KR101619140B1 - Microorganism, Pseudomonas aeruginosa. KNU-2B, producing hydroxyl fatty acid - Google Patents

Abstract

The present invention relates to a Pseudomonas aeruginosa strain KNU-2B producing a hydroxy fatty acid and a method for producing a hydroxy fatty acid using the same. The Pseudomonas aeruginosa strain KNU-2B according to the present invention is a novel microorganism and is a novel microorganism, which is superior to conventional Pseudomonas sp. Strains in that it contains hydroxy fatty acids, especially 7,10-dihydroxy-8 (E ) - octadecenoic acid (7,10-dihydroxy-8 (E) -octadecenoic acid, DOD) can be produced with high efficiency.

Description

Pseudomonas aeruginosa KNU-2B strain producing a hydroxy fatty acid {Microorganism, Pseudomonas aeruginosa. KNU-2B, producing hydroxyl fatty acid}

The present invention relates to a Pseudomonas aeruginosa strain KNU-2B producing a hydroxy fatty acid and a method for producing a hydroxy fatty acid using the same.

Hydroxy fatty acid (HFA) is a form that has at least one hydroxyl group in the central chain of a common fatty acid. The hydroxyl group added to the fatty acid chain gives the fatty acid a unique property such as high viscosity and reactivity (Chemical and Biological conversion of soybean oil for industrial products, in Fats for the Future, edited by RC Cambie, Ellis Horwood Ltd Press, Chichester, pp: 301-317). HFA is classified into mono-, di-, and tri-hydroxy fatty acids depending on the number of hydroxyl groups to which they are attached. It is classified as an epoxy-hydroxy fatty acid or oxohydro Oxo-hydroxy fatty acid, and the like.

Due to the unique properties of HFA, they can have a variety of physiologically active functions and as a result they can be applied to a wide range of industries such as pesticides, medicines, high-functional resins and fiber materials, biodegradable plastic materials, lubricants, cosmetics and paints . Ricinoleic acid or sebacic acid, which is a derivative of castor oil, has been used as a synthetic material for new functional and high-efficiency polymers. It is classified as an industrial essential material by the US government. There are also.

It has been reported that 12,13,17-trihydroxy-9 (Z) -octadecenoic acid with three hydroxyl groups in HFA has the ability to inhibit the growth of plant pathogenic fungi (Hou, CT and Forman, RJ III Growth inhibition (9,10,13-THOD, < / RTI > < RTI ID = 0.0 > 9,12,13-THOD) is known to have strong inhibitory ability against the growth of fungi that cause rice blast (Kato, T. Yamaguchi, Y. Abe, N. Uyehara, T. Nakai, T Yamanaka, S. and Harada, N. Unsaturated Hydroxy Fatty Acids, The Self-Defensive Substances in Rice Plant against Rice Blast Disease. Chem. Lett., 1984, 25, 409-412).

Although various functionalities of hydroxy fatty acids have been known to date, it is known that hydroxy fatty acids present in nature are present only in trace amounts in plants. Therefore, research was attempted to produce hydroxy fatty acids using microorganisms.

Flavobacterium sp. DS5 is capable of producing 10-hydroxy-octadecadienoic acid from oleic acid (Hou, CT 1994). Conversion of linoleic acid to 10-hydroxy-12 (Z) -octadecenoic acid by Flavobacterium sp (NRRL B-14859) , J. Am. Oil. Chem. Soc. 71: 975-978), and Pseudomonas aeruginosa PR3 is known to produce mono-, di- and tri-hydroxy fatty acids using a wider range of substrates (Kim, H. Gardner, HW and Hou, CT 10 (S) -Hydroxy-8 (E) -octadecenoic Acid, an Intermediate in the Conversion of Oleic Acid to 7,10-Dihydroxy-8 (E) -octadecenoic Acid. 8 (E) -octadecenoic Acid, an Intermediate in the Conversion < tb > ______________________________________ < tb > ______________________________________ < tb > ______________________________________ < tb > (R) -Octadecenoic Acid by Pseudomonas aeruginosa PR3. J. Ind. Microbiol. Biotechnol. 1999, 24, 167-172, Kim, H. Gardner, HW and Hou, VC. T. Production of Isomeric (9,10,13) -trihydroxy-11E (10E) -octadecenoic Acid from Linoleic Acid by Pseudomonas aeruginosa PR3, J. Ind. Microbiol. Biotechnol., 2000, 25, 109-115).

The inventors of the present invention have been studying a method for producing a hydroxy fatty acid using microorganisms, wherein a newly identified Pseudomonas aeruginosa strain KNU-2B has a higher efficiency from a vegetable oil than a conventional Pseudomonas sp. The present invention has been completed.

It is an object of the present invention to provide a novel Pseudomonas aeruginosa strain producing a hydroxy fatty acid.

It is another object of the present invention to provide a method for producing hydroxy fatty acid using the strain.

In order to achieve the above object, the present invention provides Pseudomonas aeruginosa KNU-2B strain (Accession No. KFCC11544P) producing a hydroxy fatty acid.

The present invention also provides a method for producing a hydroxy fatty acid comprising the step of culturing the Pseudomonas aeruginosa strain KNU-2B (Accession No. KFCC11544P).

The Pseudomonas aeruginosa strain KNU-2B according to the present invention is a novel microorganism and is a novel microorganism, which is superior to conventional Pseudomonas sp. Strains in that it contains hydroxy fatty acids, especially 7,10-dihydroxy-8 (E ) - octadecenoic acid (7,10-dihydroxy-8 (E) -octadecenoic acid, DOD) can be produced with high efficiency.

Brief Description of the Drawings Fig. 1 is a diagram showing a separation process of Pseudomonas aeruginosa KNU-2B strain.
FIG. 2 is a diagram showing the results of confirming the phylogeny of Pseudomonas aeruginosa strain KNU-2B through 16 sRNA analysis. FIG.
FIG. 3 is a graph showing the results of TLC analysis of products in the culture broth of a strain (A: KNU-2B strain, B: PR3 strain, Lane 1: oleic acid as a substrate, Lane 2: linoleic acid as a substrate, Lane 3: olive oil is used as a substrate).
Fig. 4 is a graph showing the results of gas chromatography analysis of crude extracts of the products in the culture medium of Pseudomonas aeruginosa PR3 strain. Fig.
Fig. 5 is a graph showing the results of gas chromatography analysis of crude extracts of products in the culture medium of Pseudomonas aeruginosa KNU-2B strain. Fig.
6 is a diagram showing the result of analysis of the structure of DOD produced by Pseudomonas aeruginosa strain KNU-2B through GC / MS.

Hereinafter, the present invention will be described in detail.

The present invention provides a Pseudomonas aeruginosa strain KNU-2B (Accession No. KFCC11544P) producing a hydroxy fatty acid.

The above strain is a mutant strain identified in the culture of Pseudomonas aeruginosa PR3 (Accession No. NRRL B-18602). The sequence of the gene (rDNA) encoding the 16sRNA of the strain is shown in SEQ ID NO: 1, It was found to be a novel strain belonging to Pseudomonas aeruginosa. Therefore, the present inventors named the novel strain obtained as Pseudomonas aeruginosa strain KNU-2B on November 21, 2012 (Accession No. KFCC11544P).

The Pseudomonas aeruginosa strain KNU-2B according to the present invention is a novel microorganism and is a novel microorganism, which is superior to conventional Pseudomonas sp. Strains in that it contains hydroxy fatty acids, especially 7,10-dihydroxy-8 (E ) -Octadecenoic acid (DOD) can be produced with high efficiency and can be industrially useful.

The present invention also provides a culture solution of Pseudomonas aeruginosa strain KNU-2B (Accession No. KFCC11544P).

The present invention also provides a method for producing a hydroxy fatty acid comprising culturing a Pseudomonas aeruginosa strain KNU-2B (Accession No. KFCC11544P).

In the present invention, the hydroxy fatty acid may be any hydroxy fatty acid having at least one hydroxyl group in the central chain of the fatty acid. Preferably, the hydroxy fatty acid has 7 carbon atoms and 10 carbon atoms on the fatty acid chain having 18 carbon atoms Dihydroxy-8 (E) -octadecenoic acid having a total of two hydroxyl groups in each case, and one double bond between carbon number 8 and carbon number 9 (7 , 10-dihydroxy-8 (E) -octadecenoic acid, DOD).

In the present invention, the substrate used for producing the hydroxy fatty acid includes vegetable oil, and preferably olive oil, safflower seed oil, soybean oil, corn oil, sesame seed oil, perilla seed oil, grape seed oil, , Canola oil, sunflower seed oil, melon seed oil, rapeseed oil, rice bran oil, and the like. The vegetable oil may be prepared by any conventional method such as solvent extraction method or pressure extraction method, which have been conventionally used for extracting oil from seeds or fruits of natural plants, and may be commercially available.

In the present invention, the culture temperature of the strain is preferably 10 ° C to 45 ° C, and the incubation time is preferably 24 hours to 240 hours, but is not limited thereto. The incubation time means the incubation time after the addition of the substrate.

In the present invention, the pH of the culture medium in which the strain is cultured is preferably 4.0 to 10.0, but is not limited thereto.

In the present invention, the carbon source of the strain may be selected from the group consisting of glucose, galactose, lactose, glycerol, xylose, sucrose, maltose or fructose fructose and the like and preferably one or more selected from the group consisting of glucose, galactose, lactose and fructose, but is not limited thereto.

In the present invention, the nitrogen source of the strain is selected from the group consisting of yeast extract, malt extract, glutamine, ammonium nitrate (NH ₄ NO ₃ ), peptone, tryptone, Ammonium, ammonium sulfate, ammonium phosphate, urea, and the like.

In one embodiment of the present invention, Pseudomonas aeruginosa strain KNU-2B (Accession No. KFCC11544P) was cultured under the following conditions in order to produce a hydroxy fatty acid with a vegetable oil as a substrate.

More specifically, Pseudomonas aeruginosa strain KNU-2B is cultured in YPD medium (1% yeast extract, 2% peptone, 2% Dextrose) at 28 ° C. The composition of the medium is substituted for the necessary carbon source in place of dextrose, if necessary, and the yeast extract and the peptone are replaced by the necessary nitrogen source. The pH of the medium is maintained at 7.0, and the strain is subcultured every 2-3 days. 1% of vegetable oil substrate is added to KNU-2B culture broth which is cultured in YPD medium for 24 hours, incubated for the necessary time, and 6N HCl is added to lower culture medium pH to 2.0 or less. Immediately after completion of the incubation, a mixture of the same amount of ethyl acetate and diethyl ether is added and extracted secondarily, and the extract is concentrated on a rotary evaporator. The culture broth of the Pseudomonas aeruginosa KNU-2B strain obtained through the above process contains a hydroxy fatty acid, especially 7,10-dihydroxy-8 (E) -octa, which is an antibacterial activity material, (7,10-dihydroxy-8 (E) -octadecenoic acid, DOD).

Hereinafter, the present invention will be described in detail with reference to examples. However, these are for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited thereto.

Example 1 Isolation and Identification of KNU-2B Strain, Mutant Strain

Pseudomonas aeruginosa PR3 (NRRL B-18602) strain was streaked and cultured, and then colony formation was observed. To this end, sterilized 1.5% potato agar was added to YDP medium (1% yeast extract, 2% peptone, 2% Dextrose), and agar plates filled with about 20 ml of plastic petridish were used . The Pseudomonas aeruginosa PR3 (NRRL B-18602) strain was cultured in a YPD medium at 28 DEG C, and the resulting culture was streaked on the agar plate prepared as described above and then cultured at 30 DEG C for 2 to 3 days. The results are shown in Fig. 1A.

As shown in Fig. 1A, it was confirmed that some of the formed colonies showed different forms from those of the conventional Pseudomonas aeruginosa PR3 strain. That is, the border around the colony was unclear and spread, and the size was relatively larger than that of the colony of Pseudomonas aeruginosa PR3 strain (circle in FIG. 1A).

The single colonies identified above were isolated and cultured in YPD medium at 28 ° C. The obtained culture solution was diluted so that the number of bacterial cells was about 100 - 200 / ml, and inoculated on another agar plate, dispersed evenly, and cultured at 30 캜 for 2-3 days. The results are shown in Fig. 1B.

As shown in Fig. 1B, it is confirmed that the colony surrounding boundary is spread uncertainly, and colony of a relatively large size is formed as compared with the colony of existing PR3 strain of Pseudomonas aeruginosa. 1C shows a single colony of Pseudomonas aeruginosa PR3 strain cultured in the same manner as described above.

The mutant strain was isolated and named as Pseudomonas aeruginosa KNU-2B, deposited on November 21, 2012 at the Korean Society for Microbiological Conservation (Accession No. KFCC11544P).

Example 2. 16SRNA Sequence Analysis of KNU-2B Strain

To analyze the genetic identification and phylogenetic relationships of KNU-2B strains isolated in Example 1, 16sRNA sequences were analyzed (SEQ ID NO: 1). Based on the results, we compared the 16 sRNA sequences of the strains registered with the National Center for Biotechnology Information (NCBI) and sorted using ClustalW. 1000 bootstrap neighbors-joining replications, and the homology was analyzed using BLAST. The results are shown in Fig.

As shown in Fig. 2, the phylogenetic analysis confirmed that KNU-2B strain belonged to Pseudomonas aeruginosa .

Example 3. Verification of production of hydroxy fatty acid by KNU-2B strain

In order to verify the ability of the KNU-2B strain isolated in Example 1 to produce hydroxy fatty acid, the following experiment was conducted.

(1) The KNU-2B strain or the previously known Pseudomonas aeruginosa PR3 strain isolated in Example 1 was cultured in YPD medium (1% yeast extract, 2% peptone, 2% Dextrose) at 28 DEG C and 200 rpm And cultured for 24 hours. Oleic acid (lane 1), linoleic acid (lane 2), or olive oil (lane 3) was added to the culture solution of each of the obtained strains at a concentration of 1%, followed by culturing for 3 days, The culture was terminated by adding 6N HCl to lower the pH of the medium to below 2.0. Immediately after completion of the incubation, a mixture of the same amount of ethyl acetate and diethyl ether was added and the mixture was extracted twice. The extract was concentrated on a rotary evaporator. This was analyzed by TLC (thin layer chromatography). TLC was coated on a glass substrate with silica gel and a mixture of toluene: dioxan: acetic acid (79: 14: 7, v / v / v) was used as a solvent for the separation of the products. After the product was separated, the product was confirmed by spotting the product by spraying 50% solution of sulfuric acid on the substrate and heating it at 100 ° C for 10 minutes or more. The results are shown in Fig.

As shown in FIG. 3, the spot generation patterns such as a, b (DOD, dihydroxy octadecenoic acid), and c in the culture extract are different from each other in KNU-2B strain and PR3 strain.

(2) The crude extract of the product, which was produced using olive oil as a substrate, was analyzed by gas chromatography. More specifically, 1 ml of diazomethane was added to 10 mg of the sample to be analyzed, and the mixture was allowed to stand at room temperature for 5 minutes. Diazomethane was removed using nitrogen gas, and 1 ml of TMSI + pyridine (1: 4, v / v) was added and left for 40 minutes. After the residual solvent was blown off with nitrogen gas, 200 μl of a solution for gas chromatography (Dichrolomethane: Methanol = 95: 5, v / v) was added, and then 1 μl of the final sample was injected into a gas chromatograph. The column used was a hydrophobic column. The analytical temperature was in the range of 100 ℃ ~ 300 ℃ and the analysis time was within 1 hour. IS stands for internal standard. The results are shown in Fig. 4 and Fig.

As shown in Fig. 4, it was confirmed that the purity of DOD produced using Pseudomonas aeruginosa PR3 strain and olive oil as a substrate was about 44% of total crude extract. As shown in Fig. 5, KNU-2B strain , And the purity of DOD produced from olive oil as a substrate was about 87% of the total crude extract and the appearance of GC peaks other than DOD was found to be different from that of Pseudomonas aeruginosa PR3.

(3) The structure of DOD produced by the Pseudomonas aeruginosa strain KNU-2B identified through the above experiment was confirmed by GC / MS. The GC / MS was carried out according to the gas chromatography method described in (2) above, wherein a column having a length of 30 m or more was used, and a mass analyzer was installed in a detector of separated molecules. The results are shown in Fig.

As shown in FIG. 6, the structure of the DOD produced by the KNU-2B strain according to the present invention has two hydroxyl groups, one in each of the 7-carbon and 10-carbon on the 18-carbon fatty acid chain, It contains one double bond between carbon and 9 carbon and confirmed that it has the same structure as the existing DOD.

(4) DOD production using KNU-2B strain was analyzed according to the type of carbon source contained in the medium. PR3 strains were prepared by the conventional known conditions (Min-Jung Suh, Ka-Yeon Baek, Beom-Soo Kim, Ching T. Hou and Hak- Ryul Kim, Production of 7,10-dihydroxy-8 (E) -octadecenoic acid The medium and production conditions were set according to olive oil by Pseudomonas aeruginosa PR3. Applied Micrbiology and Biotechnology 2011, 89 (6): 1721-1727. More specifically, 4 g dextrose, 4 g K ₂ HPO ₄ , 1 g (NH ₄ ) ₂ HPO ₄ , 1 g NH ₄ NO ₃ , 1 g yeast extract, 0.056 g FeSO ₄ .7H ₂ O, 0.1 g Each strain (KNU-2B or PR3) was inoculated into 50 ml of a medium containing MgSO ₄ and 0.01 g MnSO ₄ .7H ₂ O and cultured at 28 ° C for 24 hours. Then, 500 μl of olive oil was added and cultured for 72 hours. And extracted and analyzed according to the method of FIG. The results are shown in Table 1.

As shown in Table 1, it was confirmed that the KNU-2B strain and Pseudomonas aeruginosa PR3 strain according to the present invention exhibited different carbon source compositions. The optimal carbon source of Pseudomonas aeruginosa PR3 strain was galactose, whereas the optimum carbon source of KNU-2B strain was fructose. In addition, it was confirmed that the maximum productivity of DOD of KNU-2B strain was about 47.6% higher than the maximum productivity of DOD of Pseudomonas aeruginosa PR3 strain.

Korea Microorganism Conservation Center (Domestic) KFCC11544P 20121121

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Microorganism, Pseudomonas aeruginosa. KNU-2B, producing hydroxyl          fatty acid <130> 221 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 805 <212> DNA <213> Pseudomonas aeruginosa KNU-2B <400> 1 gtcgagcgga tgaagggagc ttgctcctgg attcagcggc ggacgggtga gtaatgccta 60 ggaatctgcc tggtagtggg ggataacgtc cggaaacggg cgctaatacc gcatacgtcc 120 ggtcggatta gctagttggt ggggtaaagg cctaccaagg cgacgatccg taactggtct gagaggatga 240 tcagtcacac tggaactgag acacggtcca gactcctacg ggaggcagca gtggggaata 300 ttggacaatg ggcgaaagcc tgatccagcc atgccgcgtg tgtgaagaag gtcttcggat 360 tgtaaagcac tttaagttgg gaggaagggc agtaagttaa taccttgctg ttttgacgtt 420 accaacagaa taagcaccgg ctaacttcgt gccagcagcc gcggtaatac gaagggtgca 480 agcgttaatc ggaattactg ggcgtaaagc gcgcgtaggt ggttcagcaa gttggatgtg 540 aaatccccgg gctcaacctg ggaactgcat ccaaaactac tgagctagag tacggtagag 600 gggtggtgga atttcctgtg tagcggtgaa atgcgtagat ataggaagga acaccagtgg 660 cgaaggcgac cacctggact gatactgaca ctgaggtgcg aaagcgtggg gagcaaacag 720 gattagatac cctggtagtc cacgccgtaa acgatgtcga ctagccgttg ggatccttga 780 gatcttagtg gcgcagctaa cgcga 805

Claims (8)

delete delete delete Olive oil and fruit sugar (fructose) by Rouge Pseudomonas, in addition to the industrial medium (Pseudomonas aeruginosa) KNU-2B strain (accession No. KFCC11544P) culturing;, 7, 10- dihydroxy -8 containing (E) - A method for producing 7,10-dihydroxy-8 (E) -octadecenoic acid (DOD). delete delete The method according to claim 4, wherein the culture temperature of the Pseudomonas aeruginosa strain KNU-2B (accession number KFCC11544P) is 10 ° C to 45 ° C. 7,10-Dihydroxy-8 (E) (7,10-dihydroxy-8 (E) -octadecenoic acid, DOD).




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