KR100620119B1 - Pseudomonas putida JYR-1 producing syn-anethole-23-epoxides and anti-anethole-23-epoxides from trans-anethole - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to Pseudomonas putida JYR-1, which biotransforms trans-anetol to neo-anetol-2,3-epoxide and anti-anetol-2,3-epoxide, and more particularly to plants It breaks down the trans-anethole produced in the biodegradation and converts it into the stereoisomers of neo-antol-2,3-epoxide and anti-antol-2,3-epoxide, and especially high concentration of trans It relates to the use of Pseudomonas putida JYR-1 [KCTC 10810BP] which can also degrade anetitol. By using the strain in the trans-anthitol bioconversion reaction, the reaction yield is higher than that of the conventional organic synthesis method, and bioconversion is possible at a low cost, thereby increasing industrial value.

Trans-Anetol, Cineantol-2,3-epoxide, Anti-Anetol-2,3-epoxide, Pseudomonas putida JYR-1

Description

Pseudomonas putida JYR-1 producing syn-anethole-2 which biotransforms trans-anetol into neo-anetol-2,3-epoxide and anti-anthitol-2,3-epoxide. , 3-epoxides and anti-anethole-2,3-epoxides from trans-anethole}

1 is a graph of the growth curve of Pseudomonas putida JYR-1 grown using 0.3% trans-antol (20 mM) as a carbon source.

Figure 2 is an HPLC chromatogram of metabolite (B) of trans-anetol by trans-anetol, para-anicin acid, para-hydroxybenzoic acid (A) and Pseudomonas putida JYR-1.

FIG. 3 is LC / Mass and UV spectra for the anti-anthitol-2,3-epoxide (II) and syn-antol-2,3-epoxide (III) metabolites of trans-anetol.

Figure 4 is a graph comparing the degradation rate of 2.5 mM trans-anetol after Pseudomonas putida JYR-1 in trans-anetol (○) and glucose (●).

Figure 5 shows the biotransformation metabolism of trans-anthitol predicted by Pseudomonas putida JYR-1.

FIG. 6 is an HPLC chromatogram of Pseudomonas putida JYR-1 grown in 100 mM trans-Anetol.

FIELD OF THE INVENTION The present invention relates to Pseudomonas putida JYR-1, which biotransforms trans-anetol to neo-anetol-2,3-epoxide and anti-anetol-2,3-epoxide, and more particularly to plants It breaks down the trans-anethole produced in the biodegradation and converts it into the stereoisomers of neo-antol-2,3-epoxide and anti-antol-2,3-epoxide, and especially high concentration of trans It relates to the use of Pseudomonas putida JYR-1 [KCTC 10810BP] which can also degrade anetitol.

There has been a great deal of interest in the synthesis pathways and the availability of the natural products derived from plants, among which secondary metabolites are used as herbs, antibiotics, insecticides, herbicides, food additives, spices. It is used in various ways. This is because, unlike metabolites derived from primary metabolism, secondary metabolites are due to the physiological activity resulting from the diversity of functional groups in their chemical structure. One example is the biotransformation of Morphine by the Pseudomonas putida strain carried out by Professor Neil Bruce of the University of Cambridge. Morphine was converted to hydromorphone by the reductase activity of Pseudomonas putida strain, and the produced dihdyromorphone was 100 times more effective than morphine. .

Secondary metabolites found in plants fall into three broad categories: alkaloid metabolism, which leads to metabolism such as nicotine and cocaine, isoprenoid metabolism, which leads to menthol and saponin, vanillin and flavone. It is a phenylpropanoid metabolic process that leads to. In particular, in the case of plants that make lignin, etc., the phenylpropanoid metabolism process is important, which can be applied to various fields due to its structural diversity.

Traditional organic synthesis methods are expensive because of low yield and high temperature and high pressure reaction for synthesizing geopolitical and optical specific materials. However, microorganisms can selectively produce the desired geopolitical and optical specific materials and save money because the reaction takes place at low temperatures and pressures.

Trans anetol itself or its metabolites, such as anesic acid, anesic alcohol, anesthel aldehyde, etc., can be utilized as various food materials as food additives and spices. Therefore, it is possible to expect a greater economic effect than that obtained from chemical synthesis by biologically degrading trans-anthitol. In particular, if the epoxide can be produced from trans anetol, it can be used as a precursor for producing various chemicals due to the active reactivity of the epoxide.

In addition, Arthrobacter strain TA13 is known as a bacterium that degrades trans-anetol, which is adsorbed by 0.1% trans-anetol to amberlite XAD-2, a type of sorbent. Cultured in the state, but the growth was insufficient.

Therefore, there is a need for new material research that decomposes high concentrations of trans-anetol.

Therefore, the present inventors have searched for a new substance capable of decomposing trans-anetol as described above. As a result, we isolated Pseudomonas putida JYR-1 capable of decomposing trans-anetol from soil, and the strain contained high concentration. It is also possible to decompose in trans-anetol, so that syn-anethole-2,3-epoxide and anti-anetol-2,3-epoxide (anti-anethole-2, The present invention was completed by confirming the bioconversion to 3-epoxide).

Thus, the present invention is directed to Pseudomonas putida JYR-1 in the reaction of trans-anetol to syn-annetol-2,3-epoxide and anti-anetol-2,3-epoxide. The purpose is to provide a method for biotransformation using [KCTC 10810BP].

The present invention is directed to Pseudomonas putida JYR-1 [KCTC] in the reaction of trans-anetol to syn-antol-2,3-epoxide and anti-anetol-2,3-epoxide. 10810BP] is characterized by the method of biotransformation.

Referring to the present invention in more detail as follows.

The present invention biotransforms the stereoisomers of neo-antol-2,3-epoxide and anti-antol-2,3-epoxide, while degrading the trans-anethole made in plants, In particular, the present invention relates to Pseudomonas putida JYR-1 (KCTC 10810BP), which can decompose high concentrations of trans-anetol.

The strain isolated in the present invention was identified as belonging to Pseudomonas putida as a result of pure separation from the soil as a strain that decomposes trans-anetol and identified as 16S rDNA sequence, which is Pseudomonas putida JYR-1 -1). In addition, the strain was deposited on May 27, 2005 at the Korea Institute of Biotechnology Research Institute Genetic Resource Center, and the accession number was assigned to KCTC 10810BP. The strain was separated from the soil and finally selected through a series of processes and identified as Pseudomonas putida, but Pseudomonas putida JYR-1 strain having physical properties that are completely different from similar strains reported so far.

The Pseudomonas putida JYR-1 strain was grown as stereo-isomers Shin-Antol-2,3-epoxide and anti-Anetol-2,3-epoxide when cultured in a minimal medium containing trans-antol. Could switch.

Accordingly, the present invention relates to the use of the Pseudomonas putida JYR-1 strain, namely syn-antol-2,3-epoxide and anti-antol-2,3-epoxide for trans-anetol. Biotransformation using Pseudomonas putida JYR-1 [KCTC 10810BP]

In the present invention, the Pseudomonas putida JYR-1 showed growth even at a high concentration of trans-antol 1.5% (100 mM), and the population increased as the concentration of trans-antol was increased up to 100 mM.

 In the present invention, Pseudomonas putida JYR-1 is grown by providing only trans-antol as a carbon source in a minimal medium (Staniner's basal minimal salt) for soil microbial culture. Pseudomonas putida JYR-1 is isolated from soil and grown in a non-light incubator at a speed of 100 to 500 rpm at 25 to 30 ° C. Incubate with 20 mM trans-antol and grow for 24 hours to obtain syn-antol-2,3-epoxide and anti-antol-2,3-epoxide and other metabolites in culture.

In addition, the present inventors found that trans-anetol is metabolized to syn-antol-2,3-epoxide and anti-anetol-2,3-epoxide as the trans-antol is degraded by Pseudomonas putida JYR-1. It was found that the final production of para-hydroxybenzoic acid via the para-anisin acid. This can predict and understand the metabolic processes of the phenylpropanoid family having a structure similar to trans-anetol. In addition, Pseudomonas putida JYR-1 can grow at high concentrations of trans-anthitol, and it is expected that industrial value is very high because a large amount of metabolites can be obtained due to high resistance to reactants. In particular, the properties of Pseudomonas putida JYR-1, which can make syn-antol-2,3-epoxide and anti-anetol-2,3-epoxide, can be used for intermediate derivative production during chemical synthesis.

Synthesis of cine epoxide is possible due to the optical activity of cin-anthitol-2,3-epoxide and anti-anthitol-2,3-epoxide obtained from the plant secondary metabolite, trans anetol by microorganisms. It is expected to show similar availability as used as synthetic precursors, and as a result it is expected to create high value-added substances without harming the environment through the joint utilization of plants / microorganisms.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Reference Example 1: Stanier's basal minimal salts buffer

1) Solution A: The following compound was dissolved in 1 L of the final volume.

69.5 g Na ₂ HPO ₄

KH ₂ PO ₄ 69.5 g

2) Solution B: The following compound was dissolved in water and mixed with a solution containing 100 ml of metal ions to make a final volume of 1 L.

Nitrilotricacetic acid 20.0 g

MgSO ₄ 28.90 g

CaCl 5.04 g

(NH ₄ ) ₆ MO ₇ O ₂₄ 4H ₂ O 18.50 mg

FeSO ₄ 7H ₂ O 198.0 mg

Nicoinic acid 100 mg

Composition of metal ion solution (per 100 ml)

0.25 g of isodium salt (EDTA)

ZnSO ₄ ㆍ 7H ₂ O 1.1 g

FeSO ₄ 7H ₂ O 0.5 g

MnSO ₄ H ₂ O 0.154 g

CuSO ₄ ㆍ 5H ₂ O 0.04 g

Co (NO ₃ ) ₂ ㆍ 6H ₂ O 0.025 g

Na ₂ B ₄ O7 10 H ₂ O 0.018 g

3) C solution: 20.0 g of (NH ₄ ) ₂ SO ₄ was dissolved in 100 ml of water.

4) Dilute 25 times A solution, 50 times B solution, and 200 times C solution to make the minimum medium.

Example 1 Isolation of Trans-Anetol Degradable Strains

Samples were taken from petroleum contaminated soil in Gyeongsangnam-do, Korea. Suspended 5 g was suspended in 100 ml medium containing 0.5% trans-antol and returned to light at a rate of 200 rpm (round per minute) at 27 ° C. Cultured in the incubator. After inoculating the culture medium in a new medium under the above conditions and repeating the culture process several times, serial dilutions were made in nutrient agar medium and then cultured at 27 ° C. After 100 colonies were obtained, they were inoculated again in a minimal medium containing 0.5% trans-anetol. One colony was grown using Trans-Anetol as the carbon source and named JYR-1.

Example 2: Strain Identification

Until a few years ago, the culture, morphology, and physiological biochemical characteristics of microorganisms were investigated and compared with Bergy's manuals. Afterwards, biolog tests were used to identify them. If you enter a sequence in a web search program such as GeneBank with a sequence, it uses a method of matching and identifying the database on the web.

Of course, it is better to perform experiments with two kinds of 16S rDNA sequencing at the same time as physiological biochemical experiment, but the sequencing of 16S rDNA is registered and updated for each strain found. The method is no longer updated because the strains identified with 16S rDNA sequences are not specified in Bergy's manual or in the biolog database. There is no situation.

In addition, the nucleotide sequence 1470 base was analyzed in the nucleotide sequence analysis of 16S rDNA to obtain 96% to 99% concordant strains on the GenBank, which may be more accurate than Bergy's manual or biolog test.

In this way, 16S rDNA of the isolate was isolated and analyzed for nucleotide sequence. Genomic DNA was extracted from the culture of the strain using a DNeasy Plant Mini Kit (Qiagen, Germany). Then, PCR (polymerase chain reaction) was performed using the following primers to obtain 16S rDNA. The primer used in the analysis was used as a universal primer, the nucleotide sequence and PCR performance conditions are as follows.

5'-primer (SEQ ID NO: 1): 5'-AGA GTT TGA TCM TGG CTC AG-3 '(p27F)

3'-primer (SEQ ID NO: 2): 5'-GYT ACC TTG TTA CGA CTT-3 '(p1525R)

PCR Reactants:

1 μl genomic DNA

100 pmol (p27F) 2 μl

100 pmol (p1525R) 2 μl

2 μl Bioneer premix

43 μl dH ₂ O

50 μl total

PCR reaction conditions (30 cycles):

Initial denaturation process 95 ℃, 15 minutes

Denaturation process 94 ℃, 1 minute

Annealing process 55 ℃, 1 minute

Extension (polymerization) process 72 ℃, 1 minute

Final extension process 72 ℃, 7 minutes

After the PCR reaction, the PCR purification kit was purified and sequence analysis of 16S rDNA was carried out to identify the strain through blast search on gin bank. As a result, Pseudomonas putida JYR- It was confirmed that 568 nucleotide sequences of 1 have 98.9% homology with the existing Pseudomonas putida, and the nucleotide sequence is shown in SEQ ID NO: 3 (GenBank Acession No. AY671902).

From the above results, the isolated strain was identified as Pseudomonas putida JYR-1 ( Pseudomonas putida JYR-1), which has similar properties to Pseudomonas putida , and it was designated as Pseudomonas putida JYR-1 in 2005. Deposited May 27, received accession number KCTC 10810BP.

Example 3: Pseudomonas putida JYR-1 Strain Culture

1) Confirmation of bioconversion ability in trans-anthitol

FIG. 1 is a graph of growth curve of purely isolated Pseudomonas putida JYR-1 in Steiner's basal minimal salt containing 20 mM trans-anthitol. Inoculated with Pseudomonas putida JYR-1 in Sterilizer's basal minimal salt containing 20 mM trans-anetol and then run at a rate of 200 rpm (round per minute) at 27 ° C. in a non-light incubator Incubated. X-axis shows incubation time after bacterial inoculation, Y-axis means absorbance at 600 nm. Pseudomonas putida JYR-1 had an absorbance increased to 2.6, and the doubling time of doubling the number of Pseudomonas putida JYR-1 cells was 8 hours. As a comparison, Pseudomonas putida KTCT 1643 and E. coli K12 were incubated in 20 mM trans-anetol under the same conditions, but did not grow.

2) Comparison of bioconversion capacity by carbon source

Figure 4 is a graph comparing the degradation rate of 2.5 mM trans-anetol after Pseudomonas putida JYR-1 in trans-anetol (○) and glucose (●). Pseudomonas putida JYR-1 was supplied with trans-anetol or glucose as a carbon source, and these were centrifuged at 10,000 × g for 15 minutes at 4 ° C, and Pseudomonas putida JYR-1 was placed on Stanner's basal minimal salt. After the suspension and centrifugation were repeated three times, the absorbance was made to 1.0 at 600 nm. 25 ml Pseudomonas putida JYR-1 was placed in a 125 ml serum vial containing 2.5 mM trans-anetol and placed in an incubator at 25 ° C. at 200 rpm to observe the rate of degradation of trans-anetol. 1 ml was extracted from the vials over time, extracted with 5 ml ethyl acetate, concentrated in a high-speed centrifuge, dissolved in methanol, and analyzed by HPLC to quantify trans-anthitol. Pseudomonas putida JYR-1 grown in trans-anetol was completely bioconverted within 25 minutes, but when grown in glucose, trans-anetol biotransformation rate was slow.

3) Biochemical Properties of Pseudomonas putida JYR-1

Various metabolic reactions of Pseudomonas putida JYR-1 were determined using Becton's BBL CRYSTAL ^™ Identification System kit. The bacterium metabolizes arabinose, mannose, and galactose, but sucrose, melibiose, rhamnose, sorbitol, mannitol, Adonitol and inositol could not metabolize. In addition, the Pseudomonas putida JYR-1 strain metabolizes the amino acids glycine and arginine, and proline nitroanilide and Γ-L-glutamyl p-nitroanilide in yellow para- The hydrolase reaction to produce nitroaniline could also be confirmed. In addition, urea decomposition reaction was shown to oxidative deaminoation of phenylalanine.

Example 4 Material Structure Analysis Isolated from Pseudomonas Putida JYR-1 Strain

1) HPLC chromatogram

Figure 2 shows the metabolites of trans-anetol by trans-anetol, para-anisic acid, para-hydroxybenzoic acid (A) and Pseudomonas putida JYR-1. HPLC chromatogram of (B). (A) combines HPLC chromatograms of trans-anetol, para-anicin acid, para-hydroxybenzoic acid. Reversed-phase C18 (5 μm particle size, 4.6 mm × 25 cm, Milford, Mass.) Was mounted on Varian ProStar HPLC (Varian, Walnut Creek, Calif.) And 20 μl of samples analyzed. H ₂ O and acetonitrile containing 1% formic acid were used as solvents. The solvent ratio was 10% acetonitrile, up to 60% in 10 minutes and 90% in 20 minutes. The amount was changed and 90 ml of acetonitrile was flowed 1 ml per minute until 30 minutes. HPLC chromatograms were obtained by measuring absorbance with a UV detector at 270 nm using a Photo Diode Array [Varian, Walnut Creek, CA]. Retention times of para-hydroxybenzoic acid and para-anicin acid were 7.8 and 12.6 minutes, respectively, and trans-antol had a retention time of 20.4 minutes. (B) was grown with Pseudomonas putida JYR-1 in a medium (Staniner's basal minimal salt) containing 20 mM (0.3%) of trans-anetol, the culture was extracted with ethyl acetate, concentrated in a vacuum high-speed concentrator and methanol Chromatogram dissolved in and analyzed by HPLC. The peaks corresponding to trans-anetol are not seen and two peaks can be seen in which para-hydroxybenzoic acid (I) and para-anicin acid (IV) have the same residence time. Liquid chloromatography / mass (LC / MS) spectroscopy and NMR spectroscopy were used to identify the remaining (II) and (III) metabolites. Para-anicin acid was most accumulated in the culture as bacteria grew, and the production ratio of (II) and (III) was approximately 1: 4.

2) LC / MS and UV Spectrum

3 shows the LC / MS and UV spectra for the (II) and (III) metabolites of trans-anthitol. LC / MS was performed using an HP1100 system using ESI ⁺ (electrospray ionization) mode and a Quattro LC triple quadruple tandem mass spectrometer (Micromass, Manchester, UK). Analytical conditions were the same as HPLC, and 20 μl of sample was injected in ESI ⁺ mode with a split 1: 3 ratio. Source temperature, desolvation temperature, cone voltage and capillary voltage were 60 ° C, 220 ° C, 26 V and 3.99 kV, respectively, and the electron multiplier voltage. ) Was 640 V, nebulizer gas and desolvation gas were flowed into high purity nitrogen at 94 l / h and 562 l / h. LC / MS analysis showed that the four peaks had molecular weights of 132.9, 165.5 and 165.3 153.3 [M + H] ⁺ at m / z, respectively, and 132.9 and 153.3 were para-hydroxybenzoic acid (138) and para-anidic acid, respectively. (152). (II) and (III) had the same molecular weight of 164, which was expected to be an epoxide in the form of oxygen-bonded prene of trans-anetol, which was separated and purified, and analyzed by NMR. .

3) NMR spectrum

Table 2 shows the ¹ H-NMR and ¹³ C-NMR data of the (II) and (III) metabolites of trans-anetol. All NMR measurements were performed at 298 K absolute temperature using a Bruker Avance 400 spectrometer system (9.4 T). The metabolites (II) showed eight peaks at ¹³ C-NMR and two peaks doubled at 112.8 ppm and 126.9 ppm, indicating that (II) consisted of 10 carbons. However, trans-antol had 10 carbons and had a molecular weight of 148 and had a molecular weight of 165.5 [M + 1] ⁺ in LC / MS. The first column of Table 2 below shows the numbers of carbons of trans-antol and the ¹ H-NMR and ¹³ C-NMR results of (II) and (III) metabolites. ¹³ C-NMR results show that C6 / C8 and C5 / C9 are in the benzene ring, C4 and C7 are single carbon bonds connected to the benzene ring, and C1 and C10 are methoxy groups. And based on the coupling constants H1 (1.03 ppm, d, J = 6.4 Hz), H2 (3.92 ppm, dd, J = 4.6 Hz, 6.4 Hz), H3 (4.50 ppm, d, J = 4.6 Hz) It was found to be in the form of an epoxide with one oxygen attached to C3. (III) metabolites showed similar results, and the binding constants of H2 and H3 were higher at (III) metabolite as 7.5 Hz, so that (II) and (III) were the stereoisomer anti-anetol-2,3-epoxide and It can be seen that it is a sin- anetol-2,3-epoxide. NMR data of metabolites (II) and (III) are shown in Table 2 below.

Example 5: Confirmation of bioconversion ability according to the concentration of trans-anthitol

Pseudomonas putida JYR-1 was incubated for 48 hours in a minimal medium containing 100 mM. 1 ml of the culture was extracted with 6 ml of ethyl acetate, concentrated with a high-speed centrifugal condenser, dissolved in methanol, and analyzed using HPLC as in Example 5, to obtain a chromatogram as shown in FIG. 6. 6 compared to B of FIG. 2 shows that the same chromatogram shows that Pseudomonas putida JYR-1 also grows at a high concentration of 100 mM trans-antol and syn-anthitol-2,3-epoxide. It can be seen that and the anti (anti)-antol-2,3-epoxide is produced.

As described above, the present invention is capable of biologically producing epoxide, which is an intermediate of industrial organic synthesis, with Pseudomonas putida JYR-1 [KCTC 10810BP], and also biotransforms its analogs in addition to trans-antol to high cost. It is expected to be used to produce natural substances.

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Claims (1)

Pseudomonas putida JYR-1 [KCTC 10810BP] was used to convert trans-anetol to syn-antol-2,3-epoxide and anti-antol-2,3-epoxide. How to use biotransformation.

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