A strain of Rhodococcus erythrorhizium and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a strain of Rhodococcus and its application. Through ARTP mutagenesis and adaptive evolution, a strain of Rhodococcus that can efficiently prepare 2,6-difluorobenzamide and other amide substances was screened ( Rhodococcus red )AR‑300, with a preservation number of CGMCC No.31246. The nitrile hydratase activity of this strain can reach 5500 U·mL ‑1 The maximum feed concentration can reach 7 mol / L. The Rhodococcus CGMCC No.31246 provided by the present invention is used for transformation to achieve the effect of rapidly producing 2,6-difluorobenzamide and other amide substances, thereby achieving the purpose of improving production efficiency, meeting environmental protection requirements, and reducing production costs, and has broad industrial application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a strain of Rhodococcus erythrorhizium and application thereof. Background Art

[0002] Nitrile compounds are a class of organic compounds containing cyano (R-CN). They are important raw materials for organic synthesis and pharmaceutical intermediates. They are widely distributed in nature and are numerous in variety. They are often found in organisms such as plants, bacteria, fungi and insects. In natural and organic synthesis, amide compounds synthesized from nitrile compounds have high polarity, high stability and structural diversity. They are widely present in drugs and bioactive molecules and are also important raw materials for organic synthesis. They are widely used in pharmaceutical synthesis, agriculture and fine chemical industries.

[0003] In traditional chemical methods, the synthesis of amides such as 2,6-difluorobenzamide includes acid-catalyzed hydrolysis of nitriles, strong base hydrolysis, and aminolysis of acids and their derivatives. The products of these hydrolysis processes need to be neutralized, consuming a large amount of acid and alkali, producing a large amount of waste salt, and requiring cumbersome separation and purification steps. The process has poor safety and serious environmental pollution.

[0004] Nitrile hydratase contained in Rhodococcus is a type of metal enzyme that can catalyze the hydration reaction of nitrile substances into amide compounds. The bioenzymatic method with nitrile hydratase (NHase) as the core is widely used in catalyzing the hydration of nitrile compounds to form amide substances. Compared with chemical hydrolysis, microbial enzyme catalysis has great advantages, such as mild conditions, high chemical and regional selectivity. Therefore, microbial enzyme catalysis has great application potential in the production of corresponding amide substances from nitrile substances.

[0005] At present, in the process of hydration reaction of nitrile substances to produce amide substances such as 2,6-difluorobenzamide by microbial method, there are problems such as low enzymatic activity and low catalytic efficiency of Rhodococcus nitrile hydratase. In addition, due to the strong toxicity of the substrate, the enzyme of the bacteria will be destroyed, causing it to be irreversibly and rapidly inactivated. Therefore, improving the enzymatic activity of nitrile hydratase to improve the catalytic efficiency and substrate feed amount is an important problem to be solved, which has important research significance in the fields of industrial production and so on. Summary of the invention

[0006] The present invention is based on the original strain Rhodococcus erythrocytes ( Rhodococcus ruber ) Based on CGMCC No.3090, a strain of Rhodococcus erythrocytes was screened through ARTP mutagenesis and adaptive evolution, which can efficiently prepare 2,6-difluorobenzamide and other amide substances. The enzyme activity of the fermentation broth of this strain was increased by 27%, and the intensity of producing difluorobenzamide was increased by 2.5 times, which is easy to cultivate on a large scale and apply in industry.

[0007] The technical route adopted by the present invention is as follows:

[0008] One of the technical solutions provided by the present invention is a strain of Rhodococcus that can efficiently prepare 2,6-difluorobenzamide and other amide substances, specifically Rhodococcus ( Rhodococcus ruber )AR-300, which was deposited on July 10, 2024 at the General Microbiology Center of China Microbiological Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, with the deposit number CGMCC No.31246.

[0009] The Rhodococcus erythrocytes ( Rhodococcusruber ) The physical and chemical properties of AR-300 are as follows: Gram-positive bacteria, the colonies on solid culture medium are spherical, moist, and orange-red in color ( Figure 1 As shown in the figure), the optimum growth pH is 6-8 and the optimum growth temperature is 28-34 ℃.

[0010] The second technical solution provided by the present invention is the red coccus ( Rhodococcusruber )Application of AR-300 in the production of amide substances;

[0011] Furthermore, the amide substances include but are not limited to: 2,6-difluorobenzamide, acrylamide, nicotinamide and cinnamamide;

[0012] Further, using Rhodococcus erythrorhizium ( Rhodococcusruber ) The method for efficiently preparing 2,6-difluorobenzamide and other amide substances by AR-300 is as follows:

[0013] (1) Transformation: Add substrate and Rhodococcus erythrocytes ( Rhodococcusruber ) AR-300 resting cells for biotransformation;

[0014] (2) Preparation of finished products: After the conversion, the feed liquid is filtered, and the obtained filter cake is washed with water and dried to obtain crude amide substances such as 2,6-difluorobenzamide. At the same time, the obtained filtrate is extracted with an organic solvent and concentrated in vacuo to obtain crude amide substances such as 2,6-fluorobenzamide. The two parts of the product are then combined and recrystallized to obtain the finished product;

[0015] Furthermore, in step (1), the substrate includes but is not limited to: 2,6-difluorobenzonitrile, acrylonitrile, nicotinonitrile and cinnamonitrile;

[0016] Furthermore, in step (1), 0.5-5% (v:v) of a solubilizing agent is also added;

[0017] Furthermore, the dissolution agent includes but is not limited to methanol, isopropanol, propylene glycol or Tween 80;

[0018] Further, the buffer system is a KH2PO4-NaOH buffer system with a pH of 6-9;

[0019] Furthermore, in the reaction system, Rhodococcus ruber ( Rhodococcusruber ) AR-300 cell concentration is 0.66-3.30 g / L;

[0020] Further, the biotransformation conditions are: rotation speed 110-220 rpm, transformation temperature 25-40 °C, transformation time 2-24 h;

[0021] Furthermore, in the reaction system, the input concentration of the substrate is not higher than 7 mol / L;

[0022] Further, the extraction solvent of the extraction treatment is ethyl acetate, chloroform, dichloromethane or butyl acetate;

[0023] Furthermore, the recrystallization solvent used in the recrystallization is at least one of methanol, ethanol, chloroform, acetone, dichloromethane, chloroform or n-hexane;

[0024] Further, the Rhodococcus erythrocytes ( Rhodococcusruber ) The preparation method of AR-300 resting cells is as follows:

[0025] (1) Seeds and fermentation culture: Take Rhodococcus erythrocytes ( Rhodococcusruber ) The AR-300 strain was inoculated into the seed culture medium, cultured at 180 rpm and 32 °C for 30 h to obtain the seed culture solution, which was then transferred to the fresh fermentation medium at a volume ratio of 3%, cultured at 180 rpm and 32 °C for 72 h to obtain the bacterial fermentation solution;

[0026] Furthermore, the seed culture medium is composed of: 5 g / L glycerol, 8-10 g / L peptone, 3-5 g / L malt extract powder, 3-5 g / L yeast powder, 1 g / L NaCl, pH 7.0-7.5;

[0027] Further, the fermentation culture medium is composed of: glucose 10-20 g / L, yeast powder 3-5 g / L, urea 6-8 g / L, KH2PO4 0.5 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, sodium glutamate 0.5-2 g / L, CoCl2·6H2O 0.01-0.02 g / L, NaCl 2 g / L, pH 7.0-7.5;

[0028] (2) Preparation of resting cells of Rhodococcus erythrorhizium: The fermentation broth was centrifuged, the supernatant was removed, and the wet bacterial cells were washed 2-3 times with KH2PO4-NaOH buffer at pH 7.5 to obtain resting cells of Rhodococcus erythrorhizium.

[0029] Beneficial effects:

[0030] 1. The Rhodococcus erythrocytes CGMCC No.31246 provided by the present invention improves the enzyme activity of the original Rhodococcus erythrocytes fermentation broth, and the nitrile hydratase activity of the strain can reach 5500 U·mL -1 The enzyme activity of the fermentation broth of the original strain was increased by 27%, which improved the catalytic efficiency of 2,6-difluorobenzonitrile to produce 2,6-difluorobenzamide and other nitrile substances to amides.

[0031] Taking the production of 2,6-difluorobenzonitrile to 2,6-difluorobenzamide as an example, under the same substrate concentration (2.5M), cell concentration and the same conversion conditions, the production intensity of this strain can reach 0.417 mol / L / h, which is 2.5 times higher than that of the original strain. After testing, the conversion rate of 2,6-difluorobenzonitrile was 100%, the selectivity for 2,6-difluorobenzamide was 100%, and no by-product 2,6-difluorobenzoic acid and other substances were produced.

[0032] Compared with the prior art, the transformation using the Rhodococcus erythrocytes CGMCC No.31246 provided by the present invention can achieve the effect of rapidly producing 2,6-difluorobenzamide and other amide substances, thereby achieving the purpose of improving production efficiency, meeting environmental protection requirements, reducing production costs, and having broad industrial application prospects.

[0033] 2. Compared with the original Rhodococcus, the total feed amount of the Rhodococcus CGMCC No.31246 provided by the present invention is increased. The maximum feed concentration of the original strain is only 3.5 mol / L, while the maximum feed concentration of CGMCC No.31246 can reach 7 mol / L, which is twice the feed concentration of the original strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 For Rhodococcus erythrocytes ( Rhodococcusruber ) Colony morphology and electron microscope images of AR-300;

[0035] in, Figure 1 A in the middle is the colony morphology; Figure 1 B is an electron microscope image.

[0036] Figure 2 Flow chart of strain mutagenesis screening.

[0037] Figure 3 For Rhodococcus erythrocytes ( Rhodococcus ruber) Comparison of bacterial concentration and nitrile hydratase activity between AR-300 and the original strain CGMCC No.3090.

[0038] Figure 4 2,6-difluorobenzonitrile, 2,6-difluorobenzamide standard and Rhodococcus erythrocytes ( Rhodococcusruber ) HPLC chromatogram of AR-300 conversion solution;

[0039] in, Figure 4 A in the figure is the HPLC chromatogram of 2,6-difluorobenzonitrile standard; Figure 4 B is the HPLC chromatogram of 2,6-difluorobenzamide standard; Figure 4 C in the middle is the HPLC chromatogram of the conversion liquid after the conversion was completed with a feed amount of 2.5 mol / L.

[0040] Figure 5 This is the high performance liquid chromatogram of the conversion liquid after the conversion is completed when the feed amount of 2,6-difluorobenzonitrile is 7 mol / L.

[0041] Figure 6 Nicotinonitrile, nicotinamide standard and Rhodococcus erythrocytes ( Rhodococcusruber ) HPLC chromatogram of AR-300 conversion solution;

[0042] in, Figure 6 A in the middle is the HPLC chromatogram of nicotinonitrile standard; Figure 6 B is the HPLC chromatogram of nicotinamide standard; Figure 6 Center C is the HPLC chromatogram of the conversion solution after the conversion is completed. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0044] The culture medium involved in the mutagenesis process of the following examples is as follows:

[0045] (1) Solid culture medium: beef extract 5 g / L, peptone 8-10 g / L, sodium chloride 5 g / L, agar powder 15-20 g / L, pH 7.0-7.5;

[0046] (2) Seed culture medium: glycerol 5 g / L, peptone 8-10 g / L, malt extract 3-5 g / L, yeast powder 3-5 g / L, NaCl 1 g / L, pH 7.0-7.5;

[0047] (3) Fermentation medium: glucose 10-20 g / L, yeast powder 3-5 g / L, urea 6-8 g / L, KH2PO4 0.5 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, sodium glutamate 0.5-2 g / L, CoCl2·6H2O 0.01-0.02 g / L, NaCl 2 g / L, pH 7.0-7.5.

[0048] The present invention is further explained below by means of specific examples.

[0049] Example 1 Mutation breeding of efficient strains

[0050] The mutagenesis screening flow chart is as follows Figure 2 As shown:

[0051] 1. ARTP mutagenesis

[0052] (1) Activation of the starting strain: Cultivate the original strain CGMCC No.3090 in a triangular flask for 24-30 h as the starting strain for ARTP mutagenesis;

[0053] (2) Preparation of starting bacterial suspension: Take 1 ml of the seed solution in step (1) and centrifuge at 12,000 rpm for 2 min. Discard the supernatant to collect the bacterial cells, wash the bacterial cells 2-3 times with 0.9% saline, resuspend and dilute 100 times to prepare a bacterial suspension with uniform cell dispersion, which will be used as the starting bacterial suspension for subsequent ARTP treatment;

[0054] (3) ARTP mutagenesis: 5-10 μL of the starting bacterial suspension prepared in step (2) was evenly coated on a sterilized metal slide and placed in an ARTP mutagenesis breeding instrument for mutagenesis. The ARTP treatment parameters were set as follows: working gas was high-purity helium, RF power was 70-120 W, gas flow rate was 8-12 slm, treatment time was 30-100 s, and irradiation distance was 2-4 mm.

[0055] (4) Post-mutagenesis culture: Wash the induced bacteria with 990 μL of 0.9% sterile saline, dilute appropriately and spread on a solid culture medium plate, and culture inverted at 28-34°C for 3-5 days.

[0056] 2. Preparation of resting cells of mutagenized strains

[0057] (1) Observe the colonies obtained by culture on solid culture medium in step 1, pick a single colony from the plate and inoculate it into a 60% glycerol tube and store it in a -80 ℃ refrigerator;

[0058] (2) The mutant strain and the original strain obtained in step (1) were streaked in a "Z" shape onto a slant culture medium and cultured at 30°C for 3-5 days;

[0059] (3) Scrape a loopful of each culture medium and inoculate it into a 250 mL Erlenmeyer flask containing 30 mL of seed culture medium. Culture at 32 °C and 180 rpm for 30 h to obtain seed solution.

[0060] (4) Inoculate the seed solution into a 250 mL triangular shake flask containing 50 mL of fermentation medium at a volume ratio of 3% and culture at 32 °C and 180 rpm for 72 h.

[0061] (5) The fermentation broth obtained in step (4) is centrifuged to remove the supernatant, and the obtained wet bacterial sludge is washed 2-3 times with a pH 7.5 KH2PO4-NaOH buffer solution, and centrifuged to remove the supernatant to obtain resting cells of Rhodococcus erythrorhizium.

[0062] 3. Screening of highly efficient mutagenic strains

[0063] The resting cells obtained in step 2 were subjected to transformation comparison. In the KH2PO4-NaOH buffer system at pH 7.5, the concentration of the input substrate 2,6-difluorobenzonitrile was 2.5 mol / L, the cell concentration was 2.37 g / L, the transformation conditions were a rotation speed of 180 rpm and a transformation temperature of 30 °C.

[0064] A strain of Rhodococcus with the best transformation effect was obtained, with a transformation time of 8 h and a production intensity of 0.313 mol / L / h, which was significantly higher than that of the original strain Rhodococcus ( Rhodococcus ruber ) Compared with CGMCC No.3090, under the same substrate concentration and bacterial concentration, the transformation time of the original strain was 15 h, and the production intensity was 0.167 mol / L / h. The production intensity of the mutated Rhodococcus strain was 1.87 times higher than that of the original strain.

[0065] Example 2 Adaptive evolution increases the feed amount of substrate for Rhodococcus rubrum strains

[0066] 1. Adaptive evolution

[0067] The induced Rhodococcus strain was selected and inoculated into a seed culture medium containing a certain concentration of 2,6-difluorobenzonitrile (DFBN) and cultured in a constant temperature shaker at 32°C and 180 rpm. Continuous culture was started with an initial DFBN concentration of 0.1% (w / v) and cultured in a constant temperature shaker at 32°C and 180 rpm. The OD 600 (cell density). When each generation was cultured to OD 2, the evolution of this generation was completed, and the adaptive evolution of this generation was considered successful. Large single colonies were picked by streaking to enter the next round of adaptive evolution. The above acclimation operation was repeated at 0.1% concentration gradient intervals until the DFBN concentration reached 1%, and the strain AR-300 was screened.

[0068] 2. Transformation of adaptively evolved strains

[0069] The adaptive evolution strain AR-300 obtained in step 1 was transformed. In the KH2PO4-NaOH buffer system with a pH of 7.5, the concentration of the substrate 2,6-difluorobenzonitrile was 2.5 mol / L, the cell concentration was 2.37 g / L, the transformation conditions were a rotation speed of 180 rpm, a transformation temperature of 30 ° C, the transformation was completed in 6 h, and the production intensity was 0.417 mol / L / h. Compared with the mutagenic Rhodococcus strain obtained in Example 1, the transformation time was shortened from 8 h to 6 h, and the production intensity was increased from 0.313 mol / L / h to 0.417 mol / L / h. The production intensity of Rhodococcus AR-300 was 1.33 times higher than that of the mutagenic Rhodococcus strain; compared with the original strain Rhodococcus ( Rhodococcus ruber ) Compared with CGMCC No.3090, under the same substrate concentration and bacterial concentration, the transformation time of the original strain was 15 h, and the production intensity was 0.167 mol / L / h. The production intensity of Rhodococcus AR-300 was 2.50 times higher than that of the original strain.

[0070] 3. Determination of the maximum substrate feed rate of Rhodococcus AR-300 and its comparison with the maximum substrate feed rate of the original strain CGMCC No.3090

[0071] (1) The obtained resting cells of Rhodococcus AR-300 and the resting cells of the original strain were subjected to gradient feeding catalysis. The bacterial concentration of each gradient was 2.37 g / L, the initial concentration of the substrate was 1 mol / L, the substrate concentration gradient interval was 0.5 mol / L, and Tween 80 was added at a volume ratio of 1% to the reaction system. The conversion system was 50 mmol / L pH 7.5 KH2PO4-NaOH buffer. The catalytic reaction was carried out at 30 °C and 180 rpm. Samples were taken at regular intervals and the conversion rate of 2,6-difluorobenzonitrile to 2,6-difluorobenzamide in the reaction system was detected by high performance liquid chromatography. The results are shown in the following table.

[0072] (2) Table 1 Conversion rate and conversion time of original strain CGMCC No.3090 at different feed rates

[0073]

[0074] (3) Table 2 Conversion rate and conversion time of strain AR-300 at different feed rates

[0075]

[0076] It can be seen from Table 1 and Table 2 that the feed concentration of the Rhodococcus CGMCC No.31246 provided by the present invention is improved compared with the original Rhodococcus. The maximum feed concentration of the original strain is only 3.5 mol / L, and the maximum feed concentration of the Rhodococcus CGMCC No.31246 can reach 7 mol / L, which is 2 times higher than that of the original strain.

[0077] Example 3 Nitrile Hydrase Activity Detection

[0078] The method for culturing bacteria in Example 1 was used to culture Rhodococcus AR-300 and CGMCC No.3090 to obtain fermentation broth.

[0079] 100 μL of fermentation broth was added to 400 μL of 125 mmol·L nicotinonitrile aqueous solution, reacted at 26 °C for 10 min, and then 500 μL of acetonitrile was added to terminate the reaction. The mixture was immediately centrifuged at 12000 rpm at 4 °C for 1 min to obtain the supernatant. The supernatant was diluted appropriately, filtered through a 0.22 μm microporous filter membrane, and detected by high performance liquid chromatography (HPLC). The enzyme activity was calculated using the standard curve.

[0080] Nitrile hydratase activity (U·mL -1 )

[0081] Among them, A NR : Nicotinamide content determined by HPLC, unit: g / L;

[0082] V 反 : Total volume of catalytic reaction system, 500 μL;

[0083] X: dilution multiple;

[0084] M NR : Niacinamide molecular weight, 122.125 g / mol;

[0085] T: reaction time, 10 min;

[0086] V 发 : The volume of fermentation liquid added, 100 μL.

[0087] Definition of enzyme activity unit (U·mL -1 ): The amount of enzyme required to catalyze the production of 1 μmol nicotinamide from the substrate nicotinonitrile per minute.

[0088] Detection method: The chromatographic column was a C18 column (150×4.6 mm), the column temperature was 40 ℃, the flow rate was 0.6 mL / min, the injection volume was 20 μL, the chromatographic retention time was 5 min, and the detection wavelength was 220 nm; the peak time of nicotinamide was 1.78 min, and the peak time of nicotinonitrile was 2.96 min.

[0089] The mobile phase was acetonitrile: ultrapure water = 1:2.

[0090] Standard solutions of nicotinonitrile and nicotinamide with different concentrations (0.25 mmol / L, 0.5 mmol / L, 1 mmol / L, 1.5 mmol / L, 2 mmol / L) were prepared with mobile phase, and the peak areas of the above standard solutions were detected by HPLC. The standard curves were calculated according to the peak areas and the concentrations of nicotinonitrile and nicotinamide standard solutions: Y=6576.6X+8.3927, R 2 =0.9996 (where Y is the concentration of nicotinonitrile and X is the peak area);

[0091] Y=5498.3X+0.2835, R 2 =0.9995 (where Y is the concentration of nicotinamide and X is the peak area). A peak area can be obtained by testing the conversion sample of unknown concentration by HPLC, and then the concentration can be obtained by substituting it into the above standard curve formula.

[0092] The calculation results are as follows Figure 3 As shown in Figure 2, the nitrile hydratase activity of the fermentation broth of Rhodococcus AR-300 can reach 5500 U·mL -1 , the starting strain Rhodococcus erythrocytes ( Rhodococcus ruber )The enzyme activity of CGMCC No.3090 fermentation broth is 4315 U·mL -1 The enzyme activity of the fermentation broth of Rhodococcus AR-300 increased by 27% compared with the fermentation broth of the original strain.

[0093] Example 4 Rhodococcus erythrocytes ( Rhodococcus ruber ) Genetic stability of AR-300

[0094] The efficient strain obtained in Example 2 ( Rhodococcus ruber )AR-300 was subcultured for 6 times, and each generation of strains was cultured according to the culture method of step 3 in Example 1, and each generation of strains was stored at -4°C in a slanted "z"-shaped streak. The results showed that ( Rhodococcus ruber ) After the AR-300 strain was subcultured for 6 times, the production intensity of each generation of the strain was maintained at about 0.417 mol / L / h, and the nitrile hydratase activity was above 5500 U / ml, with good genetic stability.

[0095] Example 5 Method for preparing 2,6-difluorobenzamide

[0096] A method for efficiently preparing 2,6-difluorobenzamide using Rhodococcus erythrorhizium AR-300, the steps are as follows:

[0097] (1) Seeds and fermentation culture: Take Rhodococcus erythrocytes ( Rhodococcusruber )AR-300 strain was inoculated into seed culture medium, cultured at 180 rpm and 32 °C for 30 h to obtain seed culture solution, which was then transferred to fresh fermentation medium at a volume ratio of 3%, cultured at 180 rpm and 32 °C for 72 h to obtain bacterial fermentation solution.

[0098] Seed culture medium: glycerol 5 g / L, peptone 8 g / L, malt extract powder 3 g / L, yeast powder 3 g / L, NaCl 1g / L, pH 7.0-7.5;

[0099] Fermentation medium: glucose 10 g / L, yeast powder 3 g / L, urea 6 g / L, KH2PO4 0.5 g / L, K2HPO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, sodium glutamate 0.5 g / L, CoCl2·6H2O 0.01 g / L, NaCl 2 g / L, pH 7.0-7.5;

[0100] (2) Preparation of resting cells of Rhodococcus erythrorhizium: centrifuge the fermentation broth, remove the supernatant, wash the wet bacterial cells 2-3 times with KH2PO4-NaOH buffer at pH 7.5, centrifuge and remove the supernatant to obtain resting cells of Rhodococcus erythrorhizium;

[0101] (3) Conversion of 2,6-difluorobenzonitrile: The reaction volume was 10 ml, the concentration of the substrate 2,6-difluorobenzonitrile was 2.5 mol / L, the concentration of Rhodococcus erythrocytes was 2.37 g / L, and Tween 80 was added at a volume ratio of 1% to the reaction system. The conversion system was 50 mmol / L KH2PO4-NaOH buffer at pH 7.5. The reaction was carried out at 32 °C and 180 rpm. The conversion was completed after 6 h.

[0102] (4) After the conversion, the liquid was sampled and tested by high performance liquid chromatography. The results were as follows: Figure 4 As shown, in this embodiment, the conversion rate of 2,6-difluorobenzonitrile reaches 100%, no by-product 2,6-difluorobenzoic acid and other substances are generated, and the selectivity for 2,6-difluorobenzamide is 100%.

[0103] (5) Preparation of finished product: The feed liquid after the conversion was filtered, the filter cake was washed with water and dried at 60 °C to obtain crude 2,6-difluorobenzamide. The filtrate was extracted with ethyl acetate three times and concentrated in vacuo to obtain crude 2,6-fluorobenzamide. The two parts of the product were combined and recrystallized from dichloromethane and n-hexane to obtain the finished product. After drying to constant weight, the mass of 2,6-difluorobenzamide was 3.82 g and the molar weight was 0.0243 mol. The mass of 2,6-difluorobenzonitrile put into the conversion system was 3.48 g and the molar weight was 0.025 mol. The mass yield was calculated to be 109.77% and the molar yield was 97.2%.

[0104] Example 6 Conversion of 2,6-difluorobenzonitrile by Rhodococcus erythrorhizium AR-300 at the highest feed amount

[0105] In this embodiment, steps (1) and (2) are the same as those in embodiment 5, and only step (3) is described below.

[0106] (3) Conversion of 2,6-difluorobenzonitrile: The reaction volume was 10 ml, the concentration of the substrate 2,6-difluorobenzonitrile was 7 mol / L, the concentration of Rhodococcus erythrocytes was 2.37 g / L, and Tween 80 was added at a volume ratio of 1% to the reaction system. The conversion system was 50 mmol / L KH2PO4-NaOH buffer at pH 7.5. The conversion was carried out at 32 °C and 180 rpm.

[0107] (4) Take samples of the converted liquid and conduct high performance liquid chromatography (HPLC) testing. The results are as follows: Figure 5 As shown, after 24 h of conversion, the final conversion rate of 2,6-difluorobenzonitrile in this example reached 97.5%. If the conversion time is further extended, the concentration of 2,6-difluorobenzamide remains unchanged, 2,6-difluorobenzonitrile is not further converted, the conversion rate no longer increases, and no by-product 2,6-difluorobenzoic acid and other substances are generated.

[0108] Example 7: Rhodococcus erythrorhizium AR-300 catalyzes acrylonitrile to produce acrylamide

[0109] In this embodiment, steps (1) and (2) are the same as those in embodiment 5, and only step (3) is described below.

[0110] (3) Conversion of acrylonitrile: The reaction volume was 10 ml, the concentration of the input substrate acrylonitrile was 5 mol / L, the concentration of the Rhodococcus erythrocytes was 2.37 g / L, and Tween 80 was added at a volume ratio of 1% to the reaction system. The conversion system was 50 mmol / L pH 7.5 KH2PO4-NaOH buffer, 30 °C, 180 rpm. After 6 h of conversion, the contents of acrylonitrile and acrylamide in the converted feed were detected by HPLC. The concentration of acrylamide reached 352.2 g / L, and the substrate conversion rate was greater than 99%. No by-product acrylic acid or other substances were generated.

[0111] Example 8: Rhodococcus erythrogenum AR-300 catalyzes nicotinonitrile to produce nicotinamide

[0112] In this embodiment, steps (1) and (2) are the same as those in embodiment 5, and only step (3) is described below.

[0113] (3) Conversion of nicotinamide: The reaction volume was 10 ml, the concentration of the substrate nicotinamide was 5 mol / L, the concentration of Rhodococcus erythrocytes was 2.37 g / L, and Tween 80 was added at a volume ratio of 1% to the reaction system. The conversion system was 50 mmol / L pH 7.5 KH2PO4-NaOH buffer, 30 °C, 180 rpm. After 9 h of conversion, the contents of nicotinamide and nicotinamide in the converted liquid were detected by high performance liquid chromatography. The results are as follows: Figure 6 As shown, the concentration of nicotinamide reached 605.2 g / L, the substrate conversion rate was greater than 99%, and no by-product nicotinic acid or other substances were generated.

[0114] Example 9: Rhodococcus erythrorhizium AR-300 catalyzes cinnamonitrile to produce cinnamamide

[0115] In this embodiment, steps (1) and (2) are the same as those in embodiment 5, and only step (3) is described below.

[0116] (3) Conversion of cinnamonitrile: The reaction volume was 10 ml. The concentration of the substrate cinnamonitrile was 2.5 mol / L. The concentration of the Rhodococcus erythrocytes was 2.37 g / L. Tween 80 was added at a volume ratio of 1% to the reaction system. The conversion system was 50 mmol / L pH 7.5 KH2PO4-NaOH buffer. The reaction was carried out at 30 °C and 180 rpm. After 10 h of conversion, the contents of cinnamonitrile and cinnamamide in the converted liquid were detected by HPLC. The concentration of cinnamamide reached 359.3 g / L. The substrate conversion rate was greater than 99.6%. No by-product cinnamic acid or other substances were generated.

[0117] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A strain of Rhodococcus erythrocytes, characterized in that: The Rhodococcus erythrocytes are specifically Rhodococcus erythrocytes ( Rhodococcus ruber )AR-300, the deposit number is CGMCC No.31246.

2. The Rhodococcus erythrocytes according to claim 1 ( Rhodococcusruber ) Application of AR-300 in the production of amide substances; characterized in that, The amide substance is: 2,6-difluorobenzamide, acrylamide, nicotinamide or cinnamamide.

3. The use according to claim 2, characterized in that Using Rhodococcus ruber ( Rhodococcus ruber ) The method for preparing amide substances by AR-300 is as follows: (1) Transformation: Add substrate and Rhodococcus erythrocytes ( Rhodococcus ruber ) AR-300 resting cells for biotransformation; (2) Preparing the finished product: After the conversion, the feed liquid is filtered, the filter cake is washed with water and dried to obtain a crude amide substance, and the filtrate is extracted with an organic solvent and concentrated in vacuo to obtain a crude amide substance, and then the two parts of the product are combined and recrystallized to obtain the finished product; The amide substance is: 2,6-difluorobenzamide, acrylamide, nicotinamide or cinnamamide.

4. The use according to claim 3, characterized in that The substrate in step (1) is: 2,6-difluorobenzonitrile, acrylonitrile, nicotinonitrile or cinnamonitrile.

5. The use according to claim 3, characterized in that In step (1), 0.5-5% of a dissolving agent is also added; the dissolving agent is methanol, isopropanol, propylene glycol or Tween 80.

6. The use according to claim 3, characterized in that The bioconversion conditions of step (1) are: rotation speed 110-220 rpm, conversion temperature 25-40 °C, and conversion time 2-24 h.

7. The use according to claim 3, characterized in that The cell concentration of Rhodococcus AR-300 is 0.66-3.30 g / L; the input concentration of the substrate is not higher than 7 mol / L.

8. The use according to claim 3, characterized in that The extraction solvent used in the extraction is ethyl acetate, chloroform, dichloromethane or butyl acetate; The recrystallization solvent used in the recrystallization is at least one of methanol, ethanol, chloroform, acetone, dichloromethane, chloroform or n-hexane.

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