Screening and application of high-yield chitosanase strain

Abstract

The invention discloses screening and application of a high-yield chitosanase strain, and belongs to the field of microbial engineering and enzyme preparations. According to the invention, a strain of bacillus cereus F3 is obtained, the enzyme activity of the strain reaches 23.40 U / mL by optimizing a fermentation process, and the fermentation enzyme production time is only 36 hours; besides, it is found that the bacillus cereus F3 is a non-inducible cell-producing chitosanase strain, colloidal chitosan and powdery chitosan do not need to be added as inducers in the culture process, and the enzyme extraction process can be simplified in industrial production. The compound preparation of bacillus cereus F3 and chitosan oligosaccharide can promote corn growth under salt stress and drought stress, has more obvious growth promoting effect on plant height and stem diameter, and can improve the chlorophyll content and antioxidant enzyme activity of corn and enhance the stress resistance of crops.

Description

Technical Field

[0001] This invention relates to the screening and application of a high-yield chitosanase strain, belonging to the field of microbial engineering and enzyme preparations. Background Technology

[0002] Chitosan is a natural alkaline polysaccharide obtained by deacetylation of chitin, possessing advantages such as good biocompatibility, biodegradability, and wide availability. However, its high molecular weight, high crystallinity, and poor water solubility significantly limit its direct utilization in agriculture, food, and medicine. Chitosanase (EC 3.2.1.132) can specifically cleave the β-1,4-glycosidic bonds of chitosan, converting it into chitooligosaccharides (COS) with a degree of polymerization of 2-8. Compared to chitosan, chitooligosaccharides possess advantages such as good water solubility, low viscosity, small molecular weight, and high cell membrane permeability. They can rapidly activate plant immunity, promote root development, regulate soil microecology, and significantly alleviate the inhibitory effects of abiotic stresses such as salt, drought, and low temperature on crop growth, thus showing great commercial potential in the field of green agricultural inputs.

[0003] Currently, the large-scale production of chitosan oligosaccharides mainly employs three technical routes: physical methods (microwave, ultrasound, gamma irradiation), chemical methods (acid hydrolysis, oxidation), and enzymatic hydrolysis. Among these, physical methods are energy-intensive and have uncontrollable product polymerization degrees; chemical methods require strong acids / bases, produce numerous byproducts, involve cumbersome post-processing, and easily generate environmentally harmful waste liquids. In contrast, enzymatic hydrolysis offers mild reaction conditions (pH 5-7, temperature 30-50℃), strong substrate specificity, high product purity, and no toxic byproducts, aligning with green manufacturing principles and becoming the preferred strategy for industrial chitosan oligosaccharide production. Nevertheless, existing chitosanase-producing strains still face challenges such as low enzyme activity, poor thermal stability and acid / alkali tolerance, long fermentation cycles, and short shelf life of liquid enzyme preparations. These shortcomings severely restrict the large-scale promotion of chitosanase and its derived chitosan oligosaccharides in the agricultural field.

[0004] More importantly, current research focuses primarily on the growth-promoting effects of chitosan oligosaccharide as a single active ingredient, while exploration of its potential synergistic growth-promoting mechanisms with enzyme-producing strains in agricultural systems and its ability to enhance overall stress resistance is still insufficient, making it difficult to meet the actual needs of modern agriculture for efficient and multifunctional biological agents.

[0005] Therefore, in order to address the problems of low enzyme activity, poor stability and limited application modes in the existing enzymatic preparation of chitosan oligosaccharides, it is urgent to screen for efficient and stable chitosanase-producing strains and systematically develop their comprehensive applications in chitosan oligosaccharide preparation and synergistic effects with strains, thereby promoting the development of chitosanase and chitosan oligosaccharides in agriculture and other related fields. Summary of the Invention

[0006] The first objective of this invention is to provide a Bacillus cereus ( Bacillus cereus F3, taxonomically named Bacillus cereus F3 was deposited at the China Center for Type Culture Collection on October 7, 2025, with accession number CCTCC NO:M 20252180.

[0007] The present invention provides Bacillus cereus that produces chitosanase and promotes crop growth. Bacillus cereus F3 strain has the following characteristics: (1) Colony characteristics: milky white, rough surface, irregular edges; (2) Morphological characteristics: Scanning electron microscopy results showed that the strain was Corynebacterium; (3) Growth characteristics: The strain enters a stable period after 70 hours and enters a decline period after 90 hours; (4) Physiological and biochemical characteristics: Gram staining result is blue-purple, indicating that it is a Gram-positive bacterium; (5) The Bacillus cereus of the present invention ( Bacillus cereus F3 combined with chitosan promotes plant growth: A second objective of this invention is to provide a microbial inoculant containing Bacillus cereus (B. cereus) of this invention. Bacillus cereus )F3.

[0008] In one embodiment of the present invention, the microbial agent is Bacillus cereus (Bacillus cereus). Bacillus cereus F3 is the main microorganism.

[0009] In one embodiment of the present invention, the bacterial agent contains Bacillus cereus (Bacillus cereus). Bacillus cereus The viable count of F3 is 10. 7 ~10 9 CFU / mL.

[0010] In one embodiment of the present invention, the compound microbial fertilizer contains Bacillus cereus (Bacillus cereus). Bacillus cereus The bacterial concentration range of F3 is 10. 7 ~10 9 CFU / mL.

[0011] In one embodiment of the present invention, the bacterial agent is prepared from Bacillus cereus (…). Bacillus cereus The seed culture of F3 was then expanded.

[0012] In one embodiment of the present invention, the bacterial agent contains Bacillus cereus of the present invention (…). Bacillus cereusThe present invention's Bacillus cereus, obtained by freeze-drying live cells of strain F3, is derived from this invention. Bacillus cereus The dried mycelium of strain F3, and the immobilized Bacillus cereus of the present invention ( Bacillus cereus F3 strain cells, Bacillus cereus of the present invention ( Bacillus cereus F3 strain fermentation broth or its lysate, or containing Bacillus cereus ( Bacillus cereus Liquid bacterial agent of strain F3, Bacillus cereus of the present invention ( Bacillus cereus Solid inoculum of strain F3, or Bacillus cereus of the present invention existing in any other form. Bacillus cereus )F3 strain.

[0013] In one embodiment of the present invention, the microbial agent also contains other excipients.

[0014] A third objective of this invention is to provide a compound microbial fertilizer, wherein the compound microbial fertilizer comprises the Bacillus cereus ( Bacillus cereus F3 or the aforementioned microbial agent.

[0015] In one embodiment of the present invention, the compound microbial fertilizer contains chitosan oligosaccharide.

[0016] In one embodiment of the present invention, the compound microbial fertilizer contains Bacillus cereus (Bacillus cereus). Bacillus cereus The viable count of F3 is 10. 7 ~10 9 CFU / mL.

[0017] In one embodiment of the present invention, the compound microbial fertilizer contains Bacillus cereus (Bacillus cereus). Bacillus cereus The bacterial concentration range of F3 is 10. 7 ~10 9 CFU / mL.

[0018] In one embodiment of the present invention, the concentration of chitosan oligosaccharide in the compound microbial fertilizer is 0.1% to 2.0%.

[0019] The fourth objective of this invention is to provide a method for promoting crop growth, wherein the method involves applying the aforementioned compound microbial fertilizer, or the aforementioned Bacillus cereus (…) during the crop growth process. Bacillus cereus F3, or the above-mentioned microbial agents.

[0020] The fifth object of the present invention is to provide a method for preparing chitosanase, wherein the method comprises using the aforementioned Bacillus cereus ( Bacillus cereus F3 or the microbial agent is inoculated into the seed culture medium, and the resulting seed liquid is inoculated into the fermentation culture medium for fermentation to produce a fermentation broth containing chitosanase.

[0021] In one embodiment of the present invention, the carbon source of the fermentation medium is one or more of glucose, sucrose, lactose, galactose, sodium carboxymethyl cellulose, mannose, fructose, mannitol, 1% colloidal chitosan, maltodextrin, soluble starch, and corn starch.

[0022] Preferably, the carbon source of the fermentation medium is sucrose and / or soluble starch.

[0023] In one embodiment of the present invention, the nitrogen source of the fermentation medium is one or more of the following: yeast extract, beef extract, peptone, tryptone ammonium sulfate, urea, fish peptone, cottonseed meal powder, corn steep liquor powder, peanut meal powder, and soybean meal powder.

[0024] Preferably, the nitrogen source of the fermentation medium is beef extract and / or yeast extract.

[0025] In one embodiment of the present invention, no other trace elements are added to the fermentation culture medium.

[0026] In one embodiment of the present invention, the fermentation culture medium uses water as a solvent and comprises the following components by mass percentage: 0.5%~1% soluble starch, 0.1%~0.5% yeast extract, and 1%~2% beef extract.

[0027] In one embodiment of the present invention, the seed culture medium uses water as a solvent and comprises the following components by mass percentage: 1%~2% tryptone, 1%~2% yeast extract, and 0.5%~1% sodium chloride.

[0028] In one embodiment of the present invention, the pH of the fermentation medium is 5.5 to 6.0.

[0029] In one embodiment of the present invention, the seed liquid is inoculated into the fermentation medium at an inoculation rate of 2-4%.

[0030] In one embodiment of the present invention, the fermentation production conditions are as follows: fermentation at a temperature of 27~37℃ and a rotation speed of 220~250 r / min for 36 h.

[0031] In one embodiment of the present invention, chitosanase is obtained by separating and purifying the fermentation broth.

[0032] In one embodiment of the present invention, the separation and purification includes ammonium sulfate precipitation, anion exchange chromatography, and dialysis concentration.

[0033] A sixth object of the present invention is to provide a chitosanase prepared by the method described above.

[0034] In one embodiment of the present invention, the chitosanase has a molecular weight of approximately 50 kDa; an optimal reaction temperature of 70°C and an optimal reaction pH of 7.0; belongs to the GH8 family of glycoside hydrolases; and has an enzyme activity retention rate of ≥80% after incubation at below 30°C for 2 h.

[0035] A seventh object of the present invention is to provide a chitosanase preparation comprising the chitosanase, as well as at least one stabilizer and at least one preservative.

[0036] In one embodiment of the present invention, the stabilizer comprises 0.1–0.5 g / L gelatin, 10–50 mmol / L NaCl, and 10–50 g / L glycerol; the preservative is 0.1%–0.2% potassium sorbate.

[0037] The eighth object of the present invention is to provide the aforementioned Bacillus cereus ( Bacillus cereus F3, or the microbial agent, or the chitosanase, in the preparation of chitosan oligosaccharides or products containing chitosan oligosaccharides.

[0038] The ninth object of the present invention is to provide the aforementioned Bacillus cereus ( Bacillus cereus F3, or the microbial agent, or the application of the chitosanase in the preparation of fertilizer.

[0039] The tenth object of the present invention is to provide the aforementioned Bacillus cereus ( Bacillus cereus The application of F3, or the microbial agent, or the microbial fertilizer, or the chitosanase in promoting crop growth and enhancing plant resistance to stress or drought stress.

[0040] Beneficial effects 1. This invention obtains a strain of Bacillus cereus from samples such as soil containing crustacean waste and sludge from shrimp and crab farms through primary screening using the plate clear zone method and secondary screening using the DNS method to measure enzyme activity. Bacillus cereus F3. This strain initially had an enzyme activity of 10.01 U / mL, and the fermentation time to produce enzyme was only 36 h, shortening the production cycle. Furthermore, it was found that Bacillus cereus (… Bacillus cereus F3 is a non-inducible chitosan-producing strain. During cultivation, there is no need to add colloidal chitosan or powdered chitosan as an inducer, which can simplify the enzyme extraction process in industrial production.

[0041] 2. This invention optimizes the fermentation process using single-factor experiments and response surface methodology. Under optimized fermentation conditions, the enzyme activity of this strain reached 20.22 U / mL, which is 2.02 times the initial enzyme activity. The optimal culture medium composition was 0.9% soluble starch, 2.0% beef extract, and 0.5% yeast extract, which further increased the enzyme activity to 23.40 U / mL, 2.34 times the initial enzyme activity. After sequential purification by 80% ammonium sulfate precipitation, dialysis, and Capto Q strong anion exchange chromatography, the enzyme activity reached a maximum of 425.54 U / mL. 3. The chitosanase provided by this invention belongs to the GH 8 family, has a molecular weight of about 50 kDa, an optimal reaction temperature of 70℃, an optimal pH of 7.0, good stability under low temperature and neutral to weakly alkaline conditions, and strong specificity for the degradation of colloidal chitosan.

[0042] 4. Using 0.30 g / L gelatin, 10 mmol / L NaCl, and 30 g / L glycerol as stabilizers, and 0.15% potassium sorbate as a preservative, the prepared enzyme preparation still maintained 84.74% enzyme activity after being refrigerated at 4℃ for 30 days, demonstrating excellent stability.

[0043] 5. Bacillus cereus ( Bacillus cereus The compound formulation of F3 and chitosan oligosaccharide can promote maize growth under salt and drought stress, with a more significant effect on promoting plant height and stem diameter. At the same time, it can increase the chlorophyll content and antioxidant enzyme activity of maize leaves, and enhance the crop's stress resistance.

[0044] Preservation of biological materials: Bacillus cereus ( Bacillus cereus F3, taxonomically named Bacillus cereus F3 strain was deposited on October 7, 2025 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20252180, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Attached Figure Description

[0045] Figure 1 This is the result of enzyme activity assay for 14 strains screened again; Figure 2 Morphological characteristics and taxonomic identification of strain F3: scanning electron microscopy and Gram staining (A), phylogenetic tree (B); Figure 3 The effects of different fermentation conditions on enzyme production by the strain are: inoculum size (A); temperature (B); pH (C); and rotation speed (D). Figure 4 The effects of different fermentation medium components on enzyme production by the strain: carbon source (A); nitrogen source ( Figure 4 B), inorganic salts ( Figure 4 C); Figure 5The response surface methodology for fermentation media includes: sucrose (A), soluble starch (B), yeast extract (C), beef extract (D), and dipotassium hydrogen phosphate (E). Figure 6 The optimal reaction temperature and thermal stability of chitosanase: enzyme activity (A); relative enzyme activity (B); Figure 7 The optimal reaction pH and pH stability of chitosanase: enzyme activity (A); relative enzyme activity (B); Figure 8 The effect of metal ions on chitosanase activity: 2 mmol / L of different metal ions (A), 5 mmol / L of different metal ions (B). Figure 9 The effect of surfactants on chitosanase activity: 2 mmol / L different surfactants (A), 5 mmol / L different surfactants (B); Figure 10 This involves the investigation of chitosanase-specific substrates; Figure 11 The effects of different treatments on plant growth under salt stress were: 50 mmol / L NaCl (A); 150 mmol / L NaCl (B); 250 mmol / L NaCl (C). Figure 12 The effects of different treatments on plant growth under drought stress were observed: 5% PEG 6000 (A); 10% PEG 6000 (B). Figure 13 The effects of different treatments on maize: plant height (A); stem diameter (B); Figure 14 The effects of different treatments on maize are as follows: chlorophyll content (A); superoxide dismutase activity (B); peroxidase activity (C); ascorbate peroxidase activity (D); glutathione peroxidase activity (E). Detailed Implementation

[0046] This invention is not limited to the embodiments described herein; the embodiments are merely illustrative and not intended to limit the scope of protection of this invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and essence of this invention are within the scope of this invention.

[0047] The culture media involved in the following examples are as follows: Enrichment medium: 1% chitosan, 0.5% yeast extract, 0.3% beef extract, pH 7.0, with water as the solvent.

[0048] Screening plates: 1% chitosan, 0.5% yeast extract, 0.3% beef extract, 2% agar, pH 7.0, with water as the solvent.

[0049] LB liquid medium: 1% tryptone, 1% yeast extract, 0.5% sodium chloride, with water as the solvent; LB solid medium: 1% tryptone, 1% yeast extract, 0.5% sodium chloride, 1.5% agar, with water as the solvent.

[0050] The test methods involved in the following embodiments are as follows: Chitosanase activity was determined using the DNS method: the 3,5-dinitrosalicylic acid (DNS) method was employed, and a standard curve was plotted using a 1 mg / mL N-acetylglucosamine standard solution. 400 µL of crude enzyme solution was placed in 2 mL EP tubes as the experimental and control groups, respectively. The control group was inactivated by boiling in a water bath for 10 min. Then, 400 µL of 10 g / L colloidal chitosan was added to each tube, and the mixture was reacted at 50℃ for 15 min. 200 µL of the reaction solution was mixed with 300 µL of DNS, boiled in a water bath for 10 min, and centrifuged at 12000 r / min for 3 min. 200 µL of the mixture was then measured at 540 nm, and the chitosan oligosaccharide content was calculated using the standard curve formula.

[0051] Chitosanase activity is defined as the amount of enzyme required to produce 1 µmol of reducing sugar in 1 min of 1 mL of crude enzyme solution under the above reaction conditions. This amount is defined as 1 unit of enzyme activity (U / mL).

[0052] The raw materials used in the following examples: In the following examples, unless otherwise specified, all solutions mentioned use water as the solvent.

[0053] The chlorophyll content detection kit, superoxide dismutase detection kit, peroxidase detection kit, ascorbic acid peroxidase detection kit, and glutathione peroxidase detection kit involved in the following examples were all purchased from Beijing Solarbio Science & Technology Co., Ltd., and chitosan was purchased from Xi'an Heshun Biotechnology Co., Ltd., and are commercially available products.

[0054] Bacillus cereus LY was purchased from Beijing Baocang Biotechnology Co., Ltd.

[0055] Example 1: Screening and Identification of Chitosanase-Producing Strains 1. Sources of chitosanase-producing strains (1) Sample collection: Strains were screened and isolated from soil containing crustacean waste (sample 1), silt collected from shrimp and crab farms in Urumqi, Xinjiang (sample 2), and sediment from Bosten Lake (sample 3).

[0056] (2) Initial screening: Take 10 g of each sample in a clean bench and mix them with 90 mL of sterile water containing glass beads. Place the mixture in a sterile Erlenmeyer flask and shake at 37℃ and 200 r / min for 3 h. After shaking, allow the mixture to stand until it separates into layers. Take 1 mL of the supernatant and inoculate it into 50 mL of enrichment medium. Incubate at 37℃ and 180 r / min for 3 days. Dilute the culture with sterile water in a serial dilution to 10⁻⁶. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 100 μL of different concentrations of dilution solution were spread on screening plates and incubated upside down at 37℃ for 48 h. Colonies that produced clear zones were picked from the screening plates and isolated and purified one by one by streaking on LB solid medium. 26 strains were obtained from the preliminary screening and preserved on slant medium.

[0057] (3) Secondary screening: The 26 strains obtained from the initial screening were inoculated into LB liquid medium and cultured at 37℃ and 180 r / min for 24 h. 10 μL of the activation solution was spread onto the screening plate and cultured upside down at 37℃ for 2 days. The diameter of the transparent zone (D) and the diameter of the colony (d) of the strains were measured respectively, and the diameter ratio (D / d) was calculated. The diameter ratio can reflect the enzyme production capacity of the strain. Strains with a D / d ratio ≥1.8 were selected to obtain strains HP-2, CY-C, F2 and F3.

[0058] The selected strains were inoculated into Erlenmeyer flasks containing 50 mL of LB liquid medium and cultured at 37℃ and 180 r / min for 12 h. The bacterial culture was then collected and inoculated into 50 mL of fermentation medium at an inoculation rate of 2% (v / v). After culturing at 37℃ and 180 r / min for 3 days, the cultured fermentation broth was obtained. The broth was centrifuged at 4℃ and 12000 r / min for 10 min, and the supernatant was collected. The enzyme activity was measured using the DNS method to determine the target strain F3 with the highest enzyme activity.

[0059] Depend on Figure 1 It can be seen that after secondary screening of the 26 bacterial strains obtained from the initial screening, 14 strains with high enzyme production were obtained. Among them, HP-1, ST-4, ST-5, F1, F2, and F3 had similar enzyme production capabilities to Bacillus cereus LY. F3 had the highest enzyme activity, at 10.01 U / mL, and F3 was finally selected as the target strain.

[0060] 2. Cultivation characteristics and morphological features The purified F3 strain was preserved on LB solid agar slant medium. Strains from the slant were picked and spread onto a glass slide for Gram staining. The colony morphology was as follows: Figure 2As shown, the colonies are white, opaque, round or nearly round, with intact edges and a slightly raised surface. Gram staining of the smear revealed the following bacterial morphology: Figure 2 As shown, the bacterium is Gram-positive. The SEM (scanning electron microscope) image is shown below. Figure 2 As shown, the strains are rod-shaped with straight ends and are either short or long chains.

[0061] 3. Growth characteristics of F3 strain The F3 strain has a production temperature range of 22℃~42℃, an optimal growth temperature of 32℃, a growth pH range of 4.5~9.0, and an optimal growth pH of 5.5; it is a facultative anaerobic bacterium.

[0062] Example 2: Classification and identification of 16S rDNA from strain F3 Molecular biological identification was performed on the F3 strain of Example 1. Its 16S rDNA gene was amplified by PCR, and sequencing was performed after passing electrophoresis. The primers used for PCR are as follows: F: 5'-AGAGTTTGATCMTGGCTCAG-3' (27F); R: 5'-GGTTACCTTGTTACGACTT-3' (1492R).

[0063] Sequencing results show the sequence as shown in SEQ ID NO.1. Figure 2 B. The 16S rDNA sequence of strain F3 was submitted to the NCBI database. Homology was compared using BLAST, and it was found to be similar to that of strain B. Bacillus cereus C23 showed the highest similarity, reaching 99%, indicating that this strain is Bacillus cereus. Bacillus cereus ).

[0064] The Bacillus cereus of the present invention ( Bacillus cereus F3, taxonomically named Bacillus cereus F3 strain was deposited on October 7, 2025 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20252180, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0065] Bacillus cereus The 16S rDNA sequence of F3 is shown below (SEQ ID NO.1):

[0066] Example 3: Bacillus cereus ( Bacillus cereus Fermentation conditions for strain F3 1. Enzyme production process before optimization Activation medium (LB liquid medium): 1% tryptone, 1% yeast extract, 0.5% sodium chloride, with water as the solvent; Fermentation medium (pH 7.0): 1% chitosan, 0.5% yeast extract, 0.3% beef extract, with water as the solvent.

[0067] Pick out Bacillus cereus ( Bacillus cereus A single colony of F3 was inoculated into an Erlenmeyer flask containing 50 mL of activation medium. After activation at 37°C and 180 r / min for 12 h, the bacterial culture was collected and inoculated into 50 mL of fermentation medium (pH 7.0) at a seed culture rate of 2% (v / v). After incubation at 37°C and 180 r / min for 3 days, the cultured fermentation broth was obtained. After centrifugation at 4°C and 12000 r / min for 10 min, the supernatant was collected and the enzyme activity was measured using the DNS method. The enzyme activity was 10.01 U / mL.

[0068] 2. Optimization of enzyme production process 2.1 Single-factor experiment: The effects of different fermentation conditions on enzyme production by strain F3 were investigated, including seed culture inoculum size (1%, 2%, 3%, 4%, 5%, 6%, v / v), fermentation temperature (17℃, 22℃, 32℃, 37℃, 42℃, 47℃), fermentation pH (4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 9.0), fermentation speed (140 r / min, 160 r / min, 180 r / min, 200 r / min, 220 r / min, 240 r / min, 260 r / min, 280 r / min), and the effects of carbon source, nitrogen source, and inorganic salts in the fermentation medium on enzyme production by strain F3. The optimal values ​​for each factor were determined.

[0069] 2.1.1 Single-factor experiments on fermentation conditions Bacillus cereus ( Bacillus cereus After the F3-activated seed culture was inoculated into 20 mL of fermentation medium (pH 7.0) at an inoculation rate of 1%~6% (v / v), it was cultured at 37℃ and 180 r / min for 24 h to obtain the cultured fermentation broth. After centrifugation at 4℃ and 12000 r / min for 10 min, the supernatant was collected and the enzyme activity was measured by the DNS method.

[0070] Bacillus cereus ( Bacillus cereusAfter the F3-activated seed culture was inoculated into 20 mL of fermentation medium with pH 4.5-9.0 at an inoculation rate of 2% (v / v), it was cultured at 37℃ and 180 r / min for 24 h to obtain the cultured fermentation broth. After centrifugation at 4℃ and 12000 r / min for 10 min, the supernatant was collected and the enzyme activity was measured by the DNS method.

[0071] Bacillus cereus ( Bacillus cereus After the F3-activated seed culture was inoculated into 20 mL of fermentation medium (pH 7.0) at an inoculation rate of 2% (v / v), it was cultured at 17℃~47℃ and 180 r / min for 24 h to obtain the cultured fermentation broth. After centrifugation at 4℃ and 12000 r / min for 10 min, the supernatant was collected and the enzyme activity was measured by the DNS method.

[0072] Bacillus cereus ( Bacillus cereus After the F3-activated seed culture was inoculated into 20 mL of fermentation medium (pH 7.0) at an inoculation rate of 2% (v / v), it was cultured at 37℃ and 140 r / min to 280 r / min for 24 h to obtain the cultured fermentation broth. After centrifugation at 4℃ and 12000 r / min for 10 min, the supernatant was collected and the enzyme activity was measured by the DNS method.

[0073] The results are as follows Figure 3 As shown, the inoculum size for the strain ( Figure 3 A) Temperature ( Figure 3 B), pH ( Figure 3 C) Rotation speed ( Figure 3 D) Single-factor experiments were conducted. Enzyme activity could reach a high level within the range of inoculum size of 3-4% (v / v), temperature of 27-37℃, pH of 5.5-6.0, and rotation speed of 220-240 r / min. The optimal values ​​were inoculum size of 3%, temperature of 32℃, pH of 5.5, and rotation speed of 240 r / min.

[0074] 2.1.2 Single-factor experiments on fermentation medium The carbon sources in the fermentation medium were set as glucose, sucrose, lactose, galactose, sodium carboxymethyl cellulose, mannose, fructose, mannitol, 1% colloidal chitosan, maltodextrin, soluble starch, and corn starch, with a mass percentage of 1% in the fermentation medium. The nitrogen source was fixed at 0.5% yeast extract. The effects of different carbon sources on Bacillus cereus (…) were investigated. Bacillus cereus The effect of F3 fermentation on enzyme production.

[0075] The nitrogen sources in the fermentation medium were set as yeast extract, beef extract, peptone, tryptone, ammonium sulfate, urea, fish peptone, cottonseed meal, corn steep liquor powder, peanut meal, and soybean meal, with a nitrogen source mass percentage of 1% in the fermentation medium; the carbon source was fixed at 0.5% sucrose. The effects of different nitrogen sources on Bacillus cereus (…) were investigated. Bacillus cereus The effect of F3 fermentation on enzyme production.

[0076] Inorganic salts potassium dihydrogen phosphate, dipotassium hydrogen phosphate, copper sulfate, magnesium sulfate heptahydrate, sodium chloride, potassium chloride, and ferrous sulfate were added to the fermentation medium, with the inorganic salts accounting for 0.20% of the total mass. The nitrogen source was fixed at 0.5% yeast extract, and the carbon source at 0.5% sucrose. The effects of different inorganic salts on *Bacillus cereus* (…) were investigated. Bacillus cereus The effect of F3 fermentation on enzyme production.

[0077] Figure 4 It showcases different types of carbon sources ( Figure 4 A) Nitrogen source ( Figure 4 B) Inorganic salts ( Figure 4 C) Effects on enzyme production by the strain: The results showed that the optimal carbon sources were sucrose and soluble starch, the optimal nitrogen sources were beef extract and yeast extract, and the optimal inorganic salt was dipotassium hydrogen phosphate. Mannose and fructose could also be used as carbon sources, but considering cost, they were not preferred options.

[0078] Figure 5 It shows different concentrations of sucrose ( Figure 5 A) Soluble starch ( Figure 5 B) Yeast extract ( Figure 5 C) Beef extract ( Figure 5 D), dipotassium hydrogen phosphate ( Figure 5 The effect of E) on enzyme production by the strain was investigated. Results showed that when sucrose concentration was 1.0%, soluble starch concentration was 1.0%, yeast extract concentration was 1.0%, beef extract concentration was 2.0%, and dipotassium hydrogen phosphate concentration was 0.2%, *Bacillus cereus* (…)… Bacillus cereus F3 fermentation produces the best enzymes.

[0079] 2.2 Response Surface Optimization: Based on the single-factor experiments, a three-factor, three-level response surface methodology was designed with temperature (A), pH (B), and rotation speed (C) as independent variables and enzyme activity as the response value to optimize the fermentation conditions; and the composition of the fermentation medium was optimized with soluble starch (A), beef extract (B), and yeast extract (C) as independent variables.

[0080] 2.2.1 Response surface methodology for fermentation conditions Table 2 Response Surface Factors and Levels

[0081] Response surface methodology was used to determine the optimal fermentation conditions: inoculum size 3%, temperature 32.5 ℃, pH 5.6, and rotation speed 242 r / min. The resulting enzyme activity was 20.22 U / mL, which was 2.02 times the initial enzyme activity.

[0082] 2.2.2 Response surface methodology for fermentation media Based on previous results regarding the effects of single C and N sources and inorganic salts on enzyme production in the strain, it was determined that C and N sources have a significant impact on enzyme production. Three C sources and three N sources with high enzyme production were selected to investigate the effects of each component at concentrations of 0.5%, 1.0%, and 1.5% on enzyme production. Four components with high enzyme production capacity—yeast extract, sucrose, soluble starch, and beef extract—were selected and combined in three-by-three combinations to obtain the optimal culture medium combination for enzyme production: soluble starch, beef extract, and yeast extract. The optimal fermentation concentrations of each component within the optimal combination were then investigated using a one-way response surface methodology.

[0083] Table 3 Response Surface Factors and Levels

[0084] Response surface methodology was used to determine the optimal fermentation medium: 0.9% soluble starch, 2% beef extract, and 0.5% yeast extract. The resulting strain had an enzyme activity of 23.40 U / mL, which was 2.34 times the initial enzyme activity.

[0085] Example 4: Isolation, purification, and property determination of chitosanase 1. Bacillus cereus ( Bacillus cereus F3 fermentation enzyme production Activation medium (LB liquid medium): 1% tryptone, 1% yeast extract, 0.5% sodium chloride, with water as the solvent; Fermentation medium (pH 5.6, g / L): 0.9% soluble starch, 0.5% yeast extract, 2.0% beef extract, with water as the solvent.

[0086] Pick out Bacillus cereus ( Bacillus cereus A single colony of F3 was inoculated into an Erlenmeyer flask containing 20 mL of activation medium. After activation at 37°C and 200 r / min for 12 h, the bacterial culture was collected and inoculated into 50 mL of fermentation medium at a seed culture rate of 3% (v / v). After incubation at 32.5°C and 242 r / min for 24 h, the cultured fermentation broth was obtained.

[0087] 2. Isolation and purification of chitosanase 2.1 Ammonium sulfate precipitation: Take Bacillus cereus ( Bacillus cereusThe fermentation broth of F3 was centrifuged at 12000 r / min for 10 min at 4℃. 20 mL of the supernatant was collected, and ammonium sulfate was slowly added at 4℃ to achieve an ammonium sulfate concentration of 80%. After standing overnight at 4℃, the mixture was centrifuged at 10000 r / min at 4℃ for 30 min. The supernatant was discarded, and the precipitate was resuspended in 5 mL of acetate-sodium acetate buffer (pH 5.6, 0.2 mol / L) to obtain the reconstituted crude enzyme solution. The reconstituted crude enzyme solution was placed in a dialysis bag and dialyzed with ultrapure water for 24 h, changing the ultrapure water every 8 h. After dialysis, the dialysate in the dialysis bag was collected, and the enzyme activity was measured to be 50.05 U / mL.

[0088] 2.2 Ion exchange chromatography: The dialysate was loaded onto a Capto Q high-performance anion exchange column (connected to an AKTA pure high-performance chromatography instrument) equilibrated with 0.1 mol / L dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 7.0). Elution was performed with 0.2 mol / L NaCl solution, and the eluent was collected to obtain the enzyme solution. The enzyme activity was measured to be 425.54 U / mL.

[0089] The purified enzyme solution was subjected to SDS-PAGE electrophoresis, and the molecular weight of the enzyme was found to be approximately 50 kDa. Mass spectrometry identification showed that the enzyme belongs to the GH8 family.

[0090] 3. Enzymatic properties analysis of chitosanase: The study investigated the optimal reaction temperature, thermal stability, optimal reaction pH, pH stability, effects of metal ions and surfactants on chitosanase activity, and the specific substrates of chitosanase.

[0091] 3.1 Effect of temperature on chitosanase activity and enzyme stability 3.1.1 Optimal reaction temperature: 0.9 mL of 1% colloidal chitosan was incubated at 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃ for 30 min each. Then, 0.5 mL of the purified enzyme solution from step 2 was added, and the reaction was allowed to proceed for 10 min. The enzyme activity was measured using the DNS assay to investigate the optimal reaction temperature for the enzymatic hydrolysis reaction and to examine the effect of temperature on the activity of chitosanase. The reaction temperature with the highest enzyme activity was taken as the optimal reaction temperature. 3.1.2 Thermal stability: The purified enzyme solution from step 2 was treated at different temperatures (25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃) for 2 h. Then, 0.9 mL of 1% colloidal chitosan and 1 mL of disodium hydrogen phosphate-citric acid buffer (pH 5.6) were added, and the reaction was carried out for 10 min. The enzyme activity was measured by the DNS assay. The enzyme activity of the untreated enzyme solution was used as a control (100%) to investigate the effect of temperature on the stability of chitosanase.

[0092] The results are as follows Figure 6 As shown, the optimal reaction temperature for chitosanase is 70℃, and the enzyme activity retention rate is over 80% after incubation at 30℃ for 2 hours.

[0093] 3.2 Effect of pH on chitosanase activity and enzyme stability Different pH buffer solutions: 0.2 M disodium hydrogen phosphate: Weigh 2.84 g disodium hydrogen phosphate and dissolve in 100 mL of water; 0.1 M citric acid: Weigh 1.92 g citric acid and dissolve in 100 mL of water; pH 4 disodium hydrogen phosphate-citric acid: 4 mL disodium hydrogen phosphate + 96 mL citric acid; pH 5 disodium hydrogen phosphate-citric acid: 23.6 mL disodium hydrogen phosphate + 76.4 mL citric acid; pH 6 disodium hydrogen phosphate-citric acid: 51.1 mL disodium hydrogen phosphate + 48.9 mL citric acid; pH 7 disodium hydrogen phosphate-citric acid: 74.7 mL disodium hydrogen phosphate + 25.3 mL citric acid; pH 8 disodium hydrogen phosphate-citric acid: 89.4 mL disodium hydrogen phosphate + 10.6 mL citric acid 3.2.1 Optimal reaction pH: The purified enzyme solution from step 2 was placed in disodium hydrogen phosphate-citric acid buffer solutions (pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0) containing 1% colloidal chitosan at different pH values. The enzyme activity was measured using the DNS assay at 37°C to investigate the effect of pH on chitosanase activity. The pH value with the highest enzyme activity was taken as the optimal reaction pH. 3.2.2 pH stability: The purified enzyme solution from step 2 was placed in disodium hydrogen phosphate-citric acid buffer solutions (pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0) containing 1% colloidal chitosan at different pH values. After co-existing at 4°C for 2 h, the enzyme activity was measured using the DNS assay method, with the enzyme activity of the untreated enzyme solution serving as a control (100%).

[0094] The results are as follows Figure 7 As shown, the optimal pH for chitosanase is 7.0, and the relative enzyme activity can be maintained above 50% when the pH is between 6.0 and 8.0.

[0095] 3.3 Effects of metal ions on chitosanase activity and enzyme stability Prepare 2 mmol / L and 5 mmol / L solutions of various metal ions (KCl, FeCl3, FeSO4, MgSO4, ZnSO4, MnSO4, CaCl2, CuSO4, CoCl2) at pH 7.0, and add them to the purified chitosanase enzyme solution. Treat at 4℃ for 30 min, and then measure the enzyme activity according to the standard enzyme activity assay method to investigate the effect of metal ions on chitosanase activity.

[0096] The results are as follows Figure 8 As shown, 2 mmol / L Mn 2+ It can increase enzyme activity by 15%~20%, 5 mmol / L Cu 2+ It can increase enzyme activity by 10%~15%, while 2 mmol / L Fe 2+ It can inhibit enzyme activity by 30% to 35%.

[0097] 3.4 Effects of surfactants on chitosanase activity and enzyme stability Prepare 2 mmol / L and 5 mmol / L solutions of various surfactants (DTT, EDTA, β-ME, SDS, CTAB, Tween 20, Tween 80, Triton X-100), pH 7.0, and add them to the purified chitosanase enzyme solution from step 2. Treat at 4℃ for 30 min, and determine enzyme activity using the DNS assay to investigate the effect of surfactants on chitosanase activity.

[0098] The results are as follows Figure 9 As shown, SDS has a significant inhibitory effect on chitosanase activity. Low concentrations of SDS have a strong inhibitory effect on enzyme activity. β-ME has a promoting effect on enzyme activity at low concentrations. The other surfactants have a slight inhibitory effect on enzyme activity at low concentrations. As the concentration increases, the promoting effect weakens and has no significant effect on enzyme activity.

[0099] 3.5 Effects of chitosanase-specific substrates on chitosanase activity and enzyme stability First, prepare 1 mol / L stock solutions of each chitosanase-specific substrate (1% chitin solution, 1% colloidal chitin, 1% chitosan solution, 1% colloidal chitosan, 1% sodium carboxymethyl cellulose solution, and 1% soluble starch solution), with a pH of 7.0. Add these solutions to the chitosanase enzyme solution purified in step 2 and treat at 55℃ for 120 min to investigate the degradation ability of chitosanase on chitosanase-specific substrates.

[0100] The results are as follows Figure 10As shown, chitosanase exhibits the best enzyme activity when using 1% colloidal chitosan as a substrate. It has no degradation effect on soluble starch and sodium carboxymethyl cellulose, but has a slight degradation effect on powdered chitosan and colloidal chitin.

[0101] Example 5: Preparation of enzyme preparations 1. Enzyme preparation The purified chitosanase solution from Example 4 was mixed with stabilizer (0.30 g / L gelatin, 10 mmol / L NaCl, 30 g / L glycerol) and preservative (0.15% potassium sorbate) in a mass ratio of 1000:0.3:0.58:30:1.5 and stirred until homogeneous to obtain the chitosanase preparation.

[0102] 2. Stability determination The enzyme preparation was refrigerated at 4°C, and samples were taken on days 1, 5, 10, 20, and 30. Enzyme activity was measured using the DNS method.

[0103] The results showed that the enzyme activity retention rate was 96.48% on day 5, 93.60% on day 10, 89.29% on day 20, and 84.74% on day 30, indicating that the enzyme preparation had good stability.

[0104] Example 6: Bacillus cereus ( Bacillus cereus Application of F3 in the preparation of microbial fertilizer 1. Preparation of microbial fertilizer: Activation medium (LB liquid medium): 1% tryptone, 1% yeast extract, 0.5% sodium chloride, with water as the solvent; Fermentation medium (pH 5.6, g / L): 0.9% soluble starch, 0.5% yeast extract, 2.0% beef extract, with water as the solvent.

[0105] Pick out Bacillus cereus ( Bacillus cereus A single colony of F3 was inoculated into an Erlenmeyer flask containing 20 mL of activation medium. After activation at 37°C and 200 r / min for 12 h, the bacterial culture was collected and inoculated into 50 mL of fermentation medium at a rate of 3% (v / v) as a seed culture. After incubation at 32.5°C and 242 r / min for 24 h, the bacterial culture was obtained.

[0106] The bacterial solution was mixed with chitosan oligosaccharide to prepare bacterial fertilizer. The OD value of the bacterial solution was around 0.9, and the chitosan oligosaccharide was obtained by enzymatic hydrolysis of 1% colloidal chitosan by chitosanase for 2 hours.

[0107] 2. Coercion Management: After disinfecting the corn seeds, they were sown and transplanted to pots containing sterilized nutrient soil and vermiculite (3:1) when they reached the three-leaf stage. Pots were prepared to simulate salt stress with 50 mmol / mL NaCl, 150 mmol / mL NaCl, and 250 mmol / mL NaCl, and pots were prepared to simulate drought stress with 5% PEG6000 and 10% PEG6000. Each simulated stress treatment included water (deionized water, control group), stress treatment only (CK), stress treatment + chitosan oligosaccharide treatment (CK+G), and stress treatment + microbial fertilizer (CK+G+J). Among them, chitosan oligosaccharide was obtained by enzymatic hydrolysis of 1% colloidal chitosan with chitosanase for 2 h, and the concentration was 0.1% to 2.0%.

[0108] 3. Index Measurement: After 30 days of treatment, the plant height and stem diameter were measured using a ruler and vernier calipers. Physiological indicators of the maize plants, such as chlorophyll content, superoxide dismutase, peroxidase, ascorbate peroxidase, and glutathione peroxidase, were measured using a kit.

[0109] Depend on Figures 11 - 13 It was found that under salt and drought stress, both single treatment with chitosan oligosaccharide and mixed treatment with chitosan oligosaccharide and bacterial solution increased plant height and stem diameter. The increased stem diameter indirectly enhanced the plant's resistance to lodging. Treatment with 10% PEG6000 inhibited the growth of Bacillus cereus (…). Bacillus cereus The growth of F3 reduced the amount of Bacillus cereus in the soil. Bacillus cereus The content of F3 was reduced, thus weakening the synergistic effect between chitosan oligosaccharide and the strain. Under 250 mmol / L NaCl salt stress, the plant height of the CK+G group and the CK+G+J group increased by 20.85% and 22.32%, respectively, and the stem diameter increased by 29.14% and 41.72%, respectively. Under 5% PEG6000 drought stress, after applying single treatment and mixed treatment, the plant height of the CK+G group and the CK+G+J group increased by 41.87% and 42.26%, respectively, and the stem diameter increased by 1.36% and 15.45%, respectively, compared with the CK group.

[0110] Depend on Figure 14 It can be seen that under salt and drought stress, both single treatment with chitosan oligosaccharide and mixed treatment with chitosan oligosaccharide and bacterial solution increased the chlorophyll content, superoxide dismutase and superoxide dismutase activities of plants. However, under 10% PEG6000 treatment, the growth-promoting effect of the mixed treatment was weaker than that of single chitosan oligosaccharide treatment, indicating that Bacillus cereus ( Bacillus cereus F3 cells, under conditions of inhibited growth, showed no promoting effect on chlorophyll, superoxide dismutase, or superoxide mirabilite. However, lower concentrations of Bacillus cereus (…) Bacillus cereusF3 combined with chitosan oligosaccharide significantly promoted the activity of ascorbic acid peroxidase and glutathione peroxidase under drought conditions, while low levels of Bacillus cereus ( Bacillus cereus F3 can still synergistically enhance the drought resistance of maize plants with chitosan oligosaccharide. Regarding the total chlorophyll content in maize plants, chitosan oligosaccharide showed a significant promoting effect under 250 mmol / L NaCl salt stress, and under drought stress, the promoting effect of chitosan oligosaccharide was even better under 5% PEG6000 stress. For superoxide dismutase (SOD), the application of chitosan oligosaccharide and the mixture had a significant promoting effect on plant growth under both 250 mmol / L NaCl salt stress and 5% PEG6000 stress. Under 250 mmol / L NaCl salt stress, the single treatment and the mixed treatment increased the SOD activity of plants by 168.13% and 224.81%, respectively; under 5% PEG6000 stress, the enzyme activity increased by 60.66% and 165.91%, respectively. Under 5% PEG6000 stress, single chitosan oligosaccharide treatment and mixed treatment increased peroxidase activity in plants by 159.25% and 256.76%, respectively; under 10% PEG6000 stress, peroxidase activity increased by 313.51% and 275.16%, respectively. For ascorbate peroxidase and glutathione peroxidase, under salt and drought stress, application of chitosan oligosaccharide and a mixture of chitosan oligosaccharide and bacteria both increased the activity of both enzymes in plants.

[0111] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A type of Bacillus cereus ( Bacillus cereus F3, its taxonomic name is Bacillus cereus F3 was deposited at the China Center for Type Culture Collection on October 7, 2025, with accession number CCTCC NO:M 20252180.

2. A microbial inoculant, characterized in that, Containing the Bacillus cereus of claim 1 ( Bacillus cereus F3; Optionally, in the microbial agent, the Bacillus cereus ( Bacillus cereus The viable count of F3 is 10. 7 ~10 9 CFU / mL.

3. A compound microbial fertilizer, characterized in that, Contains Bacillus cereus as described in claim 1 ( Bacillus cereus The microbial agent as described in claim 3 or claim 2; optionally, the compound microbial fertilizer also contains chitosan oligosaccharide.

4. The compound microbial fertilizer according to claim 3, characterized in that, The Bacillus cereus ( Bacillus cereus The viable count of F3 is 10. 7 ~10 9 CFU / mL, chitosan oligosaccharide concentration of 0.1%–2.0%.

5. A method for promoting crop growth, characterized in that, Applying Bacillus cereus as described in claim 1 during crop growth ( Bacillus cereus F3, or the microbial agent according to claim 2, or the compound microbial fertilizer according to claim 3 or 4.

6. A method for preparing chitosanase, characterized in that, The method involves using the Bacillus cereus described in claim 1 (… Bacillus cereus The microbial agent described in claim 3 or claim 2 is inoculated into a seed culture medium for cultivation, and the resulting seed liquid is inoculated into a fermentation culture medium for fermentation to produce a fermentation broth containing chitosanase; optionally, the fermentation broth is separated and purified to obtain chitosanase.

7. The method according to claim 6, characterized in that, The fermentation conditions are as follows: fermentation at a temperature of 27–37℃, pH of 5.5–6.0, and a rotation speed of 220–250 r / min for 30–40 h.

8. The method according to claim 7, characterized in that, The carbon source of the fermentation medium is sucrose and / or soluble starch, and the nitrogen source is beef extract and / or yeast extract. Preferably, the fermentation medium contains the following components by mass percentage: 0.5% to 1.0% soluble starch, 1.0% to 2.0% beef extract, 0.1% to 0.5% yeast extract, and the balance being water.

9. The Bacillus cereus of claim 1 ( Bacillus cereus F3, or the application of the microbial agent according to claim 2 in the preparation of chitosanase or chitosan oligosaccharide.

10. The Bacillus cereus of claim 1 ( Bacillus cereus The application of F3, or the microbial agent according to claim 2, or the compound microbial fertilizer according to claim 3 or 4, or the method according to claim 5 in promoting crop growth, promoting plant resistance to salt stress or drought stress.

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