Phanerochaete chrysosporium and application thereof in degradation of corn stalks for fermenting production of erucamide

Abstract

The application discloses a white-cyst Irpex lacteus and application of the white-cyst Irpex lacteus in degradation of corn stalks and fermentation production of erucic acid amide. The white-cyst Irpex lacteus is classified and named as Irpex lacteus J2, and has been preserved in the China Center for Type Culture Collection on November 24, 2023, with a preservation number of M20232318. The strain has a complete lignocellulose-degrading enzyme system, and can produce erucic acid amide by taking corn stalks as the only carbon source.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a strain of *Alternaria alternata* and its application in the fermentation of corn stalks to produce erucamide. Background Technology

[0002] Erucamide is a long-chain fatty acid amide with the chemical formula C2. 22 H 43 NO, its molecular structure contains a double bond and an amide group, these key structures determine its unique biological activity and function. As a broad-spectrum plant defense metabolite, erucic acid amide effectively inhibits the pathogenicity of various plant and animal pathogens by targeting and inhibiting the bacterial type III secretion system (T3SS), blocking the process of pathogens injecting virulence proteins into host cells. This antiviral mechanism not only provides new ideas for the development of environmentally friendly biopesticides, but also opens up new avenues for cultivating disease-resistant crops, and has important application value in agricultural disease control. Erucic acid amide has wide application value in the industrial field. As a multifunctional additive, it plays an important role in industries such as plastics, rubber, coatings, inks, textiles, and packaging. In addition, it is also used in metal processing and paper treatment to improve processing efficiency and product quality. Due to its multifunctionality, erucic acid amide has become an indispensable additive in industrial processing, driving technological innovation and product upgrading in multiple industries. Currently, erucic acid amide is produced industrially by chemical synthesis and plant extraction methods, using erucic acid as a raw material and reacting it with ammonia or amine compounds through an amidation reaction to generate erucic acid amide. Chemical extraction methods are mature and yield high outputs, but they involve high-temperature and high-pressure conditions, requiring sophisticated equipment and prone to secondary pollution. Plant extraction still faces challenges such as insufficient raw material supply and low yield of erucic acid rapeseed oil. Microbial synthesis converts fatty acids into erucic acid amides through metabolic pathways within the body; this method is mild and environmentally friendly. Therefore, screening microorganisms capable of producing erucic acid amides provides new ideas and technical support for the development of green biomanufacturing technologies. Summary of the Invention

[0003] The purpose of this invention is to provide a strain of *Alternaria alternata* and its application in the fermentation of corn stalks to produce erucamide.

[0004] The application of *Irpex lacteus* J2 in the preparation of lignocellulase is also within the scope of protection of this invention.

[0005] The lignocellulases used in this study include any one or any combination of laccase, manganese peroxidase, lignin peroxidase, exoglucanase, endoglucanase, β-glucosidase, and xylanase.

[0006] The present invention also provides the application of the aforementioned Irpex lacteus J2 in the production of erucamide from fermented corn straw.

[0007] The present invention relates to *Irpex lacteus* J2, a strain possessing a complete lignocellulose-degrading enzyme system, which produces erucamide using corn stalks as the sole carbon source. Strain F81 was deposited on November 24, 2023, at the China Center for Type Culture Collection (CCCCC), accession number M20232318, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] Irpex lacteus J2 possesses a complete enzyme system for degrading lignocellulose and the ability to secrete extracellular enzymes. Extracellular enzyme activity assays identified the activities of laccase, manganese peroxidase, lignin peroxidase, exoglucanase, endoglucanase, β-glucosidase, and xylanase. Furthermore, Irpex lacteus J2 exhibits excellent degradation activity against corn stalks and uses corn stalks as the sole carbon source to produce erucamide. Attached Figure Description

[0009] Figure 1 This is a morphological illustration of the Irpex lacteus J2 strain.

[0010] Figure 2 The image shows the degradation effect of Irpex lacteus J2 on Congo red plate.

[0011] Figure 3 Phylogenetic tree of Irpex lacteus J2.

[0012] Figure 4 The activities of laccase, lignin peroxidase, and manganese peroxidase in Irpex lacteus J2 were determined.

[0013] Figure 5 The activities of exoglucanase, endoglucanase and β-glucosidase in Irpex lacteus J2 were determined.

[0014] Figure 6 The activity of hemicellulase in Irpex lacteus J2 was determined.

[0015] Figure 7 The standard curve for erucic acid amide of Irpex lacteus J2 is shown.

[0016] Figure 8The chromatographic peak shape of erucamide in Irpex lacteus J2 is shown.

[0017] Preservation of biological materials

[0018] Name: Irpex lacteus J2;

[0019] Classification and nomenclature: Irpex lacteus;

[0020] Date of preservation: November 24, 2023;

[0021] Depository: China Center for Type Culture Collection (Wuhan, China, Wuhan University);

[0022] Accession number: CCTCC No:M20232318. Detailed Implementation

[0023] To better illustrate the objectives, complete technical approach, and advantages of this invention, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. However, the embodiments described below are only a part of the embodiments of this invention, and do not include all embodiments. Other embodiments that can be obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the protection scope of this invention.

[0024] In the embodiments described below, unless otherwise specified, the conventional methods used are all technical means recognized or commonly used by those skilled in the art, and the instruments, reagents, consumables, etc. used in the embodiments of the present invention can be obtained through legitimate commercial channels.

[0025] Example 1: Obtaining Irpex lacteus J2 of the present invention

[0026] 1. Obtaining Irpex lacteus J2

[0027] Fruiting bodies of wood-decaying fungi and decaying bark were collected from ancient trees, and the wood-decaying fungi were isolated and purified using the single-spore isolation method. Figure 1 ).

[0028] (1) Preparation of spore suspension: Rinse the fruiting bodies of wood-rotting fungi and the surface of decaying bark with sterile water or buffer solution, and collect spores. Vortex or break them up with glass beads to form a single-spore suspension, and dilute until dispersed spores are visible under a microscope.

[0029] (2) The spore suspension was serially diluted and 100 μL of the diluted solution was spread on a solid plate (200 g peeled potato, 20 g glucose, 15-20 g agar, 1000 mL distilled water, natural pH). After incubation at 30 °C, isolated colonies were picked (it is necessary to verify whether they are from single spore sources).

[0030] 2. Determination of the degradation ability of Irpex lacteus J2 on carboxymethyl cellulose

[0031] The purified strain was inoculated into 7 mm mycelial discs onto carboxymethyl cellulose medium (ammonium sulfate 2.0 g, magnesium sulfate heptahydrate 0.5 g, potassium dihydrogen phosphate 1 g, sodium chloride 0.1 g, sodium carboxymethyl cellulose 5.0 g, agar 20 g, pH 7.0, water 1000 mL) and cultured at 28℃ for 3 days. After staining with 2% Congo red for 15 min, the medium was destained with 0.9% NaCl, and the clear zones on the Congo red plates were observed. The figure shows that the strain has a very strong ability to degrade carboxymethyl cellulose. Figure 2 ).

[0032] 3. Identification of Irpex lacteus J2

[0033] Genomic DNA was extracted from strain J2, and ITS PCR amplification was performed using universal bacterial primers ITS1 and ITS4. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The ITS sequence of strain J2 is shown in Sequence Listing 1. BLAST alignment of the sequencing results was performed, and phylogenetic analysis of the obtained homologous sequences was conducted using MEGA 7.0 software. The results showed that this strain is *Irpex lacteus*, and it was named *Irpex lacteus* J2. Figure 3 ).

[0034] Irpex lacteus J2, deposited at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), on November 24, 2023, accession number CCTCC NO:M20232318.

[0035] Example 2. Enzyme activity assay of strain Irpex lacteus J2

[0036] The preserved Irpex lacteus J2 strain was first activated and cultured. Then, vigorous mycelia were selected and inoculated into a liquid culture medium (200g peeled potato, 20g glucose, 1000mL distilled water, natural pH) to prepare a seed culture. The seed culture was inoculated at a rate of 5% into a 250ml Erlenmeyer flask containing 50ml of initial enzyme-producing medium and cultured at 30℃ and 180rpm on a shaker to obtain the fermentation broth. The enzyme-producing medium formula was as follows: milled corn stalks (passed through an 80-mesh sieve) as carbon source, 10g / L; ammonium tartrate, 0.2g / L; KH₂PO₄, 2g / L; MgSO₄·7H₂O, 0.71g / L; CaCl₂, 0.1g / L; and 70mL of trace element solution. The trace element solution contained 1g / L NaCl; 0.184g / L CoCl₂·6H₂O;

[0037] FeSO4·7H2O, 0.1 g / L; ZnSO4·7H2O, 0.1 g / L; CuSO4, 0.1 g / L; H3BO3, 0.01 g / L; Na2MoO4·2H2O, 0.01 g / L; KAl(SO4)2·12H2O, 0.01 g / L; and 1.5 g / L N-triacetic acid.

[0038] Enzyme activity assay method:

[0039] (1) Laccase: 37℃, 3mL reaction system, the reaction mixture contains 2mL 0.5mmol / L ABTS, add 1mL fermentation broth to start the reaction, and measure the change in absorbance at 420nm after 3min.

[0040] (2) Lignin peroxidase: 37℃, 3mL reaction system, the reaction mixture contains 1.8mL 0.24mmol / L resveratrol and 1.1mL fermentation broth. After preheating to 37℃, add 0.1mL 6mmol / L H2O2 to start the reaction. Measure the increase in absorbance at 310nm after 3min.

[0041] (3) Manganese peroxidase: 37℃, 3mL reaction system, the reaction mixture contains 2.3mL 50mM pH8.5 acetate buffer, 0.1mL 1.6mM MnSO4 solution, 0.4mL fermentation broth, 0.1mL 1.6mM H2O2 solution is added at 37℃ to start the reaction, and the change in absorbance value at 240nm is measured in the first 3min.

[0042] (4) Exoglucanase: Mix 500 μL of crude enzyme solution with 50 μL of 1 mg / mL pNPC and 1 mg / mL gluconolactone, react at 50 °C for 30 min, and then add 150 μL of 10% Na2CO3 to terminate the reaction.

[0043] (5) Endoglucanase: Mix 50 μL of fermentation broth with 150 μL of 0.5% CMCNa, react at 50℃ for 30 min, and then add 50 μL of 1M NaOH solution and 150 μL of DNS solution to terminate the reaction.

[0044] (6) β-glucosidase: Mix 200 μL of fermentation broth with 100 μL of 1 mg / mL pNPG solution, react at 50 °C for 30 min, and add 300 μL of 10% Na2CO3 to terminate the reaction.

[0045] (7) Xylanase (hemicellulase): Mix 50 μL of fermentation broth with 100 μL of 1% xylan solution, react at 50°C for 30 min, and add 200 μL of DNS to terminate the reaction.

[0046] Strains J2 were fermented in an enzyme-producing medium at 28°C, and the activities of laccase, lignin peroxidase, and manganese peroxidase were measured at different fermentation time points. Figure 4 It can be seen that with the extension of fermentation time, the activities of laccase, lignin peroxidase, and manganese peroxidase all showed a trend of first increasing and then decreasing. Laccase activity reached its highest level on day 3, at 0.296 U / mL. Lignin peroxidase activity reached its highest level on day 7, at 1.303 U / mL. Manganese peroxidase activity reached its highest level on day 5, at 8.077 U / mL.

[0047] The activities of exoglucanase, endoglucanase, and β-glucosidase all showed a trend of first increasing and then decreasing. Exoglucanase activity reached its highest level on day 7, at 0.009 U / mL. Endoglucanase and β-glucosidase activities both reached their highest levels on day 9 of fermentation, at 0.945 U / mL and 0.013 U / mL, respectively. Figure 5 ).

[0048] Depend on Figure 6 As shown, enzyme activity changes over time, reaching its maximum value of 12.039 U / mL on day 9.

[0049] Example 3. Production of erucic acid amide from corn straw fermentation by *Irpex lacteus* J2

[0050] 1) Preparation of erucamide standard curve

[0051] Accurately weigh 1 mg of erucamide standard and dissolve it in 1 mL of hot methanol to obtain a concentration of 1 mg / mL. Dilute the standard stock solution with dichloromethane to prepare standard solutions with concentrations of 5, 10, 20, 40, 60, and 100 μg / mL, and perform the determination under the above conditions. Plot the erucamide GC-MS standard curve with concentration (x, μg / mL) on the x-axis and peak area (y) on the y-axis. Figure 7 ).

[0052] 2) Erucamide Determination Method

[0053] GC conditions: Agilent 6850; HP-5 column (30m × 0.25mm × 0.25μm), injection port temperature 290℃. Initial column temperature: 150℃, hold for 2 min, then ramp to 300℃ at 20℃ / min and hold for 3 min. Carrier gas: He, flow rate 1.0 mL / min, injection volume 1 μL. MS conditions: Ionization mode: EI, electron energy 70 eV, ion source temperature 200℃, transfer line temperature 200℃, scan range 50–400 amu, full scan mode. Solvent delay 4.0 min, run time 21 min.

[0054] 3) Processing and detection of fermentation samples of Irpex lacteus J2

[0055] The preserved Irpex lacteus J2 strain was first activated and cultured. Then, vigorous mycelia were selected and inoculated into a liquid culture medium (200g peeled potato, 20g glucose, 15-20g agar, 1000mL distilled water, natural pH) to prepare an Irpex lacteus J2 seed culture. The Irpex lacteus J2 seed culture was then inoculated at 10% (v / v) into a fermentation medium (milled corn stalks (passed through an 80-mesh sieve) as a carbon source, 10g / L; ammonium tartrate, 0.2g / L; KH2PO4, 2g / L; MgSO4·7H2O, 0.71g / L; CaCl2, 0.1g / L; and 70mL of trace element solution. The trace element solution contained 1g / L NaCl; 0.184g / L CoCl2·6H2O; Fe... The following were added: SO4·7H2O, 0.1 g / L; ZnSO4·7H2O, 0.1 g / L; CuSO4, 0.1 g / L; H3BO3, 0.01 g / L; Na2MoO4·2H2O, 0.01 g / L; KAl(SO4)2·12H2O, 0.01 g / L and 1.5 g / L N-triacetic acid. The culture was prepared in a 50 mL / 250 mL container at 30 °C and 180 rpm for 15 days. The bacterial suspension was centrifuged at 12000 rpm for 10 min, and the supernatant was used as the fermentation broth. The fermentation broth was extracted with dichloromethane at a 1:1 (v / v) ratio for 2 h, with vigorous shaking every 15 min followed by static separation. The organic phase (lower layer) was concentrated and dried using a rotary evaporator at 25–30 °C, then dissolved in 1.5 mL of dichloromethane. A suitable amount of the solution was filtered through a 0.22 μm microporous membrane and analyzed by GC / MS. Figure 8 It can be seen that the retention time of the erucamide standard is 9.439 min, and the retention time of the fermentation sample of Irpex lacteus J2 is 9.435 min, which is consistent with the erucamide standard. The erucamide content in the fermentation broth sample of Irpex lacteus J2 is 0.284 mg / L.

[0056] In summary, the *Irpex lacteus* J2 strain of the present invention can produce erucamide using corn stalks as the sole carbon source. The specific method is as follows: The preserved strain is first activated and cultured, and then vigorous mycelia are selected and inoculated into a liquid culture medium to prepare a seed culture. The seed culture is then inoculated at a rate of 5% into a fermentation medium containing 50 ml of medium (milled corn stalks as carbon source, 10 g / L; ammonium tartrate, 0.2 g / L; KH₂PO₄, 2 g / L; MgSO₄·7H₂O, 0.71 g / L; CaCl₂, 0.1 g / L; and 70 mL of trace element solution). The trace element solution contains 1 g / L of... The following ingredients were added to a 250 ml Erlenmeyer flask: NaCl (0.184 g / L), CoCl₂·6H₂O (0.1 g / L), FeSO₄·7H₂O (0.1 g / L), ZnSO₄·7H₂O (0.1 g / L), CuSO₄ (0.1 g / L), H₃BO₃ (0.01 g / L), Na₂MoO₄·2H₂O (0.01 g / L), KAl(SO₄)₂·12H₂O (0.01 g / L), and N-triacetic acid (1.5 g / L). The mixture was incubated at 30 °C and 180 rpm in a shaker. After 15 days of fermentation, the Aspergillus suspension was centrifuged at 12000 rpm for 10 min, and the supernatant was used as the fermentation broth. The fermentation broth was extracted with dichloromethane at a ratio of 1:1 (v / v) for 2 h. After vigorous shaking every 15 min, the mixture was allowed to stand and separate into layers. The organic phase (lower layer) was concentrated and dried in a rotary evaporator at a temperature of 25–30 °C. The solution was then dissolved in 1.5 mL of dichloromethane. An appropriate amount of the solution was filtered through a 0.22 μm microporous membrane and the erucamide content was determined by GC / MS.

Claims

1. A strain of *Alternaria leucocephala* J2, characterized by: This fungus is classified and named as follows: Irpex lacteus, It was deposited at the China Center for Type Culture Collection on November 24, 2023, with accession number CCTCC NO: M20232318.

2. The *Alternaria alternata* strain described in claim 1 (… Irpex lacteus Application of J2 CCTCC NO: M20232318 in the preparation of lignocellulase.

3. The *Alternaria alternata* strain described in claim 1 (… Irpex lacteus Application of J2 CCTCC NO: M20232318 in the production of erucamide from fermented corn stalks.

4. A method for producing erucic acid amide: Mature corn stalks are crushed and passed through an 80-mesh sieve, then used as the sole carbon source for the fermentation medium, followed by *Bacillus thuringiensis* (…). Irpex lacteus J2 CCTCC NO: M20232318 was inoculated into fermentation medium for fermentation culture at 30 ℃ and 150 rpm. After fermentation, the bacterial suspension was centrifuged at 12000 rpm for 10 min. The fermentation broth was mixed with dichloromethane at a volume ratio of 1:1 and extracted for 2 h. After standing and separating the layers, the lower organic phase was concentrated and dried in a rotary evaporator and then dissolved in 2 mL of dichloromethane to obtain the product erucamide.

5. The method according to claim 4, characterized in that, The fermentation medium was formulated as follows: 10 g / L of ball-milled corn stalks passed through an 80-mesh sieve as the carbon source; 0.2 g / L of ammonium tartrate; 2 g / L of KH₂PO₄; 0.71 g / L of MgSO₄·7H₂O; 0.1 g / L of CaCl₂; and 70 mL of trace element solution. The trace element solution contained 1 g / L NaCl; 0.184 g / L CoCl₂·6H₂O; 0.1 g / L of FeSO₄·7H₂O; 0.1 g / L of ZnSO₄·7H₂O; 0.1 g / L of CuSO₄; 0.1 g / L of H₃BO₃; 0.01 g / L of Na₂MoO₄·2H₂O; 0.01 g / L of KAl(SO₄)₂·12H₂O; and 1.5 g / L of N-triacetic acid.

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