Peanut asparagine-rich peptide, application and medicine thereof

Abstract

The application discloses an anti-fungal peanut asparagine-rich peptide and characterization of anti-fungal activity of the peanut asparagine-rich peptide. The peanut asparagine-rich peptide has anti-fungal activity on peanut pathogens fusarium oxysporum and rhizoctonia solani.

Description

Technical Field

[0001] This invention belongs to the field of biopesticide technology, specifically involving the chemical synthesis of an antifungal peanut-enriched asparagine peptide, exhibiting antifungal activity against peanut pathogens Fusarium oxysporum and Rhizoctonia solani. Background Technology

[0002] Plants resist pathogen infection by upregulating the expression of genes including microbial-induced plant antitoxins (Darvill AG, Albersheim P (1984) Phytoalexins and their elicitors - a defense against microbial infection in plants. Annu Rev Plant Physiol 35:243–275.), lyases, and protease inhibitors. Therefore, proteins related to pathogenesis have long been a research hotspot in agriculture (Stintzi, Alain, et al. "Plant'pathogenesis-related' proteins and their role in defense against pathogens." Biochimie 75.8 (1993):687-706; Jain, Deepti, and Jitendra Paul Khurana. "Role of pathogenesis-related (PR) proteins in plant defense mechanism." Molecular aspects of plant-pathogen interaction. Springer, Singapore, 2018. 265-281.).

[0003] Peanut (Arachis hypogaea) antimicrobial peptides represent a significant advancement in agricultural biotechnology and food safety, offering a novel and effective method for combating microbial pathogens (Zhao, Kai, et al. "Genome-wide investigation of defensin genes in peanut (Arachis hypogaea L.) reveal AhDef2.2 conferring resistance to bacterial wilt." The Crop Journal 10.3 (2022):809-819; Zhao, Kai, et al. "Genome-wide investigation of defensin genes in peanut (Arachis hypogaea L.) reveal AhDef2.2 conferring resistance to bacterial wilt." The Crop Journal 10.3 (2022):809-819.). These peptides, extracted from common legumes, offer a promising alternative to traditional antimicrobial agents, especially in the context of increasing antibiotic resistance (Mani, Saiprahalad, et al. "The updated review on plant peptides and their applications in human health." International Journal of Peptide Research and Therapeutics 28.5 (2022): 135.).

[0004] The structural and functional properties of these peptides play a crucial role in their antibacterial efficacy. Peanut antimicrobial peptides are typically small, amphiphilic molecules, enabling them to interact effectively with microbial membranes. Their mode of action primarily involves disrupting the integrity of bacterial cell membranes, leading to cell lysis and death (Terea, Hafidha, et al. Preparation and characterization of cellulose / ZnO nanoparticles extracted from peanut shells: Effects on antibacterial and antifungal activities. Biomass Conversion and Biorefinery (2023): 1-12). Aspergillus flavus is a saprophytic fungus frequently detected in oil-rich seeds. During colonization, this organism releases aflatoxins, posing a serious risk to food safety and human health. Therefore, an eco-friendly biological approach is needed to inhibit the pathogen. Experimental results showed that Aspergillus flavus spores could not germinate in potato dextrose broth when the Sub3 concentration exceeded 0.15 g L⁻¹. Morphological assessment by flow cytometry and scanning electron microscopy showed that spores shrank and developed pits after Sub3 exposure. Physiological assessment using staining with propidium iodide, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolium-carbonyl iodide, 2,7-dichlorodihydrofluorescein diacetate, and 4',6-diamidinyl-2-phenylindole revealed cell membrane damage, decreased mitochondrial membrane potential, increased intracellular reactive oxygen species levels, nuclear condensation, and increased DNA fragmentation. Furthermore, treatment with 0.1 and 0.15 g L⁻¹ Sub3 reduced mitochondrial dehydrogenase activity by 29.42% and 45.48%, respectively. In addition, peanut colonization capacity was significantly reduced compared to the control group, with spore numbers on seeds treated with Sub3 decreasing by 26.86% (0.1 g L⁻¹) and 77.74% (0.15 g L⁻¹), respectively. Sub3 may inhibit Aspergillus flavus by crossing the cell wall and targeting the cell membrane, disrupting mitochondrial energy metabolism, inducing DNA damage, and causing spore death.Therefore, Sub3 may provide a useful biocontrol strategy to control the growth of Aspergillus flavus in peanuts (Zhang, Wei, et al. Sub3 inhibits Aspergillus flavus growth by disrupting mitochondrial energy metabolism, and has potential biocontrol during peanut storage. Journal of the Science of Food and Agriculture 101.2(2021):486-496). This mechanism is quite different from the way traditional antibiotics act, which usually target specific bacterial enzymes or pathways, making these peptides potentially effective against a wider range of pathogens, including antibiotic-resistant strains.

[0005] *Fusarium harzianum* is one of the pathogens causing peanut root rot. It is a fungus that typically infects the roots of peanut plants, causing root rot and reducing plant growth and yield. This pathogen can be spread through the soil and multiplies rapidly in warm, humid environments (Erazo, Jessica Gabriela, et al. Biocontrol mechanisms of *Trichoderma harzianum* ITEM 3636 against peanut brown root rot caused by *Fusarium solani* RC 386. *Biological Control* 164(2021):104774.). *Rhizoctonia solani* is another fungus that causes problems in peanuts. It can produce toxins, one of which is called aflatoxin, which is harmful to humans and animals. Rhizoctonia solani typically thrives in warm, humid conditions and can contaminate peanuts and other grains under improper storage conditions, posing a threat to food safety (Abbas, Aqleem, et al. "Assessment of Genetic Variability and Evolutionary Relationships of Rhizoctonia solani Inherent in Legume Crops." Plants 12.13(2023):2515; "Antibacterial effect of Bradyrhizobium spp., Pseudomonas spp., and Bacillus subtilis against pathogenic fungus Rhizoctonia solani on peanut plant."18(2022).). To control these pathogens, we attempted to extract corresponding antimicrobial peptides from peanut bran and tested their antifungal effects.

[0006] Furthermore, the specificity and rapid action of these peptides are noteworthy. Unlike broad-spectrum antibiotics, which may indiscriminately target both beneficial and harmful bacteria, peanut antimicrobial peptides exhibit a degree of selectivity, minimizing negative impacts on beneficial microorganisms. This selectivity is crucial for maintaining the microbial ecological balance in various environments, including soil, water, and the gastrointestinal tracts of plants and animals. Recent studies have explored the potential applications of these peptides in various fields. In agriculture, they can be used as biocides or biocontrol agents to protect crops from bacterial infections, thereby reducing reliance on chemical pesticides. In the food industry, adding them to food packaging materials or as food preservatives can improve food safety and prevent the growth of foodborne pathogens. The biotechnological production and optimization of peanut antimicrobial peptides is currently a hot research area. Advances in genetic engineering and peptide synthesis technologies have facilitated processes for the efficient production of these peptides in host organisms or in vitro systems. In addition, efforts are underway to identify low-abundance, highly active antifungal peptides using stepwise isolation methods.

[0007] In summary, peanut antimicrobial peptides represent a promising and environmentally friendly alternative to traditional antibiotics and chemicals for combating bacterial pathogens. Their unique mechanism of action, specificity, and potential applications in agriculture and food safety make them a valuable tool in the ongoing fight against fungal infections and antibiotic resistance. Further research and development in this field will undoubtedly expand their applications and practicality across various sectors. Summary of the Invention

[0008] A highly active antifungal peptide was isolated from peanut bran and identified as peanut-rich asparagine peptide by protein sequencing. Its amino acid sequence is: NGPGPNDFNRGCGNLRNISPNPNCRPSLRANVALTHNNNNGVNPNRNVMYRVASNIPST.

[0009] Peanut-enriched asparagine peptides possess antifungal activity against peanut pathogens *Fusarium oxysporum* and *Rhizoctonia solani*. This demonstrates the long-term stable application of antifungal peanut-enriched asparagine peptides in inhibiting one or more of these pathogens.

[0010] Peanut-rich asparagine peptide is an antifungal protein found in peanuts. Peanut-rich asparagine peptide exhibits antifungal activity against peanut pathogens Fusarium oxysporum and Rhizoctonia solani.

[0011] The peanut-enriched asparagine peptide has the amino acid sequence shown in SEQ ID NO.1 of the sequence listing. The peanut-enriched asparagine peptide was prepared by a stepwise separation process using ion exchange chromatography and gel filtration chromatography, followed by desalting purification using reversed-phase column chromatography. Attached Figure Description

[0012] Figure 1 Isolation and preliminary activity identification of peanut antimicrobial peptides. A, Elution curves of peanut protein on a Sephacryl S-200 column, showing peaks A1 (elution tube 2), A2 (elution tube 6), A3 (elution tube 8), A4 (elution tube 12), and A5 (elution tube 14). B, Area of ​​the clear inhibition zone of the isolated antifungal peanut asparagine-rich peptides (A1, A2, A3, A4, and A5) against Fusarium oxysporum and Rhizoctonia solani.

[0013] Figure 2 High-performance liquid chromatography was used to analyze the purity of antifungal peanut asparagine peptides.

[0014] Figure 3 Electrospray mass spectrometry identification of antifungal peanut asparagine-rich peptides;

[0015] Figure 4 Plate culture method for analyzing fungal infection counts in rice;

[0016] Figure 5 Comparison of the antibacterial effects of peanut-rich asparagine peptides;

[0017] Figure 6 Comparative study on the inhibitory effects of peanut-rich asparagine peptides on glycoside synthase (GS) activity. Detailed Implementation

[0018] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0019] Example 1: Preparation of antifungal peanut asparagine-rich peptides

[0020] Weigh 5.0g of peanut meal (primary meal, the peanut residue remaining after the initial pressing and oil extraction), grind it repeatedly in liquid nitrogen into a fine powder (particle size less than 0.5 mm), place it in a 200mL beaker, add 100mL of protein extraction buffer (composition: 20mM Tris-HCl, pH 8.0, 10mM EDTA, 150mM NaCl, 1% dimethyl sulfoxide (DMSO), and 0.1% β-mercaptoethanol in water), and stir to mix well. Centrifuge at 10000g for 20min, and collect 60mL of supernatant.

[0021] Slowly add 767g of (NH4)2SO4 to 1L of distilled water while stirring. Adjust the pH to 7.0 with 25%-28% ammonia (and / or 95% sulfuric acid). This is a 100% saturated ammonium sulfate solution (4.1mol / L, 25℃). While stirring, add an equal volume of the 100% saturated ammonium sulfate solution to the supernatant. Stir the solution overnight (4℃) on a magnetic stirrer to allow the protein to precipitate completely. Centrifuge the protein solution at 10000g for 30min (4℃). Discard the supernatant and retain the precipitate. Dissolve the precipitate in 20ml of protein extraction buffer (composition: 20mM Tris-HCl, pH 8.0, 10mM EDTA, 150mM NaCl, 1% DMSO, 0.1% β-mercaptoethanol, and 0.2g / L sodium azide in water). Place the solution in a 40 ml dialysis bag (molecular weight cutoff 2 kDa) and dialyze with 1 L of protein extraction buffer for 24 hours (4°C). Change the dialysis buffer every 6 hours to completely remove ammonium sulfate. Collect the crude extract in the dialysis bag. Equilibrate a DEAE-Sepharose Fast Flow column (high-flow-rate ionic (DEAE) agarose gel column, inner diameter and length 2.6 × 100 cm, Merck, model 17-0709-10) with three column volumes of protein extraction buffer (composition: 20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl, 1% DMSO, and 0.1% β-mercaptoethanol in water) at a flow rate of 0.5 ml / min. 25 ml of crude extract solution was loaded onto a DEAE-Sepharose Fast Flow column (2.6 × 100 cm), and the DEAE-Sepharose Fast Flow column (2.6 × 100 cm) was equilibrated again with three column volumes of protein extraction buffer (composition: 20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl, 1% DMSO and 0.1% β-mercaptoethanol in water) at a flow rate of 0.5 ml / min. The specific procedure for eluting the column using a protein extraction buffer of 20 mM Tris-HCl (pH 8.0), 10 mM EDTA, 150 mM NaCl, 1% DMSO, and 0.1% β-mercaptoethanol, along with a 0.3 M NaCl gradient (a linear NaCl gradient from 0 to 0.3 M in the eluent), at a flow rate of 1.0 mL / min, is as follows: First, prepare the protein extraction buffer by mixing all components thoroughly and adjusting the pH to 8.0. Then, prepare the appropriate column according to the experimental type and ensure it has been equilibrated with a suitable buffer. Next, slowly load the sample containing the target protein, allowing sufficient contact between the sample and the packing material.Next, a gradient was prepared by mixing 0.3M NaCl solution with extraction buffer and eluting through the column at a flow rate of 1.0 mL / min. During this process, the eluent fraction was collected, and absorbance was monitored using a UV detector to identify the fraction containing the target protein. Finally, the collected protein fraction was subjected to necessary subsequent analysis. Protein eluent was collected from the point when the detector could detect protein until the detector could no longer detect protein, for a total of 45 mL. The collected sample was added to a gel filter column (Sephacryl S-200, 5x90 cm inner diameter and length, GE Healthcare, model 17-0584-01) equilibrated with 3 column volumes of 20 mM Tris-HCl buffer (pH 8.0) (containing 0.15 M NaCl), with 3 mL collected per column. Elution was performed with 0.5 mL / min of 20 mM Tris-HCl buffer (pH 8.0) (containing 0.3 M NaCl). From the point when the detector could detect protein, 5 mL was collected per tube, for a total of 9 tubes. By detecting absorbance at 280nm, peaks with higher protein concentrations are concentrated. Figure 1 A: Formation of peaks A1 (eluting tube 2), A2 (eluting tube 6), A3 (eluting tube 8), A4 (eluting tube 12), and A5 (eluting tube 14).

[0022] Five protein fractions were analyzed to form peaks A1 (eluted tube 2), A2 (eluted tube 6), A3 (eluted tube 8), A4 (eluted tube 12), and A5 (eluted tube 14). The antifungal activity of each fraction was analyzed. Potato dextrose agar plates (9 cm diameter, 0.3 cm thickness) were used (formulation as follows: 20 g potato extract, 2 g glucose, 1.5 g agar, 100 mL water, pH unadjusted (natural pH). Preparation method: Take 20 g fresh potatoes, wash, peel, and cut into small pieces (rectangles with sides 0.1-0.5 cm), add 80 mL water and boil for 30 min. Filter through four layers of gauze (50 mesh), add 2 g glucose and 1.5 g agar, continue heating and stirring until homogeneous, cool slightly, add water to 100 mL, and sterilize at 121℃ for 20 min). 10 μL of frozen bacterial suspensions (containing *Fusarium oxysporum* (CGMCC No. 3836) and *Rhizoctonia solani* (CICC No. 40529)) were dropped into the center of the agar plate, with a cell density of 1 × 10⁻⁶. 5 ~2×10 5CFU / g lyophilized powder (each gram of sample dissolved in 10 mL of 0.85% physiological saline). The antifungal activity of peanut protein in tubes 2, 6, 8, 12, and 14 was evaluated. A 1 cm diameter sterile circular paper plate was prepared by drawing a cross around its center. A paper plate was placed at the intersection of the cross and then at 1.5 cm intervals away from the center along the two lines, for a total of 5 paper plates (9 cm diameter). 10 μL of the eluted test protein in 20 mM PBS, pH 7.5 buffer was added to each paper plate. The control was prepared by adding only 10 μL of 20 mM PBS, pH 7.5. After incubation at 25°C for 72 hours, mycelial growth surrounded the outer disc containing the control and generated a clear zone of inhibition around the disc containing the antifungal sample. A larger clear zone indicated a higher inhibition rate. Antibacterial experiments showed that A2 in the eluted peanut asparagine peptides (eluted tube 6) inhibited Fusarium oxysporum (inhibition zone area approximately 9 cm²). 2 ) and Rhizoctonia solani (inhibition zone area approximately 7.4 cm²) 2 The samples showed significant antibacterial activity, while A1 (tube 2), A3 (tube 8), A4 (tube 12), and A5 (tube 14) exhibited very weak antibacterial activity against Fusarium oxysporum and Rhizoctonia solani, with activity less than 0.5 cm. 2 ( Figure 1 B). Therefore, A2 (the eluted tube #6) may be the ideal antibacterial peanut protein, named peanut asparagine-rich peptide, for subsequent experiments.

[0023] Figure 1 Elution curves of peanut protein on a Sephacryl S-200 column show peaks A1 (elution tube 2), A2 (elution tube 6), A3 (elution tube 8), A4 (elution tube 12), and A5 (elution tube 14). Elution was performed with 20 mM Tris-HCl buffer (pH 8.0) (containing 0.35 M NaCl) at a rate of 5 ml / min. One 5 ml tube was collected from the point where protein was detected by the detector, for a total of 18 tubes. Absorbance measurements at 280 nm revealed peaks with the highest protein concentrations: A1 (elution tube 2), A2 (elution tube 6), A3 (elution tube 8), A4 (elution tube 12), and A5 (elution tube 14).

[0024] Figure 1Isolation and antibacterial identification of peanut antimicrobial peptides. A. Elution curves of peanut peptides on a Sephacryl S-200 column, forming peaks A1 (elution tube 2), A2 (elution tube 6), A3 (elution tube 8), A4 (elution tube 12), and A5 (elution tube 14). B. Antibacterial effect of each elution peak. C. Area of ​​the clear zone of the isolated antifungal peanut asparagine-rich peptides (A1, A2, A3, A4, and A5) against Fusarium oxysporum and Rhizoctonia solani. The diameter of each inhibition zone was measured three times, and the average diameter of the inhibition zone was calculated. The area of ​​the clear zone was calculated using the area formula (S = π * (d / 2)², where π represents pi, r represents radius, d represents diameter, and S represents area).

[0025] Example 2: Identification of the molecular weight and sequence of the antifungal peanut asparagine-rich peptide obtained in Example 1

[0026] 1 ml of antifungal peanut-rich asparagine peptide was subjected to high-performance liquid chromatography (HPLC) using a reverse-phase column (Welch XB, Welch Materials, model number 1). XB-C18 (inner diameter and length C18 4.6×150mm) was purified by chromatographic desalting. The mobile phase was 40% v / v pure water (containing 0.1% trifluoroacetic acid (TFA)) - 60% v / v acetonitrile (ACN) (containing 0.1% trifluoroacetic acid). The flow rate was 1 mL / min, and detection was performed at 220 nm. The elution peak was collected, lyophilized, and then resuspended in 0.1% formic acid aqueous solution for analysis by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The molecular weight of the synthesized antifungal peanut asparagine peptide was identified by electrospray mass spectrometry. The sample was injected into the liquid chromatography system with a mobile phase of 50% H2O / 50% CAN at a flow rate of 0.2 mL / min, a protective gas nitrogen flow rate of 1.5 L / min, a collision energy of 4.5 kV, and cation mode.

[0027] Purified antifungal peanut asparagine peptides were analyzed by high performance liquid chromatography (HPLC) (Welch XBC, Welch Materials, model number 100000). The purity of XB-C18 (inner diameter and length 184.6 x 250 mm) was determined, and its molecular weight was analyzed using electrospray ionization mass spectrometry. The purity determination results by high-performance liquid chromatography (HPLC) are as follows: Figure 3 As shown: the antifungal peanut asparagine peptide exhibits a single peak at 27.1 min. Figure 2 ).

[0028] Figure 2 High-performance liquid chromatography (HPLC) analysis of the purity of antifungal peanut-rich asparagine peptides.

[0029] Electrospray mass spectrometry identification results are as follows Figure 3 As shown, the molecular weight of the protein is close to the theoretical value of 6.37 kDa.

[0030] Figure 3 Electrospray ionization mass spectrometry was used to identify an antifungal peanut-rich asparagine peptide. The labeled molecular weight was 6373.10 Da. Considering the cation mode, this is close to the theoretical molecular weight of 6372 Da.

[0031] Example 3: Sequencing of the antifungal peanut asparagine-rich peptide obtained in Example 1

[0032] Following previous literature, the Edman degradation method using phenyl isothiocyanate was employed for sequencing of peanut asparagine peptides against fungi [Sels J., Mathys J., De Coninck BM, Cammue BP, De Bolle MF. Plant pathogenesis-related (pr) proteins: A focus on pr peptides. Plant Physiol. Biochem. 2008; 46:941–950.]. Purified peanut asparagine peptides (20 μg) were dissolved in 50 μl of 0.2 M ammonium bicarbonate (pH 8.0, containing 4 M guanidine hydrochloride) and mixed with 5 ml of 45 mM dithiothreitol. Automated Edman degradation of S-carboxamide methylated peptides and detection of phenylthioacetal derivatives were performed using an automated protein sequencer (Applied Biosystems, Model 476A). In the presence of 6 mol / L HCl, cleavage occurs at the first peptide bond, yielding a peptide fragment minus the first base and the released first anilinothiazolinone (ATZ) residue. Washing away other reactants and released residues with 20 mM TrisHCl (pH 8.0) buffer allows the shortened peptide to release the second residue through another round of coupling and cleavage, and so on, until the last amino acid residue is released. Sequencing results confirmed it to be a peanut antifungal peanut-enriched asparagine peptide.

[0033] The sequence number SEQ ID No. 1 is:

[0034] The result of the three-letter amino acid code is: Asn-Gly-Pro-Asn-Pro-Asn-Asp-Phe-Asn-Arg-Gly-Cys-Gly-Asn-Leu-Arg-Asn-Ile-Ser-Pro-Asn-Pro-Asn-Cys-Arg-Pro-Ser-Leu-Arg-Ala-Asn-Val-Ala-Leu-Thr-His-Asn-Asn-Asn-Gly-Val-Asn-Pro-Asn-Arg-Val-Met-Tyr-Arg-Val-Ala-Ser-Asn-Ile-Pro-Ser-Thr.NGPNPNDFNRGCGNLRNISPNPNC RPSLRANVALTHNNNNGVNPNRNVMYRVASNIPST. (59aa)

[0035] (a) Sequence characteristics:

[0036] ●Length: 59

[0037] ●Type: Amino acid sequence

[0038] ●Chain type: Single chain

[0039] ●Topology: Linear

[0040] (b) Molecular type: protein

[0041] (c) Assumption: No

[0042] (d) Antonym: No

[0043] (e) Original source: peanut

[0044] Example 4: Control of fungal infections in rice by the asparagine-rich peptides obtained in Example 1

[0045] We first focused on the selection and treatment of rice root samples. Healthy, mature rice plants (25-35 weeks old) were selected, ensuring that each plant was uniform in size and color, and free from obvious disease or pest damage. Treatment involved immersing 5-10 cm of roots in 200 ml of physiological saline solution containing 0.1 mg / ml peanut asparagine peptide for 1 minute, followed by surface drying under sterile conditions (20°C) (10 root segment treatment groups). The control group's rice roots were treated only with 200 ml of physiological saline (immersed for 1 minute and then dried) under the same conditions. The treated samples were stored in suitable containers at 10-15°C to simulate field conditions. Next, fungal contamination was measured on the rice roots every 5 days. A portion of the roots was randomly selected from each rice plant, washed, and then immersed in 50 mL of sterile 0.1% peptone solution for 5 minutes to remove surface fungi, yielding a washing solution. The washing solution was serially diluted at dilution factors (volume factors) of 1 / 10, 1 / 100, and 1 / 1000. Each dilution (0.1 mL) was inoculated onto YPD (yeast extract peptone dextrose agar) plates (9 cm in diameter, 3-4 mm thick) and incubated at 25°C for 2 days. Colony forming units (CFU) were counted on each plate, and the CFU count was expressed as the number of samples per gram of rice root. Colony characteristics were observed under a microscope to distinguish different types of hyphae. Finally, the CFU counts of the treatment group and the control group were compared, and statistical methods were used to analyze the differences between the two groups. Data recording and analysis: The number of fungal infections in the rice roots of the treatment group and the control group was recorded and compared. Different types of hyphae were observed and distinguished under a microscope. The figure shows the results of analyzing the number of fungal infections in rice using the plate culture method. From Figure 4 It can be seen that during the 26-day storage period, the number of fungal infections in the treated group and the control group showed significantly different trends. The number of fungal infections in the control group (treated with physiological saline) remained relatively stable throughout the storage period, with a very low CFU / g (colony forming units per gram of sample), indicating a good antibacterial effect. In contrast, the number of fungal infections in the treated group (treated with asparagine peptide-enriched peptides) also remained at a low level for the first 20 days, but after about 20 days, the number of infections in the control group increased sharply, while the experimental group (treated group) remained in an inhibited state, indicating that the antibacterial effect of asparagine peptide-enriched peptides was significant after long-term storage.

[0046] Figure 4 Plate culture method was used to analyze the number of fungal infections in rice. Rice was stored at 10-15°C for 26 days. Treatment group: asparagine-enriched group. Control group: physiological saline.

[0047] Example 5: Inhibition of Fusarium oxysporum and Rhizoctonia solani by the asparagine-rich peptide obtained in Example 1.

[0048] The solid culture medium used in the antifungal studies included potato dextrose agar (PDA) for fungi (potato, 200 g; glucose, 20 g; agar, 18 g; and distilled water, 1 L). To test antifungal activity, 1 mL of fungal spore suspensions (1 x 10⁻⁶) of *Fusarium oxysporum* and *Rhizoctonia solani* were separately prepared. 7 Asparagine peptide (Spores / ml) was uniformly added to 100 mL of PDA medium and cultured at 40-50℃ to prepare solid plates (3-4 mm thick). Oxford cups (6 mm inner diameter, 8 mm outer diameter, 10 mm height) were then placed on the plates. Different concentrations of asparagine peptide (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / mL) were added to different Oxford cups, and the plates were incubated at 30℃ for 48 h. The diameter of the antibacterial zone was measured. A control group was prepared by adding 5 μL of physiological saline under the same conditions. Preliminary screening of the antifungal activity of asparagine peptide by clear zone formation showed that the IC50 values ​​of asparagine peptide against *Fusarium oxysporum* and *Rhizoctonia solani* were 11.1 and 102 μg / ml, respectively.

[0049] Figure 5 Comparison of antibacterial effects. Control group: physiological saline. Treatment group: antifungal peanut asparagine peptide solution obtained in Example 1 dissolved in physiological saline.

[0050] Example 6: Effects of the asparagine-rich peptide and amphotericin obtained in Example 1 on the glycoside synthase activity of Fusarium oxysporum and Rhizoctonia solani.

[0051] 0.1 mL of frozen *Fusarium oxysporum* and *Rhizoctonia solani* culture solutions were spread separately onto 20 mL PDA agar plates (9 cm diameter, 3-4 mm thickness) and incubated at 25°C for 5 days. The hyphae of both fungi were rinsed with 20 mL of distilled water and then lyophilized. 1.0 g of the dried hyphae was added to 4 mL of sodium acetate buffer (50 mmol / L, pH 5) and homogenized in a pre-cooled (0-4°C) mortar to obtain a homogenate. The homogenate was then centrifuged at 10,000 g for 15 minutes to obtain a supernatant containing crude glycoside synthase (GS). The protein content of the supernatant containing crude enzyme was determined using a BCA assay kit. Enzyme activity is expressed as enzyme units / mg protein. A common method for detecting glycoside synthase (GS) activity is a biochemical assay using color changes. For example, an enzymatic reaction using p-Nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate can release p-Nitrophenol (pNP), which exhibits absorbance at 405 nm and can be used for quantitative analysis. The specific experimental steps include: adding 5-10 mmol / L pNPG and 50 μL of enzyme sample to a buffer with appropriate pH and buffer strength, adjusting the total volume to 1 mL, and then incubating at 37°C for 30 minutes. The necessary equipment includes a spectrophotometer (such as a Shimadzu UV-1280) to measure the absorbance of the sample, a high-speed centrifuge (Eppendorf 5810R) to remove unreacted particles from the sample, and a constant-temperature water bath or incubator (Thermo Fisher Scientific's Precision General Purpose water bath) to maintain the reaction at a constant temperature. After the reaction is complete, the sample should be immediately transferred to a 100°C water bath for 10 minutes to stop the reaction. The reaction mixture was centrifuged at 10,000 g for 10 minutes at 4°C to remove unreacted particles and residues. The absorbance of the supernatant was measured at 405 nm using a spectrophotometer to determine the concentration of pNP. Finally, GS activity was calculated based on a standard curve, typically expressed as the amount of protein-converted substrate per unit time (units / mg protein / min). The results showed that the antifungal peanut asparagine peptide obtained in Example 1 had excellent inhibitory activity against mycoglycoside synthase (…). Figure 6 (P<0.0001). Figure 6 The results shown here are the effects of peanut asparagine peptide on glycoside synthase (GS) activity. Two bar charts represent the GS enzyme activity (in U / mg) of the control and treatment groups, respectively. The control group used physiological saline, while the treatment group was treated with a 0.1 mg / ml antifungal peanut asparagine peptide solution.

[0052] The bar chart shows that the GS enzyme activity in the control group was significantly higher than that in the treatment group. Specifically, under the experimental conditions, peanut-enriched asparagine peptide significantly reduced GS enzyme activity, indicating that this compound has the potential to inhibit this enzyme. An asterisk (****) indicates statistical significance; four asterisks typically indicate a p-value less than 0.0001, meaning the difference between the two groups is highly significant. In summary, these data suggest that, at the experimentally given concentrations, peanut-enriched asparagine peptide can significantly inhibit glycoside synthase activity, which may be important for controlling biological processes dependent on GS enzyme activity, such as the metabolic pathways of certain fungal organisms. Therefore, this peptide may be a promising candidate for developing novel antifungal agents.

[0053] Figure 6 Comparison of the inhibitory effects of peanut asparagine peptide on glycoside synthase (GS) activity. Control group: physiological saline. Treatment group: physiological saline solution of the antifungal peanut asparagine peptide (0.1 mg / ml) obtained in Example 1.

Claims

1. A peanut-enriched asparagine peptide, the amino acid sequence of which is shown in SEQ ID NO.1 of the sequence listing.

2. The application of the peanut antifungal peanut-enriched asparagine peptide according to claim 1, characterized in that, The application of the peanut-rich asparagine peptide as an antifungal active ingredient in the preparation of one or two drugs that inhibit peanut pathogens Fusarium oxysporum and / or Rhizoctonia solani.

3. A drug that is effective against one or two of the fungi Fusarium oxysporum and / or Rhizoctonia solani, wherein the antifungal peanut asparagine peptide of claim 1 is the active ingredient.

4. The medicament according to claim 3, further comprising a pharmaceutically acceptable carrier or excipient.

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