Irpex lacteus and application of Irpex lacteus in production of erucyl amide by degrading corn straw through fermentation

Abstract

The invention discloses Irpex lacteus and an application of the Irpex lacteus in production of erucyl amide by degrading corn straws through fermentation. The Irpex lacteus is classified and named as Irpex lacteus, the name of the Irpex lacteus is Irpex lacteus J2, the Irpex lacteus is preserved in the China Center for Type Culture Collection on November 24, 2023, and the preservation number of the Irpex lacteus is M20232318. The strain has a complete lignocellulose degrading enzyme system and is used for producing erucyl amide by taking corn straws as a unique carbon source.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, in particular to a strain of white sac Rhacophora and application thereof in degrading corn straw and fermenting to produce erucamide. Background Art

[0002] Erucamide is a long-chain fatty acid amide with the chemical formula C 22 H 43 NO, a chemical with a double bond and an amide group in its molecular structure, is crucial for its unique biological activity and function. As a broad-spectrum plant defense metabolite, erucamide effectively inhibits the pathogenicity of a wide range of plant and animal pathogens by inhibiting the bacterial type III secretion system (T3SS), blocking the process by which pathogens inject virulence proteins into host cells. This anti-virulence mechanism not only provides new insights into the development of environmentally friendly biopesticides but also opens new avenues for breeding disease-resistant crops, offering significant application value in agricultural disease control. Erucamide has a wide range of industrial applications. As a multifunctional additive, it plays a vital role in industries such as plastics, rubber, coatings, inks, textiles, and packaging. Furthermore, it is used in metalworking and paper processing to improve processing efficiency and product quality. Due to its versatility, erucamide has become an indispensable additive in industrial processing, driving technological innovation and product upgrades across multiple industries. Currently, erucamide is produced industrially through chemical synthesis and plant extraction. Erucamide is produced by amidation reaction of erucic acid with ammonia or amines. Chemical methods are mature and offer high yields, but they involve high temperatures and pressures, require high equipment requirements, and are prone to secondary pollution. Plant-based extraction still faces challenges such as insufficient rapeseed oil raw material and low yields. Microbial synthesis, however, involves converting fatty acids into erucamide through metabolic pathways within the body. This method is mild and environmentally friendly. Therefore, screening for microorganisms capable of producing erucamide provides new insights and technical support for the development of green biomanufacturing technologies. Summary of the Invention

[0003] The invention aims to provide a strain of white capsule rhabdocystis and application thereof in the production of erucamide by fermentation through degradation of corn straw.

[0004] The use of Irpex lacteus J2 in the preparation of lignocellulase also falls within the protection scope of the present invention.

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

[0006] The present invention also provides the use of the white sac rake tooth fungus Irpex lacteus J2 in producing erucamide by fermenting corn straw.

[0007] The present invention discloses Irpex lacteus J2, a strain that possesses a complete lignocellulose-degrading enzyme system and produces erucamide using corn straw as a sole carbon source. Strain F81 was deposited with the China Center for Type Culture Collection on November 24, 2023, with the accession number M20232318. The address of the deposit is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] Irpex lacteus J2 possesses a complete lignocellulose-degrading enzyme system and the ability to secrete it extracellularly. Extracellular enzyme activity assays have identified enzymes including laccase, manganese peroxidase, lignin peroxidase, exoglucanase, endoglucanase, β-glucosidase, and xylanase. Furthermore, Irpex lacteus J2 exhibits excellent degradation of corn stover and produces erucamide using corn stover as its sole carbon source. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0010] Figure 2 This is a diagram showing the degradation effect of Irpex lacteus J2 on Congo red plate.

[0011] Figure 3 This is the phylogenetic tree of Irpex lacteus J2.

[0012] Figure 4 To determine the activities of laccase, lignin peroxidase and manganese peroxidase of Irpex lacteus J2.

[0013] Figure 5 To determine the activities of exoglucanase, endoglucanase and β-glucosidase of Irpex lacteus J2.

[0014] Figure 6 This is the determination of hemicellulase activity of Irpex lacteus J2.

[0015] Figure 7 This is the standard curve of erucamide from Irpex lacteus J2.

[0016] Figure 8This is the chromatographic peak pattern of erucamide from Irpex lacteus J2.

[0017] Biomaterial Deposit

[0018] Name: Irpex lacteus J2;

[0019] Taxonomic nomenclature: Irpex lacteus;

[0020] Deposit date: November 24, 2023;

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

[0022] Deposit number: CCTCC No:M20232318. DETAILED DESCRIPTION

[0023] In order to better illustrate the purpose, complete technical route and advantages of the present invention, the present invention will be further described in detail below with reference to specific embodiments and drawings. However, the embodiments described below are only part of the embodiments of the present invention and do not include all embodiments. Based on the embodiments of the present invention, other embodiments that can be obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

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

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

[0026] 1. Obtaining Irpex lacteus J2

[0027] The fruiting bodies and rotten bark of wood-decaying fungi were collected from ancient trees, and the single spore separation method was used to separate and purify the wood-decaying fungi ( Figure 1 ).

[0028] (1) Preparation of spore suspension: Rinse the fruiting bodies of the wood-decaying fungus and the surface of the decayed bark with sterile water or buffer to collect the spores. Vortex or bead-beat to form a single-spore suspension and dilute until dispersed spores are visible under a microscope.

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

[0030] 2. Determination of the Degradation Ability of Irpex lacteus J2 on Carboxymethyl Cellulose

[0031] The purified strain was inoculated into a 7mm cake on a carboxymethyl cellulose medium (2.0g ammonium sulfate, 0.5g magnesium sulfate heptahydrate, 1g potassium dihydrogen phosphate, 0.1g sodium chloride, 5.0g sodium carboxymethyl cellulose, 20g agar, pH 7.0, 1000mL water), cultured at 28°C for 3 days, stained with 2% Congo red for 15 minutes, and then decolorized with 0.9% NaCl. The transparent zone on the Congo red plate was observed. As can be seen from the figure, the strain has a very strong ability to degrade carboxymethyl cellulose ( Figure 2 ).

[0032] 3. Identification of Irpex lacteus J2

[0033] The genomic DNA of strain J2 was extracted and the ITS was amplified by PCR using the universal bacterial primers ITS1 and ITS4. The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The ITS sequence of strain J2 is shown in Sequence Listing 1. The sequencing results were compared by BLAST and the homologous sequences were analyzed by phylogenetic analysis using MEGA 7.0 software. The results showed that the strain was Irpex lacteus and was named Irpex lacteus J2 ( Figure 3 ).

[0034] Irpex lacteus J2 was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China), with a deposit date of November 24, 2023, and a deposit number of CCTCC NO: M20232318.

[0035] Example 2. Determination of enzyme activity of strain Irpex lacteus J2

[0036] The preserved Irpex lacteus J2 strain was first activated and cultured, and then the vigorous mycelium was selected and inoculated into a liquid culture medium (200g of peeled potatoes, 20g of glucose, 1000mL of distilled water, natural pH) to prepare a seed solution. The seed solution was inoculated into a 250ml conical flask containing 50ml of the initial enzyme production medium at a 5% inoculum, and cultured in a constant temperature shaker at 30°C and 180rpm to obtain a fermentation broth. The formula of the enzyme production medium: ball-milled corn straw (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 contains 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 nitrilotriacetic acid.

[0038] Enzyme activity determination method:

[0039] (1) Laccase: 37°C, 3 mL reaction system, the reaction mixture contains 2 mL of 0.5 mmol / L ABTS, 1 mL of fermentation broth is added to start the reaction, and the change in absorbance is measured at 420 nm after 3 minutes.

[0040] (2) Lignin peroxidase: 37°C, 3 mL reaction system, the reaction mixture contains 1.8 mL 0.24 mmol / L resveratrol and 1.1 mL fermentation broth, preheated to 37°C, and 0.1 mL 6 mmol / L H2O2 is added to start the reaction. The increase in absorbance at 310 nm after 3 min is measured.

[0041] (3) Manganese peroxidase: 37°C, 3 mL reaction system, the reaction mixture contains 2.3 mL 50 mM pH 8.5 acetate buffer, 0.1 mL 1.6 mM MnSO4 solution, 0.4 mL fermentation broth, 0.1 mL 1.6 mM H2O2 solution is added at 37°C to start the reaction, and the change in absorbance at 240 nm is measured within the first 3 minutes.

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

[0043] (5) Endoglucanase: 50 μL of fermentation broth was mixed with 150 μL of 0.5% CMCNa, reacted at 50°C for 30 min, and then 50 μL of 1 M NaOH solution and 150 μL of DNS solution were added to terminate the reaction.

[0044] (6) β-glucosidase: 200 μL of fermentation broth and 100 μL of 1 mg / mL pNPG solution were mixed and reacted at 50°C for 30 min. 300 μL of 10% Na2CO3 was added to terminate the reaction.

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

[0046] Strain J2 was fermented in enzyme-producing medium at 28°C, and the activities of laccase, lignin peroxidase, and manganese peroxidase were determined at different fermentation time periods. Figure 4 As shown, the activities of laccase, lignin peroxidase, and manganese peroxidase all increased first and then decreased with fermentation time. 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 increasing first and then decreasing. The exoglucanase activity reached its highest level on the 7th day, which was 0.009 U / mL. The endoglucanase activity and β-glucosidase activity both reached their highest levels on the 9th day of fermentation, which were 0.945 U / mL and 0.013 U / mL respectively. Figure 5 ).

[0048] Depend on Figure 6 As shown in the figure, the enzyme activity changed with the influence of time, and the hemicellulase activity reached the maximum value of 12.039 U / mL on the 9th day.

[0049] Example 3. Production of erucamide by fermentation of corn straw 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 a mass concentration of 1 mg / mL. Dilute the standard stock solution with dichloromethane to standard solutions with mass 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 ((x, μg / mL)) with mass concentration (x, μg / mL) as the horizontal axis and peak area (y) as the vertical axis. Figure 7 ).

[0052] 2) Erucamide determination method

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

[0054] 3) Sample processing and detection of Irpex lacteus J2 fermentation

[0055] The preserved Irpex lacteus J2 strain was first activated and cultured. Strongly growing mycelia were then selected and inoculated into a liquid culture medium (200 g peeled potatoes, 20 g glucose, 15-20 g agar, 1000 mL distilled water, natural pH) to prepare an Irpex lacteus J2 seed solution. The Irpex lacteus J2 seed solution was inoculated at 10% (v / v) into a fermentation medium containing 10 g / L ball-milled corn stover (passed through an 80-mesh sieve) as a carbon source; 0.2 g / L ammonium tartrate; 2 g / L KH2PO4; 0.71 g / L MgSO4·7H2O; 0.1 g / L CaCl2; and 70 mL of a trace element solution containing 1 g / L NaCl, 0.184 g / L CoCl2·6H2O, and 70 mL trace element solution. A mixture of 50 mL / 250 mL of 1% nitric acid (0.1 g / L 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, and 1.5 g / L nitrilotriacetic acid was prepared. The suspension was incubated at 30°C, 180 rpm, and cultured for 15 days. The suspension was centrifuged at 12,000 rpm for 10 minutes. The supernatant was 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 stratification was allowed to stand. The organic phase (lower layer) was concentrated and dried on a rotary evaporator at a temperature of 25-30 ° C. It was then dissolved with 1.5 mL of dichloromethane. An appropriate 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 Irpex lacteus J2 fermentation sample is 9.435 min, which is consistent with the erucamide standard. The erucamide content in the Irpex lacteus J2 fermentation broth sample is 0.284 mg / L.

[0056] In summary, the white capsule rake tooth fungus (Irpex lacteus) J2 of the present invention can produce erucamide using corn straw as the sole carbon source. The specific method can be: first activate and culture the preserved strain, then select the vigorous growing mycelium and inoculate it into a liquid culture medium to prepare a seed solution. The seed solution is inoculated at a 5% inoculum into 50 ml of fermentation medium (ball-milled corn straw as a 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 a trace element solution. The trace element solution contains 1 g / L The mixture was incubated in a 250ml Erlenmeyer flask containing 0.184g / L CoCl2·6H2O, 0.1g / L FeSO4·7H2O, 0.1g / L ZnSO4·7H2O, 0.1g / L CuSO4, 0.1g / L H3BO3, 0.01g / L Na2MoO4·2H2O, 0.01g / L KAl(SO4)2·12H2O, and 1.5g / L nitrilotriacetic acid. The mixture was incubated in a shaker at 30°C and 180 rpm. After 15 days of fermentation, the Aspergillus suspension was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was 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 broth was allowed to stand for stratification. The organic phase (lower layer) was concentrated and dried on a rotary evaporator at a temperature of 25-30°C, and then dissolved in 1.5 mL of dichloromethane. An appropriate amount of the solution was filtered through a 0.22 μm microporous filter membrane and the erucamide content was determined by GC / MS.

Claims

1. A strain of White Sac Rake Tooth Fungus, characterized by: The white sac rake tooth fungus was classified and named Irpex lacteus, named Irpex lacteus J2, and was deposited in the China Center for Type Culture Collection on November 24, 2023, with the preservation number CCTCC NO: M20232318.

2. The white capsule rake tooth fungus Irpex lacteus J2 CCTCC NO: Application of M20232318 in the preparation of lignocellulase.

3. The white capsule rake tooth fungus Irpex lacteus J2 CCTCC NO: Application of M20232318 in the production of erucamide by fermentation of corn straw.

4. A method for producing erucamide: Mature corn stalks are pulverized and passed through an 80-mesh sieve, then used as the sole carbon source for a fermentation medium. Irpex lacteus J2CCTCC NO: M20232318 is then inoculated into the fermentation medium for fermentation at 30°C and 150 rpm. After fermentation, the bacterial suspension is centrifuged at 12,000 rpm for 10 minutes. The fermentation broth is mixed with dichloromethane in a 1:1 volume ratio and extracted for 2 hours. The mixture is allowed to stand for separation, and the lower organic phase is concentrated and dried on 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 is formulated as follows: 10 g / L ball-milled corn straw passed through an 80-mesh sieve as a carbon source; 0.2 g / L ammonium tartrate; 2 g / L KH2PO4; 0.71 g / L MgSO4·7H2O; 0.1 g / L CaCl2; and 70 mL of a trace element solution containing 1 g / L NaCl; 0.184 g / L CoCl2·6H2O; FeSO4·7H2O, 0.1g / L; ZnSO4·7H2O, 0.1g / L; CuSO4, 0.1g / L; H3BO3, 0.01g / L; Na2MoO4·2H2O, 0.01g / L; KAl(SO4)2·12H2O, 0.01g / L and 1.5g / L nitrilotriacetic acid.

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