Method for biosynthesizing three compounds olefin IMZ, CMDIA and CMI0

Abstract

The invention relates to the technical field of preparation of biodegradable materials, and particularly discloses a method for biosynthesizing three compounds olefin IMZ, CMDIA and CMI0, which comprises the following steps: culturing a white-rot fungus strain to obtain a fungus culture; adding imidaclothiz into the fungal culture, and carrying out a biotransformation reaction under a standing condition; carrying out extraction and purification treatment on the culture subjected to biotransformation, and separating to obtain target compounds olefin IMZ, CMDIA and CMIO; according to the present invention, by optimizing the degradation metabolism pathway of the white-rot fungi Phanerochaete sordida YK-624, the three target compounds olefin IMZ, CMDIA and CMIO can be synchronously generated in the single bioconversion reaction, and compared with the traditional chemical synthesis or step-by-step bioconversion process, the complex processes of multi-step reaction and separation are omitted, the production period is substantially shortened, the production cost is reduced, and the production efficiency is improved. The time cost and the operation complexity are reduced, and the overall synthesis efficiency is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of biodegradable materials, and particularly relates to a method for biosynthesizing three compounds, olefin IMZ, CMDIA, and CMI0. Background Art

[0002] Neonicotinoid insecticides have developed rapidly since their launch and have become one of the most commonly used insecticides globally. They are registered and used in more than 120 countries, accounting for one-fourth of the global insecticide usage, and have played an important role in maintaining crop yields.

[0003] Imidaclothiz (IMZ), chemically named N-(1-((2-chlorothiazol-5-yl)methyl)-1H-imidazol-2-yl)nitramide, is a neonicotinoid insecticide independently developed in China. Since its launch in 2002, due to its high efficiency and the insecticidal effect not being easily affected by temperature changes, it has gradually occupied the market and has been widely used in China. However, similar to other neonicotinoid compounds, more and more studies have shown that IMZ can cause serious harm to some non-target organisms. And in recent years, a large amount of research evidence has shown that IMZ can migrate into the environment in various ways and is frequently detected in soil, water, sediment, food, and even human urine.

[0004] The white rot fungus Phanerochaetesordida YK-624 was isolated from rotten wood on Yakushima Island, Japan, and its high-efficiency degradation ability for IMZ has been proven previously. After a large amount of cultivation, it was found that the degradation products of IMI are olefin-imidaclothiz (olefin IMZ), 1-((2-chlorothiazol-5-yl)methyl)-4,5-dihydro-1H-imidazol-2-amine (CMDIA), and 1-((2-chlorothiazol-5-yl)methyl)imidazolidin-2-one, with chemical names:

[0005] N-(1-((2-chlorothiazol-5-yl)methyl)-1H-imidazol-2-yl)nitramide, 1-((2-chlorothiazol-5-yl)methyl)-4,5-dihydro-1H-imidazol-2-amine, and 1-((2-chlorothiazol-5-yl)methyl)imidazolidin-2-one;

[0006] However, there is no method in the prior art for simultaneously synthesizing the above three compounds.

[0007] Based on this, the inventors propose a method for biosynthesizing three compounds, olefin IMZ, CMDIA, and CMI0, for simultaneously biosynthesizing the above three compounds. Summary of the Invention

[0008] The object of the present invention is to provide a method for biosynthesizing three compounds, olefin IMZ, CMDIA, and CMI0, to solve the problems presented in the above background art.

[0009] To achieve the above object, the present invention provides the following technical solution:

[0010] A method for biosynthesizing three compounds, olefin IMZ, CMDIA, and CMI0, comprising:

[0011] Culturing the white-rot fungus Phanerochaete sordida YK-624 strain to obtain a fungal culture;

[0012] Adding imidaclothiz (IMZ) to the fungal culture and performing a biotransformation reaction under static conditions;

[0013] Extracting and purifying the biotransformed culture to separate the target compounds olefin IMZ, CMDIA, and CMIO.

[0014] Preferably, the white-rot fungus is Phanerochaete sordida YK-624, and the steps for culturing the white-rot fungus include:

[0015] Inoculating the strain into a potato dextrose agar (PDA) medium for subculture and scale-up cultivation to obtain an amplified strain mycelium sheet;

[0016] Inoculating the amplified strain mycelium sheet into a liquid medium for shake cultivation to obtain the cultured white-rot fungus.

[0017] Preferably, the PDA medium contains 20% (by mass) potato leachate, 2% glucose, and 2% agar, with the remainder being water;

[0018] The liquid medium composition is 10 g glucose, 0.221 g ammonium tartrate, 1.64 g anhydrous sodium acetate, 1 g Tween 80 (added during shake cultivation), and 100 mL Kirk salt solution.

[0019] Preferably, the formulation of the Kirk salt solution is per liter: 20 g KH2PO4, 5 g MgSO4·7H2O, 1.3 g CaCl2·2H2O, 0.01 g Thiamine·HCl, 16.7 mL Kirk trace elements solution;

[0020] The formulation of the Kirk trace elements solution is per liter: 9 g Nitrilotriace, 3 g MgSO4·7H2O, 4.2 g MnSO4·H2O, 6 g NaCl, 0.6 g FeSO4·7H2O, 1.1 g CoSO4·7H2O, 1.1 g ZnSO4·7H2O, 0.6 g CaCl2·2H2O, 0.06 g CuSO4·5H2O, 0.11 g AlK(SO4)2·12H2O, 0.06 g H3BO3, 0.07 g Na2MoO4·2H2O.

[0021] Preferably, the conditions of the biotransformation reaction are:

[0022] Add thiamethoxam to the cultured white rot fungus and statically culture at 30 °C for 20 days to obtain the biotransformed culture;

[0023] The added concentration of thiamethoxam is 50 mg / L to 200 mg / L.

[0024] Preferably, the extraction treatment includes:

[0025] Centrifuge the biotransformed culture and take the supernatant;

[0026] Extract the supernatant three times with an equal volume of ethyl acetate, combine the extraction solutions and evaporate and concentrate to obtain the concentrated extraction solution.

[0027] Preferably, the steps of the purification treatment are:

[0028] Perform gradient elution on the concentrated extraction solution by silica gel chromatography to obtain the product separated by silica gel chromatography;

[0029] Further separate and purify the product separated by silica gel chromatography by preparative liquid chromatography to obtain three separated and purified products, namely Olefin IMZ, CMDIA and CMIO.

[0030] Preferably, the mobile phase for the separation and purification is a mixed solution of methanol and water, wherein the volume fraction of the methanol solution is 15% - 20%;

[0031] The chromatographic column of the liquid chromatography is a C18 reverse-phase column, 5μm, 20×250mm.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] (1) By optimizing the degradation and metabolic pathway of the white rot fungus Phanerochaete sordida YK-624, three target compounds (olefin IMZ, CMDIA, and CMIO) can be simultaneously generated in a single biotransformation reaction. Compared with traditional chemical synthesis or stepwise biotransformation processes, this technology eliminates the cumbersome processes of multiple reactions and separations, significantly shortens the production cycle, reduces time costs and operation complexity, and remarkably improves the overall synthesis efficiency. Description of the Drawings

[0034] Figure 1 1H NMR spectrum of Olefin IMZ of the present invention 1 ;

[0035] Figure 2 1H NMR spectrum of Olefin IMZ of the present invention 13 13C NMR spectrum;

[0036] Figure 3 1H NMR spectrum of CMDIA of the present invention 1 ;

[0037] Figure 4 1H NMR spectrum of CMDIA of the present invention 13 13C NMR spectrum;

[0038] Figure 5 1H NMR spectrum of CMIO of the present invention 1 ;

[0039] Figure 6 1H NMR spectrum of CMIO of the present invention 13 13C NMR spectrum. Detailed Embodiments

[0040] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0041] Example 1:

[0042] Please refer to Figures 1 to 6As shown, a method for biosynthesizing three compounds, olefin IMZ, CMDIA, and CMI0, includes:

[0043] (1) The white-rot fungus YK-624 is subcultured and expanded using a potato dextrose agar medium (PDA, composition: 20% potato, 2% glucose, 2% agar) and stored in a 4°C refrigerator for later use.

[0044] (2) Take 10 mycelium discs with a diameter of 10 mm from the PDA medium on which YK-624 is subcultured and inoculate them into 50 mL of a liquid medium. The main components (L) of the liquid medium, Kirk medium: 10 g of glucose, 0.221 g of ammonium tartrate, 1.64 g of anhydrous sodium acetate, 1 g of Tween 80 (added during shaking culture), 100 mL of Kirk salts solution.

[0045] The formula of Kirk salts solution is (L): 20 g of KH2PO4, 5 g of MgSO4·7H2O, 1.3 g of CaCl2·2H2O, 0.01 g of Thiamine·HCl, 16.7 mL of Kirk trace elements solution. The formula of Kirk trace elements solution is (L): 9 g of Nitrilotriace, 3 g of MgSO4·7H2O, 4.2 g of MnSO4·H2O, 6 g of NaCl, 0.6 g of FeSO4·7H2O, 1.1 g of CoSO4·7H2O, 1.1 g of ZnSO4·7H2O, 0.6 g of CaCl2·2H2O, 0.06 g of CuSO4·5H2O, 0.11 g of AlK(SO4)2·12H2O, 0.06 g of H3BO3, 0.07 g of Na2MoO4·2H2O.

[0046] (3) Add imidaclothiz, and incubate statically at 30°C for 20 days.

[0047] (4) Separate the liquid medium from the white-rot fungus after static incubation, add an equal volume of ethyl acetate to the separated liquid and extract it 3 times, and combine the supernatant.

[0048] (5) Rotate and evaporate to concentrate the supernatant, and perform gradient elution using silica gel chromatography. The product obtained by silica gel chromatography separation is further separated and purified by preparative liquid chromatography (chromatographic column InertSustain C18, 5 μm, 20×250 mm), and the separation mobile phase is 20% methanol.

[0049] (6) Identify the three separated and purified products. Peaks of m / z 257.9861 [M-H], - m / z 217.0314 [M+H], + and m / z 239.9974 [M+Na] + were observed by HR-ESI-TOF-MS. The speculated molecular formulas were C7H5ClN5O2S, C7H9ClN4S, and C7H8ClN3OS respectively. Combining with nuclear magnetic resonance spectroscopy 1 1H-NMR and 13 13C-NMR, the structures of the compounds were determined to be Olefin IMZ, CMDIA, and CMIO.

[0050] As can be seen from the above, by optimizing the degradation and metabolic pathway of the white-rot fungus Phanerochaete sordida YK-624, this method can simultaneously generate three target compounds (olefin IMZ, CMDIA, and CMIO) in a single biotransformation reaction. Compared with traditional chemical synthesis or stepwise biotransformation processes, this technology eliminates the cumbersome processes of multiple reactions and separations, greatly shortens the production cycle, reduces the time cost and operation complexity, and significantly improves the overall synthesis efficiency.

[0051] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for biosynthesizing three olefin compounds IMZ, CMDIA and CMIO, characterized in that: include: Cultivating white rot fungus strains to obtain fungal cultures; adding chlorothioline to the fungal culture and performing a bioconversion reaction under static conditions; The culture after bioconversion was extracted and purified to separate the target compounds olefins IMZ, CMDIA and CMIO.

2. A method for biosynthesizing three compounds of olefin IMZ, CMDIA and CMI 0 according to claim 1, characterized in that: The white rot fungus is Phanerochaete sordida YK-624, and the steps of culturing the white rot fungus include: The strain is inoculated into a potato dextrose agar (PDA) medium for subculture and expansion culture to obtain an amplified strain mycelium sheet; The amplified strain mycelium piece is inoculated into a liquid culture medium for shaking culture to obtain cultured white rot fungi.

3. A method for biosynthesizing three compounds of olefin IMZ, CMDIA and CMI 0 according to claim 2, characterized in that: The PDA culture medium comprises 20% by weight of potato extract, 2% by weight of glucose and 2% by weight of agar, and the remainder is water; The liquid culture medium consists of 10 g glucose, 0.221 g ammonium tartrate, 1.64 g anhydrous sodium acetate, 1 g Tween 80, and 100 mL Kirk salt solution.

4. A method for biosynthesizing three compounds of olefin IMZ, CMDIA and CMI 0 according to claim 3, characterized in that: The formula of the Kirk salt solution is per liter: 20 g KH2PO4, 5 g MgSO4·7H2O, 1.3 g CaCl2·2H2O, 0.01 g Thiamine·HCl, 16.7 mL Kirk trace elements solution; The formula of Kirk trace solution elements per liter is: 9g Nitrilotriace, 3gMgSO4·7H2O, 4.2gMnSO4·H2O, 6g NaCl, 0.6g FeSO4·7H2O, 1.1g CoSO4·7H2O, 1.1g ZnSO4·7H2O, 0.6g CaCl2·2H2O, 0.06g CuSO4·5H2O, 0.11g AlK(SO4)2·12H2O, 0.06g H3BO3, 0.07g Na2MoO4·2H2O.

5. The method for biosynthesizing three compounds of olefin IMZ, CMDIA and CMI 0 according to claim 1, characterized in that: The conditions of the bioconversion reaction are: Add chlorothiocarb to the cultured white rot fungi, and culture them statically at 30°C for 20 days to obtain a culture after bioconversion; The added concentration of the chlorothiophene is 50 mg / L to 200 mg / L.

6. The method of biosynthesizing three compounds of olefin IMZ, CMDIA and CMI 0 according to claim 1, characterized in that: The extraction process comprises: Centrifuging the culture after the bioconversion and taking the supernatant; The supernatant was extracted three times with equal volumes of ethyl acetate, and the extracts were combined and concentrated by evaporation to obtain a concentrated extract.

7. The method for biosynthesizing three olefin compounds IMZ, CMDIA and CMI 0 according to claim 1, characterized in that: The steps of the purification process are: Performing gradient elution on the concentrated extract by silica gel chromatography to obtain a product separated by silica gel chromatography; The product obtained by the silica gel chromatography separation was further separated and purified by preparative liquid chromatography to obtain three separated and purified products, namely Olefin IMZ, CMDIA and CMIO.

8. The method for biosynthesizing three olefin compounds IMZ, CMDIA and CMI 0 according to claim 7, characterized in that: The mobile phase for separation and purification is a mixed solution of methanol and water, wherein the volume fraction of the methanol solution is 15% to 20%; The chromatographic column of the liquid chromatography is a C18 reverse phase column, 5 μm, 20×250 mm.

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