Chitin deacetylase product and application method in agricultural disease prevention

Abstract

This invention discloses a chitin deacetylase product and its application in agricultural disease prevention. Through codon optimization, the *Rhodococcus* chitin deacetylase gene (SEQ ID NO:1) was cloned into the *Pichia pastoris* expression vector pGAPZa A, constructing the recombinant vector pGAPZaA-CDA-His, which was then transformed into the *Pichia pastoris* SMD1168 host, achieving efficient secretory expression of the enzyme. The resulting recombinant enzyme exhibits high activity and good thermostability. This invention includes a universal enzymatic deacetylation process characterized by deacetylation efficiency, applicable to chitin deacetylases from various sources. This invention also provides a method for further enzymatic hydrolysis to produce N-acetylglucosamine monomers or their oligosaccharides, and the application of the recombinant enzyme and its product in the preparation of biopesticides and the treatment of chitin-containing biomass materials.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and enzyme engineering, specifically to chitin deacetylase products and their application in agricultural disease prevention. Background Technology

[0002] Chitin is one of the most abundant aminopolysaccharides in nature, mainly derived from the exoskeletons of crustaceans such as shrimp and crabs, the cell walls of fungi, and the pupal skins of some insects. Its deacetylation product, chitosan, has wide applications in biomedicine, food industry, cosmetics, water treatment, and agriculture due to its excellent biocompatibility, biodegradability, antibacterial properties, and film-forming properties. Furthermore, further degradation products of chitosan, such as N-acetylglucosamine monomer (GlcNAc) and its oligosaccharides, are important raw materials for health supplements and drug precursors.

[0003] Currently, the industrial production of chitosan mainly employs the concentrated alkali thermochemical method. Although this method is relatively low-cost, it suffers from drawbacks such as high energy consumption, severe environmental pollution, difficulty in precisely controlling the degree of deacetylation (DD) and molecular weight of the product, and damage to the sugar chain structure, resulting in poor product uniformity.

[0004] Enzymatic deacetylation utilizes chitin deacetylase (CDA, EC 3.5.1.41), which specifically catalyzes the hydrolysis of the acetyl group of N-acetylglucosamine residues in chitin under mild conditions. Compared with chemical methods, enzymatic methods offer significant advantages such as mild reaction conditions, environmental friendliness, regular product structure, and controllable degree of deacetylation and polymerization. However, naturally derived chitin deacetylases suffer from low yield, poor stability, and low enzyme activity, severely limiting their industrial application. Achieving efficient heterologous expression of chitin deacetylases through genetic engineering is key to overcoming these bottlenecks. Existing studies have attempted to express CDA from different sources in systems such as *E. coli* and *Pichia pastoris*, but these studies generally suffer from low expression levels, low enzyme activity, or products existing in inclusion body form requiring complex renaturation. In particular, for chitin deacetylases derived from *Rhodococcus*, there are currently no reports of achieving efficient secretory expression in the *Pichia pastoris* system. Pichia pastoris expression systems (such as the SMD1168 host) have advantages such as high-density fermentation, strong secretion capacity, and post-translational modification, making them very suitable for industrial production of exogenous proteins.

[0005] Therefore, developing a chitin deacetylase product and its application in agricultural disease prevention, as well as establishing a green chitin conversion process based on this enzyme, is of great significance for promoting the industrialization of chitosan and its downstream high-value products.

[0006] Furthermore, chitosan and its low molecular weight derivatives (such as chitosan oligosaccharides) have shown great application potential in agriculture. They can act as plant immune inducers, stimulating the plant's own defense system to resist fungal, bacterial, and viral diseases; simultaneously, they have direct inhibitory effects on various plant pathogens and can serve as green, biodegradable pesticide or fertilizer carriers. However, the wide distribution of molecular weight and degree of deacetylation of chemically produced chitosan leads to unstable biological activity and large batch-to-batch variations, limiting its application in high-end biopesticides. Enzymatically produced chitosan has a uniform structure, especially chitosan oligosaccharides with specific degrees of polymerization and deacetylation prepared through specific enzymatic processes, exhibiting higher biological activity and making them ideal raw materials for developing next-generation, highly efficient, and safe biopesticides. Currently, there is a lack of mature technologies for the efficient and cost-effective enzymatic production of specific chitosan / chitosan oligosaccharide products optimized for agricultural applications.

[0007] Besides marine chitin such as shrimp and crab shells, insect-derived chitin (such as silkworm pupa skin and insect remains) is also an important chitin resource, and currently there is a lack of efficient and gentle processing methods. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a chitin deacetylase product and its application method in agricultural disease prevention. This invention solves the problems mentioned in the background art, such as high energy consumption, serious environmental pollution, difficulty in accurately controlling the degree of deacetylation (DD) and molecular weight of the product, and the damage to the sugar chain structure leading to poor product uniformity. Furthermore, it addresses the lack of mature technologies for the efficient and cost-controllable enzymatic production of specific chitosan / chitooligosaccharide products optimized for agricultural applications.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution: a nucleic acid molecule encoding chitin deacetylase, the nucleotide sequence of which is shown in SEQ ID NO: 1, or is a sequence having at least 95% identity with SEQ ID NO: 1 and encoding a protein having chitin deacetylase activity; preferably, the nucleic acid molecule is a synthetic gene that has been codon-optimized to adapt to the Pichia pastoris expression system, and its sequence is shown in SEQ ID NO: 3.

[0010] A recombinant expression vector comprising a nucleic acid molecule and an expression regulatory element operatively linked to the nucleic acid molecule; preferably, the vector is a pGAPZa A-derived vector, wherein the nucleic acid molecule is inserted via ECOR I and Not I restriction sites and a sequence encoding a 6×histidine tag is fused to the C-terminus of the coding sequence.

[0011] A recombinant Pichia pastoris engineered strain, which is obtained by transforming a recombinant expression vector into a Pichia pastoris host cell; preferably, the host cell is a Pichia pastoris protease-deficient strain SMD1168.

[0012] A recombinant chitin deacetylase, encoded by a nucleic acid molecule or produced by a recombinant Pichia pastoris engineered strain; the amino acid sequence of the recombinant enzyme is shown in SEQ ID NO: 2, and the specific activity of the enzyme against colloidal chitin is not less than 50 U / mg under the conditions of pH 7.5 and 55℃.

[0013] A method for producing chitosan involves mixing recombinant chitin deacetylase with a chitin substrate in a buffer solution and reacting at 40-60°C and pH 7.0-8.5 for 2-24 hours, then terminating the reaction and separating and purifying the chitosan. Preferably, the chitin substrate is colloidal chitin with a concentration of 1-5% (w / v).

[0014] A method for producing N-acetylglucosamine monomer or oligosaccharide thereof, comprising the following steps: S1. Use chitosan or partially deacetylated chitin; S2. Hydrolyze the product of step S1 using chitinase or chitosanase. S3. Separate and purify to obtain N-acetylglucosamine monomer or its oligosaccharide.

[0015] Furthermore, the chitin-containing biomass material is insect-derived chitin material; preferably, the insect-derived chitin material is insect pupa skin or insect remains.

[0016] A method for applying a biopesticide, wherein the biopesticide prepared from chitosan, N-acetylglucosamine monomer or its oligosaccharide is used to control fungal diseases, bacterial diseases or viral diseases of plants, or to induce systemic resistance in plants.

[0017] A biopesticide composition comprising an effective dose of chitosan, N-acetylglucosamine monomer or oligosaccharide thereof as active ingredients, and an agriculturally acceptable carrier and adjuvants.

[0018] Furthermore, the dosage form of the composition is a solvent-based, water-dispersible granule, pellet, or seed coating agent.

[0019] This invention provides a chitin deacetylase product and its application method in agricultural disease prevention, which has the following beneficial effects: 1. High expression efficiency and high enzyme activity: This invention is the first to successfully achieve high-efficiency secretory expression of chitin deacetase from Rhodococcus in Pichia pastoris SMD1168. Through codon optimization, the use of a strong constitutive GAP promoter and α-factor signal peptide, the secretory expression level of the recombinant enzyme in a 250 mL shake flask can reach over 200 mg / L, with an enzyme activity of 25-60 U / mg, significantly higher than that of Escherichia coli expression systems and many homologous or heterologous expression levels reported in the literature.

[0020] 2. Excellent enzymatic properties: The recombinant enzyme obtained in this invention exhibits excellent heat resistance and pH stability, making it suitable for industrial reaction conditions. Its optimal temperature (50-60℃) helps improve substrate solubility and reaction rate, while reducing the risk of microbial contamination.

[0021] 3. Green and environmentally friendly process: Enzymatic deacetylation completely avoids the discharge of large amounts of wastewater and waste residue from the traditional concentrated alkali process, which meets the requirements of green chemistry and sustainable development.

[0022] 4. Controllable product quality: By precisely controlling the enzyme reaction time, temperature, pH and enzyme dosage, the degree of deacetylation (DD) of the product can be flexibly customized to produce high-value-added chitosan products that meet different application requirements.

[0023] 5. Downstream application expansion: The enzyme-enzyme synergistic process (CDA combined with chitinase / chitosanase) established in this invention can realize a complete and high-value conversion chain from waste chitin to chitosan, and then to N-acetylglucosamine monomers or functional oligosaccharides.

[0024] 6. Expanding High-Value Agricultural Applications: The enzymatic production process provided by this invention allows for precise control of the degree of deacetylation in the product, thereby enabling the customized production of chitosan / chitosan oligosaccharide products with optimal inducing or inhibiting antibacterial activity. These products can serve as core active ingredients for developing novel, highly efficient, non-toxic, and residue-free biopesticides.

[0025] 7. Expanding the application of chitin treatment from insect sources: The recombinant enzyme provided by this invention can effectively treat chitin-containing biomass materials such as insect pupa skins, providing new technical means for some fields.

[0026] 8. Compared with chitin deacetylases from other sources reported in the prior art (such as Rhizopus stolonifera RsCDA1 specific activity of 5.2 U / mg and Streptomyces griseus CDA specific activity of 3.09 U / mg), the specific activity of the recombinant Rhodococcus chitin deacetylase obtained in this invention reaches 50 U / mg, which is several to more than ten times higher. More importantly, this invention achieves high-efficiency secretory expression of 200 mg / L through the Pichia pastoris system, with a total fermentation broth activity of 10,000 U / L, demonstrating good prospects for industrial application. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the plasmid map of the recombinant expression vector pGAPZaA-CDA-His of this invention; Figure 2 The expression and purification results of recombinant chitin deacetylase were analyzed by SDS-PAGE.

[0028] In this invention, the term "strict conditions" refers to hybridization and washing conditions conventionally used in the art. For example, in one embodiment, the strict conditions are: overnight hybridization at 65°C in a hybridization solution of 6×SSC (0.9 M NaCl, 0.09 M sodium citrate), 5×Denhardt's solution, 0.5% SDS, and 100 μg / mL denatured salmon sperm DNA; followed by washing twice at room temperature with 2×SSC and 0.1% SDS for 15 minutes each, and twice at 65°C with 0.5×SSC and 0.1% SDS for 15 minutes each. Those skilled in the art can select other equivalent strict conditions based on known methods. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto.

[0030] Unless otherwise specified, all reagents and materials used in the examples are commercially available products.

[0031] 1. The nucleotide sequence of the nucleic acid molecule encodes a protein with chitin deacetylase activity and is selected from the following group: (a) The sequence shown in SEQ ID NO: 1; (b) A sequence that has at least 95% identity with the sequence shown in SEQ ID NO: 1 and encodes a protein having chitin deacetylase activity; (c) A sequence that can hybridize with the complementary strand of sequence (a) or (b) under strict conditions and encodes a protein with chitin deacetylase activity.

[0032] An isolated protein, which is a polypeptide with chitin deacetylase activity, wherein the amino acid sequence of the polypeptide is selected from the following group: (a) The sequence shown in SEQ ID NO: 2; (b) A sequence that has at least 95% identity with the sequence shown in SEQ ID NO: 2 and has chitin deacetylase activity.

[0033] A complex enzyme preparation comprising the isolated protein described above and at least one other coenzyme or adjuvant.

[0034] An enzyme with chitin deacetylase activity, derived from Rhodococcus, exhibiting a specific activity of not less than 50 U / mg for colloidal chitin under pH 7.5 and 55℃ conditions.

[0035] A method for producing deacetylated chitin products includes a step of deacetylation of chitin raw material under the action of an enzyme, wherein the enzyme is a chitin deacetylate enzyme, and the reaction conditions enable the deacetylation efficiency to reach more than 85%.

[0036] Among them, chitin deacetylase is encoded by the above-mentioned protein or by the above-mentioned nucleic acid molecule.

[0037] Enzymes possess one or more of the following characteristics: (a) Its optimal reaction pH is 6.0-8.0; (b) Its optimal reaction temperature is 40℃-60℃; (c) Its Michaelis constant (Km) for chitin is not higher than 5 mM.

[0038] Chitin deacetylase is the protein mentioned above.

[0039] Among them, the chitosan or N-acetylglucosamine products prepared by the above method.

[0040] The aforementioned nucleic acid molecule is a synthetic gene that has been codon-optimized to adapt to the Pichia pastoris expression system, and its nucleotide sequence is shown in SEQ ID NO: 3.

[0041] A recombinant expression vector comprising the aforementioned nucleic acid molecule and an expression regulatory element operatively linked to the nucleic acid molecule.

[0042] The expression regulatory elements include promoters, secretory signal peptides, and selection markers.

[0043] The vector is a pGAPZa A-derived vector, the promoter is a GAP promoter, the secretory signal peptide is an α-factor signal peptide, the selection marker is the Zeocin resistance gene, the nucleic acid molecule is inserted through ECOR I and Not I restriction sites, and a sequence encoding a polyhistidine tag is fused to the C-terminus of the coding sequence.

[0044] A recombinant host cell comprising the aforementioned recombinant expression vector.

[0045] The host cell is a yeast cell; preferably a Pichia pastoris cell; more preferably a Pichia pastoris protease-deficient strain SMD1168.

[0046] A method for producing recombinant chitin deacetylase includes: (a) Recombinant host cells cultured under conditions suitable for protein expression; (b) Isolate and / or purify chitin deacetylase from the culture.

[0047] In step (b), purification is performed using a polyhistidine tag via metal chelate affinity chromatography.

[0048] Among them, the recombinant chitin deacetase produced by the above method.

[0049] Among them, the application of recombinant chitin deacetylase in catalyzing the deacetylation reaction of chitin, colloidal chitin or chitin oligosaccharides.

[0050] Among them, the application of recombinant chitin deacetylase in the treatment of chitin-containing biomass materials.

[0051] Among them, the chitin-containing biomass materials are insect-derived chitin materials.

[0052] Among them, the chitinous material derived from insects is insect pupa skin or insect remains.

[0053] A method for producing N-acetylglucosamine monomer or oligosaccharides thereof, comprising: (i) Chitosan or partially deacetylated chitin is obtained using the above method; (ii) Enzymatically hydrolyze the product of step (i) using chitinase or chitosanase; (iii) Separate and purify N-acetylglucosamine monomer or its oligosaccharide from the enzymatic hydrolysis product.

[0054] A composition comprising the above-described recombinant chitin deacetase, as well as a chitin substrate and / or a reaction buffer.

[0055] Among them, biological pesticides are used to control plant pathogenic fungi, bacteria or viruses, or to induce systemic resistance in plants.

[0056] A biopesticide composition comprising chitosan or N-acetylglucosamine product as the active ingredient, or N-acetylglucosamine monomer and its oligosaccharides produced by the above method, as well as an agriculturally acceptable carrier and adjuvants.

[0057] The composition is in the form of a soluble concentrate, a water-dispersible granule, a pellet, or a seed coating agent.

[0058] Example 1: Codon optimization and synthesis of the chitin deacetylase gene from Rhodococcus pyogenes Based on the conserved sequence (reference sequence) of the *Rhodococcus* chitin deacetase gene in the NCBI database, PCR primers were designed for cloning and verification, yielding the gene sequence shown in SEQ ID NO: 1. To improve expression efficiency in *Pichia pastoris*, Sangon Biotech (Shanghai) Co., Ltd. was commissioned to synthesize the entire gene and optimize the codons (the optimization principle was to use Pichia pastoris-preferred codons). The optimized gene sequence is shown in SEQ ID NO: 3. ECOR I and Not I restriction sites were introduced at the 5' and 3' ends of the sequence, respectively, and a sequence encoding a 6× histidine tag was inserted before the stop codon.

[0059] Example 2: Construction of the recombinant expression vector pGAPZaA-CDA-His 1. The pGAPZa A empty vector purchased from Invitrogen was digested with ECOR I and Not I, and the linearized vector fragment was recovered.

[0060] 2. The synthesized optimized CDA gene fragment (SEQ ID NO: 3, containing ECOR I and Not I sites at both ends) with homologous arms was ligated to the linearized pGAPZa A vector by reacting with homologous recombinase at 50°C for 30 minutes.

[0061] 3. The ligation product was transformed into E. coli DH5α competent cells, plated on LB plates containing 25 μg / mL Zeocin, and incubated overnight at 37°C.

[0062] 4. Select single colonies for colony PCR and plasmid double enzyme digestion identification. Send positive clones for sequencing verification. Name the correctly sequenced recombinant plasmid pGAPZaA-CDA-His (see structural diagram). Figure 1 ).

[0063] Example 3: Construction and screening of recombinant Pichia pastoris engineered strains 1. The pGAPZaA-CDA-His plasmid was linearized with Sac I and electroporated into Pichia pastoris SMD1168 competent cells.

[0064] 2. Spread the electroporated cells onto YPDS plates containing 100 μg / mL Zeocin and incubate at 30°C for 2–3 days.

[0065] 3. Pick multiple transformants and inoculate them into 5 mL of YPD medium (containing 100 μg / mL Zeocin), and culture at 30℃ and 250 rpm for 48 hours.

[0066] 4. Centrifuge to collect the culture supernatant and perform SDS-PAGE analysis ( Figure 2The strains with the highest expression levels were screened using both Western blot (using anti-His tag antibody) and Western blot (using anti-His tag antibody).

[0067] 5. The selected high-expression strain was named Pichia pastoris SMD1168 / pGAPZaA-CDA-His, and multiple generations were performed to verify its genetic stability.

[0068] Example 4: Fermentation production and purification of recombinant chitin deacetylase 1. Inoculate the high-expression strain into BMGY medium and incubate at 30℃ and 250 rpm until the OD600 reaches 6-10.

[0069] 2. Collect the bacterial cells by centrifugation, resuspend them in BMMY medium (or continue constitutive expression using BMGY), and express them at 28℃ and 250 rpm for 72 hours.

[0070] 3. After fermentation, centrifuge at 4℃ and 8000×g for 10 minutes, and collect the supernatant, which is the crude enzyme solution.

[0071] 4. After filtering the crude enzyme solution through a 0.45 μm filter membrane, the sample was loaded onto a Ni-NTA affinity chromatography column that had been pre-equilibrated with equilibration buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0).

[0072] 5. Perform staged elution with elution buffer containing 50 mM and 250 mM imidazole, and collect the 250 mM imidazole elution peak.

[0073] 6. Dialyze the eluent in PBS buffer (pH 7.4) to desalt, then freeze-dry or concentrate and store at -80°C. The purification process is as follows: Figure 2 As shown, a single protein band was obtained. The specific activity of the purified recombinant enzyme was determined to be 50 U / mg.

[0074] Example 5: Production of chitosan using recombinase 1. Prepare a 2% (w / v) colloidal chitin suspension (dissolved in 50 mM acetate-sodium acetate buffer, pH 7.5).

[0075] 2. Add purified recombinant chitin deacetase to 10 mL of the above substrate solution to a final concentration of 5 U / mL.

[0076] 3. Place the reaction system in a 45°C water bath shaker and shake at 200 rpm for 12 hours.

[0077] 4. After the reaction is complete, place the mixture in a boiling water bath for 10 minutes to inactivate the enzyme.

[0078] 5. After cooling, adjust the pH to 8-9 with 1 M NaOH to precipitate chitosan.

[0079] 6. Centrifuge to collect the precipitate, wash with deionized water until neutral, and freeze-dry to obtain a white, flaky chitosan product.

[0080] 7. The degree of deacetylation (DD%) of the product was determined by acid-base titration, and the result was 93.5%. FT-IR analysis showed that at 1650 cm⁻¹... -1 (Amide I band) and 1320 cm -1 The characteristic peak at (amide III band) is significantly weakened, while at 1590 cm⁻¹... -1 A strong absorption peak appeared at (-NH2), confirming successful deacetylation.

[0081] Example 6: Production of N-acetylglucosamine monomer (GlcNAc) using recombinant enzymes 1. Take 0.5 g of the chitosan prepared in Example 5 and dissolve it in 50 mL of 1% (v / v) acetic acid solution.

[0082] 2. Add commercial chitosanase (10 U / g substrate) and react at 45°C and pH 7.5 for 6 hours.

[0083] 3. After the reaction solution is decolorized with activated carbon and filtered, it is separated and purified by gel permeation chromatography (GPC) or preparative high performance liquid chromatography (HPLC).

[0084] 4. The main components were collected, concentrated, and freeze-dried to obtain a white powder. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) identified it as an N-acetylglucosamine monomer.

[0085] Example 7: Determination of Enzymatic Properties 1. Optimal pH: Enzyme activity was measured in different buffer systems with pH ranges from 4.0 to 8.0, and the optimal pH was determined to be 7.5.

[0086] 2. Optimal temperature: Enzyme activity was measured in the range of 30-70℃, and the optimal temperature was determined to be 55℃.

[0087] 3. pH stability: The residual enzyme activity was measured after the enzyme was placed in different pH buffers at 4°C for 24 hours. The stability was greater than 90% in the pH range of 6.0-10.0.

[0088] 4. Thermal stability: The enzyme was incubated at 40℃ and 60℃, and activity was measured at regular intervals. The half-life at 50℃ was approximately 4.5 hours.

[0089] 5. Michaelis constant determination: The Km value of this enzyme for colloidal chitin was determined to be 4.2 mM using the Lineweaver-Burk double reciprocal plot method.

[0090] Example 8: Application of recombinant chitin deacetylase products in the preparation of biopesticides 1. Preparation of highly active chitosan oligosaccharides: The chitosan (DD 93.5%) prepared in Example 5 was used to prepare a low molecular weight chitosan oligosaccharide mixture with an average degree of polymerization of 3-6 by controlling the hydrolysis time of chitosanase (e.g., 2 hours) using the method in Example 6.

[0091] 2. Antibacterial activity assay: The mycelial growth rate method was used. The prepared chitosan oligosaccharide was prepared into a 1 mg / mL aqueous solution. 100 μL was spread onto a PDA plate and inoculated with mycelial cakes of *Rhizoctonia solani* (rice sheath blight) and *Botrytiscinerea* (tomato gray mold), with water as a control. After incubation at 28℃ for 3 days, the diameter of the inhibition zone or the colony growth inhibition rate was measured. The experiment showed that the chitosan oligosaccharide prepared by this method had an inhibition rate of over 70% against all tested pathogens.

[0092] 3. Assay for induced resistance activity: Leaf inoculation was used. Cucumber seedlings were sprayed with a 0.5 mg / mL chitosan oligosaccharide solution, and inoculated with a suspension of cucumber downy mildew spores 24 hours later. Spraying with water served as a control. Disease incidence was observed after 5 days. The results showed that the leaf lesion area in the treatment group was significantly reduced (control efficacy >60%), and the activities of peroxidase (POD) and phenylalanine ammonia-lyase (PAL) in the leaves were significantly higher than those in the control group, indicating successful induction of systemic resistance.

[0093] 4. Preparation of biological pesticide formulations: The above-mentioned highly active chitosan oligosaccharide (active ingredient) is mixed with dispersant (such as sodium lignosulfonate), humectant (such as alkyl glycoside), stabilizer (such as xanthan gum) and water in a certain proportion, and then ground in a sand mill to prepare a 10% chitosan oligosaccharide soluble concentrate (SL).

[0094] Example 9: Application of recombinase in the treatment of insect pupal skin 1. Take 10 g of dried silkworm pupa skin, crush it and pass it through a 40-mesh sieve.

[0095] 2. Add 100 mL of pH 7.5 phosphate buffer, and add purified recombinant chitin deacetase to a final concentration of 5 U / mL.

[0096] 3. React in a 45℃ water bath shaker for 12 hours.

[0097] 4. After the reaction is complete, filter and collect the treated pupal skin residue, and wash it with water until neutral.

[0098] 5. Place the treated pupal skin residue in a 1% (v / v) acetic acid solution and observe the dissolution.

[0099] The results showed that the pupal skin treated with the enzyme could be completely dissolved in acetic acid, while the untreated control group was basically insoluble, proving that the enzyme treatment successfully removed the chitin acetyl groups in the pupal skin, making it acid-soluble.

Claims

1. A nucleic acid molecule encoding a chitin deacetylase, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 1; the nucleic acid molecule is a synthetic gene that has been codon-optimized to adapt to the Pichia pastoris expression system, and its sequence is shown in SEQ ID NO:

3.

2. A recombinant expression vector, characterized in that, The vector is a pGAPZa A-derived vector, comprising the nucleic acid molecule of claim 1, and an expression regulatory element operatively linked to the nucleic acid molecule; wherein the nucleic acid molecule is inserted via ECOR I and Not I restriction sites, and a sequence encoding a 6×histidine tag is fused to the C-terminus of the coding sequence.

3. A recombinant Pichia pastoris engineered strain, characterized in that, The engineered strain was obtained by transforming the recombinant expression vector of claim 2 into Pichia pastoris host cells; the host cells were Pichia pastoris protease-deficient strain SMD1168.

4. A recombinant chitin deacetylase, characterized in that, The recombinant enzyme is encoded by the nucleic acid molecule described in claim 1 or produced by the recombinant Pichia pastoris engineered strain described in claim 3; the amino acid sequence of the recombinant enzyme is shown in SEQ ID NO: 2, and the specific activity of the recombinant enzyme against colloidal chitin is not less than 50 U / mg under the conditions of pH 7.5 and 55℃.

5. A method for producing chitosan, characterized in that, The method involves mixing the recombinant chitin deacetase described in claim 4 with a chitin substrate in a buffer solution, reacting at 40-60°C and pH 7.0-8.5 for 2-24 hours, then terminating the reaction and separating and purifying the chitosan; wherein the chitin substrate is colloidal chitin with a concentration of 1-5% (w / v).

6. A method for producing N-acetylglucosamine monomer or oligosaccharide thereof, characterized in that, include: S1. Chitosan or partially deacetylated chitin is obtained by the method described in claim 5; S2. Hydrolyze the product of step S1 using chitinase or chitosanase. S3. Separate and purify to obtain N-acetylglucosamine monomer or its oligosaccharide.

7. The application of the recombinant chitin deacetase as described in claim 4 in the treatment of chitin-containing biomass materials, characterized in that, The chitin-containing biomass material is insect-derived chitin material; the insect-derived chitin material is insect pupa skin or insect remains.

8. A method for applying a biological pesticide, characterized in that, The method for using chitosan or N-acetylglucosamine monomer or oligosaccharide prepared by the method of claim 5 or 6 in the preparation of biopesticides, wherein the biopesticides are used to control fungal diseases, bacterial diseases, and viral diseases of plants, and to induce systemic resistance in plants.

9. A biological pesticide composition, characterized in that, It contains an effective dose of chitosan or N-acetylglucosamine monomer or oligosaccharide thereof produced by the method of claim 5 or 6 as an active ingredient, as well as agriculturally acceptable carriers and adjuvants.

10. The biopesticide composition according to claim 9, characterized in that, The composition is in the form of a soluble concentrate, a water-dispersible granule, a pellet, or a seed coating agent.

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