JP4716785B2 - Method for producing 3-halo-1,2-propanediol and microorganism used therefor - Google Patents

Description

  The present invention relates to a method for producing 3-halo-1,2-propanediol useful as a raw material or intermediate for pharmaceutical intermediates and a microorganism used therefor.

The following general formula (1)

As an enzyme that dehalogenates and / or opens an epoxide ring, the genus Pseudomonas, Corynebacterium, Microbacterium, and Aureobacterium Alternatively, those derived from microorganisms belonging to the genus Agrobacterium are known. For example, from the action of dehalogenase of microorganisms belonging to the genus Corynebacterium, Microbacterium, Oreobacterium, Agrobacterium, optically active 3-halo- from 1,3-dihalo-2-propanol A method for producing 1,2-propanediol (Patent Documents 1 to 3), a method for producing optically active 3-halo-1,2-propanediol from epihalohydrin (Patent Documents 2, 4, and 5), and Agrobacterium-derived A method for producing optically active 3-halo-1,2-propanediol using a dehalogenase gene recombinant microorganism (Patent Document 6) is known.
In addition to these, Pseudomonas genus and Alkaligenes genus are utilized to assimilate the (S) isomer or (R) isomer from 3-halo-1,2-propanediol, and the remaining (R) isomer or (S) (S) -3-Chloro-1,2-propanediol by hydrolyzing (R) -1-acyloxy-3-chloro-2-propanol (Patent Document 7) Patent document 8) is known.
However, the enzymes used in the above-mentioned methods are Pseudomonas, Corynebacterium, Microbacterium, Aureobacterium or Agrobacterium Only enzymes derived from microorganisms belonging to the genus.
Japanese Patent Laid-Open No. 02-291280 JP 05-219965 A Japanese Patent Laid-Open No. 04-94689 Japanese Patent Laid-Open No. 02-283295 Japanese Patent Laid-Open No. 04-94690 Japanese Patent Laid-Open No. 10-210981 JP 2004-41076 A Japanese Patent Laid-Open No. 11-103878

  An object of the present invention is to provide a method for producing 3-halo-1,2-propanediol using a microorganism and a microorganism used therefor.

That is, the present invention includes the following inventions.
A compound represented by the following general formula (1) can be converted into 3-halo-1,2-propanediol represented by the following general formula (2), which belongs to the genus Rhizobium or the genus Leifsonia. The microorganism to which it belongs.

The microorganism described above which is Rhizobium radiobacter MRC01 strain (FERM AP-2052), the microorganism described above as Leifsonia sp. MRC03 strain (FERM AP-20453), and any one of the above A 3-halo-1,2-propanediol is recovered by contacting a microorganism or a treated product thereof with a compound represented by the following general formula (1) to recover 3-halo-1,2-propanediol represented by the following general formula (2) A method for producing propanediol.

  According to the present invention, it is possible to provide a method for producing 3-halo-1,2-propanediol using microorganisms and the microorganisms used therefor.

As the microorganism according to the present invention, Rhizobium having the ability to convert the compound represented by the general formula (1) into the compound represented by the general formula (2) (3-halo-1,2-propanediol). ) Microorganisms belonging to the genus or microorganisms belonging to the genus Leifsonia, preferably Rhizobium radiobacter MRC01 strain (hereinafter referred to as “MRC01 strain”) and Leifsonia sp. MRC03 strain (Hereinafter referred to as “MRC03 strain”).
The taxonomic properties of the MRC01 and MRC03 strains are described below.

(Taxonomic properties)
The MRC01 strain and MRC03 shares, have been deposited at National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center (Higashi, Tsukuba, Ibaraki, 1-1-1 Central 6) in March 2005 9 date, the deposit number MRC01 strain is FERM P-20453 and MRC03 strain is FERM P-20453 .

Regarding the MRC01 strain and the MRC03 strain, the nucleotide sequence of the 16S rRNA gene (469 bp for the MRC01 strain and 506 bp for the MRC03 strain) was determined (MicroSeq 500 16S) by entrusting to NCIMB Japan Co., LTD. rDNA Bacterial Sequencing Kit (Applied Biosystems, CA, USA) was used, and a molecular phylogenetic tree based on the 16S rRNA gene base sequence was prepared for phylogenetic analysis. The determined base sequence of 16S rRNA gene of MRC01 strain is shown in SEQ ID NO: 1. The base sequence of the 16S rRNA gene of the MRC03 strain is shown in SEQ ID NO: 2.
Using the obtained 16S rRNA gene base sequence, a homology search was performed to determine the top 10 homology homologues. Further, using the 16S rRNA genes of the top 10 strains and the MRC03 strain that were searched, a molecular phylogenetic tree was prepared by the proximity binding method, and related species and attribution classification of the MRC01 strain and the MRC03 strain were examined. MicroSeq Microbial Identification System Software 1.4.1 was used for homology search and phylogenetic tree preparation.

Results of the homology analysis, the nucleotide sequence of 16S rRNA gene of MRC01 strain was consistent with the 16S rRNA gene of Rhizobium radiobacter (Rhizobium radiobacter) 100% homology. Further, it was consistent with 16S rRNA gene such as Rhizobium sp. GH-2001 strain. The base sequence of the 16S rRNA gene of the MRC03 strain showed the highest homology to the 16S rRNA gene of Leifsonia poae with a homology rate of 98.4%. In addition, a 16S rRNA gene derived from Leifsonia was detected. In the analysis using the 16S rRNA gene , when the homology rate was 97% or more with respect to the reference strain, it may be the same species, but the two base sequences did not completely match. Therefore, it was judged that the strain is closely related to Leifsonia poae but is systematically different.
From the above, the MRC01 strain was determined to be a strain belonging to Rhizobium radiobacter. In addition, the MRC03 strain was determined to be a strain belonging to the genus Leifsonia.
The method for culturing the microorganism according to the present invention including the MRC01 strain or the MRC03 strain may be any method as long as the microorganism can grow.

The medium used for the culture may be either a natural medium or a synthetic medium as long as the microorganism according to the present invention can grow. As the carbon source, for example, sugars such as glucose, sucrose, maltose and fructose, organic acids such as acetic acid, citric acid and fumaric acid or salts thereof, alcohols such as ethanol and glycerol can be used. As the nitrogen source, for example, various inorganic and organic acid ammonium salts can be used in addition to general natural nitrogen sources such as peptone, meat extract, yeast extract and amino acids. In addition, inorganic acids such as sulfuric acid, hydrochloric acid, phosphoric acid and boric acid or salts thereof, inorganic salts containing sodium, magnesium, potassium, calcium, etc. that can be used by the microorganisms used, iron, manganese, zinc, cobalt, nickel trace metal salts In addition, vitamins such as vitamins B1, B2, C, and K are appropriately added as necessary as a microorganism growth promoter. Furthermore, an enzyme having the ability to convert a compound represented by the general formula (1) into a compound represented by the general formula (2) (3-halo-1,2-propanediol) by adding an inducer to the medium Can increase the amount of enzyme. Examples of the inducing agent include compounds represented by the general formula (1). For example, 1,3-dichloro-2-propanol, 1,3-dibromo-2-propanol, epichlorohydrin, epibromohydrin, 3-chloro-1,2-propanediol, 3-bromo-1,2 -Propanediol and the like.
The microorganism according to the present invention is usually cultured at 10 to 50 ° C., preferably 25 to 45 ° C. under aerobic conditions such as shaking culture or aeration and agitation culture. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like, and may be set to pH 2 to 11, preferably pH 5 to 9, particularly preferably pH 7 to 8. The culture time is preferably 1 to 96 hours, particularly preferably 8 to 72 hours.

As the microorganism according to the present invention, a microorganism obtained by culturing is used as a catalyst. Microorganisms catalyze the conversion of general formula (1) to general formula (2).
The form of its use is not particularly limited as long as it shows the catalytic activity, and it can be obtained by microbial cells, culture supernatants, treated cells or conventional methods obtained from the culture solution as it is or by collection from the culture, such as centrifugation. It can be used in the form of immobilized cells. Examples of treated products include cells treated with acetone, toluene, etc., disrupted cells, cell-free extracts, crude enzymes, purified enzymes, genetically engineered microorganisms, and the like. Extracts, crude enzymes, purified enzymes or genetically engineered microorganisms are preferred.
Here, the genetically engineered microorganism is a microorganism in which a gene encoding the enzyme is incorporated into a host cell so that the enzyme can be expressed. The usage form of the genetically engineered microorganism is not particularly limited as described above. In general, enzyme activity may be increased in the form of a treated microbial cell product, crude enzyme, purified enzyme, or genetically engineered microorganism as compared with microbial cell, microbial cell culture solution, or the like.
A substrate generally used in the present invention is represented by the general formula (1).

  The halogen atom is preferably fluorine, chlorine, bromine or iodine, particularly preferably chlorine or bromine. Specifically, 1,3-difluoro-2-propanol, 1,3-dichloro-2-propanol, 1,3-dibromo-2-propanol, 1,3-diiodo-2-propanol, epifluorohydrin, Examples include epichlorohydrin, epibromohydrin and epiiodohydrin. 1,3-dichloro-2-propanol, 1,3-dibromo-2-propanol, epichlorohydrin and epibromohydrin are preferred.

  When the compound represented by the general formula (1) is brought into contact with the microorganism or a treated product thereof, 3-halo-1,2-propanediol represented by the following general formula (2) is generated.

  For example, 3-fluoro-1,2-propanediol, 3-chloro-1,2-propanediol, 3-bromo-1,2-propanediol, 3-iodo-1,2-propanediol and the like. Optically active substances such as optically active 3-chloro-1,2-propanediol and optically active 3-bromo-1,2-propanediol are particularly preferred.

  The “contact” may be any state in which the enzyme catalyst in the culture or the treated product functions as an enzyme and the enzyme catalyst and the substrate react with each other. The conditions for functioning as an enzyme are, for example, pH 4-10, preferably pH 6-9, temperature 5-50 ° C., preferably 10-40 ° C. Or it is preferable to make the processed material and a substrate contact.

A reaction for obtaining a 3-halo-1,2-propanediol represented by the general formula (2) by bringing a microorganism or a treated product thereof into contact with the compound represented by the general formula (1) is shown below.
The concentration of the substrate represented by the general formula (1) in the reaction solution is preferably 0.01 to 15 (W / V)%. Within this range, it is preferable from the viewpoint of enzyme stability. The substrate concentration is particularly preferably 10% or less.
The substrate can be added to the reaction solution all at once or in divided portions. It is preferable from the viewpoint of the accumulation property of 3-halo-1,2-propanediol that the substrate concentration is made constant by divided addition.

  The solvent for the reaction solution is preferably water or a buffer solution. Examples of the buffer solution include a buffer solution composed of a salt such as phosphoric acid, boric acid, citric acid, glutaric acid, malic acid, malonic acid, o-phthalic acid, succinic acid, or acetic acid, and a Tris buffer. Liquid or Good buffer solution. This solvent is preferable because it is in the vicinity of the optimum pH of enzyme activity of 4 to 10. The solvent is particularly preferably water.

The reaction time is appropriately selected according to the substrate concentration, the enzyme amount of the microorganism or its processed product, or other reaction conditions. The reaction time is preferably set so that the reaction time is 0.1 to 120 hours. The reaction time is particularly preferably 0.1 to 48 hours. Within this range, it is preferable from the viewpoint of productivity.
In addition, the order of adding the microorganism or the processed product and the substrate to the reaction system is not limited. For example, a method of adding a substrate to a culture solution or a microorganism obtained by centrifugation, or a treated product thereof, a method of adding a substrate to the culture solution at the time of culturing the microorganism, and a method of reacting simultaneously with the culture are included.

The 3-halo-1,2-propanediol represented by the general formula (2) according to the present invention can be recovered by the following method. When the culture or a mixture of the processed product and the product compound contains microbial cells or cells, the microbial cells or cells are removed by centrifugation or the like, and then filtration, concentration, various chromatography, extraction, activated carbon treatment and It applies to usual methods, such as distillation, alone or in appropriate combination. Alternatively, the mixture may be directly applied to usual methods such as filtration, concentration, various types of chromatography, extraction, activated carbon treatment and distillation alone or in appropriate combination.
For example, microbial cells are removed from the reaction solution by centrifugation, followed by extraction with an organic solvent, and removal of the solvent from the organic phase under reduced pressure to obtain a 3-halo-1,2-propanediol syrup. Can do. Further, these syrups can be further purified by distillation under reduced pressure.
As described above, according to the method for producing 3-halo-1,2-propanediol represented by the general formula (2) according to the present invention, the compound represented by the general formula (2) is represented by the compound represented by the general formula (1). 3-halo-1,2-propanediol can be produced efficiently.

Hereinafter, the present invention will be specifically described by way of examples.
The quantification and optical purity of 3-halo-1,2-propanediol were determined using gas chromatography under the following analysis conditions.

<Quantification of 3-halo-1,2-propanediol>
Sample preparation method: Dissolving the sample in an equal amount of ethyl acetate Device: Column oven GC-17A manufactured by Shimadzu Corporation
Integrator C-R5A manufactured by Shimadzu Corporation
Column: DB-5 J & W Scientific carrier: He purity 99.995% or more Make-up gas: He purity 99.995% or more Detector gas: Hydrogen purity 99.995% or more Column temperature: 50 ° C to 10 ° C increments Warm and hold at 250 ° C for 5 minutes Injection temperature: 250 ° C
Detector temperature: 250 ° C
Linear velocity: 35 cm / s
Retention time: 10 min

<Optical purity of 3-halo-1,2-propanediol>
Sample preparation method: Extraction from the reaction solution with an equal amount of methylene chloride, derivatization with p-toluenesulfonate, removal of the solvent under reduced pressure to obtain a syrup, 3-chloro-1,2- in the syrup Propanediol is dissolved in 200 μl of carrier, and optical isomers are analyzed by high performance liquid chromatography.
Apparatus: Column oven CTO-6AUV manufactured by Shimadzu Corporation
Pump PU-1580 made by Jusco
Integrator Chromatocoder 21J made by sic
UV 1575 made by UV Jusco
Column: CHIRALCEL OC Daicel Chemical Industries, Ltd. Carrier: Hexane / 2-propanol = 98.5 / 1.5 (volume ratio)
Column temperature: 40 ° C
Flow rate: 2.5mL / min
Wavelength: 235nm
Retention time: S body 20min
R body 26min
The optical purity (enantiomeric excess;% ee) was calculated from the peak areas of HPLC of (S) -3-halo-1,2-propanediol and (R) -3-halo-1,2-propanediol as follows: It can be calculated by the following formula.
When R> S: Optical purity of R-form (% ee) = (R−S / R + S) × 100
In the case of S> R: Optical purity of S-form (% ee) = (S−R / R + S) × 100
S: Peak area of (S) -3-halo-1,2-propanediol R: Peak area of (R) -3-halo-1,2-propanediol

[Preparation Example 1]
Medium (medium composition; glucose 1%, peptone 0.5%, meat extract 0.3%, yeast extract 0.3%) was adjusted to pH 7.0, dispensed 100 ml each in a 500 ml Erlenmeyer flask, and 15 ° C. at 121 ° C. Sterilized by minute. After returning the medium to room temperature, 0.8 ml of 25 (w / v)% 3-chloro-1,2-propanediol aqueous solution sterilized with a membrane filter was added.
[Preparation Example 2]
A medium was prepared in the same manner as in Preparation Example 1, except that 0.1 ml of epichlorohydrin was added instead of the aqueous 3-chloro-1,2-propanediol solution.

[Example 1]
100 μl of Leifsonia sp. MRC03 strain (FERM AP-20453) was inoculated with Pipetman into the medium prepared in Preparation Example 1, and cultured with shaking at 30 ° C. for 48 hours. The culture was centrifuged (15000 × g) to collect the cells. To this was added 100 ml of 100 mM Tris-HCl (pH 8.0) buffer. Thereafter, the cells were washed by centrifugation (15000 × g). Thereafter, the cells were suspended in 100 ml of a 780 mM 1,3-dichloro-2-propanol solution (1M phosphate buffer pH 7.0), and the reaction was shaken at 30 ° C. for 23 hours.
After the reaction, the reaction solution was centrifuged (15000 × g) to remove the cells. The produced 3-chloro-1,2-propanediol in the supernatant was quantified by gas chromatography, and the yield from the substrate was determined. As a result, 600 mM 3-chloro-1,2-propanediol was produced. Further, 50 ml of methylene chloride was added to the supernatant for extraction, derivatized with p-toluenesulfonate, and then the solvent was removed under reduced pressure to obtain a syrup. 3-Chloro-1,2- Propanediol was dissolved in 200 μl of carrier, and optical isomers were analyzed by high performance liquid chromatography. The optical purity of 3-chloro-1,2-propanediol was R-form 83% ee.

[Example 2]
Epichlorohydrin was added to 35 ml of 1 M Tris-HCl buffer (pH 8.0) to a final concentration of 110 mM, and then the same procedure as in Example 1 was performed except that the cells were suspended.
As a result, 86 mM 3-chloro-1,2-propanediol was produced. The optical purity of 3-chloro-1,2-propanediol was R-form 32% ee.

[Example 3]
The medium prepared in Preparation Example 2 was inoculated with Rhizobium radiobacter MRC01 strain (FERM AP-20452) and cultured with shaking at 30 ° C. for 48 hours. The culture was centrifuged (15000 × g) to collect the cells. To this, 100 ml of 100 mM phosphate buffer (pH 7.0) was added, and the cells were washed by centrifugation (15000 × g). Bacteria were suspended in 50 ml of the same buffer to obtain a bacterial solution. Thereafter, 50 ml of the bacterial solution was mixed with 100 ml of a 156 mM 1,3-dichloro-2-propanol solution (1M phosphate buffer pH 7.0), and the reaction was shaken at 20 ° C. for 30 minutes. After the reaction, the reaction solution was centrifuged (15000 × g) to remove the cells. As a result of analysis in the same manner as in Example 1, 156 mM 3-chloro-1,2-propanediol was produced. The optical purity of 3-chloro-1,2-propanediol was 95% ee in the S form.

[Example 4]
The cells were washed in the same manner as in Example 3 except that the cells were washed with 100 ml of 100 mM Tris-HCl buffer. Bacteria were suspended in 50 ml of the same buffer to obtain a bacterial solution. Except that epichlorohydrin was added to 35 ml of 1 M Tris-HCl buffer (pH 8.0) to a final concentration of 110 mM, 50 ml of the above bacterial solution was mixed, and the reaction was shaken at 30 ° C. for 23 hours. In the same manner as in Example 3. As a result, 70 mM 3-chloro-1,2-propanediol was produced. The specific rotation of the syrup was S-form optical purity of 5% ee.

[Example 5]
The same procedure as in Example 1 was performed, except that washing was performed 3 times with 140 ml of 100 mM Tris-HCl (pH 8.0) buffer. What was obtained by centrifuging (15000 × g) the bacteria obtained by culturing in 50 ml of the same buffer solution was suspended, and the cells were disrupted by ultrasonic waves. The obtained cell disruption solution was centrifuged and the supernatant was recovered. This supernatant was used as a crude enzyme solution. 1,3-Dichloro-2-propanol was added to the crude enzyme solution to a final concentration of 50 mM, and the reaction was shaken at 20 ° C. for 50 minutes. As a result of analysis, 36 mM 3-chloro-1,2-propanediol was produced. The optical purity was 87% ee and (R) -3-chloro-1,2-propanediol was obtained.

[Example 6]
Cells of Rhizobium radiobacter MRC01 strain (FERM AP-20442) were obtained in the same manner as in Example 3. A crude enzyme solution was obtained in the same manner as in Example 5. 1,3-Dichloro-2-propanol was added to the obtained crude enzyme solution to a final concentration of 50 mM, and the reaction was shaken at 20 ° C. for 30 minutes. As a result of analysis, 46 mM 3-chloro-1,2-propanediol was produced. It was (S) -3-chloro-1,2-propanediol having an optical purity of 96% ee.

Claims (2)

Rhizobium radiobacter MRC01 strain (FERM P-20451) . Production of 3-halo-1,2-propanediol represented by the following general formula (2), which comprises a step of bringing the microorganism according to claim 1 or a treated product thereof into contact with a compound represented by the following general formula (1) Method.