CN1786179A - Microbiological preparation method of S-(+)-2,2-dimethyl cyclo propyl formamide - Google Patents
Microbiological preparation method of S-(+)-2,2-dimethyl cyclo propyl formamide Download PDFInfo
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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
Technical Field
The invention relates to a method for producing racemic 2, 2-dimethylcyclopropanecarboxamide by catalyzing racemic 2, 2-dimethylcyclopropanecarbonitrile with two new microorganisms screened from soil and sewage, and producing S- (+) -2, 2-dimethylcyclopropanecarboxamide by selectively hydrolyzing racemic 2, 2-dimethylcyclopropanecarboxamide.
Background
Nitrile compounds are widely distributed in nature and are also important C1Sources convertible to amines, imines, amides, amidines, carboxylic acids and carbonylsThe traditional chemical method for hydrolyzing nitrile compounds has large energy consumption and a plurality of byproducts, and needs to be carried out under the condition of strong acid or strong alkali, so that the method is only limited to nitrile compounds without easily hydrolyzed groups and has no selectivity. On the contrary, the conversion of nitrile compounds into corresponding amides and acids by microorganisms has the advantages of mild conditions, little environmental pollution, realization of chemoselectivity, regioselectivity, stereoselectivity, and the like. At present, microorganism-derived nitrile hydratase has been successfully applied to biocatalytic production of acrylamide, nicotinamide, and the like.
The microbial degradation of nitrile substances includes three ways of oxidation, reduction, hydrolysis and the like. The latter can in turn be divided into two classes according to the enzyme systems involved in the hydrolysis reaction: one is nitrilase (e.c. 3.5.5.1), which directly hydrolyzes nitriles to carboxylic acids and ammonia; another approach is to hydrolyze the nitrile to the amide by the action of nitrile hydratase (E.C.4.2.1.84), which, if amidase (E.C.3.5.1.4) is also present in the microorganism, leads to the further formation of carboxylic acid and ammonia, and the reaction is carried out in two steps.
S- (+) -2, 2-dimethylcyclopropanecarboxamide is an important intermediate of cilastatin. Cilastatin, the chemical name of which is (+) - (Z) -7- [ (2R) - (2-amino-2-carboxyethyl) sulfur]-2- [ (1S) - (2, 2-dimethylcyclopropanecarboxamido)]-2-heptenoic acid, is the first kidney dehydropeptidase inhibitor applied to clinical application, can effectively prevent the kidney dehydropeptidase in vivo from degrading imipenem and enhance the antibacterial activity of imipenem.
Robins et al (US Patent 5273903, 5427934) have reported that the synthesis of a compound from racemic 2, 2-dimethylcyclopropanecarboxamide by the optically selective amidase-containing microorganism Comamonas acidovarans a: 18(DSM No.6315), Commonas acidovanansTG 308(DSM No.6552), Pseudomonas sp.NSAK: 42(DSM No.6433), Bacterium sp.VIII: II (DSM No.6316) kinetic resolution, conversion of R- (-) -2, 2-dimethylcyclopropanecarboxamide therein 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 to the corresponding ethyl ester, S- (+) -2, 2-dimethylcyclopropanecarboxylic acid ethyl ester therein was selectively hydrolyzed to the corresponding amide and ethanol by an optically selective lipase, and S-amide was prepared by the ammonification of S- (+) -2, 2-dimethylcyclopropanecarboxylic acid (WO 2004/005241A 1).
Mei-xing Wang et al (Journal of Molecular Catalysis B: Enzymatic, 2002, 18: 267-272) have reported the preparation of R- (-) -2, 2-dimethylcyclopropanecarboxamide and S- (+) -2, 2-dimethylcyclopropanecarboxylic acid using racemic 2, 2-dimethylcyclopropanecarbonitrile as a substrate and Rhodococcus sp.AJ270 as a catalyst.
However, reports that racemic 2, 2-dimethylcyclopropanecarbonitrile is taken as a substrate, corresponding amide is prepared by a microbial catalysis method, and then the amide is selectively hydrolyzed to produce S- (+) -2, 2-dimethylcyclopropanecarboxamide are not seen at home and abroad. Starting from the same substances, the amide is prepared by a chemical method through refluxing dimethylcyclopropanecarbonitrile in 6mol/L KOH aqueous solution for 8-16 h. However, this process involves strong alkali and high-temperature and high-pressure reaction conditions, which cause environmental pollution, and the by-product 2, 2-dimethylcyclopropanecarboxylic acid (J.Am.chem.Soc, 1957, 79: 3467-3469) is inevitably produced therein. Therefore, the 2, 2-dimethyl cyclopropane carboxamide is catalytically synthesized by a microbiological method, provides raw materials for further kinetic resolution, and is selectively hydrolyzed, so that the method has economic and social values.
Disclosure of Invention
The present invention provides novel microorganisms selected from soil and sewage that convert racemic 2, 2-dimethylcyclopropanecarbonitrile to the corresponding amides, and novel microorganisms that selectively hydrolyze the amides, respectively. Secondly, a method for producing amides by transformation with the novel microorganism and producing optically active amides by selective hydrolysis is provided.
Relates to the synthesis principle of 2, 2-dimethyl cyclopropane carboxamide and S-2, 2-dimethyl cyclopropane carboxamide, and the reaction formula is as follows:
2, 2-dimethylcyclopropanecarbonitrile 2, 2-dimethylcyclopropanecarboxamide
2, 2-dimethyl Cyclopropanecarboxamide S-2, 2-dimethyl Cyclopropanecarboxamide R-2, 2-dimethyl Cyclopropanecarboxylic acid
The microorganism for producing nitrile hydratase provided by the invention is Rhodococcus equi (Rhodococcus equi) of Rhodococcus, the strain is preserved in China Center for Type Culture Collection (CCTCC) 10 months and 10 days in 2005, and the preservation number is CCTCC No. M205114.
The new strain is characterized as follows:
colony morphology: culturing at 30 deg.C for 2 days, wherein the colony on beef extract peptone plate is round, has a diameter of 2 mm, and has a convex lens shape, light red color, regular edge, and smooth surface.
Cell morphology: is in the shape of a straight thin rod, 0.3-0.8 μm multiplied by 1.5-8.0 μm; cells appeared as single pairs, no sporulation, no motility.
The strain is characterized by positive catalase, positive nitrate reduction, negative pyrazinamine enzyme, negative pyrrolidone arylamine enzyme, positive alkaline phosphatase, negative β -glucuronidase, negative β -galactosidase, positive α -glucosaccharase, negative N-acetyl- β -glucosaminidase, negative esculetin hydrolysis, negative urease and negative gelatin hydrolysis.
The microorganism for producing the optical selectivity amidase is Delftia tsuruhatensis (Chinese name can not be found) bacteria of Delftia, which is deposited in China center for type culture collection, CCTCC for short, 10 months in 2005, and the collection number is CCTCC No. M205115.
The new strain is characterized as follows:
colony morphology: culturing at 30 deg.C for 3 days, wherein the colony on beef extract peptone plate is round, has a diameter of 3 mm, and has a tip-shaped bulge, and neat and smooth edge.
Cell morphology: the shape of a slightly bent rod is 1.4-4.0 mu m long and 0.6-1.3 mu m wide, and the rod appears singly or in pairs, does not form spores and can move.
Physiological and biochemical characteristics: the new strains screened from the soil are gram negative, arginine double hydrolase positive, oxidase positive, catalase positive, esterase (Tween 80) positive and cytochrome oxidase positive. Glucose produces acid negatively, fructose produces acid positively. Nitrate and nitrite reduction is negative. Rhamnose, N-acetylglucosamine, ribose, inositol, sucrose, glycogen, D-glucose, saligenin, D-melibiose, L-fucose, D-sorbitol, L-arabinose, L-serine, as a carbon source, could not be utilized. 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 can be used as a carbon source. Taken together with the above results, this strain was identified as Delftia tsuuhatensis.
The microorganism of the present invention is obtained by screening according to the following procedures:
1) and inoculating the collected soil sample and sewage sample into an enrichment culture medium, culturing for 4 days on a shaking table at 30 ℃ and 150rpm, taking 1ml of culture solution to transfer into a fresh enrichment culture medium after the culture medium becomes turbid, and culturing for 4 days. Repeating the above steps for 4-5 cycles. 1-2.5 g/L of 2, 2-dimethylcyclopropanecarbonitrile (for screening CCTCC No. M205114) or 2, 2-dimethylcyclopropanecarboxamide (for screening CCTCC No. M205115) is used as a unique nitrogen source in the enrichment medium, and the formula of other substances is as follows (g/L): na (Na)2HPO4·12H2O：1.0～2.5，KH2PO4：1.0～2.0，MgSO4·7H2O：0.2～0.5，FeSO4·7H2O：0.01～0.05，CoCl2：0.01～0.05，CaCl2: 0.01-0.06, prepared by tap water, and adjusting the pH value to 6.0-8.0 by using 1.0mol of hydrochloric acid or NaOH solution.
2) And (4) diluting the enrichment culture solution for the last time, coating the enrichment culture solution on a plate culture medium, and selecting a single colony to be stored on an inclined plane. The plate culture medium is an enrichment culture medium added with 1.5 to 2.0 percent of agar.
3) The single colony picked is inoculated to an induction fermentation medium, and the components are as follows (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, caprolactam 1.0-2.0, and natural pH (sterilization for 20min at 121 ℃); culturing for 48-72 h, suspending in a phosphoric acid buffer system after centrifugation, adding 2, 2-dimethylcyclopropanecarbonitrile or amide as a substrate, and converting; the amounts of product and substrate were analyzed 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 injection port, the detector and the column box are respectively 180 ℃, 220 ℃ and 120 ℃, and the split ratio is 50: 1. The contents of R-and S-2, 2-dimethylcyclopropanecarboxamide in the conversion product were determined by chiral liquid chromatography. Firstly, amide is derived into N, N-benzhydryl amide, and is separated on a Chiralcel OD column, wherein the mobile phase is cyclohexane/2-acetone, the ratio is 9: 1, and the flow rate is 0.4 ml/min.
Primary culture: the screened microorganisms are inoculated into a seed culture medium for primary culture. The seed culture medium comprises the following components (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 (sterilization for 20min at 121 ℃). The requirement of the seed culture medium is to have a proper C/N ratio and minimize the growth of microorganismsThe lag phase of (3). Suitable carbon sources are also glycerol, maltose, fructose, sucrose, mannitol, and the like; suitable nitrogen sources are peptone, beef extract, soybean powder, ammonium sulfate, and the like. The pH value of the culture medium is 6-8, and the culture temperature is 20-50 ℃. After culturing in the seed culture medium for 24-48 h, inoculating a fermentation induction culture medium can be prepared.
Induction: since the nitrile hydratase contained in the microorganism of the invention is an inducible enzyme, the enzyme activity is high only after induction. The amidase contained in the microorganism is a constitutive enzyme, induction is not needed, but the enzyme activity can be greatly improved by adding a proper chemical substance as an activator, such as caprolactam, isobutyronitrile, 2-dimethylcyclopropanecarboxamide and the like, wherein the amount of the activator is 1.0-2.0 g/L of the culture medium. Inoculating the seed liquid into an induction culture medium according to a certain proportion, wherein the induction culture medium is generally prepared by adding a proper inducer on the basis of the seed culture medium, the inducer comprises one of 2, 2-dimethylcyclopropanecarbonitrile, 2-dimethylcyclopropanecarboxamide, acrylamide, caprolactam, acetamide and acrylonitrile, and the proper amount of the inducer is 1.0-2.0 g/L of the induction culture medium. After 60-80 h of induction, collecting thalli through centrifugation or filtration, and providing a catalyst for the next step of biotransformation.
And (3) biotransformation: the biotransformation process uses the cells or cell treated matters of the microorganisms Rhodococcus equiCCTCC No. M205114 and Delftia tsuuhatensis CCTCC No. M205115 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 the conversion is 20-40 ℃. The amount of the substrate in the conversion system is in the range of 0.08-2% (wt/v, weight to volume ratio, the same applies hereinafter), and the optimum range is 0.1-2% (wt/v). The 2, 2-dimethylcyclopropanecarbonitrile can be completely converted into corresponding racemic amide by taking Rhodococcus equi CCTCC No. M205114 cells as a catalyst and reacting for 0.5-4 h. After removing cells by centrifugation, cells of Delftia tsuruhatensis CCTCC No. M205115 were added to the transformation solution, and R- (-) -2, 2-dimethylcyclopropanecarboxamide in the transformation solution was further converted into the corresponding acid to prepare S- (+) -2, 2-dimethylcyclopropanecarboxamide. Because the solubility of the 2, 2-dimethyl cyclopropane formamide in water is low, the conversion solution is subjected to decompression evaporation after being extracted by ethyl acetate, the obtained solid is dissolved in hot methanol and is placed in an ice bath overnight, and the separated crystal is dried in an evaporation dish after being filtered, so that the optically pure S- (+) -2, 2-dimethyl cyclopropane formamide is obtained.
The method takes 2, 2-dimethylcyclopropanecarbonitrile as a substrate, synthesizes the 2, 2-dimethylcyclopropanecarboxamide by a biotransformation method, and has the advantages of mild reaction conditions, simple reaction process, higher yield and less environmental pollution. Meanwhile, newly screened microorganisms are directly added into the conversion solution in the step as a catalyst,and the racemic amide is hydrolyzed in an optical selectivity mode to generate S- (+) -2, 2-dimethyl cyclopropane formamide, so that the extraction steps of intermediate products are reduced, the yield and the corresponding body excess value are high and reach more than 43% and 98% respectively.
Detailed description of the preferred embodiments
The following examples are presented to enable one of ordinary skill in the art to more fully understand the present invention.
Preparing a culture medium:
(1) the composition and the proportioning range of the enrichment medium are as follows (g/L): na (Na)2HPO4·12H2O：1.0～2.5，KH2PO4：1.0～2.0，MgSO4·7H2O：0.2～0.5，FeSO4·7H2O：0.01～0.05，CoCl2：0.01～0.05，CaCl2: 0.01-0.06, 2, 2-dimethylcyclopropanecarbonitrile (screening CCTCC No. M205114) or 2, 2-dimethylcyclopropanecarboxamide (screening CCTCC No. M205115): 1.0-2.5, prepared by tap water, and adjusting the pH value to 6.0-8.0 by using 1.0mol of hydrochloric acid or NaOH solution.
(2) The seed culture medium comprises the following components in percentage by weight and volume, 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.
(3) CCTCC No. M205114 fermentation medium containing inducer, the contents of each component are weight volume percentage, 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，FeSO40.0l～0.05，CoCl20.01～0.05，CaCl20.01 to 0.06 percent, 1.0 to 2.0 percent of inducer, prepared by tap water, and the pH value is adjusted to 6.0 to 8.0 by 1.0mol of hydrochloric acid or NaOH solution. The inducer is selected from any one of 2, 2-dimethylcyclopropanecarbonitrile, 2-dimethylcyclopropanecarboxamide, acrylamide, caprolactam, acetamide and acrylonitrile.
(4) 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 1.0mol of hydrochloric acid or NaOH solution. The activator is selected from any one of isobutyronitrile, caprolactam and 2, 2-dimethyl cyclopropane formamide.
The following examples were taken from any of the above ranges of components.
Example 1: isolation of nitrile hydratase-containing microorganisms
Adding 1g of soil sample and a proper amount of glass beads into 9ml of 0.9% physiological saline, and shaking uniformly to obtain a uniform soil suspension; 0.5ml of soil suspension is sucked and inoculated into a 250ml triangular flask filled with 30ml of enrichment medium, the flask is placed in a shaking table at 30 ℃ and 150rpm for 4 days, when the enrichment medium is turbid, 0.5ml of soil suspension is sucked and transferred into a fresh medium, and the culture is continued for 4 days; repeating the steps for 4-5 cycles, diluting the enrichment solution with multiple gradients, and coating the enrichment solution on a separation plate to obtain a single colony;
the enrichment medium uses 2, 2-dimethylcyclopropanecarbonitrile as a sole nitrogen source, and the composition and formula (g/L) of this example: na (Na)2HPO4·12H2O：2.5，KH2PO4：2，MgSO4·7H2O：0.5，FeSO4·7H2O：0.01，CoCl2：0.01，CaCl2: 0.06, 2, 2-dimethylcyclopropanecarbonitrile: 1.5, preparing the mixture by using tap water, and adjusting the pH value to 7.0;
inoculating the single colony to an enrichment medium, culturing for 48h and 72h, sampling, and detecting whether 2, 2-dimethylcyclopropanecarboxamide is generated or not by using a gas chromatography; the product with amide formation is the required microorganism Rhodococcus equi CCTCC No. M205114.
Example 2: isolation of optically selective amidase-containing microorganisms
The other steps were the same as in example 1 except that the nitrogen source in the enriched medium was changed to 2, 2-dimethylcyclopropanecarboxamide (1.5 g/L); detecting the corresponding body excess value (e.e value) of the product S- (+) -2, 2-dimethyl cyclopropane formamide by using chiral liquid chromatography, wherein the e.e value is more than 98 percent, and the product is the required microorganism Delftia tsuuhatensis CCTCC No. M205115.
Example 3: production of 2, 2-dimethylcyclopropanecarboxamide
The strain Rhodococcus equi CCTCC No. M205114 was placed in a 250ml triangular flask containing 30ml of seed medium, the composition of which was as follows (g/L)): 10.0 percent of glucose, 5.0 percent of yeast extract, 2.0 percent of peptone, 1.0 percent of NaCl and K2HPO41.0，KH2PO41.0，MgSO40.2，FeSO40.01，CoCl20.01，CaCl20.05, natural pH (121 ℃, sterilization for 20min), culturing at 30 ℃ for 2 days to obtain seed liquid;
inoculating the seed solution into an induction fermentation culture medium containing an inducer caprolactam, wherein the inoculation amount is 5% (V/V); the components of the fermentation medium are as follows (g/L): grapeGlucose 10.0, yeast extract 5.0, peptone 2.0, NaCl 1.0, K2HPO41.0，KH2PO41.0，MgSO40.2，FeSO40.01，CoCl20.01，CaCl20.05, 1.0 caprolactam, pH6.0 (sterilization for 20min at 121 ℃), fermentation culture for 60h at 20 ℃;
the fermentation liquor is centrifuged to obtain thalli, the thalli is washed by 0.9% physiological saline, the thalli is suspended in 10mM phosphate buffer (1000ml) to ensure that the pH value is 7.0 and the OD value of a buffer system is 1, 2-dimethylcyclopropanecarbonitrile is added to ensure that the concentration of a substrate is 0.95g/L, the thalli is converted for 0.5h in a water bath shaking table at the temperature of 30 ℃, gas chromatography detection shows that the substrate is completely converted, the thalli is centrifugally removed from the conversion liquor, and the supernatant is evaporated under reduced pressure, so 0.93g of the product 2, 2-dimethylcyclopropanecarboxamide is finally obtained, and the yield is 97.8%.
Example 4: production of 2, 2-dimethylcyclopropanecarboxamide
Seed solutions were prepared as in example 3;
inoculating the seed solution into an induction fermentation culture medium containing an inducer 2, 2-dimethylcyclopropanecarbonitrile, wherein the inoculation amount is 5% (V/V); the components of the fermentation medium are as follows (g/L): 10.0 percent of glucose, 5.0 percent of yeast extract, 2.0 percent of peptone, 1.0 percent of NaCl and K2HPO41.0，KH2PO41.0，MgSO40.2，FeSO40.01，CoCl20.01，CaCl20.05, 1.0 of 2, 2-dimethylcyclopropanecarbonitrile, pH 7.0 (sterilization is carried out for 20min at 121 ℃), the temperature is 30 ℃, and fermentation culture is carried out for 72 h;
after the cells were washed with 0.9% physiological saline, they were suspended in 50mM Tris-hydrochloric acid buffer (1000ml) so that the pH was 8.9 and the OD of the buffer system was 4, and 2, 2-dimethylcyclopropanecarbonitrile was added so that the concentration of the substrate was 4.75 g/L; transforming in a water bath shaker at 20 ℃ for 1.0h, and centrifuging to remove thalli; the conversion solution was evaporated under reduced pressure to give 4.55g of 2, 2-dimethylcyclopropanecarboxamide as a final product in a yield of 95.7%.
Example 5: production of 2, 2-dimethylcyclopropanecarboxamide
Seed solutions were prepared as in example 3;
inoculating the seed liquid into an induction fermentation culture medium containing an inducer acrylamide, wherein the inoculation amount is 5% (V/V); the components of the fermentation medium are as follows (g/L): 10.0 percent of glucose, 5.0 percent of yeast extract, 2.0 percent of peptone, 1.0 percent of NaCl and K2HPO41.0，KH2PO41.0，MgSO40.2，FeSO40.01，CoCl20.01，CaCl20.05, 1.0 acrylamide, pH 8.0 (sterilizing at 121 ℃ for 20min), temperature 40 ℃, fermenting and culturing for 80 h;
after the cells were washed with 0.9% physiological saline, they were suspended in 100mM citric acid buffer (1000ml) so that the pH was 6.0 and the OD value of the buffer system was 7, and 2, 2-dimethylcyclopropanecarbonitrile was added so that the substrate concentration was 19 g/L; transforming for 2.0h in a water bath shaker at 30 ℃, and centrifuging to remove thalli; the conversion solution was evaporated under reduced pressure to give 17.91g of 2, 2-dimethylcyclopropanecarboxamide as a final product in a yield of 94.2%.
Example 6: production of S- (+) -2, 2-dimethylcyclopropanecarboxamide
The slant activated strain Delftia tsuruhatensis CCTCC No. M205115 was inoculated into a 250ml flask containing 30ml of seed medium of the following composition (g/L): 10.0 parts of mannitol, 5.0 parts of yeast extract, 2.0 parts of peptone, 1.0 part of NaCl and K2HPO41.0，KH2PO41.0，MgSO40.2，FeSO40.01，CaCl20.05, natural pH (121 ℃, sterilization for 20min), and culturing at 30 ℃ for 48 h;
inoculating the seed liquid into a fermentation culture medium containing caprolactam as an activator, wherein the inoculation amount is 5% (V/V), and the fermentation culture medium comprises the following components (g/L): 10.0 parts of mannitol, 5.0 parts of yeast extract, 2.0 parts of peptone, 1.0 part of NaCl and K2HPO41.0，KH2PO41.0，MgSO40.2，FeSO40.01，CaCl20.05, 1.0 caprolactam, pH6.0 (sterilizing at 121 ℃ for 20min), fermenting and culturing for 80 h;
centrifuging the fermentation liquid to obtain thallus, washing with 0.9% physiological saline, suspending in 10mM phosphate buffer (1000ml) to make pH 7.0 and OD value of buffer system 5, adding racemic 2, 2-dimethylcyclopropanecarboxamide to make substrate concentration 1.14 g/L; converting for 4h in a water bath shaker at 20 ℃; centrifuging the conversion solution to remove thalli, adjusting the pH value of the supernatant to 12 by using sodium hydroxide, and extracting S- (+) -2, 2-dimethyl cyclopropane formamide (containing a small amount of 2, 2-dimethyl cyclopropane formamide) by using ethyl acetate with the same volume; distilling ethyl acetate under reduced pressure to obtain white powder; the white powder was dissolved in hot methanol for recrystallization to give 0.55g S- (+) -2, 2-dimethylcyclopropanecarboxamide in 48.2% yield with an e.e value greater than 98%.
Example 7: production of S- (+) -2, 2-dimethylcyclopropanecarboxamide
Seed solutions were prepared as in example 6;
inoculating the seed liquid into a fermentation culture medium containing isobutyronitrile as an activator, wherein the inoculation amount is 5% (V/V), and the fermentation culture medium comprises the following components (g/L): 10.0 parts of mannitol, 5.0 parts of yeast extract, 2.0 parts of peptone, 1.0 part of NaCl and K2HPO41.0，KH2PO41.0，MgSO40.2，FeSO40.01，CaCl20.05, isobutyronitrile at 1.0, pH 7.0 (sterilization at 121 ℃ for 20min), and fermentation culture for 72 h;
after the cells were washed with 0.9% physiological saline, they were suspended in 50mM Tris-hydrochloric acid buffer (1000ml) so that the pH was 8.9 and the OD of the buffer system was 10, and racemic 2, 2-dimethylcyclopropanecarboxamide was added so that the concentration of the substrate was 5.7 g/L; converting for 9h in a water bath shaker at 30 ℃; extracting with ethyl acetate, and recrystallizing to obtain 2.71g S- (+) -2, 2-dimethyl cyclopropane formamide with yield of 47.5% and e.e value greater than 98%.
Example 8: production of S- (+) -2, 2-dimethylcyclopropanecarboxamide
Seed solutions were prepared as in example 6;
inoculating the seed liquid into a fermentation medium containing 2, 2-dimethyl cyclopropane formamide serving as an activating agent, wherein the inoculation amount is 5% (V/V), and the fermentation medium comprises the following components (g/L): 10.0 parts of mannitol, 5.0 parts of yeast extract, 2.0 parts of peptone, 1.0 part of NaCl and K2HPO41.0，KH2PO41.0，MgSO40.2，FeSO40.01，CaCl20.05, 1.0 of 2, 2-dimethyl cyclopropane formamide, pH 8.0 (sterilization for 20min at 121 ℃), and fermentation culture for 60 h;
the cells were washed with 0.9% physiological saline, suspended in 100mM citric acid buffer (1000ml) so that the pH was 6.0 and the OD of the buffer system was 14, and racemic 2, 2-dimethylcyclopropanecarboxamide was added so that the substrate concentration was 22.8 g/L; converting for 13h in a water bath shaker at 40 ℃; extracting with ethyl acetate, and recrystallizing to obtain 5.52g S- (+) -2, 2-dimethyl cyclopropane formamide with yield of 48.2% and e.e value greater than 98%.
Example 9: combined conversion of racemic 2, 2-dimethylcyclopropanecarbonitrile to S- (+) -2, 2-dimethylcyclopropanecarboxamide with Rhodococcus equi CCTCC No. M205114 and Delftiatsurhautensis CCTCC No. M205115
Rhodococcus equi CCTCC No. M205114 was cultured in example 3;
cell culture of Delftia tsuuhatensis CCTCC No. M205115 is described in example 6;
rhodococcus equi CCTCC No. M205114 cells were washed with 0.9% physiological saline, suspended in 10mM phosphate buffer (1000ml) with an OD of 4, and 2, 2-dimethylcyclopropanecarbonitrile was added to give a substrate concentration of 4.75 g/L; transforming in a water bath shaker at 30 ℃ for 1.0h, and centrifuging to remove thalli; then, the bacterial cells of Delftia tsuruhatensis CCTCC No. M205115 washed by 0.9% physiological saline are added into the transformation liquid and transformed for 4 hours in a water bath shaker at 30 ℃; extracting with ethyl acetate, and recrystallizing to obtain 2.68g S- (+) -2, 2-dimethyl cyclopropane formamide with yield of 47% and e.e value greater than 98%.
Example 10: combined conversion of racemic 2, 2-dimethylcyclopropanecarbonitrile to S- (+) -2, 2-dimethylcyclopropanecarboxamide with Rhodococcus equi CCTCC No. M205114 and Delftiatsurhautensis CCTCC No. M205115
Rhodococcus equi CCTCC No. M205114 was cultured in example 3;
cell culture of Delftia tsuuhatensis CCTCC No. M205115 is described in example 6;
rhodococcus equi CCTCC No. M205114 cells were washed with 0.9% physiological saline, suspended in 50mM Tris-HCl phosphate buffer (1000ml) with an OD of 8, and 2, 2-dimethylcyclopropanecarbonitrile was added to give a substrate concentration of 9.5 g/L; transforming for 2.0h in a water bath shaker at 30 ℃, and centrifuging to remove thalli; then, the bacterial cells of Delftia tsuruhatensis CCTCC No. M205115 washed by 0.9% physiological saline are added into the transformation liquid and transformed for 5h in a water bath shaker at 30 ℃; extracting with ethyl acetate, and recrystallizing to obtain 5.41g S- (+) -2, 2-dimethyl cyclopropane formamide with yield of 47.4% and e.e value 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.
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