KR102131549B1 - Production of 5,15-dihydroxy eicosapentaenoic acid by new bacterial lipoxygenase - Google Patents

Abstract

The present invention relates to a bacteria-derived novel lipoxygenase, a method of producing 5,15-dihydroxy eicosapentaenoic acid by using the same, and a method for producing the same. According to the present invention, 5,15-dihydroxy eicosapentaenoic acid may be biosynthesized with high productivity and a high yield by an eco-friendly method that has not been reported before. Thus, 5,15-dihydroxy eicosapentaenoic acid produced by the present invention may be usefully utilized in various fields, such as drugs, food, cosmetics, etc. and is expected to be involved, as a signal transduction material, in various physiological activity functions in the human and animal body.

Description

Production of 5,15-dihydric acid eicosapentaenoic acid by a novel lipoxygenase derived from bacteria{PRODUCTION OF 5,15-DIHYDROXY EICOSAPENTAENOIC ACID BY NEW BACTERIAL LIPOXYGENASE}

The present invention relates to a novel lipoxygenase derived from bacteria, a method for producing 5,15-eicosapentaenoic acid using the same, and a composition for producing the same.

Hydroxyl fatty acids are substances that exist in nature and are present in the lipids of many organisms, such as animals, plants, insects, and microorganisms, and are generally substances that have hydroxyl groups. Because it has a hydroxyl group, it has excellent reactivity and is used industrially as a raw material. Also, due to the action of the hydroxyl group, it is used as a raw material for cosmetics due to a decrease in surface tension and an increase in antifungal activity. In particular, hydroxylated fatty acids in animals are used as precursors for signal transduction, and only a single substance is involved in various physiological activities. Lipid mediator, a type of signal transduction substance, is an important substance involved in various physiological functions such as immune response and homeostasis regulation in animals including humans. Enic acid is mainly converted by lipoxygenase acting on polyunsaturated fatty acids.

Functional experiments on 5,15-dihydroxyeicosapentaenoic acid (5,15-diHEPE) have been studied in recent years, but little has been discovered. 5,15-Eicosapentaenoic acid is a physiologically active substance produced by human macrophages and is known to relieve inflammation and promote tissue repair as exposed to pathogenic bacteria. 5,15-Eicosapentaenoic acid has an anti-inflammatory reaction and autoimmunity, and is also involved in removing external invaders through activating phagocytosis of white blood cells against external invaders such as bacteria and viruses. In addition, it is derived from the acute inflammation relieving step, and confirmed strong anti-inflammatory and early inflammatory action. It is known that the treatment of this substance has the ability to potentially reduce and relieve inflammation. It has the potential as a next-generation medical substance in place of chemically synthesized medicines with side effects, and can be used as a research material in a wide range of fields such as medicine, biology, and biotechnology, but a biological production method has not been developed.

Lipoxygenase (LOX), which synthesizes hydroxyl fatty acids, is a dioxygenase and is an oxidase, but is characterized by not having a heme, but instead an iron-containing enzyme. Characteristically, a polyunsaturated fatty acid having one or more cis, cis-1,4-pentadiene is used as a substrate to catalyze stereospecificity and reaction specificity through a dioxygenation reaction.

The known lipoxygenase of bacteria has only one location specificity to the substrate, so it contains only one hydroxyl group, and in the case of lipoxygenase having a deoxygenation function, it can be found in eukaryotes such as rabbits and soybeans. It has been found, but no lipoxygenase having a bacteria-derived deoxygenation function has been reported.

On the other hand, 5,15-eicosapentaenoic acid has not been developed biological production method, and was produced using a chemical synthesis method (Biochwmca et Biophysics Acta 917 (1987) 398-405). However, the chemical synthesis method is a method of synthesizing using an organic solvent, heavy metals such as tin and lead, and carcinogens that are highly toxic and harmful to the human body. There was a limit in that it caused environmental pollution.

As a result, the present inventors have tried diligently to synthesize 5,15-dioxydipentaenoic acid in an eco-friendly method, and as a result, discovered a new dual position-specific lipoxygenase derived from microorganisms that has not been previously reported and used it While constructing the synthetic route, the present invention was completed by confirming that 5,15-dihydroeicosapentaenoic acid was biosynthesized with high yield in an eco-friendly manner without generating by-products from polyunsaturated fatty acids.

Accordingly, the main object of the present invention is to provide a dual position-specific lipoxygenase gene, protein, recombinant vector, and transformant that converts unsaturated fatty acids to 5,15-dihydroeicosapentaenoic acid.

Another object of the present invention is to provide a method for producing 5,15-dihydro-eicosapentaenoic acid in a biotransformation process using the dual position-specific lipoxygenase or transformant and a composition for producing the same. .

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention, 5,15-epoxygenase derived from Arcanium violaceum ( Archangium violaceum ) or a gene encoding the same, as an active ingredient, 5,15-eicosa dihydrate It provides a composition for the production of 5,15-dihydroxyeicosapentaenoic acid.

The present invention also provides a method for producing 5,15-dihydroxyeicosapentaenoic acid by bioconversion by treating a dual position-specific enzyme on a substrate.

The present invention also provides a recombinant vector comprising a 5, 15-lipoxygenase gene consisting of the nucleotide sequence of SEQ ID NO: 2.

The present invention also provides a transformant transformed with the recombinant vector.

The present invention also provides a method for producing 5,15-dihydroxyeicosapentaenoic acid by treating the transformant with a substrate.

The present invention also provides a lipoxygenase gene consisting of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

The present invention also provides a lipoxygenase consisting of the amino acid sequence of SEQ ID NO: 1.

According to the present invention, a method for producing 5,15-dihydroeicosapentaenoic acid using a lipoxygenase gene, a protein, a recombinant vector and a transformant derived from Archangium violaceum and The composition for production can be provided.

According to the present invention, since 5,15-dihydroxyacetic pentaenoic acid can be produced with high productivity and high yield in an environmentally friendly manner not reported to date, it can be usefully used in various industries such as medicine, food, and cosmetics. Can.

The 5,15-dihydroeicosapentaenoic acid prepared according to the present invention is a signal transducer and is expected to be involved in various physiologically active functions in animals including humans.

FIG. 1 shows a synthetic route of 5,15-dihydroeicosapentaenoic acid produced upon treatment of lipoxygenase from Archangium violaceum with eicosapentaenoic acid.
Figure 2 is an HPLC chromatogram confirming the production of 5,15-eicosapentaenoic acid dihydrate.
Figure 3 is a diagram proceeding the material identification of 5,15-eicosapentaenoic acid using LC-MS/MS.
FIG. 4 shows the amount of 5,15-dihydroeicosapentaenoic acid generated over time when eicosapentaenoic acid is treated with all cells containing lipoxygenase derived from Archangium violaceum . It is a graph confirming (■: eicosapentaenoic acid, ●: 5,15-eicosapentaenoic acid dioxide, ▼: 5-eicosapentaenoic acid hydroxide).
FIG. 5 shows that when eicosapentaenoic acid is treated with all cells containing lipoxygenase derived from Archangium violaceum , the concentration of cysteine changes to 5,15-eicosapentaenoic acid. It is a graph showing the effect on activity.
FIG. 6 shows that when eicosapentaenoic acid is treated with all cells containing lipoxygenase derived from Archangium violaceum , the concentration change of eicosapentaenoic acid is 5,15-Eicosa Dihydrate It is a graph showing the effect on the concentration of pentaenoic acid produced.
FIG. 7 is a graph showing the effect of changes in concentration of whole cells containing lipoxygenase derived from Archangium violaceum on the production concentration of 5,15-eicosapentaenoic acid.

Hereinafter, the present invention will be described in detail.

The present inventors have conducted continuous research to prepare 5,15-eicosapentaenoic acid more effectively through a bioconversion process. Specifically, the present inventors cloned a lipoxygenase derived from Arcanium violaceum to produce a recombinant expression vector and a microorganism transformed therefrom, and produced whole cells using the same, followed by By treating the substrate with an environmentally friendly bioconversion method, it was confirmed that 5,15-eicosapentaenoic acid can be produced with high productivity and high yield, and the present invention was completed.

Thus, the present invention provides a composition for the production of 5,15-dihydro-eicosapentaenoic acid comprising a lipoxygenase derived from Archangium violaceum or a gene encoding it as an active ingredient.

In the present specification, 5,15-dihydroxyeicosapentaenoic acid (5,15-dihydroxyeicosapentaenoic acid; 5,15-diHEPE) is a compound represented by Formula 1 below, and the IUPAC name is 5,15-dihydroxy- 6E,8Z ,11Z,13E,17Z -eicosapentaenoic acid). As described above, the dioxide can be used as a research material in various disciplines such as medicine, biology, and biotechnology, but a biological production method has not been developed.

[Formula 1]

In this specification, 5,15-lipoxygenase (5,15-lipoxygenase) is a site-specific enzyme, and forms a hydroxyl group only at positions 5 and 15 in unsaturated fatty acids having 20 or more carbon atoms, such as eicosapentaenoic acid. In addition, if the substrate is treated with a dual position-specific enzyme, it undergoes a double-dioxide process, and forms 15-hydroxyeicosapentaenoic acid from eicosapentaenoic acid and then forms a hydroxyl group at carbon number 5 to form 5,15- Eicosapentaenoic acid is formed. The 5,15-lipoxygenase of the present invention is a lipoxygenase derived from Archangium violaceum , to date, a dual position-specific lipoxygenase has been found in eukaryotic organisms such as rabbits and soybeans. However, a dual site-specific lipoxygenase derived from bacteria has not been found.

In one embodiment of the present invention, the lipoxygenase derived from the Archangium violaceum is not only composed of the amino acid sequence of SEQ ID NO: 1. Functional equivalents thereof, ie, mutant enzymes capable of achieving the desired effect of the present invention by one or more substitutions, deletions, additions, etc. to the sequence are included in the scope of the present invention. The lipoxygenase derived from the Arcanium biorasium may be a product expressed from a gene consisting of the nucleotide sequence of SEQ ID NO: 2.

In one embodiment of the present invention, the composition is an unsaturated fatty acid having 20 to 22 carbon atoms as a substrate, in particular, an unsaturated fatty acid having at least one cis, cis-1,4 pentadiene and having 20 to 22 carbon atoms, preferably It may be intended to treat a substrate containing eicosapentaenoic acid (EPA).

In another aspect, the present invention provides a method for producing 5,15-dihydroxyeicosapentaenoic acid by bioconversion by treating a dual position-specific enzyme on a substrate.

In one embodiment of the present invention, the dual site-specific enzyme may be a lipoxygenase derived from Archangium violaceum , which consists of the amino acid sequence of SEQ ID NO: 1, Mutant enzymes capable of achieving the desired effect of the present invention by one or more substitutions, deletions, additions, and the like are all included in the scope of the present invention. The lipoxygenase may be a product expressed from a gene consisting of the nucleotide sequence of SEQ ID NO: 2.

In one embodiment of the present invention, the method is an unsaturated fatty acid having 20 to 22 carbon atoms as a substrate, in particular, an unsaturated fatty acid having at least one cis, cis-1,4 pentadiene and having 20 to 22 carbon atoms, preferably Eicosapentaenoic acid (EPA) may be used, and the substrate is preferably used in a concentration of 1 mM to 5 mM in terms of improving the concentration of dioxide, but is not limited thereto.

In one embodiment of the present invention, the method may further improve the production yield of dioxide by further treating cysteine as a reducing agent, thereby reducing epoxide to dioxide. In one embodiment of the present invention, when using eicosapentaenoic acid as a substrate, treatment with the cysteine at a concentration of 100 mM to 300 mM improves the production concentration of 5,15-eicosapentaenoic acid. Preferably, but not limited to.

In addition, it is preferable that the method is carried out in a pH range of 7.5 to 9.5, and an EPPS buffer solution may be used as a reaction solvent to maintain the pH condition.

In addition, the enzymatic reaction is preferable in terms of improving the production concentration of 5,15-eicosapentaenoic acid to be performed at a temperature of 15°C to 35°C, but is not limited thereto.

In another aspect, the present invention provides a recombinant vector comprising a gene encoding a lipoxygenase from Archangium violaceum .

In the present invention, it is preferable that the recombinant vector uses an expression vector for efficient expression of a gene encoding 5,15-lipoxygenase derived from the Arcanium biorasium. In the present invention, "expression vector" is a recombinant vector capable of expressing a target peptide in a suitable host cell, and refers to a gene construct containing essential regulatory elements operatively linked to express a gene insert. The expression vector of the present invention is an element generally contained in a suitable expression vector, and includes expression control elements such as a promoter, an operator, and an initiation codon. The initiation codon and termination codon are generally considered to be part of the nucleotide sequence encoding the polypeptide, and must be in action and in frame with the coding sequence when the gene construct is administered. The promoter of the vector can be constitutive or inducible.

In addition, it may include a signal sequence for the release of the fusion polypeptide in order to promote the separation of the protein from the cell culture. Specific initiation signals may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG initiation codon and adjacent sequences. In some cases, an exogenous translational control signal should be provided that may include an ATG initiation codon. These exogenous translational control signals and initiation codons can be a variety of natural and synthetic sources. Expression efficiency can be increased by the introduction of suitable transcription or translation enhancing factors.

As the expression vector, any conventional expression vector can be used. For example, plasmid DNA, phage DNA, and the like can be used. Specific examples of plasmid DNA include commercial plasmids such as pUC18 and pIDTSAMRT-AMP. Other examples of plasmids that can be used in the present invention include E. coli-derived plasmids (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis-derived plasmids (pUB110 and pTP5) and yeast-derived plasmids (YEp13, YEp24 and YCp50). Specific examples of phage DNA include λ-phages (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11 and λZAP). In addition, animal viruses such as retrovirus, adenovirus or vaccinia virus, and insect viruses such as baculovirus can also be used. Since the expression vector and expression of the protein are different depending on the host cell, such an expression vector may be used by selecting the host cell that is most suitable for the purpose.

In the present invention, the gene encoding the 5,15-lipoxygenase was introduced into the plasmid vector pET 28(+) to produce a recombinant expression vector.

In another aspect, the present invention provides a transformant transformed with a recombinant vector comprising the 5,15-lipoxygenase gene.

The transformant of the present invention is obtained by introducing the recombinant vector of the present invention into a suitable host. Types of host include Escherichia genus, Pseudomonas genus, Ralstonia genus, Alcaligenes genus, Comamonas genus, Burkholderia genus, Agrobacterium, Flavoacterium, Vibrio, Enterobacter, Rhizobium, Gluconobacter, Acinetobacter ), Moraxella, Nitrosomonas, Aeromonas, Paracoccus, Bacillus, Clostridium, Lactobacillus (Lactobacillus), Corynebacterium, Arthrobacter, Achromobacter, Micrococcus, Mycobacterium, Streptococcus ) Genus, Streptomyces genus, Actinomyces genus, Nocardia genus, Methylobacterium genus and other bacteria. In addition, yeasts such as the genus Saccharomyces and the genus Candida, as well as various fungi, may be mentioned as hosts.

For example, when a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention is capable of autonomously replicating itself in a host, and at the same time, a DNA containing a promoter, a 5,15-lipoxygenase gene, and It is desirable to have a configuration necessary for expression such as transcription termination sequence.

As a method for introducing recombinant DNA into bacteria, the calcium chloride method or the electroporation method (Method Enzymol., 194, 182-187 (1990)), the spheroplast method (Proc. Natl. Acad. Sci. USA, 84, 1929) -1933 (1978)), lithium acetate method (J.Bacteriol., 153, 163-168 (1983)) and the like can be used.

In addition, the recombinant vector contains a fragment for expression suppression with various functions for suppressing or amplifying or inducing expression, a marker for selection of transformants, a gene resistant to antibiotics, or secretion from outside the cell. It is also possible to have another gene encoding a signal for the purpose.

The production of 5,15-lipoxygenase according to the present invention is, for example, by culturing a transformant obtained by transforming a host by a recombinant vector having a gene encoding it, and in a culture (cultured cell or culture supernatant). This is done by generating and accumulating 5,15-lipoxygenase, a gene product, and obtaining an enzyme from the culture.

The method for culturing the transformant of the present invention may be a conventional method used for culturing a host.

In addition, as the culture method, any method used for culturing ordinary microorganisms, such as a batch type, a flow batch type, a continuous culture, and a reactor type, can be used. As a medium for transformants obtained by using bacteria such as E. coli as a host, a complete medium or a synthetic medium, for example, LB medium, M9 medium, etc. may be mentioned. In addition, the culture temperature can be accumulated and recovered in 5,15-lipoxygenase by culturing in the above-mentioned range of red temperature.

In the present invention, the transformant was produced by transforming the plasmid vector pET 28(+) into which the 5,15-lipoxygenase gene of the present invention was introduced into E. coli ER 2566 strain.

In another aspect, the present invention provides a recombinant vector comprising (1) lipoxygenase and a gene; (2) culturing the microorganism transformed with the recombinant vector; (3) Inducing the expression of a lipoxygenase gene, and (4) providing a method for producing the expressed recombinant protein and whole cells containing the same.

The whole cell containing the lipoxygenase is i) centrifuging the culture medium of the microorganism to recover the primary whole cell; ii) washing the recovered whole cells with a saline solution; iii) a second centrifugation of the washed whole cells to remove the supernatant and obtaining whole cells; And iv) washing the whole cells recovered with the second time with physiological saline. Specifically, the recovery of all cells in step i) may be performed in a range of about 13,000 g using a device known in the art such as a centrifuge, and the washing of all cells in step ii) with a sodium chloride solution of 0.9% or less It is suitable to perform.

In another aspect, the present invention provides a method for producing 5,15-eicosapentaenoic acid using whole cells containing lipoxygenase.

It is preferable to induce the expression of the recombinant enzyme gene by culturing E. coli transformed with the recombinant vector containing the lipoxygenase gene, and then recover and use the strain containing the expressed enzyme protein.

In addition, it is preferable to use a fatty acid as a substrate, and more preferably, it is preferable to use eicosapentaenoic acid. In addition, the concentration of the substrate is preferably a concentration of 1 mM to 5 mM, and more preferably around 3 mM.

In addition, the enzymatic reaction is preferably performed in the range of pH 7.5 to pH 9.5, more preferably in the range of about pH 8.5. In order to maintain these pH conditions, an EPPS buffer solution can be used as a reaction solvent.

In addition, the enzymatic reaction is preferably carried out in a temperature range of 15°C to 35°C, more preferably in a temperature range of around 20°C. By maintaining these conditions, it is possible to improve the activity of producing 5,15-dihydroxydiacid.

In addition, the enzymatic reaction is preferably performed at a cysteine concentration of 100 mM to 300 mM, and more preferably, the cysteine concentration is preferably at a concentration range of about 250 mM.

In addition, the time of the whole cell reaction can be appropriately adjusted according to a conventional method.

In another embodiment of the present invention, the method is preferred to treat the concentration of cells with lipoxygenase, an enzyme that produces dihydroxylated fatty acids from unsaturated fatty acids, in the range of 0.1 g/L to 1 g/L, but is not limited thereto. It is not.

In another aspect, the present invention provides a lipoxygenase gene consisting of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

In the present invention, the nucleotide sequence may be any nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, preferably may be made of the nucleotide sequence of SEQ ID NO: 2. The gene is derived from Archangium violaceum .

In another aspect, the present invention provides a lipoxygenase consisting of the amino acid sequence of SEQ ID NO: 1. The full-length cDNA of the lipoxygenase encodes a polypeptide of 681 amino acids.

The lipoxygenase consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention includes a variant, and the variant means a protein having a sequence having 90% or more homology to the lipoxygenase consisting of the amino acid sequence of SEQ ID NO: 1 do.

The 5,15-lipoxygenase derived from the Archangium violaceum of the present invention can produce 5,15-eicosapentaenoic acid in high yield without generating by-products using unsaturated fatty acid as a substrate. . Referring to the following examples, the 5,15-lipoxygenase of the present invention produced 1.45 mM 5,15-eicosapentaenoic acid with 3 mM eicosapentaenoic acid as a substrate and a conversion yield of 48.3% per hour. Has been shown. This compares the enzyme activity with the known lipoxygenase from Burkholderia thailandensis , while the activity of the lipoxygenase derived from Burkholderia thailandensis is 22.3 umol/min/mg, whereas the present invention It was confirmed that the activity of the lipoxygenase derived from Arcanium biorasium was about 2 times higher at 46.6 umol/min/mg.

As described above, according to the present invention, by using the Arcanium violaceum ( Archangium violaceum )-derived lipoxygenase, using 5,15-dihydroeicosapentaenoic acid with high productivity and existing heavy metals and organic It is expected to be useful in various industries such as medicine, food, and cosmetics, because it can be produced in a higher yield without producing an environmentally friendly method and no harmless by-products than a material obtained by chemical synthesis using a solvent.

The 5,15-dihydroeicosapentaenoic acid, prepared according to the present invention, is a signal transduction agent and is expected to be involved in various physiologically active functions in animals including humans.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Construction of recombinant expression vectors and transforming microorganisms for expression of 5,15-lipoxygenase

Arcanium violaceum ( Archangium violaceum ) derived from the present invention to prepare a lipoxygenase, A gene encoding lipoxygenase was first isolated from the gene of the Archangium violaceum strain, and a recombinant expression vector was constructed to overexpress it.

Specifically, the gene base sequence and the amino acid sequence are already specified in the German Center for Biological Resources (Germany). Archangium violaceum DSM 52838 was purchased and screened, and Arcanium biora was used to perform polymerase chain reaction (PCR) based on the DNA sequence of lipoxygenase derived therefrom. The genomic DNA of Ariumium violaceum was extracted and used as a template for PCR, and primers based on the DNA sequence of lipoxygenase were devised to perform polymerase chain reaction (PCR). . NdeI and SalI were used as restriction enzymes for each gene, and the cloning method was a Gibson assembly method, and the primers were as follows. The primer sequence of the dual position-specific 5, 15-lipoxygenase from Archangium violaceum is F: AGC GGC CTG GTG CCG CGC GGC AGC CAT ATG ATG CGT TCC ATT CCC TCC CTG CCC CAG AAT ( SEQ ID NO: 3), R: ATT CTG GGG CAG GGA GGG AAT GGA ACG CAT CAT ATG GCT GCC GCG CGG CAC CAG GCC GCT (SEQ ID NO: 4). In addition, to clone each gene into pET 28a, a vector was prepared using PCR, and then ligated using an isothermal buffer to produce recombinant 5, 15-lipoxygenase.

Subsequently, the recombinant expression vector obtained as described above was transformed into E. coli ER 2566 strain purchased from New England Biolabs (Hertfordshire, UK) by a conventional transformation method, and the transformed microorganisms were treated with 20% glycerin solution. After addition, the cells were cultured for the production of 5,15-dihydroxydiacid and stored frozen at -80°C before use.

Expression of 5,15-lipoxygenase

For protein expression of enzymes, the transformed microorganisms stored frozen in Example 1 were aspirated at 200 rev/min in a flask with 500 ml of LB (Difco, Sparks, MD, USA) medium and 20 μg/ml of kanamycin. Cultured at 37 °C under conditions. When the absorbance of bacteria reached 0.6 to 0.8 at 600 nm, a final concentration of 0.1 mM IPTG was added to induce protein expression of enzymes, and the culture was incubated with stirring at 150 rev/min at 16° C. for 16 hours. .

E. coli cells containing cultured 5, 15-lipoxygenase were collected and used. In addition, the lipoxygenase produced by over-expressing as described above was washed twice with 0.85% sodium chloride (NaCl) by centrifuging the culture medium of the transformed strain at 6,000xg for 30 minutes at 4°C, followed by washing with 5,15- It was used as a recombinant cell to produce dihydroxy fatty acids.

Construction of a synthetic route for dioxide from animal fatty acids using 5,15-lipoxygenase

Eicosapentaenoic acid was used as a substrate in order to construct a synthetic route of 5,15-dihydroxydiethylacosapentaenoic acid using the 5,15-lipoxygenase. As for 5,15-dihydroeicosapentaenoic acid, 0.5 g/L of whole cells containing lipoxygenase was used for 3 mM eicosapentaenoic acid, and cysteine concentration was 250 mM, pH 8.5, and 30 minutes at 20°C. During the whole cell reaction was performed.

As a result, a synthetic route as shown in FIG. 1 was constructed. In addition, the generated 5,15-dioxide fatty acid was confirmed by HPLC (FIG. 2), and 5,15-dioxide fatty acid was confirmed through mass identification (FIG. 3).

Synthesis of Dioxide using 5,15-lipoxygenase and cysteine

The amount of dioxide produced by the 5,15-lipoxygenase enzyme in a single reaction was confirmed in the reactant to which cysteine was added by the recombinant E. coli expressing the 5,15-lipoxygenase. As a substrate, 250 mM cysteine was added to the initial reaction of 3 mM eicosapentaenoic acid, and the whole cell was reacted at pH 8.5 for 20 minutes at a temperature of 5°C. The production concentration of ensan over time was measured.

As a result, as shown in FIG. 4, 5,15-dihydroxyhydric acid eicosapentaenoic acid of about 1.45 mM as a substrate using 3 mM eicosapentaenoic acid as a substrate by a single reaction of 5,15-lipoxygenase. Was produced, and the conversion yield of 5,15-eicosapentaenoic acid per hour was 48.3%.

Confirmation of optimal conditions for bioconversion activity using 5,15-lipoxygenase

5-1. Confirmation of bioconversion activity according to changes in cysteine concentration

Confirming the effect of cysteine concentration change on 5,15-dihydroeicosapentaenoic acid production activity when all cells containing Arcangium biolasium-derived lipoxygenase are treated with eicosapentaenoic acid To this end, cysteine was not added to 3 mM eicosapentaenoic acid, or it was added at the beginning of the reaction by varying the concentration in the range of 100 to 300 mM, and the reaction was performed at pH 8.5 and 20°C for 30 minutes.

As a result, as shown in FIG. 5, it was confirmed that when cysteine was added, 5,15-eicosapentaenoic acid production activity was higher than when cysteine was not added. This is due to the fact that a large amount of hydroxyeicosapentaenoic acid, a primary product, is generated by the addition of cysteine, and thus passes through a 5,15-dihydroeicosapentaenoic acid pathway, rather than the previously known hepoxylin pathway. On the other hand, it was confirmed that the optimal addition concentration of cysteine is 150 to 300 mM, preferably 250 mM.

5-2. Confirmation of bioconversion activity according to changes in the concentration of eicosapentaenoic acid

When all the cells containing lipoxygenase derived from Arcanium biorasium were treated with eicosapentaenoic acid, the concentration change of eicosapentaenoic acid was 5,15-dihydrodihydroeicosapentaenoic acid. In order to confirm the effect, eicosapentaenoic acid and 250 mM cysteine having different concentrations in the range of 1 to 5 mM were added at the beginning of the reaction, and the reaction was performed at pH 8.5 and 20°C for 30 minutes.

As a result, as shown in Figure 6, it was confirmed that the optimal substrate concentration is 2 to 5 mM, preferably 3 mM.

5-3. Confirmation of bioconversion activity according to changes in the concentration of lipoxygenase

When treating all the cells containing the lipoxygenase derived from Arcanium biorasium with eicosapentaenoic acid, it was confirmed that the effect of the enzyme concentration change on the production concentration of 5,15-dihydro-eicosapentaenoic acid In order to do this, 0.1 to 0.9 g/L of whole cells and 250 mM of cysteine were added to the beginning of the reaction to 3 mM eicosapentaenoic acid, and the reaction was performed at pH 8.5 and 20° C. for 30 minutes. .

As a result, as shown in [Fig. 7], it was confirmed that the optimum concentration of all cells containing lipoxygenase was 0.1 to 0.7 g/L, preferably 0.5 g/L.

As described above, the present invention uses 5,15-lipoxygenase derived from Archangium violaceum to act as a lipid mediator in animals including humans. It relates to a method for producing a fatty acid using a bioconversion method. Specifically, the substrate is specifically 5 using a biotransformation method using transformed cells containing 5,15-lipoxygenase derived from Archangium violaceum and using eicosapentaenoic acid. Since it can produce ,15-dihydrated fatty acids, it not only overcomes the problems of the conventional chemical method with environmentally friendly conditions, but also the production of lipid regulators not previously reported and specific production by bioconversion methods have great significance. .

As described above, since a specific part of the contents of the present invention has been described in detail, for those skilled in the fatty acid industry and the medical industry, this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. The point will be clear. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University Industrial Cooperation Corp <120> PRODUCTION OF 5,15-DIHYDROXY EICOSAPENTAENOIC ACID BY NEW BACTERIAL LIPOXYGENASE <130> 1065007 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 681 <212> PRT <213> Artificial Sequence <220> <223> Archangium violaceum <400> 1 Met Arg Ser Ile Pro Ser Leu Pro Gln Asn Asp Ser Pro Ala Gln Gln 1 5 10 15 Glu Leu Arg Leu Ala Thr Leu Thr Gln Glu Arg Gln Val Tyr Ala Tyr 20 25 30 Asn Phe Asp Ser Arg Leu Ser Pro Leu Gly Ile Ala Ala Gln Val Pro 35 40 45 Arg Gln Asp Asn Phe Ser Phe Val Trp Leu Asp Gly Val Ala Thr Thr 50 55 60 Gly Leu Gln Leu Leu Gly Asn Val Leu Ala Ile Gly Ala Lys Leu Phe 65 70 75 80 Asp Asn Gly Glu Ala Ile Gln Asn Asp Ala Gly Leu Ser Glu Leu Glu 85 90 95 Leu Arg Arg Val Glu Gly Thr Tyr Arg Glu Leu Thr Asp Gly Phe Ser 100 105 110 Arg Leu Ser Asp Arg Thr Ser Arg Leu His Gln Ala Pro Arg Ser Pro 115 120 125 Ala Val Thr Leu Ile Ala Asp Val Ile Glu Arg Pro Arg Thr Leu Leu 130 135 140 Lys Ala Ala Gly Gln Lys Leu Glu Glu Thr Ala Gly Val Ala Ile Val 145 150 155 160 Ser Glu Leu Ala Thr Gln Ala Ala Leu Asp Ala Pro Ala Ile Gly Gln 165 170 175 Asp Ile Leu Asp Leu Ile Asp Thr Leu Leu Lys Lys Leu Leu Ser Glu 180 185 190 Ala Gly Asp Leu Phe Leu Lys Tyr Leu Gly Leu Tyr Gly Lys Ala Ala 195 200 205 Ser Leu Asp Asn Tyr Thr Ala Gln Phe Ser Leu Leu Gln Pro Pro Ser 210 215 220 Val Ala Ser Asn Tyr Glu Thr Asp Leu Ile Phe Ala Arg Met Arg Leu 225 230 235 240 Ala Gly Pro Asn Pro Ala Leu Leu Gln Gly Ile Ala Ala Leu Pro Glu 245 250 255 Lys Phe Pro Val Thr Asp Ala Gln Tyr Gln Ser Val Met Gly Ser Gln 260 265 270 Asp Thr Leu Ala Arg Ala Gly Gln Glu Gly Arg Leu Tyr Leu Leu Asp 275 280 285 Tyr Ala Val Phe Gln Gly Ile Pro Thr Gly Gln Thr Ser Gly Gly Gln 290 295 300 Lys Tyr Ile Glu Ala Pro Leu Ala Leu Phe Ala Val Pro Ala Ala Asn 305 310 315 320 Gln Ala Asp Arg Lys Leu Arg Ala Val Ala Ile Gln Cys Ser Gln Thr 325 330 335 Pro Gly Arg Ser Asn Pro Ile Phe Thr Pro Ala Asp Gly Thr Ser Trp 340 345 350 Ser Leu Ala Arg Leu His Val Gln Val Ala Asp Gly Asn Tyr His Glu 355 360 365 Leu Ile Ala His Leu Gly Leu Thr His Leu Ile Leu Glu Glu Phe Thr 370 375 380 Leu Ser Thr His Arg Gln Leu Ala Pro Gln His Pro Leu Tyr Leu Leu 385 390 395 400 Leu Thr Pro His Phe Gln Gly Thr Leu Ala Ile Asn Asn Ala Ala Glu 405 410 415 Thr Ser Leu Ile Ala Pro Gly Gly Pro Val Asp Gln Leu Leu Ala Gly 420 425 430 Glu Ile Lys Ala Ser Thr Gln Val Ser Ile Gln Ala Val Ala Asn Gln 435 440 445 Ser Ile Asn Gln Ser Phe Leu Pro Arg Ala Leu Ala Ala Arg Gly Val 450 455 460 Asp Asp Ala Ser Lys Leu Pro Asp Tyr Pro Tyr Arg Asp Asp Ala Leu 465 470 475 480 Leu Leu Trp Asn Asp Ile Arg Ala Trp Val Ser Asp Tyr Leu Ala Ile 485 490 495 Tyr Tyr Asn Asp Asp Ala Ala Val Arg Ser Asp Tyr Glu Leu Gln Ala 500 505 510 Trp Val Thr Glu Leu Ala Ser Pro Asp Ala Gly Lys Leu Lys Asp Val 515 520 525 Thr Glu Gly Gly Gly Gly Ile Gln Thr Phe Glu Tyr Leu Val Asp Leu 530 535 540 Thr Thr Tyr Ile Ile Phe Thr Ala Ser Val Gln His Ala Ala Val Asn 545 550 555 560 Phe Pro Gln Arg Thr Val Met Ser Tyr Thr Pro Ala Leu Pro Leu Ala 565 570 575 Ala Tyr Ala Pro Ala Pro Thr Ser Val Glu Glu Leu Pro Pro Ser Thr 580 585 590 Glu Leu Thr His Leu Pro Pro Leu Gln Met Ala Phe Leu Gln Gln Ala 595 600 605 Val Thr Phe Gly Leu Gly Asn Val Tyr Phe Thr Arg Leu Gly Gly Tyr 610 615 620 Asp Thr Tyr Leu Arg Glu Pro Trp Phe Ala Asp Ala Arg Val Trp Pro 625 630 635 640 Ala Leu Glu Val Phe Gln Lys Arg Leu Arg Ala Thr Glu Arg Glu Ile 645 650 655 Gly Gln Arg Asn Leu Ser Arg Ile Pro Tyr Glu Thr Leu Leu Pro Thr 660 665 670 Ala Ile Pro Gln Ser Ile Asn Ile Glx 675 680 <210> 2 <211> 2043 <212> DNA <213> Artificial Sequence <220> <223> Archangium violaceum <400> 2 atgcgttcca ttccctccct gccccagaat gactctcccg cccagcagga gctgcgcctc 60 gccacgctca cacaagagcg gcaggtctac gcctacaact tcgacagccg gctgagtccg 120 ctggggatcg cggcccaggt cccccgtcag gacaacttct ccttcgtctg gttggatggc 180 gtcgccacca ccggcctgca gctgctgggc aacgtgctgg ccatcggcgc gaagctcttc 240 gacaacgggg aggcgatcca gaacgacgcc ggcctgtccg agctggagct ccgcagggtc 300 gaggggacct accgggagct gaccgacgga ttcagccggc tgtcggaccg gacctcccgg 360 ctccaccagg cgccgcgctc cccggcggtg acgctcatcg ccgatgtcat cgagcgcccc 420 cggacgctgc tcaaggcggc gggccagaag ctggaggaga ccgccggtgt cgccatcgtc 480 tccgagctcg ccacccaggc cgcgctggat gcgcccgcca tcgggcagga catcctcgac 540 ctcatcgaca ccctgttgaa gaagctgttg tccgaggccg gagatctgtt cctgaagtac 600 ctggggctgt acggcaaggc ggcgtcgctc gacaactaca cggcccagtt ctcgctgctg 660 cagcctccgt cggtcgccag caattacgag accgacctca tcttcgcgcg catgcggctg 720 gccgggccca acccggcgct gctccagggc atcgcggccc tccccgagaa gttcccggtg 780 acggacgcgc agtaccagtc cgtgatgggc tcgcaggaca ccctggcccg cgcgggccag 840 gaaggtcggc tgtacctgct ggactacgcc gtcttccagg gcatccccac cggacagacg 900 tcgggcggac agaagtacat cgaagcgccg ctcgccctgt tcgccgtgcc cgccgcgaac 960 caggccgacc ggaagctgcg cgcggtggcc atccagtgct cgcagacgcc gggccgctcc 1020 aaccccatct tcaccccggc ggacggcacc tcctggagcc tggcgcgcct gcacgtgcag 1080 gtggcggatg gcaactacca cgagctcatc gcgcacctgg gcctcacgca cctgattctg 1140 gaagagttca ccctgtccac gcaccggcag ctcgcgcccc agcacccgct ctacctgctg 1200 ctgacgccgc acttccaggg caccctggcc atcaacaacg ccgcggagac gtccctcatc 1260 gcgcccggtg gcccggtgga ccagctgctc gccggtgaga tcaaggcctc cacccaggtg 1320 agcatccagg ccgtggccaa ccaatccatc aaccagtcgt tcctgccccg ggcgctggcg 1380 gcccgcgggg tggacgacgc ctcgaagctg ccggactacc cgtaccgcga cgacgcgctg 1440 ctgctctgga acgacatccg cgcctgggtg agcgactacc tggccatcta ctacaacgat 1500 gacgcggcgg tgcggagcga ctacgagctg caggcctggg tcacggagct cgccagcccc 1560 gacgccggca agctgaagga tgtgacggag ggcggagggg gcatccagac cttcgagtat 1620 ctggtggacc tgaccaccta catcatcttc acggcgagcg tgcagcacgc ggcggtcaac 1680 ttcccgcagc gcacggtcat gagctacacc ccggcgctgc cgctggcggc ctacgctccc 1740 gcgcccacct ccgtggaaga gctgccgccg agcacggagc tgacccacct gccgccgttg 1800 cagatggcct tcctgcagca ggcggtcacc ttcgggctgg gcaacgtgta cttcacccgg 1860 ctgggcggct acgacacgta cctgcgcgag ccctggttcg cggacgcgcg cgtgtggccc 1920 gccctggagg tcttccagaa gcgcctgcgc gccacggagc gggagatcgg ccagcgcaac 1980 ctgtcgcgca tcccctacga gacgctgctg cccacggcga ttccgcagag catcaacatc 2040 taa 2043 <210> 3 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 agcggcctgg tgccgcgcgg cagccatatg atgcgttcca ttccctccct gccccagaat 60 60 <210> 4 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 attctggggc agggagggaa tggaacgcat catatggctg ccgcgcggca ccaggccgct 60 60

Claims (10)

5,15-lipoxygenase consisting of the amino acid sequence of SEQ ID NO: 1 from Archangium violaceum ; Or 5,15-dihydroxyeicosapentaenoic acid (5,15-dihydroxyeicosapentaenoic acid) production composition comprising a gene consisting of the nucleotide sequence of SEQ ID NO: 2 as an active ingredient.
delete delete According to claim 1,
The composition is a composition characterized in that it further comprises eicosapentaenoic acid (eicosapentaenoic acid) as a substrate.
5,15- by bioconversion by treating 5,15-lipoxygenase consisting of the amino acid sequence of SEQ ID NO: 1 from Archangium violaceum as a substrate to eicosapentaenoic acid as a substrate Method of producing 5,15-dihydroxyeicosapentaenoic acid.
delete The method of claim 5,
The method is to use eicosapentaenoic acid as a substrate in the range of 1 mM to 5 mM.
The method of claim 5,
The method is characterized in that to add cysteine at a concentration of 100 mM to 300 mM.
The method of claim 5,
The method is that the reaction is carried out in the range of pH 7.5 to 9.5.
The method of claim 5,
The method is that the reaction is carried out in a temperature range of 20 ℃ to 35 ℃.

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