CN1786179A - Microbiological preparation method of S-(+)-2,2-dimethyl cyclo propyl formamide - Google Patents

Abstract

The invention discloses a new S-(+)-2,2-dimethyl ring abicin formamide microorganism making method. It includes using new microorganism rhodococcuss equi CCTCC No.M 205114 to catalyze racemize 2, 2-dimethyl ring abicin formonitrile to produce racremize 2,2-dimethyl ring abicin formamide, and using new microorganism delftia tsuruhatensis CCTCC No.M 205115 to selectively catalyze and hydrolyze racemize 2,2-dimethyl ring abicin formamide to produce S-(+)-2,2-dimethyl ring abicin formamide, or uniting CCTCC No.M 205114 and CCTCC No.M 205115 to transform racemize 2, 2-dimethyl ring abicin formonitrile to make S-(+)-2,2-dimethyl ring abicin formamide. Its yield is more than 47%; and e.e value is more then 98%.

Description

The microbial preparation method of S-(+)-2,2-dimethylcyclopropanamide

                      

The present invention relates to a kind of new two kinds of microorganisms screened from soil and sewage, which are used to catalyze racemic 2,2-dimethylcyclopropanecarbonitrile to produce racemic 2,2-dimethylcyclopropanamide , and then selectively hydrolyzing racemic 2,2-dimethylcyclopropylcarboxamide to produce S-(+)-2,2-dimethylcyclopropylcarboxamide.

Background technique

Nitrile compounds are a class of substances widely distributed in nature, and are also an important source of C ₁ , which can be converted into amines, imines, amides, amidines, carboxylic acids and carbonyl compounds. Traditional chemical hydrolysis of nitrile compounds can The consumption is large, the by-products are many, and it needs to be carried out under the condition of strong acid or strong base, so it is only limited to those nitrile compounds that do not contain easily hydrolyzed groups, and it is not selective. On the contrary, the conversion of nitrile compounds into corresponding amides and acids by microorganisms has the advantages of mild conditions, less environmental pollution, and the ability to achieve chemoselectivity, regioselectivity, and stereoenantioselectivity. At present, microbial source nitrile hydratase has been successfully applied to the biocatalytic production of acrylamide, nicotinamide, etc.

The microbial degradation of nitriles has three pathways: oxidation, reduction and hydrolysis. The latter can be divided into two types according to the different enzyme systems involved in the hydrolysis reaction: one is nitrilase (nitrilase, E.C.3.5.5.1), which directly hydrolyzes nitrile into carboxylic acid and ammonia; Under the action of enzymes (nitrole hydratas, E.C.4.2.1.84), nitriles are hydrolyzed into amides. If amidases (amidase, E.C.3.5.1.4) still exist in microorganisms, carboxylic acids and ammonia are further generated under the action of the microorganisms. The reaction requires two step complete.

S-(+)-2,2-Dimethylcyclopropylcarboxamide is an important intermediate of cilastatin. Cilastatin, the chemical name is (+)-(Z)-7-[(2R)-(2-amino-2-carboxyethyl)sulfur]-2-[(1S)-(2,2-di Methylcyclopropanamide)]-2-heptenoic acid, is the first renal dehydropeptidase inhibitor in clinical application, which can effectively prevent imipenem from being degraded by renal dehydropeptidase in vivo, and enhance imipenem Antibacterial activity of South.

K.Robins et al. (US Patent 5273903, 5427934) have reported that starting from racemic 2,2-dimethylcyclopropylcarboxamide, through the microorganism Comamonas acidovorans A: 18 (DSM No. 6315), Comamonas acidovoransTG 308 (DSM No.6552), Pseudomonas sp.NSAK: 42 (DSM No.6433), Bacterium sp.VIII: II (DSM No.6316) kinetic split, the R-( -)-2,2-Dimethylcyclopropanecarboxamide is converted to the corresponding acid to prepare S-(+)-2,2-dimethylcyclopropanecarboxamide. LIM, Kwang-Min et al. reported that racemic 2,2-dimethylcyclopropanecarboxylic acid was first derivatized into the corresponding ethyl ester, and S-(+)-2 was selectively hydrolyzed by optically selective lipase, Ethyl 2-dimethylcyclopropanecarboxylate is the corresponding amide and ethanol, and the S-amide is prepared by adding hydrogenation of S-(+)-2,2-dimethylcyclopropanecarboxylic acid (WO2004/005241 A1).

Mei-Xiang Wang et al. (Journal of Molecular Catalysis B: Enzymatic, 2002, 18: 267-272) reported that racemic 2,2-dimethylcyclopropanenitrile was used as a substrate, and Rhodococcus sp.AJ270 was used as a catalyst To prepare R-(-)-2,2-dimethylcyclopropanamide and S-(+)-2,2-dimethylcyclopropanecarboxylic acid.

However, there is no racemic 2,2-dimethylcyclopropanecarbonitrile as a substrate at home and abroad. The corresponding amide is prepared by microbial catalysis, and then the amide is selectively hydrolyzed to produce S-(+)-2. A report on 2-dimethylcyclopropanamide. Starting from the same substance, the chemical method prepares the amide by refluxing dimethylcyclopropanenitrile in 6mol/L aqueous KOH solution for 8-16h. But this process involves the reaction conditions of strong alkali and high temperature and high pressure, which will pollute the environment, and will inevitably produce by-product 2,2-dimethylcyclopropanecarboxylic acid (J.Am.Chem.Soc, 1957, 79:3467-3469). Therefore, the catalyzed synthesis of 2,2-dimethylcyclopropanecarboxamide by microbial method provides raw materials for further dynamic resolution, and then its selective hydrolysis has dual value of economy and society.

Contents of the invention

The present invention respectively provides new microorganisms screened from soil and sewage that can convert racemic 2,2-dimethylcyclopropanecarbonitrile into corresponding amides, and new microorganisms that selectively hydrolyze amides . Secondly, the method of using the new microorganism to transform and produce amides and to produce optically active amides through selective hydrolysis is provided.

Involving 2,2-dimethylcyclopropanamide and S-2,2-dimethylcyclopropanamide synthesis principle reaction formula is as follows:

2,2-Dimethylcyclopropanecarbonitrile 2,2-Dimethylcyclopropanecarboxamide

2,2-Dimethylcyclopropanamide S-2,2-Dimethylcyclopropanamide R-2,2-Dimethylcyclopropanecarboxylic acid

The microorganism producing nitrile hydratase provided by the invention is Rhodococcus equi (Rhodococcus equi) of the genus Rhodococcus (Rhodococcus), and this bacterial strain has been preserved in China Type Culture Collection Center on October 10, 2005, referred to as CCTCC, and the preservation number It is CCTCC No.M205114.

The new strain is characterized as follows:

Colony morphology: cultured at 30°C for 2 days, the colony on the beef extract peptone plate culture is round, with a diameter of 2 mm, and the raised shape is convex mirror-like, light red, with neat and smooth edges.

Cell morphology: straight thin rod-shaped, 0.3-0.8μm×1.5-8.0μm; cells appear in single pairs, without spore formation, and do not move.

Physiological and biochemical characteristics: catalase positive, nitrate reduction positive, pyrazinase negative, pyrrolidone arylaminease negative, alkaline phosphatase positive, β-glucuronidase negative, β-galactosidase negative, α - Glucosidase positive, N-acetyl-β-glucosaminidase negative, escin hydrolysis negative, urease negative, gelatin hydrolysis negative. Glucose, ribose, xylose, mannitol, malonate, lactose, sucrose, and liver cannot be utilized. Based on the above results, the strain was identified as Rhodococcus equi.

The microorganism producing optically selective amidase provided by the present invention is the Delftia tsuruhatensis (Chinese name not found) bacterium of the genus Delftie (Delftie), and this bacterial strain has been preserved in Chinese Type Culture on October 10, 2005 The depository center, referred to as CCTCC, has a deposit number of CCTCC No.M 205115.

The new strain is characterized as follows:

Colony morphology: cultured at 30°C for 3 days, the colony on the beef extract peptone plate culture is round, with a diameter of 3 mm, the shape of the bulge is pointed, and the edges are neat and smooth.

Cell morphology: Slightly curved rod-shaped, 1.4-4.0 μm long, 0.6-1.3 μm wide, appearing singly or in pairs, without spore formation, and able to move.

Physiological and biochemical characteristics: new strains screened from soil, Gram negative, arginine dihydrolase positive, oxidase positive, catalase positive, esterase (Tween 80) positive, cytochrome oxidase positive . Glucose acid production negative, fructose acid production positive. Nitrate and nitrite reduction were negative. Cannot utilize rhamnose, N-acetylglucosamine, ribose, inositol, sucrose, glycogen, D-glucose, salicin, D-melibiose, L-fucose, D-sorbitol, L-arabino Sugar, L-serine as carbon source. Itaconic acid, suberate, malonate, acetate, DL-lactate, L-alanine, 5-keto-gluconate, 3-hydroxy-benzoate, Mannitol, propionate, caprate, valerate, citrate, histidine, 3-hydroxy-butyrate, L-proline, terephthalate are carbon sources. Based on the above results, the strain was identified as Delftia tsuruhatensis.

The microorganisms involved in the present invention are screened through the following procedures:

1) Inoculate the collected soil and sewage samples into the enrichment medium, and culture them on a shaker at 30°C and 150rpm for 4 days. After the medium becomes turbid, transfer 1ml of the culture solution to the fresh enrichment culture culture medium for another 4 days. Repeat this for 4 to 5 cycles. The enrichment medium was enriched with 1-2.5g/L 2,2-dimethylcyclopropanecarbonitrile (for screening CCTCC No.M 205114) or 2,2-dimethylcyclopropaneamide (for screening CCTCC No. .M 205115) is the only nitrogen source, and other substances are added as follows (g/L): Na ₂ HPO ₄ 12H ₂ O: 1.0～2.5, KH ₂ PO ₄ : 1.0～2.0, MgSO ₄ 7H ₂ O: 0.2 to 0.5, FeSO ₄ ·7H ₂ O: 0.01 to 0.05, CoCl ₂ : 0.01 to 0.05, CaCl ₂ : 0.01 to 0.06, prepared with tap water, and adjusted to pH 6.0 to 8.0 with 1.0 mol of hydrochloric acid or NaOH solution.

2) Dilute the enriched culture solution for the last time and spread it on the plate medium, pick a single colony and store it on the slant. The plate medium is enriched medium with 1.5%-2.0% agar added.

3) Pick a single colony and inoculate it into the induction fermentation medium. The components are as follows (g/L): glucose 10.0-20.0, yeast extract 5.0-10.0, peptone 2.0-5.0, NaCl 1.0-3.0, K ₂ HPO ₄ 1.0- 2.0, KH ₂ PO ₄ 1.0~2.0, MgSO ₄ 0.2~0.5, FeSO ₄ 0.01~0.05, CoCl ₂ 0.01~0.05, CaCl ₂ 0.05~0.1, caprolactam 1.0~2.0, natural pH (sterilized at 121°C for 20 minutes) ; Cultivate for 48h to 72h, centrifuge and suspend in phosphate buffer system, add 2,2-dimethylcyclopropanecarbonitrile or amide as a substrate for conversion; analyze the amount of product and substrate by gas chromatography. The specific operating conditions are as follows: the gas chromatograph is GC-14C, the capillary column is HP-1, the temperatures of the inlet, detector and column oven are 180°C, 220°C, and 120°C respectively, and the split ratio is 50:1. The content of R- and S-2,2-dimethylcyclopropylcarboxamide in the converted product was determined by chiral liquid chromatography. First, the amide was derivatized into N,N-benzhydrylamide, and separated on a Chiralcel OD column, the mobile phase was cyclohexane/2-acetone, the ratio was 9:1, and the flow rate was 0.4ml/min.

Initial culture: The screened microorganisms are inoculated into the seed medium for initial culture. The composition of the seed medium is as follows (g/L): glucose 10.0-20.0, yeast extract 5.0-10.0, peptone 2.0-5.0, NaCl 1.0-3.0, K ₂ HPO ₄ 1.0-2.0, KH ₂ PO ₄ 1.0-2.0, MgSO ₄ 0.2-0.5, FeSO ₄ 0.01-0.05, CoCl ₂ 0.01-0.05, CaCl ₂ 0.05-0.1, natural pH (sterilized at 121°C for 20 minutes). The requirement for the seed medium is to have a suitable C/N ratio to shorten the lag period of microbial growth as much as possible. Suitable carbon sources include glycerin, maltose, fructose, sucrose, mannitol, etc.; suitable nitrogen sources include peptone, beef extract, soybean powder, ammonium sulfate, etc. The pH of the medium should be between 6 and 8, and the culture temperature should be 20°C to 50°C. After culturing in the seed medium for 24-48 hours, it can be prepared to be inserted into the fermentation induction medium.

Induction: Since the nitrile hydratase contained in the microorganism of the present invention is an inducible enzyme, it needs to be induced to have a high enzyme activity. And the amidase that the microorganism of the present invention comprises is a constituent enzyme, does not need induction, but adding suitable chemical substance also can greatly improve enzyme activity as activator, as caprolactam, isobutyronitrile, 2,2-dimethylcyclopropane Formamide, etc., the amount of the activator is 1.0-2.0 g/L of the culture medium. The seed liquid is inoculated into the induction medium at a certain ratio, and the induction medium is generally based on adding a suitable inducer on the basis of the seed medium, and these inducers include 2,2-dimethylcyclopropanecarbonitrile, 2, One of 2-dimethylcyclopropanamide, acrylamide, caprolactam, acetamide, and acrylonitrile. The appropriate amount of the inducer is 1.0-2.0 g/L of the induction medium. After 60-80 hours of induction, the cells are collected by centrifugation or filtration to provide catalysts for the next step of biotransformation.

Biotransformation: The process of biotransformation uses the cells or cell treatments of microorganisms Rhodococcuss equiCCTCC No.M 205114 and Delftia tsuruhatensis CCTCC No.M 205115 of the present invention as catalysts, and suitable systems for biotransformation include phosphate buffer solution, Tris-hydrochloric acid buffer solution, citric acid buffer solution, etc. The pH range of the buffer system is 6.5-9.0. The temperature range during conversion is 20-40°C. The range of the substrate amount in the conversion system is 0.08-2% (wt/v, weight to volume ratio, the same below), and the optimal range is between 0.1-2% (wt/v). Using the cells of Rhodococcus equi CCTCC No.M 205114 as a catalyst, 2,2-dimethylcyclopropanecarbonitrile can be completely converted into the corresponding racemic amide after 0.5-4 hours of reaction. After removing the cells by centrifugation, add the cells of Delftia tsuruhatensis CCTCC No.M205115 to the transformation solution, and further convert the R-(-)-2,2-dimethylcyclopropanamide into the corresponding acid to prepare S-(+)-2,2-Dimethylcyclopropylcarboxamide. Due to the low solubility of 2,2-dimethylcyclopropanamide in water, the conversion solution was extracted with ethyl acetate, evaporated under reduced pressure, and the obtained solid was dissolved in hot methanol, placed in an ice bath overnight, and precipitated The crystals were filtered and dried in an evaporating dish to obtain optically pure S-(+)-2,2-dimethylcyclopropylcarboxamide.

In the present invention, 2,2-dimethylcyclopropanecarbonitrile is used as a substrate to synthesize 2,2-dimethylcyclopropanecarboxamide through a biotransformation method, the reaction conditions are mild, the reaction process is simple, and the yield is high. Environmental pollution is less. At the same time, by directly adding the newly screened microorganisms into the transformation solution of this step as a catalyst, the optically selective hydrolysis of the racemic amide generates S-(+)-2,2-dimethylcyclopropylcarboxamide, reducing The extraction step of the intermediate product is eliminated, and the yield and the excess value of the corresponding body are relatively high, reaching 43% and above 98% respectively.

Specific implementation plan

The following examples can enable those skilled in the art to understand the present invention more fully.

Prepare medium:

(1) The composition and ratio range of enrichment medium is (g/L): Na ₂ HPO ₄ ·12H ₂ O: 1.0～2.5, KH ₂ PO ₄ : 1.0～2.0, MgSO ₄ ·7H ₂ O: 0.2 ～0.5, FeSO ₄ ·7H ₂ O: 0.01～0.05, CoCl ₂ : 0.01～0.05, CaCl ₂ : 0.01～0.06, 2,2-dimethylcyclopropanecarbonitrile (screening CCTCC No.M 205114) or 2, 2-Dimethylcyclopropylcarboxamide (screening CCTCC No.M 205115): 1.0-2.5, prepared with tap water, and adjusted to pH 6.0-8.0 with 1.0 mol of hydrochloric acid or NaOH solution.

(2) Seed medium, the content of each component is weight volume percentage, that is, g/L: glucose 10.0-20.0, yeast extract 5.0-10.0, peptone 2.0-5.0, NaCl 1.0-3.0, K ₂ HPO ₄ 1.0- 2.0, KH ₂ PO ₄ 1.0-2.0, MgSO ₄ 0.2-0.5, FeSO ₄ 0.01-0.05, CoCl ₂ 0.01-0.05, CaCl ₂ 0.05-0.1, pH natural.

(3) CCTCC No.M 205114 Inducer-containing fermentation medium, the content of each component is weight volume percentage, that is, g/L: glucose 10.0-20.0, yeast extract 3.0-5.0, peptone 2.0-5.0, NaCl 1.0 ～3.5, K ₂ HPO ₄ 1.0～2.0, KH ₂ PO ₄ 1.0～2.0, MgSO ₄ 0.2～0.5, FeSO ₄ 0.01～0.05, CoCl ₂ 0.01～0.05, CaCl ₂ 0.01～0.06, inducer 1.0～2.0, It is prepared with tap water, and the pH value is adjusted to 6.0-8.0 with 1.0 mol of hydrochloric acid or NaOH solution. The inducer is selected from any one of 2,2-dimethylcyclopropanecarbonitrile, 2,2-dimethylcyclopropanecarboxamide, acrylamide, caprolactam, acetamide, and acrylonitrile.

(4) Prepare CCTCC No.M 205115 fermentation medium containing activator, the content of each component is weight volume percentage, that is, g/L: mannitol 10.0-20.0, yeast extract 5.0-5.0, peptone 2.0-5.0, NaCl 1.0～3.5, K ₂ HPO ₄ 1.0～2.0, KH ₂ PO ₄ 1.0～2.0, MgSO ₄ 0.2～0.5, FeSO ₄ 0.01～0.05, CaCl ₂ 0.01～0.06, activator 1.0～2.0, prepared with tap water, with 1.0mol hydrochloric acid or NaOH solution to adjust the pH value to 6.0-8.0. The activator is selected from any one of isobutyronitrile, caprolactam and 2,2-dimethylcyclopropylcarboxamide.

The following examples are all selected from any set of specific data in the scope of the above-mentioned components.

Embodiment 1: contain the isolation of nitrile hydratase microorganism

Add 1g of soil sample and appropriate amount of glass beads to 9ml of 0.9% physiological saline, shake well to make a uniform soil suspension; draw 0.5ml of soil suspension and inoculate it into a 250ml Erlenmeyer flask containing 30ml of enrichment medium Place in 30°C, 150rpm shaker for 4 days. After the enrichment solution becomes turbid, transfer 0.5ml to fresh medium and continue to cultivate for 4 days; after repeating this for 4 to 5 cycles, Dilute the enrichment solution into multiple gradients and spread it on the separation plate to obtain a single colony;

The enrichment medium uses 2,2-dimethylcyclopropanenitrile as the sole nitrogen source, and the composition and formula (g/L) of this example: Na ₂ HPO ₄ ·12H ₂ O: 2.5, KH ₂ PO ₄ : 2. MgSO ₄ 7H ₂ O: 0.5, FeSO ₄ 7H ₂ O: 0.01, CoCl ₂ : 0.01, CaCl ₂ : 0.06, 2,2-dimethylcyclopropanecarbonitrile: 1.5, prepared with tap water, pH adjusted is 7.0;

Pick a single colony and inoculate it into the enrichment medium, take samples after 48h and 72h of culture, and use gas chromatography to detect whether there is 2,2-dimethylcyclopropanamide; if there is amide formation, it is the required Microorganism Rhodococcus equi CCTCC No.M 205114.

Embodiment 2: Contain the separation of photoselective amidase microorganism

Only the nitrogen source in the enrichment medium is changed to 2,2-dimethylcyclopropanamide (1.5g/L), and other steps are the same as in Example 1; the product S-(+ )-2,2-dimethylcyclopropanamide corresponding body excess value (e.e value), e.e value exceeds 98%, that is the required microorganism Delftia tsuruhatensis CCTCC No.M 205115.

Embodiment 3: the production of 2,2-dimethylcyclopropanamide

Put the bacterial strain Rhodococcus equi CCTCC No.M 205114 into a 250ml Erlenmeyer flask containing 30ml of seed medium. The composition of the seed medium is as follows (g/L): glucose 10.0, yeast extract 5.0, peptone 2.0 , NaCl 1.0, K ₂ HPO ₄ 1.0, KH ₂ PO ₄ 1.0, MgSO ₄ 0.2, FeSO ₄ 0.01, CoCl ₂ 0.01, CaCl ₂ 0.05, natural pH (121°C, sterilized for 20min), cultured at 30°C for 2 days , to obtain the seed solution;

Inoculate the seed liquid into the induction fermentation medium containing inducer caprolactam, and the inoculum size is 5% (V/V); the components of the fermentation medium are as follows (g/L): glucose 10.0, yeast extract 5.0, peptone 2.0, NaCl 1.0, K ₂ HPO ₄ 1.0, KH ₂ PO ₄ 1.0, MgSO ₄ 0.2, FeSO ₄ 0.01, CoCl ₂ 0.01, CaCl ₂ 0.05, caprolactam 1.0, pH 6.0 (121°C, sterilized for 20min), temperature 20°C, Fermentation culture 60h;

The fermentation broth was centrifuged to obtain the thalline, washed with 0.9% normal saline, suspended in 10mM phosphate buffer (1000ml), the pH was 7.0, the OD value of the buffer system was 1, and 2,2-dimethylcyclo Acetonitrile, so that the concentration of the substrate is 0.95g/L, transformed in a water-bath shaker at 30°C for 0.5h, detected by gas chromatography, the substrate has been completely transformed, the transformed liquid was centrifuged to remove the bacteria, and evaporated under reduced pressure In the supernatant, 0.93 g of the product 2,2-dimethylcyclopropylcarboxamide was finally obtained, with a yield of 97.8%.

Embodiment 4: the production of 2,2-dimethylcyclopropanamide

The preparation of seed liquid is with embodiment 3;

Seed liquid is inoculated into the induction fermentation medium that contains inducer 2,2-dimethylcyclopropanecarbonitrile, and the inoculum size is 5% (V/V); The composition of fermentation medium is as follows (g/L): Glucose 10.0, Yeast Extract 5.0, Peptone 2.0, NaCl 1.0, K ₂ HPO ₄ 1.0, KH ₂ PO ₄ 1.0, MgSO ₄ 0.2, FeSO ₄ 0.01, CoCl ₂ 0.01, CaCl ₂ 0.05, 2,2-Dimethylcyclopropane Formaldehyde 1.0, pH 7.0 (at 121°C, sterilize for 20 minutes), temperature 30°C, ferment for 72 hours;

After the bacterial cells were washed with 0.9% physiological saline, they were suspended in 50mM Tris-hydrochloric acid buffer (1000ml), the pH was 8.9, and the OD value of the buffer system was 4, and 2,2-dimethylcyclopropanecarbonitrile was added , so that the concentration of the substrate was 4.75g/L; transformed in a water-bath shaker at 20°C for 1.0h, and centrifuged to remove the bacteria; under reduced pressure, evaporate the transformation solution to finally obtain the product 2,2-dimethylcyclopropane Formamide 4.55g, yield 95.7%.

Embodiment 5: the production of 2,2-dimethylcyclopropanamide

The preparation of seed liquid is with embodiment 3;

Inoculate the seed liquid into the induction fermentation medium containing inducer acrylamide, the inoculum size is 5% (V/V); the components of the fermentation medium are as follows (g/L): glucose 10.0, yeast extract 5.0, peptone 2.0 , NaCl 1.0, K ₂ HPO ₄ 1.0, KH ₂ PO ₄ 1.0, MgSO ₄ 0.2, FeSO ₄ 0.01, CoCl ₂ 0.01, CaCl ₂ 0.05, acrylamide 1.0, pH 8.0 (121°C, sterilized for 20min), temperature 40 ℃, fermentation culture for 80h;

Thalline is suspended in the citric acid buffer (1000ml) of 100mM after washing with 0.9% physiological saline, makes pH 6.0, and the OD value of buffer system is 7, adds 2,2-dimethylcyclopropanecarbonitrile, Make the concentration of the substrate 19g/L; transform in a water-bath shaker at 30°C for 2.0h, centrifuge to remove the bacteria; evaporate the transformation solution under reduced pressure, and finally obtain the product 2,2-dimethylcyclopropylcarboxamide 17.91 g, yield 94.2%.

Embodiment 6: the production of S-(+)-2,2-dimethylcyclopropylcarboxamide

Put the Delftia tsuruhatensis CCTCC No.M 205115 through the slant-activated bacterial species into a 250ml Erlenmeyer flask with 30ml seed culture medium one by one. The composition of the seed culture medium is as follows (g/L): mannitol 10.0, yeast extract 5.0, peptone 2.0, NaCl 1.0, K ₂ HPO ₄ 1.0, KH ₂ PO ₄ 1.0, MgSO ₄ 0.2, FeSO ₄ 0.01, CaCl ₂ 0.05, natural pH (sterilization at 121°C for 20 minutes), culture at 30°C for 48 hours;

Inoculate the seed liquid into the fermentation medium containing caprolactam as an activator, the inoculum size is 5% (V/V), and the components of the fermentation medium are as follows (g/L): mannitol 10.0, yeast extract 5.0, peptone 2.0 , NaCl 1.0, K ₂ HPO ₄ 1.0, KH ₂ PO ₄ 1.0, MgSO ₄ 0.2, FeSO ₄ 0.01, CaCl ₂ 0.05, caprolactam 1.0, pH 6.0 (sterilized at 121°C for 20 minutes), fermented for 80 hours;

The fermentation broth was centrifuged to obtain the bacterial cells, washed with 0.9% normal saline, suspended in 10mM phosphate buffer (1000ml) to make the pH 7.0, and the OD value of the buffer system was 5, adding racemic 2,2-di Methylcyclopropanamide, so that the concentration of the substrate is 1.14g/L; transform in a water-bath shaker at 20°C for 4h; centrifuge the transformed liquid to remove the bacteria, and adjust the pH of the supernatant to 12 with sodium hydroxide, Extract S-(+)-2,2-dimethylcyclopropylcarboxamide (with a small amount of 2,2-dimethylcyclopropylcarboxamide) with an equal volume of ethyl acetate; distill the ethyl acetate under reduced pressure Finally, a white powder was obtained; the white powder was dissolved in hot methanol and recrystallized to obtain 0.55g of S-(+)-2,2-dimethylcyclopropylcarboxamide, the yield was 48.2%, and the e.e value was greater than 98%.

Embodiment 7: the production of S-(+)-2,2-dimethylcyclopropylcarboxamide

The preparation of seed liquid is with embodiment 6;

The seed liquid is inoculated into the fermentation medium containing isobutyronitrile as an activator, the inoculum size is 5% (V/V), and the components of the fermentation medium are as follows (g/L): mannitol 10.0, yeast extract 5.0, Peptone 2.0, NaCl 1.0, K ₂ HPO ₄ 1.0, KH ₂ PO ₄ 1.0, MgSO ₄ 0.2, FeSO ₄ 0.01, CaCl ₂ 0.05, Isobutyronitrile 1.0, pH 7.0 (sterilized at 121°C for 20 minutes), fermented for 72 hours ;

After the bacteria were washed with 0.9% normal saline, they were suspended in 50mM Tris-hydrochloric acid buffer (1000ml) to make the pH 8.9, and the OD value of the buffer system was 10. Add racemic 2,2-dimethylcyclo Propyl formamide, so that the concentration of the substrate was 5.7g/L; transformed in a water-bath shaker at 30°C for 9h; extracted with ethyl acetate and recrystallized to obtain 2.71g of S-(+)-2,2-dimethyl Cyclopropylcarboxamide, the yield is 47.5%, and the e.e value is greater than 98%.

Embodiment 8: the production of S-(+)-2,2-dimethylcyclopropylcarboxamide

The preparation of seed liquid is with embodiment 6;

The seed liquid is inoculated into the fermentation medium containing activator 2,2-dimethylcyclopropanamide, the inoculum size is 5% (V/V), and the composition of the fermentation medium is as follows (g/L): manna Alcohol 10.0, yeast extract 5.0, peptone 2.0, NaCl 1.0, K ₂ HPO ₄ 1.0, KH ₂ PO ₄ 1.0, MgSO ₄ 0.2, FeSO ₄ 0.01, CaCl ₂ 0.05, 2,2-dimethylcyclopropylcarboxamide 1.0, pH 8.0 (121°C, sterilized for 20 minutes), fermented for 60 hours;

After the bacteria were washed with 0.9% normal saline, they were suspended in 100mM citrate buffer (1000ml) to make the pH 6.0, and the OD value of the buffer system was 14. Add racemic 2,2-dimethylcyclopropane Formamide, so that the concentration of the substrate was 22.8g/L; transformed in a water-bath shaker at 40°C for 13h; extracted with ethyl acetate and recrystallized to obtain 5.52g S-(+)-2,2-dimethyl Cyclopropylcarboxamide, the yield is 48.2%, and the e.e value is greater than 98%.

Embodiment 9: Rhodococcuss equi CCTCC No.M 205114 and Delftiatsuruhatensis CCTCC No.M 205115 combined conversion racemic 2,2-dimethylcyclopropanecarbonitrile to produce S-(+)-2,2-dimethylcyclopropane Formamide

The bacterium culture of Rhodococcus equi CCTCC No.M 205114 sees embodiment 3;

The bacterial cell culture of Delftia tsuruhatensis CCTCC No.M 205115 is shown in Example 6;

The cells of Rhodococcus equi CCTCC No.M 205114 were washed with 0.9% normal saline, suspended in 10mM phosphate buffer (1000ml), the OD value of the buffer system was 4, and 2,2-dimethylcyclopropane was added Nitrile, so that the concentration of the substrate is 4.75g/L; transformed in a water-bath shaker at 30°C for 1.0h, and centrifuged to remove the bacteria; Dosing into the transformation liquid, transforming in a water-bath shaker at 30°C for 4h; extracting with ethyl acetate and recrystallizing to obtain 2.68g of S-(+)-2,2-dimethylcyclopropylcarboxamide, to obtain The rate is 47%, and the e.e value is greater than 98%.

Embodiment 10: Rhodococcuss equi CCTCC No.M 205114 and Delftiatsuruhatensis CCTCC No.M 205115 combined conversion racemic 2,2-dimethylcyclopropanecarbonitrile to produce S-(+)-2,2-dimethylcyclopropane Formamide

The bacterium culture of Rhodococcus equi CCTCC No.M 205114 sees embodiment 3;

The bacterial cell culture of Delftia tsuruhatensis CCTCC No.M 205115 is shown in Example 6;

Rhodococcus equi CCTCC No.M 205114 cells were washed with 0.9% normal saline, suspended in 50mM Tris-hydrochloric acid phosphate buffer (1000ml), the OD value of the buffer system was 8, and 2,2-dimethyl Cyclopropanecarbonitrile, so that the concentration of the substrate is 9.5g/L; transformed in a water-bath shaker at 30°C for 2.0h, and centrifuged to remove the bacteria; then Delftia tsuruhatensis CCTCC No.M 205115 washed with 0.9% normal saline The bacteria were added to the transformation solution and transformed in a water-bath shaker at 30°C for 5 hours; extracted with ethyl acetate and recrystallized to obtain 5.41g of S-(+)-2,2-dimethylcyclopropylmethyl Amide, the yield is 47.4%, and the e.e value is greater than 98%.

Claims (6)

1. A two-step microorganism preparation method of S- (+) -2, 2-dimethyl cyclopropane formamide is characterized in that the microorganism is Rhodococcus equi (Rhodococcus equi) of Rhodococcus (Rhodococcus equi) screened from soil and sewage, CCTCC No. M205114 and Delftia tsuruhatensis bacteria of Delftia, CCTCC No. M205115,firstly, CCTCC No. M205114 is used for catalyzing racemic 2, 2-dimethyl cyclopropane carbonitrile to produce racemic 2, 2-dimethyl cyclopropane formamide, and then CCTCC No. M205115 is used for selectively hydrolyzing racemic 2, 2-dimethyl cyclopropane formamide to produce S- (+) -2, 2-dimethyl cyclopropane formamide; or the S- (+) -2, 2-dimethyl cyclopropane carboxamide is produced by jointly converting racemic 2, 2-dimethyl cyclopropanecarbonitrile by using CCTCC No. M205114 and CCTCC No. M205115.

2. The preparation method of claim 1, wherein the CCTCC No. M205114 catalyzes racemic 2, 2-dimethylcyclopropanecarbonitrile to produce racemic 2, 2-dimethylcyclopropanecarboxamide, and the method comprises the following steps:

(1) preparing a seed culture medium, wherein the contents of all components are weight volume percent, namely g/L: 10.0-20.0% of glucose, 5.0-10.0% of yeast extract, 2.0-5.0% of peptone, 1.0-3.0% of NaCl and K₂HPO₄1.0～2.0，KH₂PO₄1.0～2.0，MgSO₄0.2～0.5，FeSO₄0.01～0.05，CoCl₂0.01～0.05，CaCl₂0.05-0.1, and natural pH;

(2) preparing a CCTCC No. M205114 fermentation medium containing an inducer, wherein the contents of all components are weight volume percent, namely g/L: 10.0-20.0% of glucose, 3.0-5.0% of yeast extract, 2.0-5.0% of peptone, 1.0-3.5% of NaCl and K₂HPO₄1.0～2.0，KH₂PO₄1.0～2.0，MgSO₄0.2～0.5，FeSO₄0.01～0.05，CoCl₂0.01～0.05，CaCl₂0.01-0.06 wt%, 1.0-2.0 wt% inducer, prepared with tap water, and adjusting pH to 6.0-8.0 with 1.0M hydrochloric acid or NaOH solution;

(3) preparation of CCTCC No. M205114 thalli: inoculating the CCTCC No. M205114 activated by the inclined plane into a seed culture medium, and culturing under the following conditions: culturing at the pH of 5.0-9.0 and the temperature of 20-50 ℃ for 24-48 h to obtain seed liquid; inoculating the seed liquid into a fermentation medium containing an inducer, wherein the culture conditions are as follows: performing induced culture for 60-80 h at the temperature of 20-40 ℃ and the initial pH of 6.0-8.0; centrifuging or filtering and collecting the induction culture solution to obtain thalli;

(4) biotransformation to prepare 2, 2-dimethylcyclopropanecarboxamide: washing the bacteria with 0.9% physiological saline, suspending the bacteria in phosphate buffer solution, Tris-hydrochloric acid buffer solution or citric acid buffer solution, wherein the pH of the buffer solution is 6.5-9.0, adding a substrate 2, 2-dimethylcyclopropanecarbonitrile, and the concentration of the substrate is 0.8-20 in g/L; the conversion temperature is 20-40 ℃, and the time is 0.5-4 h;

(5) extraction of 2, 2-dimethylcyclopropanecarboxamide: the conversion solution is centrifuged to remove thalli, and the supernatant is evaporated under reduced pressure to obtain the product 2, 2-dimethyl cyclopropane formamide.

3. The method according to claim 2, wherein the inducer is one of 2, 2-dimethylcyclopropanecarbonitrile, 2-dimethylcyclopropanecarboxamide, acrylamide, caprolactam, acetamide, and acrylonitrile.

4. The preparation method of claim 1, wherein the CCTCC No. M205115 selectively hydrolyzes racemic 2, 2-dimethylcyclopropanecarboxamide to produce S- (+) -2, 2-dimethylcyclopropanecarboxamide, and the method comprises the following steps:

(1) preparing a seed culture medium, wherein the contents of all components are weight volume percent, namely g/L: 10.0-20.0% of glucose, 5.0-10.0% of yeast extract, 2.0-5.0% of peptone, 1.0-3.0% of NaCl and K₂HPO₄1.0～2.0，KH₂PO₄1.0～2.0，MgSO₄0.2～0.5，FeSO₄0.01～0.05，CoCl₂0.01～0.05，CaCl₂0.05-0.1, and natural pH;

(2) preparing a CCTCC No. M205115 fermentation medium containing an activator, wherein the contents of all components are in percentage by weight and volume, namely g/L: 10.0-20.0 mannitol, 5.0-5.0 yeast extract, 2.0-5.0 peptone, 1.0-3.5 NaCl, K₂HPO₄1.0～2.0，KH₂PO₄1.0～2.0，MgSO₄0.2～0.5，FeSO₄0.01～0.05，CaCl₂0.01-0.06 wt%, 1.0-2.0 wt% of activator, prepared with tap water, and adjusting pH to 6.0-8.0 with 1.0M hydrochloric acid or NaOH solution;

(3) preparing CCTCC No. M205115 thalli: inoculating the CCTCC No. M205115 activated by the inclined plane into a seed culture medium, wherein the culture conditions are as follows: culturing at the pH of 6.0-8.0 and the temperature of 20-40 ℃ for 24-48 h to obtain seed liquid; inoculating the seed liquid into a fermentation culture medium, wherein the culture conditions are as follows: culturing for 60-80 h at 20-40 ℃ and initial pH of 6.0-8.0; centrifuging or filtering and collecting the induction culture solution to obtain thalli;

(4) biotransformation of S- (+) -2, 2-dimethylcyclopropanecarboxamide: washing the above-mentioned fungus body with 0.9% physiological saline, suspending in phosphate buffer solution, or Tris-hydrochloric acid buffer solution, or citric acid buffer solution, the pH of buffer solution is 6.5-9.0, adding racemic 2, 2-dimethyl cyclopropane formamide, the concentration of substrate 2, 2-dimethyl cyclopropane formamide is expressed by g/L as 1.14-22.8; the conversion temperature is 20-40 ℃, and the time is 4-13 h;

(5) extracting S- (+) -2, 2-dimethyl cyclopropane formamide: centrifuging the conversion solution to remove thalli, adjusting the pH value to 12 by using sodium hydroxide, extracting by using ethyl acetate with the same volume, distilling under reduced pressure, and recrystallizing by using methanol to obtain the S- (+) -2, 2-dimethyl cyclopropane formamide.

5. The method according to claim 4, wherein the activator is one of caprolactam, isobutyronitrile, and 2, 2-dimethylcyclopropanecarboxamide.

6. The preparation method of claim 1, 2 or 4, wherein the CCTCC No. M205114 and the CCTCC No. M205115 are used for jointly converting racemic 2, 2-dimethylcyclopropanecarbonitrile to produce S- (+) -2, 2-dimethylcyclopropanecarboxamide, and the method comprises the following steps:

(1) washing the prepared CCTCC No. M205114 strain with 0.9% physiological saline, suspending the strain in a phosphate buffer solution, a Tris-hydrochloric acid buffer solution or a citric acid buffer solution, wherein the pH of the buffer solution is 6.5-9.0, adding a substrate 2, 2-dimethylcyclopropanecarbonitrile, and the concentration of the substrate is expressed by g/L as 4.75-9.5; the conversion temperature is 20-40 ℃, and the time is 0.5-4 h; centrifuging to remove thallus;

(2) washing the prepared CCTCC No. M205115 strain with 0.9% physiological saline, adding the strain-removed strain-containing 2, 2-dimethylcyclopropanecarboxamide conversion solution, and continuing to convert for 4-6 h at the conversion temperature of 20-40 ℃;

(3) and extracting the conversion solution by ethyl acetate, and recrystallizing to obtain the S- (+) -2, 2-dimethyl cyclopropane carboxamide.