Application of pycnoporus sanguineus in oxidizing abamectin
By using the hemoglobin JPS-W5 activator to oxidize avermectin under specific conditions, the problems of insufficient selectivity of existing oxidant and difficult to control reaction conditions are solved, and efficient and environmentally friendly preparation of oxidized avermectin is achieved.
Patent Information
- Application Number
- CN202411993731.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-03-28
AI Technical Summary
The oxidants used in existing avermectin are not selective, with safety risks and environmental pollution problems, and the reaction conditions are difficult to control.
The yield and reaction efficiency of oxidized avermectin were improved by activating the strain and oxidizing reaction under specific conditions.
The high yield of avermectin oxidized, mild reaction conditions, no pollution, low cost and easy operation are achieved.
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Figure CN119709914A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of chemical industry, and in particular to application of Pycnoporus sanguineus in oxidizing avermectin. Background Art
[0002] Emamectin benzoate (emamectin benzoate) is a new type of highly effective antibiotic insecticide synthesized from fermentation product avermectin B1 as the starting material. Emamectin benzoate has the characteristics of super high efficiency, low toxicity, no residue, and no pollution. It is a biological pesticide advocated by the state, safe and environmentally friendly, and has broad development prospects. The research on the synthesis technology of emamectin benzoate mainly uses avermectin as the raw material, and obtains the product through six steps of protection, oxidation, amination, reduction, deprotection and salt formation. At present, the oxidants used in the oxidation of avermectin mainly include Jones reagent and manganese dioxide, etc. However, there are still many problems in the use of existing oxidants in the oxidation of avermectin, including the following: (1) Low selectivity: In the oxidation reaction, it is difficult to completely control the oxidant to oxidize only the C4″ hydroxyl group, and excessive oxidation or unexpected oxidation reactions may occur at other sites; (2) The safety and pollution of the oxidant are difficult to control: Some commonly used oxidants have safety risks during use and storage, and the post-treatment of these oxidants will generate a large amount of waste, causing environmental pollution; (3) The reaction conditions are difficult to control: The temperature, time, amount of oxidant and other conditions of the oxidation reaction have a great influence on the reaction results; therefore, it is of great significance to develop an oxidant with high selectivity, mild reaction conditions and less "three wastes". Summary of the invention
[0003] In view of the above problems, the present invention provides an application of Pycnoporus sanguineus in oxidizing avermectin. The Pycnoporus sanguineus JPS-W5 provided by the present invention has high laccase activity, and the application of Pycnoporus sanguineus JPS-W5 in the reaction of oxidizing avermectin has the characteristics of high product yield, mild reaction conditions, no pollution, low cost and simple operation.
[0004] In order to solve the above technical problems, the technical solution provided by the present invention is:
[0005] The first aspect of the present invention provides an application of Pycnoporus sanguineus in oxidizing avermectin. The Pycnoporus sanguineus is Pycnoporus sanguineus JPS-W5, and its preservation number is CGMCC No.40368.
[0006] Compared with the prior art, the invention provides P. sanguinis JPS-W5, which is an active bacterium selected and bred by heavy ion irradiation using P. sanguinis ACCC51180 as the original bacterium, and uses propanol as a reaction substrate for oxidation reaction research, and selects P. sanguinis JPS-W5 as a high-yield strain. The conversion rate of propanol oxidized by P. sanguinis JPS-W5 is increased by 54.6% compared with the conversion rate of propanol oxidized by P. sanguinis ACCC 51180, and the yield is increased by 59.2%. In addition, the invention finds through research that the laccase activity of the mutant strain P. sanguinis JPS-W5 is significantly higher than that of the starting bacterium P. sanguinis ACCC51180, and the application of P. sanguinis JPS-W5 in the reaction of oxidizing avermectin has the characteristics of high product yield, mild reaction conditions, no pollution, low cost and simple operation.
[0007] In a second aspect, the present invention provides a method for oxidizing avermectin, comprising the following steps:
[0008] S1, protecting the 5-hydroxyl group of avermectin to obtain the 5-hydroxyl protected avermectin shown in formula I;
[0009] S2, activating the Pycnoporus sanguineus JPS-W5 to obtain an activated bacterial solution;
[0010] S3, under the conditions of a carbon source solution and an organic solvent, the activated bacterial solution and the 5-hydroxyl-protected avermectin shown in formula I are subjected to an oxidation reaction to obtain an oxidation product shown in formula II;
[0011]
[0012] The method for oxidizing avermectin provided by the present invention can further improve the growth and metabolic capacity of Pycnoporus sanguineus JPS-W5 by limiting the addition of a carbon source in the reaction, thereby improving the activity of Pycnoporus sanguineus JPS-W5 in producing a large amount of laccase, and greatly improving the yield of oxidized avermectin; and the present invention also limits the reaction temperature, promotes Pycnoporus sanguineus JPS-W5 to reach a maximum laccase production amount, and further improves the yield of oxidized avermectin.
[0013] It should be further explained that, preferably, in S1, the preparation of the 5-hydroxyl-protected avermectin shown in formula I can be prepared by conventional preparation methods.
[0014] Preferably, in S2, the concentration of live bacteria of Pycnoporus sanguinosa JPS-W5 in the activated bacterial solution is 10 7 CFU / mL-10 9 CFU / mL.
[0015] The number of live bacteria in the activated bacterial solution is small, and the rate of the product is low. The number of live bacteria is large, and the viscosity of the reaction solution increases, which not only reduces the reaction rate but also affects the yield of the oxidized avermectin product. The present invention further improves the yield of the reaction product by limiting the number of live bacteria of Pycnoporus sanguinosa JPS-W5 in the activated bacterial solution.
[0016] Preferably, in S2, the culture medium used for activation is liquid PDA culture medium.
[0017] Further preferably, in S2, the formula of the liquid PDA medium (g / L) is: 200 potatoes, 20 glucose, KH 2 PO 4 1.5, MgSO 4 1. Water 1000 mL, pH 3.
[0018] Further preferably, the basal culture medium needs to be sterilized at 121° C. for 15 min.
[0019] Preferably, in S3, the carbon source in the carbon source solution is glucose.
[0020] Preferably, in S3, the organic solvent is dimethyl sulfoxide.
[0021] Preferably, in S3, the mass volume ratio of the activated bacterial solution to the carbon source in the carbon source solution is 1 mL: (0.2-0.6) g, and the concentration of the carbon source solution is (70-90) g / L 。
[0022] Preferably, in S3, the volume ratio of the activated bacterial solution to the organic solvent is 1:(0.1-0.3).
[0023] Preferably, in S3, the mass volume ratio of the activated bacterial solution and the 5-hydroxyl-protected avermectin is 1 mL: (0.1-0.3) g.
[0024] Preferably, in S3, the temperature of the oxidation reaction is 50°C-55°C, the time is 8d-12d, and the pH is 4-6.
[0025] It should be further explained that in S3, the carbon source solution needs to be further sterilized at high temperature and cooled to room temperature before use.
[0026] Preferably, the high temperature sterilization temperature is 121° C. and the time is 15 minutes.
[0027] Further preferably, in S2, after the oxidation reaction, the process further comprises: separating the reaction liquid after the oxidation reaction into solid and liquid, adjusting the pH of the obtained liquid phase to 12, standing for phase separation, extracting with ethyl acetate, drying the organic phase, filtering, and concentrating to obtain the oxidation product shown in Formula II. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is a statistical diagram showing the relationship between the lethality and lethal dose of Pycnoporus sanguineus ACCC51180 in Example 1 of the present invention;
[0029] Figure 2 This is a screening diagram for high-yield strains induced by irradiation in Example 1 of the present invention. DETAILED DESCRIPTION
[0030] The technical solution of the present invention will be described clearly and completely below. It should be understood that the specific embodiments described herein can enable those skilled in the art to have a more comprehensive understanding of the present invention, but the present invention is not limited to the scope of the embodiments.
[0031] Bacteria, reagents and culture media:
[0032] Pycnoporus sanguineus ACCC 51180: purchased from China Agricultural Microbiological Culture Collection Center, with the collection number of ACCC 51180;
[0033] The formula of solid PDA medium (g / L) is: potato 200, glucose 20, KH 2 PO 4 1.5, MgSO 4 1. Agar 20, water 1000mL, natural pH;
[0034] The formula of liquid PDA medium (g / L) is: potato 200, glucose 20, KH 2 PO 4 1.5, MgSO 4 1. 1000 mL of water, pH adjusted to 3;
[0035] Activation of bacteria:
[0036] The strain of Pycnoporus sanguineus ACCC 51180 was inoculated on solid PDA medium, cultured at 28°C for 7 days, then transferred to liquid PDA medium for shaking culture, cultured at 28°C at a speed of 150 r / min for 7 days, and then taken out for subsequent experiments.
[0037] Determination of laccase activity:
[0038] The reaction system was 3 mL, including 0.5 mL DMP, 2.4 mL phosphate-citrate buffer solution (20 mmol / L, pH = 3) and 0.1 mL enzyme solution. The OD absorption value was measured at 470 nm. 1 μmol of DMP was oxidized in 1 min to generate 3,5,3',5'2-tetramethoxydiphenylquinone [ε 470=49.6 L / (mmol·cm)] is defined as 1 enzyme activity unit (U).
[0039] The preparation method of 5-hydroxyl-protected avermectin comprises the following preparation steps:
[0040] In a 500mL three-necked flask, add 50g (0.0334mol) of avermectin, add 200mL of dichloromethane to dissolve under stirring, place in a low-temperature cold trap, cool to -20°C, add 13.09g of allyl chloroformate (0.050mol), stir for 60min before the reaction is terminated, extract the reaction solution with ethyl acetate (200ml×3), and adjust the pH of the extract phase to 7 with a saturated sodium carbonate solution. After concentration, 62.29g of 5-hydroxy-protected avermectin was obtained with a yield of 96%.
[0041] In order to better illustrate the present invention, further examples are given below.
[0042] Example 1
[0043] This example is used to illustrate the acquisition and preservation of Pycnoporus sanguineus JPS-W5.
[0044] 1. Obtaining strains
[0045] (1) Treatment of irradiated samples
[0046] The ACCC 51180 cultured on a slant medium stored at 4°C was activated and subcultured. A 2 cm 2 The bacterial block was placed in 5 mL of physiological saline and fully ground with a grinder. A uniform bacterial suspension was prepared with liquid PDA medium and dispensed into sterilized 50 mm small plates, 1 mL per plate. The plates were sealed with sealing film to prevent the bacterial suspension from overflowing. The prepared bacterial suspension was immediately irradiated or stored at 4 °C for 12 h.
[0047] (2) Selection of irradiation method and dose
[0048] The heavy ion beam used was 12 C 6+ The irradiation method was injection, the bacterial suspension was located at the Bragg peak, and the doses were 20Gy, 30Gy, 40Gy, and 50Gy. Each dose was divided into three parallels, and a blank control was also set up.
[0049] (3) Determination of lethality and lethal dose after radiation mutagenesis
[0050] The unirradiated blank control and the bacterial suspensions of each dose group after irradiation were spread on plate culture medium respectively, and the bacterial suspensions were diluted and colonies were counted. The ratio of the number of colonies before and after irradiation was statistically calculated, and then the lethality was calculated and the lethal dose was determined. Each dose was measured three times, and the average value was taken as the basis for calculating the lethality.
[0051] (4) Lethality and lethal dose of ACCC 51180 injected
[0052] The dose range of 20Gy-50Gy was selected for injection irradiation. The relationship between irradiation dose and mortality rate is as follows: Figure 1 As shown in the figure, within the dose range shown in the figure, the lethality rate increases with the increase of dose, the lethality rate is 97% at 40Gy, and the lethality rate is 100% at 50Gy, so the lethal dose of Pycnoporus sanguineus ACCC 51180 after carbon ion implantation irradiation is between 40Gy and 50Gy.
[0053] (5) Preservation of bacterial strains
[0054] The bacterial suspension before and after irradiation was stored in a refrigerator at 4°C.
[0055] (6) Activation of bacteria
[0056] Take 0.2 mL of bacterial suspension, add 5 mL of sterile water to make a suspension, inoculate it into a 250 mL Erlenmeyer flask containing 50 mL of basal medium, and culture it in a shaker at 30 °C and 160 r / min for 72 h; take out the proliferation solution and dilute it to 10 -5 CFU / mL-10 -7 CFU / mL, use a pipette to accurately draw 100 μL of each concentration, inoculate on the plate separation medium by spreading method, culture at 30℃ for 72h, pick out single colonies with large transparent circle and thick colonies, streak inoculate on the slant preservation medium, culture at 30℃ for 48h, seal with sterile liquid paraffin, and store in a 4℃ refrigerator.
[0057] (7) Culture of bacteria
[0058] Take the slant-preserved strains and use sterile saline to prepare bacterial suspensions before and after irradiation (so that the bacterial content is about 10 6 Accurately pipette 10 mL of the bacterial suspension into 100 mL of proliferation medium and culture at 30°C, 160 rpm for 48 h.
[0059] (8) Preparation of propionic acid
[0060] Add 50mL of distilled water and 2g of glucose to a 100mL conical flask, stir and dissolve, sterilize at high temperature (121℃, 15min), cool to room temperature, add 5mL of proliferation solution before and after irradiation and 0.5g of propanol to the reaction bottle respectively, heat to 37℃ for reaction, take samples, and track the reaction progress by GC; after the reaction, centrifuge the bacterial solution, filter the supernatant, add 5% NaOH solution to alkalize to pH>10, extract, separate the organic phase, and determine the conversion rate of alcohol by GC; acidify the aqueous phase with 5% hydrochloric acid to pH<2, centrifuge, extract with ethyl acetate, dry with anhydrous magnesium sulfate, filter, and concentrate to obtain propionic acid; bp140-141℃; 1 HNMR (CDCl 3 ,600MHz)δ:9.26(s,1H),2.41(q,J=7.6Hz,2H),1.17(t,J=7.6Hz,3H); IR(KBr)v,cm -1 :2988(OH),1716(C=O).
[0061] (9) Positive mutation rate of surviving strains after injection of irradiation
[0062] The oxidation of propanol to propionic acid by the irradiated bacterial suspension was used as an indicator. The conversion rate and yield of propionic acid were determined. The strains with a fermentation yield 30% higher than that of the unirradiated strain were defined as positive mutants, and the percentage of positive mutants in the surviving strains was calculated. The results are shown in Figure 2 As shown, with the increase of irradiation dose, the proportion of positive mutants in surviving bacteria increased, reaching 45% at 40 Gy.
[0063] The irradiated bacterial suspension was diluted and spread on the plate, and the single colony was isolated and fermented in a shake flask. Each strain was repeated 3 times. After the propanol oxidation propionic acid fermentation was completed, the conversion rate and yield were determined. High-yield strains were selected according to the yield. A total of 20 surviving strains were isolated at a dose of 40Gy. There were 9 strains with a 40% increase in yield. From them, a strain with a conversion rate of 92.5% and a yield of 95.4% was selected (numbered JPS-W5), which was 54.6% higher than the conversion rate of the starting strain of 37.9%; the yield of the starting strain of 36.2% was increased by 59.2%;
[0064] 2. Comparison of laccase activity among strains
[0065] Pycnoporus sanguineus ACCC 51180 and Pycnoporus sanguineus JPS-W5 were cultured at 55°C for 8 days, with a pH value of 3. Under exactly the same culture conditions, the laccase activity of the starting strain and the variant strain was compared as follows:
[0066] Strain name Laccase activity (U / g) Pycnoporus sanguineus ACCC 51180 6.8 Pycnoporus sanguineus JPS-W5 53.1
[0067] By comparison, the laccase activity of the mutant Pycnoporus sanguineus JPS-W5 was significantly higher than that of the original strain Pycnoporus sanguineus ACCC 51180.
[0068] 3. Preservation of strains
[0069] The above-obtained strain Pycnoporus sanguineus JPS-W5 was deposited in the China Center for Type Culture Collection on December 26, 2022, with the deposit number CGMCC No.40368.
[0070] Example 2
[0071] This embodiment provides a method for preparing oxidized avermectin, comprising the following steps:
[0072] S1. Activate Pycnoporus sanguineus JPS-W5 with liquid PDA medium to obtain a live bacterial concentration of 10 7 CFU / mL of activated bacterial solution; wherein the formula of liquid PDA medium (g / L) is: potato 200, glucose 20, KH 2 PO 4 1.5, MgSO 4 1. 1000 mL of water, pH to 3 ；
[0073] S2. Add 500 mL of distilled water and 40 g of glucose to a 1000 mL conical flask, stir and dissolve, sterilize at high temperature (121°C, 15 min), cool to room temperature, add 100 mL of activated bacterial solution, then add 20 mL of dimethyl sulfoxide (DMSO) and 10 g of 5-hydroxy-protected avermectin shown in formula I, heat to 50°C for oxidation reaction, control the pH during the reaction to be 4, shake flask fermentation for 8 days, centrifuge the bacterial solution, filter the supernatant, add 5% NaOH solution by mass to alkalize to pH 12, stand for phase separation, adjust the pH of the aqueous phase to <2 with 5% hydrochloric acid by mass, centrifuge, extract with ethyl acetate, dry over anhydrous magnesium sulfate, filter, and concentrate to obtain the oxidation product shown in formula II, wherein the purity of the oxidation product shown in formula II is 96.3%, and the yield is 90%.
[0074]
[0075]
[0076] Example 3
[0077] This embodiment provides a method for preparing oxidized avermectin, comprising the following steps:
[0078] S1. Activate Pycnoporus sanguineus JPS-W5 with liquid PDA medium to obtain a live bacterial concentration of 10 8CFU / mL of activated bacterial solution; wherein the formula of liquid PDA medium (g / L) is: potato 200, glucose 20, KH 2 PO 4 1.5, MgSO 4 1. Water 1000 mL, pH 3 ；
[0079] S2. Add 600 mL of distilled water and 20 g of glucose to a 1000 mL conical flask, stir and dissolve, sterilize at high temperature (121°C, 15 min), cool to room temperature, add 100 mL of activated bacterial solution, then add 10 mL of dimethyl sulfoxide (DMSO) and 20 g of 5-hydroxy-protected avermectin shown in formula I, heat to 55°C for oxidation reaction, control the pH during the reaction to be 5, shake flask fermentation for 10 days, centrifuge the bacterial solution, filter the supernatant, add 5% NaOH solution by mass to alkalize to pH 12, stand for phase separation, adjust the pH of the aqueous phase to <2 with 5% hydrochloric acid by mass, centrifuge, extract with ethyl acetate, dry over anhydrous magnesium sulfate, filter, and concentrate to obtain the oxidation product shown in formula II, wherein the purity of the oxidation product shown in formula II is 95.4%, and the yield is 91%.
[0080]
[0081] Example 4
[0082] This embodiment provides a method for preparing oxidized avermectin, comprising the following steps:
[0083] S1. Activate Pycnoporus sanguineus JPS-W5 with liquid PDA medium to obtain a live bacterial concentration of 10 9 CFU / mL of activated bacterial solution; wherein the formula of liquid PDA medium (g / L) is: potato 200, glucose 20, KH 2 PO 4 1.5, MgSO 4 1. Water 1000 mL, pH 3 ；
[0084] S2. Add 300 mL of distilled water and 60 g of glucose to a 1000 mL conical flask, stir and dissolve, sterilize at high temperature (121°C, 15 min), cool to room temperature, add 100 mL of activated bacterial solution, then add 30 mL of dimethyl sulfoxide (DMSO) and 30 g of 5-hydroxy-protected avermectin shown in formula I, heat to 52°C for oxidation reaction, control the pH during the reaction to be 6, shake flask fermentation for 12 days, centrifuge the bacterial solution, filter the supernatant, add 5% NaOH solution by mass to alkalize to pH 12, stand for phase separation, adjust the pH of the aqueous phase to <2 with 5% hydrochloric acid by mass, centrifuge, extract with ethyl acetate, dry over anhydrous magnesium sulfate, filter, and concentrate to obtain the oxidation product shown in formula II, wherein the purity of the oxidation product shown in formula II is 96.1%, and the yield is 89%.
[0085]
[0086]
[0087] Comparative Example 1
[0088] This comparative example provides a method for preparing oxidized avermectin, which differs from Example 2 in that:
[0089] The sanguineous pycnoporus JPS-W5 was replaced with an equal amount of sanguineous pycnoporus ACCC 51180, and the other components, contents and preparation methods were the same as those in Example 1; the results showed that the purity of compound 2 was 86% and the yield was 41%. 。
[0090] The above-described embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. An application of Pycnoporus sanguineus in oxidized avermectin, characterized in that: The Pycnoporus sanguineus is Pycnoporus sanguineus JPS-W5, and its preservation number is CGMCC No.40368.
2. A method for oxidizing avermectin, characterized in that: The following steps are involved: S1, protecting the 5-hydroxyl group of avermectin to obtain the 5-hydroxyl protected avermectin shown in formula I; S2, activating the Pycnoporus sanguineus JPS-W5 according to claim 1 to obtain an activated bacterial solution; S3, under the conditions of a carbon source solution and an organic solvent, the activated bacterial solution and the 5-hydroxyl-protected avermectin shown in formula I are subjected to an oxidation reaction to obtain an oxidation product shown in formula II; 3. The method for oxidizing avermectin as claimed in claim 2, characterized in that: In S2, the concentration of live bacteria of Pycnoporus sanguinosa JPS-W5 in the activated bacterial solution is 10 7 CFU / mL-10 9 CFU / mL.
4. The method for oxidizing avermectin as claimed in claim 3, characterized in that: In S3, the mass volume ratio of the activated bacterial solution to the carbon source in the carbon source solution is 1 mL: (0.2-0.6) g, and the concentration of the carbon source solution is (70-90) g / L.
5. The method for oxidizing avermectin as claimed in claim 3, characterized in that: In S3, the volume ratio of the activated bacterial solution to the organic solvent is 1:(0.1-0.3).
6. The method for oxidizing avermectin as claimed in claim 3, characterized in that: In S3, the mass volume ratio of the activated bacterial solution and the 5-hydroxyl-protected avermectin is 1 mL: (0.1-0.3) g.
7. The method for oxidizing avermectin as claimed in claim 2, characterized in that: In S3, the carbon source in the carbon source solution is glucose.
8. The method for oxidizing avermectin as claimed in claim 2, characterized in that: In S3, the organic solvent is dimethyl sulfoxide.
9. The method for oxidizing avermectin as claimed in claim 2, characterized in that: In S3, the temperature of the oxidation reaction is 50°C-55°C, the time is 8d-12d, and the pH is 4-6.
10. The method for oxidizing avermectin according to claim 2, characterized in that: In S3, after the oxidation reaction, the process further includes: separating the reaction liquid after the oxidation reaction into solid and liquid, adjusting the pH of the obtained liquid phase to 12, standing for phase separation, extracting with ethyl acetate, drying the organic phase, filtering, and concentrating to obtain the oxidation product shown in Formula II.