KR20080023561A - Method for manufacturing (s)-dimethyl cyclopropane carboxylic acid by using rhodococous erythropolis strain - Google Patents

Abstract

A method for producing (s)-dimethyl cyclopropane carboxylic acid as a core intermediate in the production of antibiotics is provided to improve production yield and economical efficiency of the acid through the bioconversion of Rhodococcus erythropolis strain by culturing the strain under mild conditions, so that economical pollution is inhibited. A method for producing (s)-dimethyl cyclopropane carboxylic acid comprises the steps of: culturing Rhodococcus erythropolis strain(KCCM 25544) producing nitrile-decomposing enzymes such as nitrilase, nitrile hydratase and amidase in a medium to obtain the nitrile-decomposing enzymes; reacting the produced nitrile-decomposing enzymes with 10-1150 mM of a substrate dimethyl cyclopropane cyanide in organic solvent selected from methanol, ethanol, dimethyl formamide, dioxane, isopropanol and acetone at 15-30 deg. C and pH 6.0-8.0. Further, a concentration of the methanol is adjusted at 5 to 20%(v/v).

Description

Method for manufacturing (s) -dimethyl cyclopropane carboxylic acid by using Rhodococous erythropolis strain}

And Figure 1 is analyzed the activity of nitrile lyase according to the pH (A) and a temperature (B) in u (Rhodococous erythropolis) strains in Cocos Erie used also according to the invention (S) - dimethyl cyclopropane carboxylic isomer of the acid It is expressed as a ratio.

Figure 2 is a u strain 10 g / ℓ gyuncheryang, 10% Coconut Erie used also in the invention (v / v) methanol, pH 7.0 and in 50 mM phosphate buffer conditions 100 mM dimethyl bicyclo culture was reacted with propane cyanide When the (s) -dimethyl cyclopropane carboxylic acid (●) is produced and its stereospecific ratio (o) is shown.

Using dimethyl cyclopropane carboxylic acid u (Rhodococous erythropolis) to Cocos Erie used relates to the production method, and more particularly to as the ((s) -dimethyl cyclopropane carboxylic acid) strain comprising (S) using microorganisms The present invention relates to a method for stereotyping specific mass production of (S) -dimethyl cyclopropane carboxylic acid, which is a key intermediate of antibiotic production.

Nitrile has two different bioconversion stages in the microbial body. These two processes have different enzymes acting on them, first of which is nitrile, which is an enzyme that converts nitrile to carboxylic acid and ammonia in one step. That is, nitrile hydratase and amidase, which primarily decompose nitrile to amide and second decompose amide to carboxylic acid and ammonia.

These nitrile and amido hydroxy latte rise Days is at the same time, also used as a Cocos Erie u (Rhodococcus erythropolis), also Cocos ekwi (R. equi), also Cocos also Cruz (R. rhodochrous microorganisms that require a two-step reaction which acts ), also Cocos in R312 (Rhodococcus sp. R312), also Cocos in AJ270 (Rhodococcus sp. AJ270), also Cocos in CGMCC 0497 (Rhodococcus sp. CGMCC 0497 ), fans, toy Oh endo blood urticae (Pantoea endophytica), coma Pseudomonas Testo Stephen Ronnie (Comamonas testosteroni), Bacillus arms Leaders (Bacillus pallidus), micro tumefaciens in AJ115 (Microbacterium sp. AJ115), micro tumefaciens Imperial (M. imperiale), Candida Pharma other (Candia famata), Candida gwil rear hormone di (C. guilliermondi), and the like Cri Sat Rhodococcus genus (Crytococcus sp.) and torulra article Ruti varnish (Rhodotorula glutinis) also.

(S) - dimethyl cyclopropane carboxylic acid ((s) -dimethyl cyclopropane carboxylic acid) is an important intermediate of pharmaceutical production as a key raw material for antibiotics of "cilastatin". Such chiral complex (S) -dimethyl cyclopropane carboxylic acid has a stereospecificity of the compound in one step reaction at room temperature and atmospheric pressure when produced using a biocatalyst. In contrast, the chemical synthesis method has a limitation in that a racemic mixture is produced by a multi-step reaction under high temperature and high pressure conditions. In addition, chemical synthesis can subsequently increase the cost of purification and furthermore lead to environmental pollution due to the organic solvents and by-products generated during this purification process. For this reason it is absolutely necessary to develop a method for the selective production of isomers of dimethyl cyclopropane carboxylic acid using biocatalysts.

Therefore, the present inventors have continued efforts to develop an economical biosynthetic method of medicines, and as a result, when microorganisms containing nitrile enzymes are incubated with a dimethyl cyclopropane cyanide substrate and an organic solvent, Reaction confirmed that only (S) -dimethyl cyclopropane carboxylic acid can be produced in high yield without a separate purification process, the optimum reaction conditions (organic solvent, concentration, pH, temperature) in the bioconversion reaction, hourly production and their stereospecificity ratio and u (Rhodococous erythropolis) is irradiated with such substrate specificity of the strain (S) to Cocos Erie used also as the above microorganism by providing a method for the dimethyl cyclopropane carboxylic acid mass-produced at a low price The present invention has been successfully completed.

It is an object of the present invention to provide a method for producing a core intermediate of antibiotics, (S) -dimethyl cyclopropane carboxylic acid using a microorganism.

In order to achieve the above object, the present invention provides a (S) -dimethyl cyclopropane carboxylic acid (( s ) -dimethyl cyclopropane carboxylic acid) as a bioconversion reaction using a microorganism, a substrate and an organic solvent to produce nitrile enzymes It provides a manufacturing method to obtain.

The invention also u (Rhodococous erythropolis) strains in Cocos Erie also used in producing the nitrile lyase: provides (accession No. KCCM 25544).

Hereinafter, the present invention will be described in detail.

The present invention provides a production method for obtaining ( S ) -dimethyl cyclopropane carboxylic acid (( s ) -dimethyl cyclopropane carboxylic acid) by a bioconversion reaction using microorganisms.

Specifically, the present invention is a bioconversion reaction using microorganisms producing nitrile degrading enzymes, adding substrates and organic solvents in the culture, and adjusting conditions such as their type, concentration, temperature, time and pH (S). A production method for obtaining dimethyl cyclopropane carboxylic acid is provided.

A microorganism producing the nitrile degrading enzyme is also a Cocos Erie used by police to as Cocos Erie used preferably, and the use of (Rhodococous erythropolis) strain KCCM 25544 Indianapolis ( Rhodococous erythropolis KCCM 25544) It is more preferred to use strains.

A microorganism having an activity of nitrile-decomposing enzyme is also used as a Cocos Erie Indianapolis addition also it may specify a variety of strains, or the like in Cocos (Rhodococcus sp.) Strains and Pseudomonas species (Pseudomonas sp.) Strains. In the present invention, in particular , the Rhodococcus erythropolis KCCM25544 strain was found to be 96% due to the stereospecificity of (S) -dimethyl cyclopropane carboxylic acid and was the highest, and various strains of bioconversion were established using this strain.

Further, also the culture medium of a microorganism, including the police (Rhodococous erythropolis) strains in Cocos Erie used is 1 to 10 g / ℓ glucose, 0.1 to 3 g / ℓ yeast extract, 0.1 - 1 g / ℓ one potassium phosphate, 0.1 to Preference is given to a medium consisting of 1 g / l potassium diphosphate, 0.1 to 1 g / l magnesium sulfate and 1 to 10 g / l epsilon-caprolactam.

In addition, the substrate is preferably dimethyl cyclopropane cyanide (dimethyl cyclopropane cyanide), the concentration of the dimethyl cyclopropane cyanide is preferably adjusted to the range of 10 to 1,150 mM, it is adjusted to the range of 10 to 200 mM More preferred. In addition, various kinds of substrates can be used (see Table 2).

In addition, the organic solvent is preferably at least one selected from the group consisting of methanol, dimethyl formamide, dioxane, isopropanol and acetone, and more preferably at least one selected from the group consisting of methanol, ethanol and isopropanol.

In addition, when methanol is used as the organic solvent, the concentration thereof is preferably adjusted in the range of 5 to 20% (v / v), more preferably in the range of 5 to 15% (v / v).

In addition, the bioconversion reaction is preferably made in the pH range 6.0 to 8.0, more preferably in the pH range 7 to 8.

In addition, the bioconversion reaction is preferably made in the temperature range of 15 ° C to 30 ° C, more preferably in the temperature range of 20 to 25 ° C.

The present invention also provides a strain of Rhodococous erythropolis .

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Characterization of Microorganisms Producing Nitrile Degrading Enzymes

The present invention also were not found at all in the conventional bar using, for example, police KCCM 25544 strain was used as a Cocos Erie the characterization of microorganisms in order to separate the microorganism producing the age krill enzymes. The microbial strain was cultured for 150 hours using a medium prepared for the purpose of expressing nitrile degrading enzyme, and cells were collected therefrom.

Specifically, the microbial cells were centrifuged at 6,000 × g for 30 minutes at 4 ° C. and washed twice with 0.85% NaCl. In addition, the substrate concentration in the reaction conditions of the microbial cells and dimethyl cyclopropane cyanide is in the range of 10 to 200 mM (v / v) pH 7 to 8 to 20 to 30 ℃ as an organic solvent in methanol 5 to 15% (v / v). Also of the present invention to Cocos Erie used Indianapolis strain in propane cyanide range of substrate concentrations from 10 to 200 mM (v / v) in dimethyl sayikeu (es) in dimethyl sayikeu were excellent in production yield of the acid, It was confirmed that the experimental group to which 10% (v / v) methanol was added exhibited 2.12 times more enzyme activity than the experimental group to which the substrate had no hydrophobicity.

Example 2. Rhodococos Eritrospolis  Investigation of nitrate conversion activity according to organic solvents of strains

(One) Rhodococos Eritrospolis  ( Rhodococous erythropolis ) Strain Culture

In the present invention, as the derivative without the use of conventional strain culture medium in order to obtain u (Rhodococous erythropolis) strains used in Cocos Erie also the expression of nitrile lyase increase epsilon-caprolactam was used for containing the culture medium. First , the Rhodococ Eristhropolis strain was inoculated into a test tube containing 3 ml of nutrient broth, and then seeded in a 250 ml flask containing 50 ml of the same medium, followed by seed culture. The spawn culture was again cultured for the expression of nitrile enzyme (1-10 g / L glucose, 0.1-3 g / L yeast extract, 0.1-1 g / L potassium phosphate, 0.1-1 g / L) The incubation was carried out by inoculating a 2 L flask containing 500 mL of potassium phosphate, 0.1-1 g / L magnesium sulfate and 1-10 g / L epsilon caprolactam). At this time, the stirring rate in the culture process was adjusted to 200 rpm, the culture temperature was maintained at 28 ℃. In addition, the culture solution was centrifuged at 6,000 × g for 30 minutes at 4 ° C., and then washed twice with 0.85% NaCl to obtain cells.

(2) In the present invention, in order to investigate the culture effect of the organic solvent added when performing the bioconversion reaction, the enzymatic activity of nitrile as shown in Table 1 using 100 mM dimethyl cyclopropane cyanide substrate Measured. In addition, the enzyme reaction was performed in a 50 mM phosphate buffer solution containing 10 g / l of the cell weight and then reacted with each 10% (v / v) organic solvent for 2 hours at pH 7.0 and a temperature of 20 ° C for 0.2 M hydrochloric acid. Was added again to stop the reaction.

In addition, the Rhodococcus erythropolis strain was removed by centrifugation, and only the supernatant was recovered therefrom, extracted with dimethyl chloride, followed by a chiral capillary column equipped with a silica gel. This equipped gas chromatography (GC) was used to measure the enzyme activity of the cells. At this time, the detector used a flame ionization detector, and the temperature of the column, the detector, and the inlet was set at 270 ° C, 250 ° C, and 250 ° C, respectively.

As a result, it was found that the enzyme activity of the Rhodococcus erythropolis strain of the present invention was the highest when methanol, ethanol and isopropanol were added, and the production yield was also increased. In the present invention, methanol was used as an organic solvent generally used.

Organic solvent Relative activity (%) Methanol 100 Dimethyl formamide 97 Butanol 18 ethanol 101 Dimethyl chloride 19 Dioxane 87 toluene 53 Ethyl acetate 35 2-propanol 38 Isopropanol 103 Acetone 83

Example  3. Rhodokocos Eritrospolis  Investigation of substrate specificity of strains

The present invention is also to investigate the substrate specificity of u strain used as a Cocos Erie, Cocos also by the same procedure as in the (1) in Example 2 Erythropolis strains were cultured. At this time, the reaction was performed in 50 mM phosphate buffer solution containing 100 mM substrate and 10 g / L and 10% (v / v) methanol for 4 hours at pH 7.0 and 20 ° C., followed by addition of 0.2 M hydrochloric acid. The reaction was stopped.

As a result, as shown in Table 2, Rhodococos Erythropolis strains showed very broad substrate specificity.

temperament Relative activity (%) temperament Relative activity (%) Dimethyl cyclopropane cyanide 100 4-Aminobenzylcyanide 171 Aromatic nitrile 4-Fluorophenylacetonitrile 6 2-Aminobenzonitrile 0 Enzonitrile 118 3-Aminobenzonitrile 0 Mandelonitrile 0 4-Aminobenzonitrile 258 Heterocyclic nitrile 2-Bromobenzonitrile 179 2-Cyanopyridine 93 3-Bromobenzonitrile 130  3-Cyanopyridine 137 2-Chlorobenzonitrile 202 4-Cyanopyridine 44 3-Chlorobenzonitrile 11 Alipathic nitrile 4-Chlorobenzonitrile 232 - Saturated 2-Fluorobenzonitrile 48 Isovaloronitrile 136 3-Fluorobenzonitrile 0 Propionitrile 238 4-Fluorobenzonitrile 135 Butyro nitrile 236 M -Tolunitrile 10 Isobutyro nitrile 228 O -Tolunitrile 59 - Saturated dinitrile P -Tolunitrile 35  Malononitrile 0 2-Chlorobenzylcyanide 43  Succinonitrile 185 3-Chlorobenzylcyanide 45  Glutaronitrile 277 4-Chlorobenzylcyanide 84  Sebaconitrile 153 2-Cyanophenol 0  Adiponitrile 168 3-Cyanophenol 9 Fumaronitrile 352 4-Cyanophenol 0 - Unsaturated 3-Phenylpropionitrile 196 Acrylonitrile 209 Crotononitrile 105

Example 4. Rhodococos Eritrospolis  Investigation of bioconversion activity according to pH and temperature changes of strains

The present invention is also to investigate the substrate specificity of u strain in Cocos Erie used, the second embodiment also strains the police to Cocos Erie used the same procedure as (1) was cultured in order.

In the present invention, in order to confirm the activity and stereospecificity of the nitrile degrading enzyme of the Rhodococcus erythropolis cells according to the pH and temperature changes, the enzyme and the substrate were reacted at various pH and temperature conditions and the enzyme activity was compared.

First, in order to investigate the effect of pH, 100 mM dimethyl cyclopropane cyanide and 10 g / l cells, 10% (v / v) methanol and 50 mM phosphate buffer solution was reacted in the range of pH 6.0 to pH 8.0, 50 mM citric acid buffer solution containing 100 mM dimethyl cyclopropane cyanide, 10 g / L cells and 10% (v / v) methanol was reacted at a pH ranging from 4.5 to 6.0. In addition, 50 mM boric acid buffer solution was reacted in the range of pH 8.0 to pH 9.5, at which time the reaction temperature is 20 ℃, the reaction time was adjusted to 4 hours. Then, 0.2 M hydrochloric acid was added to terminate the reaction, and nitrile decomposition activity was measured. The results are shown in FIG. 1 (A).

Second, to investigate the effect of temperature, the reaction was carried out in a 50 mM phosphate buffer solution containing 100 mM dimethyl cyclopropane cyanide, 10 g / L cells, and 10% (v / v) methanol in the temperature range of 10 ° C to 45 ° C. Were reacted at pH 7.0 for 4 hours, at which time 0.2 M hydrochloric acid was added to terminate the reaction, and nitrile degradation activity was measured. The results are shown in FIG. 1 (B).

As a result, the optimum pH and temperature at which (S) -dimethyl cyclopropane carboxylic acid is produced were found to be pH 8.0 and 25 ° C., respectively. However, the highest ratio of stereospecificity was found to be pH 7.0, and the hourly production of (S) -dimethyl cyclopropane carboxylic acid was measured at pH 7.0 with the highest stereospecificity and at a temperature of 20 ° C. This is because absolute production is important, but steric specificity is important for reducing production costs. Indeed, the ratio of stereospecificity will greatly affect the cost of purification thereafter.

And Figure 1 is analyzed the activity of nitrile lyase according to the pH (A) and a temperature (B) in u (Rhodococous erythropolis) strains in Cocos Erie used also according to the invention (S) - dimethyl cyclopropane carboxylic isomer of the acid It is expressed as a ratio. In Figure 1 (A) is citric acid buffer solution; ○ is phosphate buffer solution; Δ is a boric acid buffer solution; And □ represent stereospecific ratios, in Figure 1 (B) is the activity of the Rhodococos erythropolis strain with temperature; And □ represent stereospecific ratios.

Example 5. Rhodococos Eritrospolis  Investigation of Production of (S) -Dimethyl Cyclopropane Carboxylic Acid Using Strains

Dimethyl bicyclo u strain, the Cocos Erie used also by the same procedure as in the (1) in Example 2 to examine the productivity of propane carboxylic acid comprising (S) using a police strain in Cocos Erie used also Incubated. In addition, the bioconversion reaction was sufficiently performed in a 50 mM phosphate buffer solution containing 100 mM dimethyl cyclopropane cyanide, 10 g / L cells and 10% (v / v) methanol for up to 64 hours at pH 7.0. It was made. In addition, 0.2 M hydrochloric acid was added to terminate the reaction, and then nitrile degradation activity was measured. The results are shown in FIG. 2.

Figure 2 is the present invention also Cocos Erie write Indianapolis strain 10 g / ℓ as gyuncheryang of, 10% (v / v) methanol, pH 7.0 50 mM in phosphate buffer conditions 100 mM dimethyl cyclopropane-cyanide and reacted to the culture When the amount of dimethyl cyclopropane cyanide (▲) and thus the (s) -dimethyl cyclopropane carboxylic acid (●) and (s) -dimethyl cyclopropane carboxylic acid (○) and (al) -dimethyl It shows the stereospecific ratio ((circle)) of cyclopropane carboxylic acid.

As a result, as shown in Figure 2, when the reaction of the dimethyl cyclopropane cyanide and the Rhodococos erythropolis strain (S) -dimethyl cyclopropane carboxylic acid showed a conversion rate of 45% or more after 64 hours, Stereospecificity ratio was found to be 90%. Therefore, it can be seen that the production method of (S) -dimethyl cyclopropane carboxylic acid through the biocatalyst of the present invention is a very excellent method.

As described above, the present invention uses a strain of Rhodococous erythropolis as a microorganism to produce stereospecifically mass-produced (S) -dimethyl cyclopropane carboxylic acid, which is a key intermediate of antibiotic production. relates to, and the u strain was added to dimethyl cyclopropane cyanide (dimethyl cyclopropane cyanide) substrate and an organic solvent to adjust the concentration, temperature and composition of the culture medium and the culturing process to Cocos Erie used also with a nitrile degrading enzyme ( Since only S) -dimethyl cyclopropane carboxylic acid can be produced in high yield, it not only overcomes the problem of racemic mixing in an environmentally friendly condition compared to the conventional chemical method, but also has a very gentle bioconversion process, thus producing costs. Efficiency can be maximized and production efficiency can be maximized. Therefore, stereospecific (S) -dimethyl cyclopropane carboxylic acid produced in large quantities at low cost can be usefully used for the production of pharmaceuticals such as antibiotic cilastatin.

It will be apparent to those skilled in the art that various modifications or variations are possible within the spirit and scope of the invention. Accordingly, the appended claims should be construed broadly according to the spirit and scope of the present invention.

Claims (10)

A process for obtaining (s) -dimethyl cyclopropane carboxylic acid (s) -dimethyl cyclopropane carboxylic acid by a bioconversion reaction using microorganisms, substrates and organic solvents that produce nitrile degrading enzymes. The method of claim 1, Microorganism producing the nitrile degrading enzyme is also a u (Rhodococous erythropolis) strains used in Cocos Erie, preferably also in Cocos Erie used Indianapolis KCCM 25544 ( Rhodococous erythropolis KCCM 25544) production method characterized in that the strain. The method according to claim 1 or 2, The microorganisms producing the nitrile enzymes include 1 to 10 g / L glucose, 0.1 to 3 g / L yeast extract, 0.1 to 1 g / L potassium monophosphate, 0.1 to 1 g / L potassium diphosphate, 0.1 to 1 A production method characterized by culturing with a culture medium consisting of g / l magnesium sulfate and 1 to 10 g / l epsilon-caprolactam. The method of claim 1, The substrate is a manufacturing method, characterized in that dimethyl cyclopropane cyanide (dimethyl cyclopropane cyanide). The method of claim 4, wherein The substrate dimethyl cyclopropane cyanide is a manufacturing method characterized in that the concentration is adjusted to the range of 10 to 1,150 mM. The method of claim 1, The organic solvent is selected from the group consisting of methanol, ethanol, dimethyl formamide, dioxane, isopropanol and acetone, and preferably selected from the group consisting of methanol, ethanol and isopropanol. The method of claim 6, Methanol used as the organic solvent is characterized in that the concentration is adjusted to 5 to 20% (v / v) range. The method of claim 1, The bioconversion reaction is a production method characterized in that the pH range from 6.0 to 8.0. The method of claim 1, The bioconversion reaction is a production method, characterized in that made in the temperature range 15 ℃ to 30 ℃. Rhodocokos of the first paragraph Erie write to Indianapolis (Rhodococous erythropolis ) strain (Accession Number: KCCM 25544).

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