transfer the conical flask to 28 ℃, and induce expression at 120 rpm for 15 h.

[0033] 5. Chitinase derived from tea plant was isolated and purified from the expression product and named chitinase Cschit1B. 5.1 After the expression was induced, the bacterial cells were collected by centrifugation at 6000 g for 10 min at 4 ℃. They were then resuspended in 30 mL of Tris-HCl buffer (20 mM Tris, 200 mM NaCl, 1 mM DTT, 1 mM EDTANa2·2H2O) and sonicated for 30 min with a 3 s interval and a 4 s pause. The power was set to 350 W. 5.2 Centrifuge the crushed homogenate at 4℃, 5000 g for 15 min, and retain the supernatant; 5.3 Remove the stopper and cap from the amylose column, allowing the 20% ethanol preservation solution to flow down naturally at maximum flow rate. Flush the column with ultrapure water for two column volumes to remove as much residual ethanol as possible. Flush the column with Tris-HCl-maltose elution buffer (Tris-HCl buffer, 3.6 g / L maltose) for at least two column volumes to wash the amylose column. Then flush the column with Tris-HCl buffer for at least two column volumes to equilibrate the amylose column. To prevent protein denaturation, the entire procedure should be performed at low temperature. 5.4 Add the supernatant to the starch column and control the flow rate at 6-8 s / gtt to ensure that the protein and the packing material are fully combined. After all the supernatant has passed through the column, wash off the impurities with Tris-HCl buffer for three column volumes. Finally, wash off two column volumes with Tris-HCl-maltose elution buffer. Collect the eluent in a centrifuge tube to obtain the enzyme solution containing chitinase CsChit1B. Add the collected enzyme solution to an ultrafiltration tube with MW=300,000 and concentrate the enzyme solution at 5000 g. Collect the concentrated enzyme solution and store it on ice. 5.5 Add the elution buffer to 5×SDS-PAGE Loading Buffer (SDS-PAGE protein loading buffer (5×)) and mix. Boil in boiling water for 5 min and perform SDS-PAGE (polyacrylamide gel electrophoresis). Stain the gel with Coomassie Brilliant Blue, add acetic acid and ethanol to decolorize, and observe the expression and purification of the protein.

[0034] The results of SDS-PAGE determination are as follows: Figure 3 As shown, from Figure 3 It can be seen that the theoretical size of the chitinase Cschit1B from tea plant is about 70 kDa (including the MBP tag), with no obvious contaminating protein bands, indicating high purity.

[0035] The protein sequence of chitinase CsChit1B is shown in SEQ ID NO.4: MRFCILVLFSIISLLGATAQDISSLISRDLFDQMLKHRNDASCPGNGFYTYDAFVAAAKSFGGFGTTGDTDTRKREIAAFLAQTSHETTGGWPSAPDGPYAWGYCHVREQNPAGDYCSPSQEWPCAPGKQYYG RGPIQISHNYNYGPAGKAIGSDLLGNPDLVATDPTISFKTAFWFWMTPQSPKPSCHDVITGSWTPSSADTSAGRVPGYGVITNIINGGLECGKGSNAQAEDRIGFYKRYCDLFGVGYGNNLDCNNQQPFA (SEQ ID NO.4) 6. Performance testing of chitinase CsChit1B derived from tea tree 6.1 Enzyme Activity Assay Chitinase can catalyze the hydrolysis of chitin to produce N-acetyl-D-glucosamine (GlcNAc). Based on this reaction, colloidal chitin was used as a substrate and catalyzed by the chitinase Cschit1B derived from tea trees for a certain period of time. Then, the concentration of the product GlcNAc was determined by DNS reagent, thereby calculating the enzyme activity.

[0036] The specific steps are as follows: (1) Take 50 μL of the enzyme solution containing chitinase Cschit1B obtained in step 5.4 and add 50 μL of colloidal chitin. React at 50 °C for 45 min. After the reaction, centrifuge at 6000 g for 2 min. Take 80 μL of the supernatant and add 50 μL of DNS reagent. Boil at 100 °C for 6 min to develop color. Cool to room temperature. Take 20 μL of the supernatant and add 80 μL of Tris-HCl buffer to determine OD. 540 Absorbance at nm; for the blank control, the enzyme solution was replaced with the elution solution used for eluting proteins, and all other parts were kept the same, with 3 replicates for each; (2) The standard curve was determined using N-acetylglucosamine (GlcNAc) as the standard concentration gradient (0.05~1 g / L). The results of the concentration standard curve determination are as follows: Figure 4 As shown, the reducing sugar concentration was calculated based on the standard curve; Enzyme activity (U / mL) refers to the enzyme activity that produces 1 μL of GlcNAc per hour under optimal conditions per microliter of enzyme solution. The formula for calculating enzyme activity is as follows: ; In the formula, C is the concentration of GlcNAc (μg / 100μL) calculated based on the standard curve. V 总 The total volume of the colorimetric reaction is (μL). D represents the enzyme solution dilution factor; t is the enzyme-catalyzed reaction time (h); V 酶 The volume (μL) of enzyme solution added to the reaction system.

[0037] (3) Determine the enzyme activity of chitinase Cschit1B under different temperature conditions. Under Tris-HCl caching conditions, the following grouping design is performed: Experimental group: 10 μL Cschit1B + 15 μL colloidal chitin + 5 μL Tris-HCl buffer; Control group: 15 μL buffer + 15 μL colloidal chitin.

[0038] Both the control and experimental groups were set up in triplicate, and the reactions were carried out at 0 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, and 70 ℃ for 50 min, respectively. The enzyme activity was measured at each reaction, and the reaction temperature at which the enzyme activity was highest was taken as the optimum reaction temperature. The enzyme activity at the highest point was defined as 100%. The results of the determination of chitinase Cschit1B activity under different temperature conditions are as follows: Figure 5 As shown, by Figure 5 It can be seen that the enzyme activity of chitinase Cschit1B is highest at 30℃, that is, the optimal temperature for catalysis is 30℃.

[0039] (4) Determine the enzyme activity of chitinase CsChit1B at different pH values. Colloidal chitin was resuspended in Tris-HCl buffers of different pH values. Using colloidal chitin as a substrate, enzyme activity was measured at 50 °C. The pH of the reaction buffer at which the enzyme activity was highest was taken as the optimal pH, and the enzyme activity at which the highest activity was highest was defined as 100%. The results of the determination of chitinase CsChit1B activity at different pH values ​​are as follows: Figure 6 As shown, by Figure 6 It can be seen that the enzyme activity of chitinase CsChit1B is highest at pH 7.0, that is, the optimal pH for catalysis is 7.0; In addition, by Figure 5 and Figure 6 It can be seen that the relative activity of chitinase CsChit1B is >80% in the temperature range of 10~60 ℃ and >85% in the pH range of 5.0-10.0. This indicates that chitinase CsChit1B has catalytic activity in a wide range of temperature and pH, and has potential practical application value.

[0040] 6.2 Test on the degradation ability of tea tree-derived chitinase on mycelial powders from different fungi, chitosan with different degrees of deacetylation, and shrimp shells. (1) Weigh out 0.1 g of each of the following pathogenic fungi isolated from diseased tea leaves at the experimental base of Anhui Agricultural University in Hefei, Anhui Province: Colletotrichum camelliae mycelium powder, Pseudopestalotiopsis spp. mycelium powder, shrimp shell powder, chitosan with a degree of deacetylation of 70%, 80%, 90%, and greater than 95%, and place them in 10 mL of Tris-HCl buffer to prepare a 1% (W / V) colloid (suspension) for later use.

[0041] (2) Under the Tris-HCl buffer condition, the following grouping design is performed: Experimental group: 15 μL CsChit1B + 20 μL of each substrate; Control group: 15 μL Tris-HCl buffer + 20 μL of each substrate; Both the control and experimental groups were set up in triplicate, and the enzyme activity was measured at 50 °C to compare the decomposition activity of chitinase CsChit1B on different substrates.

[0042] Chitinase CsChit1B exhibits various substrate degradation activities, such as Figure 7 As shown in Table 1, the results indicate that the chitinase CsChit1B prepared in this invention exhibits highly efficient degradation capabilities for different fungal mycelial powders, shrimp shells, and chitosan with different degrees of deacetylation. In particular, it shows the highest relative enzyme activity (184.79%) for chitosan with a degree of deacetylation of 90%, demonstrating broad-spectrum substrate specificity.

[0043] Table 1. Statistical analysis of the degradative activity of chitinase CsChit1B on different substrates.

[0044] 6.3 Relative enzyme activity test of chitinase from tea plant source under different metal ion conditions (1) Weigh the corresponding mass of inorganic salts according to Table 2, place them in 5 mL centrifuge tubes, add 5 mL of deionized water to dissolve them, and prepare stock solutions containing different metal ions with a concentration of 1.25 mM. Seal them for later use.

[0045] Table 2. Statistics on the amount of inorganic salts required for the preparation of stock solutions containing different metal ions (1.25 mM)

[0046] (2) Under the Tris-HCl buffer condition, the following grouping design is performed: Experimental group: 10 μL CsChit1B + 10 μL colloidal chitin + 80 μL corresponding metal ion stock solution; Control group: 90 μL buffer + 10 μL colloidal chitin; Both the control and experimental groups were set up in triplicate, and the enzyme activity was measured at 50 °C. The relative enzyme activity of chitinase Cschit1B under different metal ion conditions was compared, and the enzyme activity of the control group was defined as 100%.

[0047] The relative enzyme activity of chitinase Cschit1B under different metal ion conditions is as follows: Figure 8 As shown, from Figure 8 It can be seen that Fe 2+ Co 2+ and Mn 2+ The chitinase CsChit1B prepared in this invention exhibits activating activity, with relative enzyme activities of 119%, 130%, and 159%, respectively; while Mg 2+ Zn 2+ K + These exhibit varying degrees of inhibitory effects, among which Mg... 2+ The inhibitory effect was most significant, with relative enzyme activity decreasing to 65%.

[0048] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A chitinase derived from tea trees, characterized in that, The chitinase CsChit1B derived from tea plants was obtained by recombinant expression and purification of the tea plant chitinase gene fragment CsChit1B.

2. The chitinase derived from tea trees according to claim 1, characterized in that, The nucleotide sequence of the chitinase gene fragment CsChit1B is shown in SEQ ID NO.

1.

3. The chitinase derived from tea trees according to claim 1, characterized in that, The protein sequence of the chitinase CsChit1B derived from the tea plant is shown in SEQ ID NO.

4.

4. The use of a chitinase derived from tea plants as described in any one of claims 1-3 in at least one of the following, characterized in that, (1) Inhibits the activity of Colletotrichum camelliae or Pseudopestalotiopsis spp. strains; (2) Degrading shrimp and crab shells or crustacean waste; (3) Degradation of chitosan with different degrees of deacetylation.

5. A method for preparing chitinase derived from tea trees, characterized in that, Includes the following steps: Step 1: Clone the chitinase gene fragment CsChit1B from the tea plant, with the nucleotide sequence shown in SEQ ID NO.1; Step 2: Ligate the chitinase gene fragment CsChit1B to the expression vector to construct a recombinant expression plasmid; Step 3: Transform the recombinant expression plasmid into host cells, clone and express it to obtain a recombinant engineered strain; Step 4: Induce the expression of the recombinant engineered strain to obtain the expression product; Step 5: Separate and purify the expressed product to obtain the chitinase derived from the tea plant.

6. The method for preparing chitinase from tea plants according to claim 5, characterized in that, The sequence of the forward primer for cloning the chitinase gene fragment CsChit1B is shown in SEQ ID NO. 2, and the sequence of the reverse primer is shown in SEQ ID NO.

3.

7. The method for preparing chitinase from tea plants according to claim 5, characterized in that, The recombinant expression vector is pMAL-c2x-CsChit1B.

8. The method for preparing chitinase from tea plants according to claim 5, characterized in that, The host cell used for cloning is Escherichia coli DH5α; the host cell used for expression is Escherichia coli Rosetta (DE3).

9. The method for preparing chitinase from tea plants according to claim 5, characterized in that, IPTG was used to induce expression. The induction conditions were: IPTG final concentration 0.5 mM, 28 ℃, 120 rpm for 15 h.

10. The method for preparing chitinase from tea plants according to claim 5, characterized in that, The separation and purification process employs a linear starch column affinity chromatography. Specifically, after induction of expression, the bacterial cells are collected, sonicated, and the supernatant is taken. The supernatant is then adsorbed onto a linear starch column using affinity chromatography, followed by elution with a TrisHCl buffer containing maltose. The eluent is collected to complete the separation and purification.

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