Application of Bacillus megaterium in the synthesis of 5-aminobenzimidazole-2-carbamic acid methyl ester

Abstract

The invention belongs to the technical field of organic synthesis, and discloses an application of Bacillus megaterium in the synthesis of 5-aminobenzimidazole-2-methyl carbamate. The present invention adopts Bacillus megaterium JOM-01 to reduce 5-nitrobenzimidazole-2-methyl carbamate to 5-aminobenzimidazole-2-methyl carbamate, and provides a nitro reduction green biosynthetic pathway. The synthesis method has the advantages of less three wastes, low production cost, mild production conditions, strong selectivity, easy operation, relatively high yield, and conducive to promotion and application. The present invention is applicable to the preparation field of albendazole and its intermediates.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis and relates to application of Bacillus megaterium in synthesizing 5-aminobenzimidazole-2-methyl carbamate. Background Art

[0002] Albendazole belongs to the benzimidazole class of drugs. Its chemical name is 5-propylthiobenzimidazole-2-carbamic acid methyl ester. It is a class of highly effective broad-spectrum anthelmintics and is widely used in clinical practice to treat various helminthic infectious diseases.

[0003] At present, the production of albendazole at home and abroad mainly adopts the synthesis route using o-nitroaniline as raw material. In addition, the route of synthesizing albendazole by nitration reaction, reduction reaction, diazotization, substitution reaction and alkylation using cheap and readily available carbendazole as raw material is also gaining increasing attention. Among them, the synthesis of 5-aminobenzimidazole-2-carbamic acid methyl ester by reduction reaction is a key link in this synthesis route. The reduction reactions widely used in process production mainly include metal reduction method, sulfide reduction method and catalytic hydrogenation method. However, the above three reduction methods generally have the disadvantages of serious environmental pollution, harsh reaction conditions, generation of sulfur-containing impurities, cumbersome separation and purification steps, high catalyst cost, high investment in specific equipment or safety risks. Summary of the invention

[0004] In view of the deficiencies in the reduction reaction of synthesizing 5-aminobenzimidazole-2-carbamic acid methyl ester in the prior art, the present invention provides the application of Bacillus megaterium JOM-01 in synthesizing 5-aminobenzimidazole-2-carbamic acid methyl ester. Specifically, under the catalytic reduction of Bacillus megaterium JOM-01, methyl benzimidazole-2-carbamic acid methyl ester (i.e., carbendazim) is reduced to 5-aminobenzimidazole-2-carbamic acid methyl ester. The synthesis method has the advantages of low production cost, mild reaction conditions, green environmental protection, and conducive to popularization and application. Furthermore, the synthesis method of 5-aminobenzimidazole-2-carbamic acid methyl ester can be applied to the synthesis of albendazole.

[0005] In order to achieve the above-mentioned object of the invention, the embodiment of the present invention adopts the following technical solution: In a first aspect, the present invention provides a use of Bacillus megaterium in the synthesis of 5-aminobenzimidazole-2-carbamic acid methyl ester, wherein the Bacillus megaterium is Bacillus megaterium ( Bacillus megaterium )JOM-01, its deposit number is CGMCC No.26268.

[0006] With the rapid development of productivity, people have higher demands on microbial strains, which is difficult to achieve by blindly screening from nature and randomly inducing mutations of strains. Purposeful transformation of microorganisms is an inevitable trend, and heavy ion irradiation technology has gradually deepened in the field of microbial mutagenesis. Biocatalytic reduction of nitro compounds has become an effective method for preparing amines, such as Bacillus licheniformis ( Bacillus licheniformis )、Nocardia( Nocardia ), Escherichia coli ( Escherichia coli ) have been studied in the nitro reduction reaction, but there are few reports on the biocatalytic reduction of nitro by Bacillus megaterium obtained by heavy ion mutagenesis of Bacillus cereus. According to literature reports, the reduction process of Bacillus megaterium is achieved through the catalytic action of enzymes, such as: in the process of nitrite degradation in water, Bacillus megaterium can produce nitrite reductase; nitrate assimilating Bacillus megaterium has the assimilating nitrate reductase gene, which can be used to improve secondary salinized soil by bioenzymatic method.

[0007] The Bacillus megaterium JOM-01 provided by the present invention is obtained by heavy ion beam irradiation mutagenesis using Bacillus cereus as the starting strain, and is proved to belong to Bacillus megaterium, It was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on December 26, 2022. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The Bacillus megaterium JOM-01 provided by the present invention can reduce 5-nitrobenzimidazole-2-carbamic acid methyl ester to 5-aminobenzimidazole-2-carbamic acid methyl ester.

[0008] In a second aspect, the present invention provides a method for synthesizing 5-aminobenzimidazole-2-carbamic acid methyl ester, the method comprising: reacting 5-nitrobenzimidazole-2-carbamic acid methyl ester with Bacillus megaterium ( Bacillus megaterium ) JOM-01 is reduced to 5-aminobenzimidazole-2-carbamic acid methyl ester, wherein the preservation number of the Bacillus megaterium JOM-01 is CGMCC No.26268.

[0009] The equation for the synthesis method of the 5-aminobenzimidazole-2-methyl carbamate is as follows: .

[0010] Compared with the environmental problems caused by conventional chemical synthesis, the present invention adopts microbial transformation to provide a green biosynthetic pathway of nitro reduction. The synthesis method has the advantages of less three wastes, low production cost, mild production conditions, strong selectivity, easy operation, relatively high yield and conducive to promotion and application. The reaction yield can reach 76.6% to 81.2%.

[0011] Preferably, the reduction reaction temperature is 30° C. to 40° C., and the reaction time is 10 h to 20 h.

[0012] Preferably, the method comprises the following steps: inoculating Bacillus megaterium JOM-01 seed liquid into a fermentation medium to obtain bacterial cells; and enzymatically hydrolyzing the bacterial cells and reacting the cells with 5-nitrobenzimidazole-2-carbamic acid methyl ester to obtain 5-aminobenzimidazole-2-carbamic acid methyl ester.

[0013] Preferably, the viable bacterial count of the Bacillus megaterium JOM-01 seed solution is 1.5×10 8 CFU / mL~5×10 8 CFU / mL, the inoculation amount is 3%~10%.

[0014] Preferably, during the reduction, the initial concentration of 5-nitrobenzimidazole-2-carbamic acid methyl ester is 0.23 mol / L to 0.29 mol / L.

[0015] Preferably, during the reduction reaction, the pH of the reaction solution is 7-8.

[0016] In a third aspect, the present invention provides application of the synthesis method of 5-aminobenzimidazole-2-carbamic acid methyl ester provided in the second aspect in the preparation of albendazole.

[0017] Preferably, in the above application, methyl benzimidazole-2-carbamate is used as a substrate, and albendazole is prepared by nitration reaction, reduction reaction, diazotization reaction, substitution reaction and alkylation reaction. The preparation method of albendazole specifically comprises the following steps: S1. Nitration reaction The methyl benzimidazole-2-carbamate is reacted with nitric acid to obtain methyl 5-nitrobenzimidazole-2-carbamate; The molar ratio of methyl benzimidazole-2-carbamate to nitric acid is 0.20:0.75-0.20:1.05; the reaction temperature of the nitration reaction is -5°C to 25°C, and the reaction time is 2h to 6h. S2. Reduction reaction According to the synthesis method of 5-aminobenzimidazole-2-carbamic acid methyl ester provided in the second aspect, 5-aminobenzimidazole-2-carbamic acid methyl ester is prepared using 5-nitrobenzimidazole-2-carbamic acid methyl ester as a raw material; S3. Diazotization and substitution reactions Sodium nitrite and 5-aminobenzimidazole-2-carbamic acid methyl ester are subjected to diazotization reaction at 10°C to 40°C to obtain a diazotization reactant; The diazotized reactant is subjected to a substitution reaction with sodium sulfide to obtain 5-mercaptobenzimidazole-2-carbamic acid methyl ester; S4. Alkylation reaction Albendazole is prepared by alkylating 5-mercaptobenzimidazole-2-carbamic acid methyl ester with a halopropane at 35° C. to 45° C.; wherein the halopropane includes 1-bromopropane; The molar ratio of the benzimidazole-2-carbamic acid methyl ester to the monohalogenated propane is 1:1 to 1:1.2.

[0018] The reaction equation of the preparation method of albendazole in the present invention is as follows:

[0019] The present invention applies the synthesis method of 5-aminobenzimidazole-2-carbamic acid methyl ester provided in the second aspect to the preparation of albendazole. The preparation method of albendazole has the advantages of relatively low production cost, mild reaction conditions, green environmental protection, and convenience for industrial production, and has both good environmental and economic benefits, and is in line with the concept of modern green chemical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is the HPLC figure of detecting albendazole in Example 5 of the present invention. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] The raw materials and reagents used in the present invention, unless otherwise specified, are conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0024] Carbendazim (content 98% or more) in the present invention was purchased from Guiyang Zhongjing Technology Co., Ltd., and other reagents used were analytically pure or chromatographically pure and purchased from domestic reagent companies; Bacillus cereus XK-4 was provided by Shandong Sukehan Bioengineering Co., Ltd.; Bacillus megaterium (CGMCC 1.1741) was provided by Shandong Runwo Biotechnology Co., Ltd.; Basic culture medium: 1L, peptone 10g, yeast extract 5g, NaCl 10g, balance water, pH 7.0-7.2; Preservation medium: Based on 1L, add 10g agar powder to the ingredients of the basic medium; Seed culture medium: 1L, beef extract 10g, peptone 10g, NaCl 10g, the balance is water, pH 7.0-7.2; Fermentation medium: 1L: 10g glucose, 5g yeast extract, 1g magnesium sulfate, the balance is water, pH 7.5.

[0025] The 5-nitrobenzimidazole-2-carbamic acid methyl ester in the embodiment of the present invention is prepared by the inventor, and the specific preparation method is: in a 500mL three-necked bottle connected to a condenser and a stirrer, the temperature is controlled by placing it in a cold well and a water bath, and 39g (0.2mol) of methyl benzimidazole-2-carbamic acid and 0.75mol of concentrated sulfuric acid (98%) are added to the above-mentioned triangular flask, and the reaction temperature is controlled to -5°C~5°C, and 0.95mol of fuming nitric acid (98%) is added dropwise, and the addition is completed, stirred, and the reaction temperature is controlled to 15°C. After reacting for 5h, the reactant is poured into ice water, and a light yellow solid is precipitated. After suction filtration, washing, and drying, 5-nitrobenzimidazole-2-carbamic acid methyl ester is obtained. It is calculated that the yield of the reaction is 92.1%.

[0026] Example 1 In this example, Bacillus cereus XK-4 was used as the starting strain, and a mutagenized strain was obtained by heavy ion beam irradiation. Bacillus megaterium JOM-01 was obtained after screening and identification. The specific contents are as follows: 1. Bacteria activation Weigh 2g of Bacillus cereus XK-4 powder, add 5mL of sterile water to make a suspension, inoculate it into a 50mL sterilized basal medium flask (capacity 250mL), and culture it in a shaker at 30℃ and 160rpm for 24h. Take out the proliferation solution and dilute it into five gradients (10 -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5 ), use a pipette to accurately draw 100 μL of each concentration, inoculate on the plate separation medium by spreading method, and culture at 30℃ for 24 hours. Pick out single colonies with large transparent circles and thick colonies, and streak inoculate on slant preservation medium. After constant temperature culture at 30℃ for 48 hours, seal with sterile liquid paraffin and store in a 4℃ refrigerator.

[0027] 2. Preparation of Seed Bacteria Solution Pick a ring of activated single colonies and place them in the seed culture medium. Culture them at 30°C and 180 rpm for 16-18 hours. The bacterial count is 10 8 CFU / mL of bacterial solution was used as the seed bacterial solution.

[0028] 3. Irradiation mutagenesis Heavy ion beam 12 C 6+ The radiation parameters are: energy 80MeV / u, 12 C 6+ It passes through a 50μm stainless steel window, a 20μm mylar film and 1.3m of air. The heavy ion beam has a range of 16mm and a peak position of 15.5mm in water.

[0029] The small dishes containing seed solution were placed in the irradiation tray designed and manufactured by the Institute of Modern Physics, Chinese Academy of Sciences in order, and then placed in the heavy ion beam irradiation breeding experimental platform under safe conditions. The irradiation parameters corresponding to the irradiation were set in the control system, and the heavy ion beam with an energy of 80MeV / μ provided by the shallow irradiation terminal of the heavy ion accelerator was used. 12 C 6+ The irradiation treatment was carried out by 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. The mutagenized strains were subjected to high-throughput screening.

[0030] 4. High-throughput screening Seed culture: Use a 24-well deep-well cell culture plate to rejuvenate the mutant strain obtained in part 3, transfer the activated bacterial solution to the seed culture medium at a 10% inoculation rate, and culture at a constant temperature of 30°C and 180 r / min.

[0031] Fermentation culture: 200 μL of seed solution after constant temperature culture for 8h~9h was transferred to a 24-well deep-well cell culture plate containing 2mL of fermentation medium. After constant temperature culture for 24h at 30℃ and 180r / min, a mixed solution of 5-nitrobenzimidazole-2-carbamic acid methyl ester and DMF in a mass ratio of 1:1 was added. After shaking culture for 10h, the organic solvent DMF was evaporated and the solution was extracted with ethyl acetate (20mL / time×3 times) to obtain 5-aminobenzimidazole-2-carbamic acid methyl ester. The yield of 5-aminobenzimidazole-2-carbamic acid methyl ester was calculated by thin layer scanning, and positive mutants were screened. A total of 3 positive mutant strains were screened, among which the JOM-01 strain had the highest yield of 81.5%. After further culture and transformation verification in a shake flask, the yield of the target product of the JOM-01 strain was still the highest. The JOM-01 strain was further studied and preserved.

[0032] Microscopic examination showed that the cells of the JOM-01 strain were rod-shaped, with blunt ends, arranged singly or in short chains. The cell size was 1.2-1.5×2.0-4.0μm, and Gram staining was positive. The spores were oval or oblong, about 1.0-1.2×1.5-2.0μm in size, and were mesogenic or subterminal. The JOM-01 strain was sent to the General Microbiology Center of the China Association for the Preservation of Microbiological Cultures for preservation, and further identification showed that the strain was Bacillus megaterium ( Bacillus megaterium ), its accession number is CGMCC No.26268.

[0033] Example 2 This embodiment provides a method for synthesizing 5-aminobenzimidazole-2-methyl carbamate, the method comprising the following steps: 400 mL of fermentation medium was placed in a 2000 mL three-necked flask with a stirring device, sterilized at high temperature and cooled to room temperature, and Bacillus megaterium JOM-01 seed solution I was inoculated into the fermentation medium at a rate of 5%, and cultured at 30°C and 180 r / min for 16 h to obtain a fermentation broth; in a 4°C environment, the fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was discarded to obtain the bacterial cells; a 0.5% lysozyme solution (prepared from 20 mg of lysozyme powder with an activity of 2000 U / mg and water) was added to the bacterial cells, and after keeping warm at 37°C for 40 min, 200 mL of water was added, and the pH was adjusted to 7.5 with alkaline water, and 17.69 g of 5-nitrobenzimidazole-2-carbamic acid methyl ester (80 mmol) and 80 mL of The solution after DMF mutual dissolution was reacted at a speed of 300 rpm and 35 ° C for 16 hours, and the organic solvent was evaporated, extracted with ethyl acetate (3×200 mL), and the organic layer was washed with a saturated NaCl solution (3×100 mL), dried over anhydrous Na2SO4, filtered, and the solvent was concentrated under reduced pressure; separated by silica gel chromatography, eluted with ethyl acetate-95% ethanol (volume ratio of 80:1), and the eluate was collected, concentrated, and dried to obtain 12.42 g of a light yellow solid, which was 5-aminobenzimidazole-2-carbamic acid methyl ester. After detection and calculation, the yield of the reaction of 5-aminobenzimidazole-2-carbamic acid methyl ester provided in this embodiment was 81.2%, and the purity was 95.2%.

[0034] The preparation method of Bacillus megaterium JOM-01 seed solution I is as follows: the activated bacterial solution of Bacillus megaterium JOM-01 in part 4 of Example 1 is transferred to the seed culture medium at a 10% inoculation rate, and cultured at a constant temperature of 30°C and 180 r / min for 9 hours to obtain a viable bacterial count of 3.5×10 8 CFU / mL of Bacillus megaterium JOM-01 seed solution Ⅰ.

[0035] Example 3 This embodiment provides a method for synthesizing 5-aminobenzimidazole-2-methyl carbamate, the method comprising the following steps: 400 mL of fermentation medium was placed in a 2000 mL three-necked flask with a stirring device, sterilized at high temperature and cooled to room temperature, and Bacillus megaterium JOM-01 seed solution II was inoculated into the fermentation medium at a rate of 3%, and cultured at 35°C and 220 r / min for 20 h to obtain the fermentation broth; in a 4°C environment, the fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was discarded to obtain the bacterial cells; a 0.5% lysozyme solution (prepared with 20 mg of lysozyme powder with an activity of 2000 U / mg and water) was added to the bacterial cells, and after keeping warm at 37°C for 30 min, 200 mL of water was added, and the pH was adjusted to 7 with alkaline water, and 15.48 g of 5-nitrobenzimidazole-2-carbamic acid methyl ester (70 mmol) and 100 mL of The solution after DMF dissolution was reacted at a speed of 300 rpm and 30°C for 20 hours, and then the organic solvent was evaporated and extracted with ethyl acetate (3×200 mL). The organic layer was washed with a saturated NaCl solution (3×100 mL), dried over anhydrous Na2SO4, filtered, and the solvent was concentrated under reduced pressure; separated by silica gel chromatography, eluted with ethyl acetate-95% ethanol (volume ratio of 80:1), and the eluate was collected, concentrated, and dried to obtain 10.61 g of a light yellow solid, which was 5-aminobenzimidazole-2-carbamic acid methyl ester. After detection and calculation, the yield of the reaction was 79.3% and the purity was 95.5%.

[0036] The preparation method of Bacillus megaterium JOM-01 seed solution II in this embodiment is as follows: the activated bacterial solution of Bacillus megaterium JOM-01 in part 4 of Example 1 is transferred to the seed culture medium at a 5% inoculation rate, and cultured at a constant temperature of 35°C and 220 r / min for 10 h to obtain a viable bacterial count of 5.0×10 8 CFU / mL of Bacillus megaterium JOM-01 seed solution Ⅱ.

[0037] Example 4 This embodiment provides a method for synthesizing 5-aminobenzimidazole-2-methyl carbamate, the method comprising the following steps: 400 mL of fermentation medium was placed in a 2000 mL three-necked flask with a stirring device, sterilized at high temperature and cooled to room temperature, and Bacillus megaterium JOM-01 seed solution III was inoculated into the fermentation medium at a rate of 10%, and cultured at 32°C and 200 r / min for 24 h to obtain fermentation broth; in a 4°C environment, the fermentation broth was centrifuged at 8000 rpm for 20 min, and the supernatant was discarded to obtain bacterial cells; 0.5% lysozyme solution (prepared with 20 mg lysozyme powder with an activity of 2000 U / mg and water) was added to the bacterial cells, and after keeping warm at 37°C for 30 min, 200 mL of water was added and the pH was adjusted to 8 with alkaline water, and 22.11 g of 5-nitrobenzimidazole-2-carbamic acid methyl ester (100 mmol) and 200 mL of The DMF-miscible solution was reacted at a speed of 300 rpm and 40°C for 10 hours, and then the organic solvent was evaporated and extracted with ethyl acetate (3×200 mL). The organic layer was washed with a saturated NaCl solution (3×100 mL), dried over anhydrous Na2SO4, filtered, and the solvent was concentrated under reduced pressure; separated by silica gel chromatography, eluted with ethyl acetate-95% ethanol (volume ratio of 80:1), the eluate was collected, concentrated, and dried to obtain 14.65 g of a light yellow solid. The obtained light yellow solid was 5-aminobenzimidazole-2-carbamic acid methyl ester. After detection and calculation, the yield of the reaction was 76.6% and the purity was 95.1%.

[0038] The preparation method of Bacillus megaterium JOM-01 seed solution III is as follows: the activated bacterial solution of Bacillus megaterium JOM-01 in part 4 of Example 1 is transferred to the seed culture medium at an inoculation rate of 8%, and cultured at a constant temperature of 32°C and 200 r / min for 8 hours to obtain a viable bacterial count of 1.5×10 8 CFU / mL of Bacillus megaterium JOM-01 seed solution III.

[0039] Example 5 The present embodiment provides a method for preparing albendazole, which comprises using 5-aminobenzimidazole-2-carbamic acid methyl ester prepared by the synthesis method of 5-aminobenzimidazole-2-carbamic acid methyl ester provided by Embodiment 2, Embodiment 3 or Embodiment 4 of the present invention as a raw material, and performing diazotization reaction, substitution reaction and alkylation reaction to prepare albendazole. The present embodiment takes 5-aminobenzimidazole-2-carbamic acid methyl ester prepared by Embodiment 4 as an example for explanation, and the specific contents are as follows: (1) Diazotization reaction and substitution reaction 14.65 g (76.6 mmol) of 5-aminobenzimidazole-2-carbamic acid methyl ester prepared in Example 4 was dissolved in 100 mL of 2 mol / L hydrochloric acid and 50 mL of water. The solution temperature was controlled at 0°C, and 120 mL of 0.83 mol / L sodium nitrite solution was added dropwise. After about 1 hour of dripping, the reaction solution temperature was controlled to 5°C and reacted for 2 hours. A drop of the reaction solution was taken to make the starch potassium iodide test paper turn blue, and the diazotization reaction was completed.

[0040] 60 mL of sodium sulfide solution was added dropwise to the above diazotization reaction product at 0-5°C, and the temperature was raised to 20 ℃ , react for 4 hours, then add 30mL, 2mol / L hydrochloric acid solution to make it acidic to Congo red test paper, filter out the precipitated solid, place the obtained solid in a silica gel chromatography column, elute with ethyl acetate-95% ethanol (volume ratio of 80:1), collect the eluate, concentrate and dry to obtain 15.17g of 5-mercaptobenzimidazole-2-carbamic acid methyl ester. After detection and calculation, the yield of the target product in this step is 88.8% and the purity is 96.3%. The sodium sulfide solution is prepared by dissolving 20 g of Na2S·9H2O in 60 mL of water and cooling the solution for later use.

[0041] (2) Alkylation reaction Dissolve 6g (0.15mol) of sodium hydroxide in 100mL of water, slowly add 7g (0.03mol) of the above-prepared 5-mercaptobenzimidazole-2-carbamic acid methyl ester at room temperature, stir for 10min, add 0.5g of activated carbon for decolorization and filtration, add 0.5g (1.6mmol) of tetrabutylammonium bromide to the filtrate, and then add 25mL of methanol and a mixture of 3.7g (0.03mol) of bromopropane, react at 45℃ for 5h, white solids are continuously precipitated during the reaction, cool, filter, wash the filter cake with water and formic acid to neutrality, and place it in a silica gel chromatography column, elute with ethyl acetate-95% ethanol (volume ratio of 80:1), collect the eluate, concentrate and dry to obtain 7.56g of white crystals. The white crystals are albendazole after liquid phase detection, and its liquid chromatogram is as follows Figure 1 As shown in the figure, the yield of crude albendazole in this step is 90.8% and the purity is 96.5%.

[0042] Comparative Example 1 This comparative example provides a method for synthesizing 5-aminobenzimidazole-2-carbamic acid methyl ester, which is basically the same as that in Example 2, except that in this comparative example, "Bacillus megaterium JOM-01 seed solution I" is replaced with "Bacillus cereus XK-4 seed solution", wherein the viable cell count of the Bacillus cereus XK-4 seed solution is also adjusted to 3.5×10 8The remaining reaction step parameters were consistent with those in Example 2 and will not be described in detail.

[0043] The synthetic method provided in this comparative example was determined to obtain 6.61 g of the target product 5-aminobenzimidazole-2-carbamic acid methyl ester. The reaction yield in this step was calculated to be 43.0% and the purity was 95.2%.

[0044] Comparative Example 2 This comparative example provides a method for synthesizing 5-aminobenzimidazole-2-carbamic acid methyl ester, which is basically the same as that in Example 2, except that in this comparative example, "Bacillus megaterium JOM-01 seed solution I" is replaced with "Bacillus megaterium CGMCC1.1741 seed solution", wherein the viable cell count of Bacillus cereus XK-4 seed solution is also adjusted to 3.5×10 8 The remaining reaction step parameters are consistent with those in Example 2 and will not be described in detail.

[0045] The synthetic method provided in this comparative example was determined to obtain 4.91 g of the target product 5-aminobenzimidazole-2-carbamic acid methyl ester. The reaction yield in this step was calculated to be 32.1% and the purity was 95.7%.

[0046] By comparing the results of Comparative Examples 1-2 with those of Example 2, it can be seen that, compared with the starting strain Bacillus cereus XK-4 or conventional Bacillus megaterium, the Bacillus megaterium JOM-01 obtained by heavy ion irradiation mutagenesis used in the present invention can significantly improve the reaction yield when synthesizing 5-aminobenzimidazole-2-carbamic acid methyl ester.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of Bacillus megaterium in the synthesis of 5-aminobenzimidazole-2-carbamic acid methyl ester, characterized in that: The Bacillus megaterium is the Bacillus megaterium with the deposit number of CGMCC No.26268 ( Bacillus megaterium )JOM-01.

2. A method for synthesizing 5-aminobenzimidazole-2-methyl carbamate, characterized in that: 5-Nitrobenzimidazole-2-carbamic acid methyl ester was synthesized by Bacillus megaterium ( Bacillus megaterium ) JOM-01 is reduced to 5-aminobenzimidazole-2-carbamic acid methyl ester, wherein the preservation number of the Bacillus megaterium JOM-01 is CGMCC No.26268.

3. The synthetic method of 5-aminobenzimidazole-2-methyl carbamate as claimed in claim 2, characterized in that: The reduction reaction temperature is 30°C to 40°C, and the reaction time is 10h to 20h.

4. The synthetic method of 5-aminobenzimidazole-2-methyl carbamate as claimed in claim 2, characterized in that: The method specifically comprises the following steps: inoculating Bacillus megaterium JOM-01 seed liquid into a fermentation medium to obtain bacterial cells; and enzymatically hydrolyzing the bacterial cells and reacting the cells with 5-nitrobenzimidazole-2-carbamic acid methyl ester to obtain 5-aminobenzimidazole-2-carbamic acid methyl ester.

5. The method for synthesizing 5-aminobenzimidazole-2-methyl carbamate according to claim 4, wherein: The viable bacterial count of the Bacillus megaterium JOM-01 seed solution was 1.5×10 8 CFU / mL~5×10 8 CFU / mL, the inoculation amount is 3%~10%.

6. The method for synthesizing 5-aminobenzimidazole-2-methyl carbamate according to claim 2, wherein: During the reduction, the initial concentration of 5-nitrobenzimidazole-2-carbamic acid methyl ester is 0.23 mol / L to 0.29 mol / L.

7. The method for synthesizing 5-aminobenzimidazole-2-methyl carbamate according to claim 2, characterized in that: During the reduction, the pH of the reaction solution is 7-8.

8. Application of the synthetic method of 5-aminobenzimidazole-2-methyl carbamate as claimed in any one of claims 2 to 7 in the preparation of albendazole.

9. The use according to claim 8, characterized in that: Taking benzimidazole-2-carbamic acid methyl ester as a substrate, albendazole is prepared through nitration reaction, reduction reaction as described in any one of claims 2 to 6, diazotization reaction, substitution reaction and alkylation reaction.

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