KR101856308B1 - Novel oleate hydratase 2 and uses thereof - Google Patents

Novel oleate hydratase 2 and uses thereof Download PDF

Info

Publication number
KR101856308B1
KR101856308B1 KR1020160025454A KR20160025454A KR101856308B1 KR 101856308 B1 KR101856308 B1 KR 101856308B1 KR 1020160025454 A KR1020160025454 A KR 1020160025454A KR 20160025454 A KR20160025454 A KR 20160025454A KR 101856308 B1 KR101856308 B1 KR 101856308B1
Authority
KR
South Korea
Prior art keywords
acid
oleic acid
hydrolase
hydroxyoctadec
fatty acid
Prior art date
Application number
KR1020160025454A
Other languages
Korean (ko)
Other versions
KR20170104015A (en
Inventor
박진병
오덕근
이정후
강우리
Original Assignee
이화여자대학교 산학협력단
건국대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 이화여자대학교 산학협력단, 건국대학교 산학협력단 filed Critical 이화여자대학교 산학협력단
Priority to KR1020160025454A priority Critical patent/KR101856308B1/en
Priority to PCT/KR2016/011224 priority patent/WO2017119576A1/en
Publication of KR20170104015A publication Critical patent/KR20170104015A/en
Application granted granted Critical
Publication of KR101856308B1 publication Critical patent/KR101856308B1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01053—Oleate hydratase (4.2.1.53)

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The present invention relates to a method for producing hydroxy fatty acids from unsaturated fatty acids in a high yield by using oleophilic acid hydratase 2 derived from Stannotrophomonas maltophilia and recombinant Escherichia coli expressing the same, Hydroxy fatty acids can be produced at a high rate and yield, and the produced hydroxy fatty acids can be used for the production of various fragrances, and also for the synthesis of polymers and the production of emulsifiers.

Description

Oleic acid hydratase 2 and its use {Novel oleate hydratase 2 and uses thereof}

The present invention relates to the use of Stenotrophomonas The present invention relates to a method for producing hydroxy fatty acids from unsaturated fatty acids in a high yield by using maltophilia- derived oleic acid hydrolase 2 and recombinant Escherichia coli expressing the same. More specifically, the present invention relates to a method for producing oleic acid hydrolase, A polynucleotide containing the polynucleotide, an expression vector containing the polynucleotide, a transformant into which the expression vector has been introduced, a method for producing the oleosin hydrolase 2 using the transformant, and a method for producing the oleosan hydrolase 2 or the transformant To a method for producing a hydroxy fatty acid from an unsaturated fatty acid.

Hydroxy fatty acids (hydroxy fatty acid) are found in nature, such as triglycerol, wax, cervicoside, and other lipids present in plants, insects and microorganisms. Hydroxylated fatty acid is an important industrial substance as a precursor of lactone which constitutes spice. It is used as a starting material of resin, wax, nylon, plastic, lubricant and coating material because it has better reactivity than fatty acid other than hydroxylated fatty acid. . Particularly, 10-hydroxystearic acid, which is one of the hydroxycarboxylic acids, is used as a lubricant and is known to be an important material for producing sebacic acid and azelic acid used for synthesizing plasticizers and resins (Biotechnology Advances 31, 2013, 1473-1485).

Hydroxylated fatty acids that can be used in various fields as described above can be produced through biological conversion using microorganisms. For example, a process for producing 10-hydroxystearic acid using oleic acid hydrolase and its enzyme-containing recombinant microorganism Patent Application No. 10-2011-0061911), a process for producing 10,12-dihydrous stearic acid from ricinoleic acid by fatty acid hydrolase (Korean Patent Application No. 10-2012-0108758), immobilization (Korean Patent Application No. 10-2014-0054686), production of 13-hydroxy fatty acid by 13-linolenic acid hydrolase derived from Lactobacillus acidophilus, A method for producing delta-decaractone using the ripper, and a composition thereof (Korean Patent Application No. 10-2015-0127223).

However, the use of 10-hydroxyoctadec-12-enoic acid, 10-hydroxyoctadec-6,12-dienostearic acid (10-hydroxyoctadec-6, 12-dienoic acid, 10-hydroxystearic acid and the like are not only produced in a low yield but also are inexpensive in mass production. For example, conventional microorganisms that produce the 10-hydroxy fatty acid include Flavobacterium sp . NRRL B-14859 ), Nocardia cholesterolicum , and lactic acid bacteria. However, these microorganisms not only produce the above-mentioned 10-hydroxy fatty acid in a low yield, but also produce a by-product such as 10-kedo stearic acid. In recent years, stenotrophomonas maltophilia , Macrococcus caseolyticus and oleate hydratases derived from Lysinibacillus fusiformis can be used to produce 10-hydroxystearic acid at a high concentration, but hydroxyoctadec-12-enoic acid The production yield of 10-hydroxyoctadec-6,12-dienostric acid is known to be very low.

Under these circumstances, efforts have been made to develop a method for producing hydroxy fatty acids from unsaturated fatty acids at a high yield. As a result, it has been found that a novel oleic acid hydration, which exhibits more excellent activity than the conventional oleic acid hydrolase from Stentonfomonas maltophila strain, The present inventors completed the present invention by confirming that the enzyme 2 was excavated and the hydroxy fatty acid could be produced at a high yield and rate when the digested enzyme was used.

One object of the present invention is to provide oleic acid hydrolase 2 consisting of the amino acid sequence of SEQ ID NO: 2.

It is another object of the present invention to provide a polynucleotide comprising a nucleotide sequence encoding oleic acid hydrolase 2.

It is still another object of the present invention to provide an expression vector comprising the polynucleotide.

It is still another object of the present invention to provide a transformant into which the above expression vector has been introduced.

It is still another object of the present invention to provide a method for producing oleic acid hydrolase 2 using the transformant.

It is still another object of the present invention to provide a method for producing a hydroxy fatty acid from unsaturated fatty acid using the oleosin hydrolase 2 or the transformant.

In carrying out various studies to develop a method for producing hydroxy fatty acid with high yield by using oleate hydratase 2, the inventors of the present invention have found that when Stenotrophomonas maltophilia ), which is a novel oleic acid hydrolase, was identified. Using the above-mentioned oleosin hydrolase 2 or the transformed recombinant Escherichia coli expressing it, 10-hydroxyoctadec-12-eno Hydroxyoctadec-12-enoic acid, 10-hydroxyoctadec-6,12-dienoic acid, 10-hydroxyoctadec-12,15 10-hydroxy fatty acids such as 10-hydroxyoctadec-12,15-dienoic acid, 10-hydroxypalmitic acid or 10-hydroxystearic acid, Or 9-hydroxynonanoic acid was obtained at a high yield and at a high yield. The oleic acid hydrolase 2 is novel and has a remarkably high reaction activity and production rate as compared with conventionally used oleate hydrolyzing enzyme derived from Stenotrophomonas maltophilia. Therefore, the present invention is significant. The oleic acid hydrolase 2 and the method for producing the hydroxy fatty acid from the unsaturated fatty acid at a high rate and in a high yield have not been known at all and have been developed for the first time by the present inventors.

In order to achieve the above object, the present invention provides oleic acid hydrolase 2 composed of the amino acid sequence of SEQ ID NO: 2 as one embodiment.

The term " oleosin hydrolase " in this case is known as an enzyme which catalyzes the conversion of oleic acid used as a substrate to 10-hydroxystearic acid as one of the fatty acid hydrolases. The oleic acid hydrolase can produce various hydroxy fatty acids by using various unsaturated fatty acids including oleic acid as a substrate. In the present invention, a novel oleic acid hydrolase 2 distinguished from the conventional oleic acid hydrolase was identified.

In the present invention, the oleic acid hydrolase 2 may be derived from Stenotrophomonas maltophilia strain. Specifically, the oleic acid hydratase 2 may include an amino acid sequence of SEQ ID NO: 2, Hydroxyoctadec-12,15-dienoic acid, 10-hydroxypalmitolic acid or 10-hydroxyoctadec-12-enoic acid, 10-hydroxyoctadec-6,12- Hydroxystearic acid and the like, or a novel oleic acid hydrolase capable of producing 9-hydroxynonanoic acid. As long as it can exhibit the enzyme activity that mass-produced the hydroxy fatty acid , And all peptides to which various amino acid sequences have been added at the N-terminal or C-terminal of the amino acid sequence of SEQ ID NO: 2. In addition, it may further comprise a targeting sequence, a tag, an amino acid sequence designed for specific purposes to increase the stability of the labeled residue, half-life or peptide.

The oleic acid hydrolase 2 provided in the present invention is similar to the conventional oleic acid hydrolase in that it produces a hydroxy fatty acid from an unsaturated fatty acid, but the 10-hydroxyoctadec-12-enoic acid, 10-hydroxyocta Hydroxyoctadec-12,15-dienoic acid, 10-hydroxypalmitoleic acid or 10-hydroxystearic acid, such as 10-hydroxyoctadec-12,15-dienoic acid, Which is distinguished from the conventional oleic acid hydrolase.

According to the present inventors' findings, the oleic acid hydrolase 2 provided by the present invention is obtained from oleic acid by 10-hydroxystearic acid, palmitoleic acid by 10-hydroxypalmitoleic acid, linoleic acid hydroxyoctadec-12,15-dienostearic acid, gamma-linolenic acid) from 10-hydroxyoctadec-12-enoic acid, alpha-linolenic acid, In the production of 10-hydroxyoctadec 6.12-dienostearic acid, the reaction activity and the production rate of oleic acid hydrazide 2 in the production of oleic acid, palmitoleic acid, linoleic acid, alpha-linolenic acid or gamma- (Table 1), which was 45%, 10%, 80%, 90% and 130% higher than the hydration enzyme.

In addition, it was confirmed that the production rate of 10-hydroxystearic acid using oleic acid as a substrate was 20% higher than that of the conventional oleic acid hydrolase (Fig. 4) using E. coli containing oleic acid hydrolase or oleic acid hydrolase It was confirmed that the production rate of 10-hydroxyoctadec-12-enoic acid as a substrate was 5 times higher than that of the conventional oleosinase (Fig. 5), and that linoleic acid, which is a fatty acid decomposition product derived from soybean oil, It was confirmed that the production rate of 10-hydroxyoctadec-12-enoic acid was four times higher than that of the conventional oleic acid hydrolase. The oleic acid hydrolase 2 having such characteristics has not been known from any strains so far and has been discovered for the first time by the present inventors.

It has been reported that the oleic acid hydrolase derived from Stenotrophomonas maltophilia strain has a relatively excellent enzyme activity as compared with oleic acid hydrolase derived from other strains (J Biotechnol., 158: 17-23 ), The oleic acid hydrolase 2 of the present invention exhibits relatively excellent activity as compared with the conventional oleic acid hydrolase derived from Stenton phomonas maltophilia strains. Therefore, the oleic acid hydrolase 2 of the present invention can be produced by the conventional method Which is relatively higher than that of oleic acid hydrolase.

In another aspect, the present invention provides a polynucleotide comprising a nucleotide sequence encoding the oleosin hydrolase 2.

 In the present invention, the nucleotide sequence constituting the polynucleotide may be a nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 2 or a variety of amino acids which may be added at the N- or C-terminus of the amino acid sequence of SEQ ID NO: The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 may be added to the 5'-terminal or 3'-terminal of the nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 2, The nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence that can be added to the N-terminus or C-terminus of the amino acid sequence of SEQ ID NO: 2 is the 5'-terminal of the nucleotide sequence of SEQ ID NO: 1 Or added at the 3'-end. Specifically, it may be composed of the nucleotide sequence of SEQ ID NO: 1 encoding the amino acid sequence of SEQ ID NO: 2.

In addition, as long as it is capable of encoding a protein capable of expressing oleic acid hydrolase 2, a polynucleotide comprising a nucleotide sequence homologous to the above nucleotide sequence may be included in the category of the polynucleotide provided in the present invention. Specifically, , 90% or more homology, and more specifically, a polynucleotide including a nucleotide sequence having 95% or more homology.

In addition, the polynucleotide sequence encoding a protein capable of representing oleic acid hydrolase 2 may have the nucleotide sequence of SEQ ID NO: 1, or may be constituted or constituted essentially. The expression " having, consisting essentially of, or consisting of ", as used herein, does not exclude the addition of a meaningless sequence to or from the nucleotide sequence of SEQ ID NO: 1 or a mutation that may occur naturally, or a silent mutation thereof, In the case of "having, essentially consisting of, or consisting of the nucleotide sequence of SEQ ID NO: 1" in the present invention as long as it has the same or corresponding activity with the protein encoded by the nucleotide sequence of SEQ ID NO: . ≪ / RTI >

On the other hand, the polynucleotide may be mutated by substitution, deletion, insertion, or a combination of one or more bases. When the nucleotide sequence is prepared by chemically synthesizing, it is possible to use a method well known in the art, for example, a method described in Engels and Uhlmann, Angew Chem IntEd Engl., 37: 73-127, 1988 , Triesters, phosphites, phosphoramidites and H-phosphate methods, PCR and other auto primer methods, and oligonucleotide synthesis on solid supports.

In another aspect, the present invention provides an expression vector comprising the polynucleotide.

The term "expression vector" as used herein refers to a gene construct comprising a gene insert that is capable of expressing a target protein in an appropriate host cell, and an essential regulatory element operably linked to the expression of the gene insert. The expression vector includes expression control elements such as an initiation codon, a termination codon, a promoter, an operator, etc. The initiation codon and termination codon are generally regarded as part of the nucleotide sequence encoding the polypeptide, and when the gene product is administered, And must be in coding sequence and in frame. The promoter of the vector may be constitutive or inducible.

The term " operably linked " means a state in which a nucleic acid sequence encoding a desired protein or RNA and a nucleic acid expression control sequence are functionally linked to perform a general function. For example, a nucleic acid sequence encoding a promoter and a protein or RNA may be operatively linked to affect the expression of the coding sequence. The operative linkage with an expression vector can be produced using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art.

In addition, the expression vector may include a signal sequence for the release of oleosin hydrolase 2 to facilitate the separation of the protein from the cell culture broth. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translational control signal, which may include the ATG start codon, should be provided. These exogenous translational control signals and initiation codons can be of various natural and synthetic sources. Expression efficiency can be increased by the introduction of suitable transcription or translation enhancers. In addition, the expression vector may further comprise a protein tag which can be optionally removed using endopeptidase, in order to facilitate the detection of oleic acid hydrolase 2.

In the present invention, the expression vector is not particularly limited as long as it can express the polynucleotide comprising the oleic acid hydrolase 2 gene to produce oleic acid hydrolase 2 provided in the present invention. In mammalian cells (for example, Cloning and / or expression of the polynucleotide in eukaryotic or prokaryotic cells, including mammalian cells, such as yeast cells, yeast cells, insect cells or bacterial cells (e. G., E. coli, etc.)), monkeys, rabbits, rats, hamsters, mouse cells, And may be a vector comprising at least one selectable marker operatively linked to a suitable promoter so that the polynucleotide can specifically be expressed in a host cell, The plasmids (pUC18, pBAD, pIDTSAMRT-AMP, pET 28 (+) a and the like), the plasmids derived from Escherichia coli (pYG601BR322, derived plasmids (pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24 and YCp50), lambda-phage (Charon4A, Charon21A, EMBL3, EMBL4, lambda gt10, pUC115, pUC118 and pUC119), Bacillus subtilis-derived plasmids ,? gt11 and? ZAP), retrovirus, adenovirus, vaccinia virus, baculovirus, and the like. Most specifically a pET 28 (+) a plasmid vector. Since the amount of expression of the protein and the expression of the expression vector are different depending on the host cell, it is preferable to select and use the host cell most suitable for the purpose.

The host cell to which the expression vector provided in the present invention can be introduced is not particularly limited as long as it can produce the peptide by expressing the polynucleotide, but bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium and the like; Yeast cells such as Saccharomyces cerevisiae, and ski-inspected caromyces pombe; Fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, Bowmanella cells; Or plant cells.

In yet another embodiment, the present invention provides a transformant expressing the above expression vector.

The term " transformant " in the present invention means a cell or microorganism that has been introduced into the interior of a host cell by using an expression vector and then mutated to express the target protein, wherein the polynucleotide encoding the target protein is expressed. Specifically, the transformant may be a recombinant Escherichia coli expressing oleic acid hydrolase 2. More specifically, it may be Escherichia coli ER 2566 pET28 (+) a-OhyA2 or Escherichia coli BL21 (DE3) pET28 (+) a-OhyA2.

The transformed is one which can produce two oleic hydration enzyme of the present invention showing the effect which can be can be carried out by various methods, increase the variety of cellular activity at a high level, is not particularly limited to, CaCl 2 precipitation method , CaCl 2 The Hanahan method, the electroporation method, the calcium phosphate precipitation method, the protoplast fusion method, the agitation method using the silicon carbide fiber, and the Agrobacterium-mediated transformation method are used for the precipitation method by using a reducing material called DMSO (dimethyl sulfoxide) A transformation method using PEG, dextran sulfate, lipofectamine, and a dry / suppression-mediated transformation method.

According to another aspect of the present invention, there is provided a method for producing oleic acid hydrolase 2 of the present invention using the above transformant.

Specifically, the method for producing oleic acid hydrolase 2 of the present invention comprises the steps of: (a) culturing the transformant to obtain a culture; And (b) recovering the oleoresinase 2 of the present invention from the culture.

In the present invention, the term " culture product " is a substance obtained as a result of culture of a strain, and may include a medium, a strain to be cultured, and a substance secreted from the cultured microorganism. Such as inorganic nutrients, amino acids, vitamins, nucleic acids and / or other ingredients which may generally be contained in the culture medium, in addition to the nutrient sources necessary for culturing the cells, for example, carbon sources, nitrogen sources and the like.

According to another aspect of the present invention, there is provided a method for producing a hydroxy fatty acid comprising the step of treating an oleic acid hydratase 2 or a transformant expressing it with an unsaturated fatty acid to hydrate.

In the present invention, the term " hydration reaction " means a reaction in which a hydroxy group and a hydrogen ion intercalate into a carbon-carbon double bond. In the present invention, the hydration reaction may be caused by oleic acid hydrolase 2 or a transformant expressing it.

The hydration reaction of the oleosin hydratase 2 may be performed under acidic conditions of pH 5.0 to pH 6.8. Specifically, the hydration reaction may be performed at a pH of from 5.5 to pH 6.5, more specifically, from pH 5.8 to pH 6.2. The inventors of the present invention have confirmed that about 80% or more of oleic acid hydrolase 2 is reacted when a hydration reaction is carried out under pH conditions of pH 5.0 to pH 6.8 (FIG. 1A). On the other hand, there is a problem that the reaction activity of oleic acid hydrolase 2 decreases as the pH decreases below pH 5.0 or exceeds pH 6.8.

The hydration reaction of oleic acid hydrolase 2 may be performed at a temperature of 30 ° C to 45 ° C. Specifically, it may be performed at a temperature condition of 32 ° C to 42 ° C, more specifically, be performed at a temperature condition of 32 ° C to 38 ° C. The present inventors have confirmed the effect of exhibiting a reaction activity of oleic acid hydrolase 2 of about 80% or more when the hydration reaction is carried out at a temperature of 30 ° C to 45 ° C (FIG. 1b). On the other hand, there is a problem that the reaction activity of oleic acid hydrolase 2 decreases as the temperature decreases below 30 ° C or exceeds 45 ° C.

When the hydration reaction is carried out, methanol, ethanol, 2-propanol, dimethyl sulfoxide (DMSO), butanol, acetone or toluene may be added to the reaction product.

The dimethylsulfoxide may be carried out by further adding a concentration of 12% (v / v) or less of the final reactant. Specifically, it may be performed by additionally adding a concentration of 1% to 10% (v / v), and more specifically, may be performed by further adding a concentration of 2% to 8% Specifically, a concentration of 4 to 6%. The inventors of the present invention confirmed that about 80% or more of the oleic acid hydrolase 2 was reacted when dimethylsulfoxide was further added at a concentration of 12% or less to perform the hydration reaction (FIG. 2B). On the other hand, when a hydration reaction is performed by further adding dimethyl sulfoxide at a concentration exceeding 12%, there is a problem that the reaction activity is gradually reduced.

In one embodiment of the present invention, it has been found that, when dimethyl sulfoxide is added after various kinds of organic solvents are added and hydration reaction is performed, the reaction product is produced most in comparison with other organic solvents ( 2a). The reaction activity was highest when 5% dimethylsulfoxide was added by performing a hydration reaction after adding the dimethylsulfoxide concentration differently (FIG. 2b).

The hydration reaction may be performed at a concentration of oleic acid hydrolase 2 of 1.5 g / L to 3.0 g / L.

Specifically, the hydration reaction may be carried out at a concentration of 2.0 to 3.0 g / L, more specifically 2.3 g / L to 2.7 g / L, Most specifically at 2.5 < RTI ID = 0.0 > g / L. ≪ / RTI > The present inventors confirmed the effect of producing 5 g / L of 10-hydroxystearic acid when the hydration reaction was carried out at a concentration of oleic acid hydrolase 2 of 1.5 g / L to 3.0 g / L. On the other hand, when the hydration reaction was carried out in excess of 3.0 g / L, the yield of 10-hydroxystearic acid was not increased.

In one embodiment of the present invention, the oleic acid hydration reaction was performed with various concentrations of oleic acid hydrolase 2 to find that the yield of 10-hydroxy fatty acid was the highest when the concentration of oleic acid hydrolase 2 was 2.5 g / L there was.

As used herein, the term " Unsaturated fatty acids " refers to fatty acids in which a carbon-carbon double bond is present as a chain compound having an unsaturated bond and a carboxy group of carbon-carbon in one molecule. One having a double bond is called monoenoic acid, and dienic acid, trienoic acid, tetraenoic acid, pentaenoic acid, and hexaenoic acid are present in nature. Dienic acids and more are collectively referred to as polyenic acids, and more than tetraenoic acid is called an unsaturated fatty acid in fish oil. The position of the double bond is indicated by the number of carbons attached to the carbon atom from the carboxy group, but the fatty acid existing in nature shows a constant arrangement.

In the present invention, the unsaturated fatty acid is selected from the group consisting of oleic acid, palmitoleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, Or a combination of two or more thereof.

In the present invention, the unsaturated fatty acid may be a fatty acid decomposed product obtained by treating fatty acid hydrolytic enzyme (TLL) in soybean oil. Specifically, the fatty acid degradation product may be linoleic acid or oleic acid, but is not limited thereto.

The term " hydroxy fatty acid " in the present invention is referred to as a hydroxylated fatty acid, and exists in a small amount in a plant in a natural state and exists as a medium decomposition substance of a fatty acid in an animal. In the present invention, the hydroxy fatty acid is selected from the group consisting of 10-hydroxystearic acid, 10-hydroxypalmitolic acid, 10-hydroxyoctadec-12-enoic acid, 10-hydroxyoctadec-6,12- Hydroxyoctadec-12,15-dienoic acid, or 9-hydroxoyfatty acid.

In one embodiment of the present invention, 10-hydroxystearic acid from oleic acid, 10-hydroxypalmitolic acid from palmitoleic acid, 10-hydroxyoctadec-12-enoic acid from linoleic acid, Hydroxyoctadec-12,15-dienostearate from alpha-linolenic acid, 10-hydroxyoctadec-12,15-dienostearic acid, gamma-linolenic acid and 10-hydroxyoctadec-6,12-dienostearic acid with conventional oleic acid hydrolases, (Table 1). ≪ tb > < TABLE >

In another embodiment of the present invention, 10-hydroxystearic acid, 10-hydroxyoctadec-12-enoic acid produced from oleic acid, 10-hydroxyoctadec-12-enoic acid produced from linoleic acid, and decomposed product of soybean oil using oleic acid- 10-hydroxystearic acid and 10-hydroxyoctadec-12-enoic acid were produced in high yield from phosphoryl linoleic acid and oleic acid, and compared to the production rate of recombinant E. coli expressing conventional oleic acid hydrolase, (Figs. 4 to 6).

In the present invention, the 9-hydroxy fatty acid may be 9-hydroxynonanoic acid.

In one embodiment of the present invention, 10-hydroxy fatty acid was produced using recombinant Escherichia coli expressing oleic acid hydrolase 2 from linoleic acid and oleic acid, which are fatty acid degradation products produced by reacting soybean oil with TLL, and 10-hydroxy It was found that the fatty acid can produce 9-hydroxynnonanoic acid by using Escherichia coli expressing ADH and BVMO (Fig. 7).

The term " cultivation " in the present invention means a method of growing a microorganism under a moderately artificially controlled environmental condition. In the present invention, the method for culturing the transformant may be carried out by a method well known in the art. Specifically, the culture is not particularly limited as long as it can be produced by expressing the oleic acid hydrolase 2 of the present invention. However, the culture may be continuously cultured in a batch process or an injection batch or a repeated fed batch process can do.

The medium used for the culture may be a medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, phosphorus, inorganic compound and the like. The carbon sources that can be used include glucose and xylose mixed sugar as main carbon sources, and sugar and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, Oils and fats such as oils and the like, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Nitrogen sources that may be used include inorganic sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or their decomposition products, defatted soybean cake or decomposition products thereof . These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate and the corresponding sodium-containing salts as a source. Potassium which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used.

The term " bioconversion " in the present invention means a method of converting an initial substance into a desired substance using an enzyme or a transformant expressing the enzyme.

In the present invention, the bioconversion process may be performed by treating oleic acid hydrolase 2 or recombinant E. coli expressing the unsaturated fatty acid or a fatty acid degradation product containing the same with 10-hydroxystearic acid, 10-hydroxypalmitolic acid, Hydroxyoctadec-12-enoic acid, 10-hydroxyoctadec-6,12-dienostearic acid, 10-hydroxyoctadec-12,15-dienoic acid or 9-hydroxynonanoic acid It can be interpreted as a process.

The method for producing hydroxy fatty acids from unsaturated fatty acids using the oleic acid hydrolase 2 provided in the present invention can produce hydroxy fatty acids at a high rate and yield as compared with conventional oleic acid hydrolases, Can be used for the production of various fragrances, and can also be useful for the synthesis of polymers and for the production of emulsifiers.

1 (a) is a graph showing the reaction activity of oleate hydratase 2 according to the reaction pH.
1 (b) is a graph showing the reaction activity of oleic acid hydrolase 2 according to the reaction temperature.
FIG. 2 (a) is a graph showing the reaction activity of oleic acid hydrolase 2 according to the type of organic solvent added to the reaction solution.
2 (b) is a graph showing the reaction activity of oleic acid hydrolase 2 according to the concentration of dimethyl sulfoxide (DMSO) in the organic solvent added to the reaction solution.
3 is a graph showing the reaction activity depending on the concentration of oleic acid hydrazide 2 added to the reaction solution.
4 is a graph comparing the production rates of 10-hydroxystearic acid produced from oleic acid upon biotransformation using oleic acid hydrolase 2 and conventional oleic acid hydrolase (O: The concentration of oleic acid in the reaction solution when the oleic acid hydrolase 2 is used, the concentration of 10-hydroxystearic acid in the reaction solution when using the oleic acid hydrazine 2, the oleic acid concentration of the reaction solution when using the conventional oleic acid hydrolase, : 10-hydroxystearic acid concentration of reaction solution when using conventional oleic acid hydrolase).
FIG. 5 is a graph showing the activity of 10-hydroxyoctadec-12-enoic acid produced from linoleic acid at the time of bioconversion using recombinant E. coli expressing oleic acid hydrolase 2 and recombinant Escherichia coli expressing conventional oleic acid hydrolase 10: hydroxyoctadec-12-enoic acid) (o: the concentration of linolenic acid in the reaction solution when using recombinant E. coli expressing oleosinolytic enzyme 2, and: the recombinant E. coli expressing oleic acid hydrolase 2 ?: The concentration of 10-hydroxyoctadec-12-enoic acid in the reaction solution when used,?: The concentration of linolenic acid in the reaction solution when using conventional recombinant Escherichia coli,?: A recombinant Escherichia coli 10-hydroxyoctadec-12-enoic acid concentration of the reaction solution when using < RTI ID = 0.0 >
6 is a graph showing the production rate of 10-hydroxy fatty acid produced from soybean oil during bioconversion using recombinant Escherichia coli expressing lipolytic enzyme (TTL) and oleic acid hydrolase 2 (○: linolenic acid concentration ,?: Concentration of 10-hydroxyoctadec-12-enoic acid in the reaction solution,?: Concentration of oleic acid in the reaction solution,?: Concentration of 10-hydroxystearic acid in the reaction solution).
FIG. 7 is a graph showing the results of TTL, the expression of recombinant Escherichia coli, alcohol dehydrogenase and Bayer-Villiger monooxygenase (BVMO) expressing oleic acid hydrolase 2 from soybean oil during bioconversion Hydroxyoctadec-12-enoic acid concentration of the reaction solution, and?: The concentration of 10-hydroxyoctadec-12-enoic acid in the reaction solution,?: The production rate of 9-hydroxynonanoic acid : Concentration of oleic acid in the reaction solution;?: Concentration of 10-hydroxystearic acid in the reaction solution;?: Concentration of 9-hydroxy nonanoic acid in the reaction solution).

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.

Example  1. Oleic acid hydrolase 2 gene ( oleate hydratase  2 genes, ohyA2 ) And the production of transformed recombinant E. coli

In order to prepare an expression vector containing oleic acid hydrolase 2 and recombinant Escherichia coli, Stenotrophomonas maltophilia ) strain 2 was synthesized and the following method was performed using the synthesized gene.

(SEQ ID NO: 3): 5'-CATATGAGCCAGCCCACCGCAC-3 'and reverse direction (SEQ ID NO: 4): 5'-CTCGAGTCAGGGCGCGCGCCGCCTG-3') based on the DNA sequence of the synthesized oleic acid hydrolase 2, The prepared primers were subjected to polymerase chain reaction (PCR) to amplify the nucleotide sequence of the corresponding gene. The amplified oleic acid hydrolase 2 gene was inserted into the plasmid vector pET 28 (+) a (manufactured by Novagen) using restriction enzymes Nde I and Xho I to prepare pET 28 (+) a-OhyA2.

The recombinant expression vector thus obtained was transformed into Escherichia coli ER 2566 or BL21 (DE3) strain, and the transformant Escherichia coli was stored frozen in a 20% glycerin solution.

Example  2. Preparation of oleic acid hydrolase 2

To prepare oleic acid hydrolase 2 from recombinant E. coli prepared by the method of Example 1, the E. coli was cultured and oleic acid hydrolase 2 was purified to prepare a sample containing oleic acid hydrolase 2.

The recombinant Escherichia coli ER 2566 of Example 1 was cultured in a 500 ml LB (Difco, Sparks, MD, USA) medium and a flask supplemented with kanamycin at 50 占 퐂 / mL at 37 占 폚 Lt; / RTI > When the absorbance of the bacteria reached from 0.6 to 0.8 at 600 nm, IPTG was added to a final concentration of 0.1 mmol / L to induce the expression of oleic acid hydrolase, and the culture was stirred for 12 hours at 16 DEG C at 150 rev / min Lt; / RTI >

The culture was centrifuged at 6,000 x g for 30 minutes at 4 ° C, and then washed twice with 0.85% NaCl and 50 mM sodium phosphate (NaH 2 PO 4 ), 300 mM sodium chloride, 10 mM imidazole, , A 0.1 mM proteolytic enzyme inhibitor (phenylmethylsulfonyl fluoride) was added, and the cell solution was disrupted with a sonicator. The cell lysate was centrifuged at 13,000 xg for 20 minutes at 4 < 0 > C, and only the cell supernatant was isolated. The cell supernatant was applied to fast protein liquid chromatography (Bio-Rad Laboratories, Hercules, Calif., USA) to obtain an active fraction, and the active fraction was labeled with a histidine tag (His- Was used to purify oleic acid hydrolase 2, and a sample containing the purified oleic acid hydrolase 2 was prepared.

Experimental Example  1. Analysis of optimal reaction conditions for oleic acid hydrolase 2

Experimental Example  1.1: Reaction pH

To investigate the effect of pH on the reaction activity of the oleic acid hydrate 2 produced in Example 2, oleic acid hydration was carried out under acidic conditions of pH 5 to pH 7 using oleic acid as a substrate.

0.14 g / L oleic acid was added to a 50 mM citrate / phophate buffer buffer solution to conduct oleic acid hydration. The reaction activity was relatively measured by measuring the yield of 10-hydroxystearic acid.

As a result of the experiment, oleic acid hydrolase 2 exhibited a reaction activity of about 80% or more from pH 5 to pH 6.5, and showed the maximum reaction activity particularly at pH 6.0 (FIG.

Experimental Example  1.2: reaction temperature

In order to investigate the effect of the reaction temperature on the reaction activity of the oleic acid hydrolase 2 prepared in Example 2, oleic acid hydration was performed at 25 ° C to 50 ° C using oleic acid as a substrate.

The oleic acid and citrate / phosphate buffer solutions were added to the same concentration as in Experimental Example 1.1, and oleic acid hydration was performed at each temperature for 10 minutes. The reaction activity was relatively measured by measuring the yield of 10-hydroxystearic acid.

As a result of the experiment, oleic acid hydrolase 2 exhibited a reaction activity of 80% or more at 30 ° C to 45 ° C, and exhibited the maximum reaction activity at pH 35 ° C (FIG. 1b).

Experimental Example  1.3: Organic solvent  Kinds

In order to investigate the effect of organic solvent on the reaction activity of oleic acid hydrazine 2, oleic acid hydration reaction was performed after various organic solvents were added to the reaction solution.

The oleic acid hydration reaction was carried out by adding 5 or 10% (v / v) of methanol to a 50 mM citrate / phosphate buffer solution (pH 6.0) containing 0.14 g / L of oleic acid and 0.005 g / , Ethanol, 2-propanol, dimethyl sulfoxide (DMSO), butanol, acetone, and toluene were added to the reaction mixture. The reaction was terminated by adding ethyl acetate in the same amount as the amount of the liquid. The reaction activity was compared by measuring the yield of 10-hydroxystearic acid.

As a result of the experiment, the reaction solution to which 5% (v / v) of methanol and 2-propanol, 5 and 10% (v / v) of dimethylsulfoxide was added, showed that the reaction activity of oleic acid hydrolase 2 was higher , And the reaction activity of the reaction solution added with dimethylsulfoxide was found to be higher than that of other organic solvents (Fig. 2A).

Experimental Example  1.4: Dimethyl sulfoxide  density

Since the reaction activity of oleic acid hydrolase 2 was the highest when dimethylsulfoxide was added in the organic solvent in Experimental Example 1.3, the concentration of dimethylsulfoxide, which can optimize the reaction activity of oleic acid hydrolase 2, , And 0 to 12.5% of dimethylsulfoxide was added to the reaction solution to conduct an oleic acid hydration reaction.

The oleic acid hydration reaction was carried out in the same manner as described in Experimental Example 1.3 by adding various concentrations of dimethylsulfoxide. The reaction activity was relatively measured by measuring the yield of 10-hydroxystearic acid.

As a result of the experiment, when the dimethylsulfoxide was added so as to be 5% (v / v), the reaction activity of oleic acid hydrolase 2 was the highest, as in the case of Experimental Example 1.3 (Fig. 2B).

Experimental Example 1.5: Unsaturated fatty acid type

To investigate the effect of unsaturated fatty acids on the reaction activity and production rate of oleic acid hydrolase 2, oleic acid, palmitoleic acid linoleic acid, alpha-linolenic acid, Or gamma-linolenic acid as a substrate to perform hydration reaction.

The hydration reaction was carried out in the same manner using the same concentrations of substrate, oleic acid hydrolase 2 and citrate / phosphate buffer solution described in Experimental Example 1.3 (Table 1).

Comparison of inactivity according to substrate of oleic acid hydrolase 2
temperament
product Inactivity of oleic acid hydrolase 2 (umol / mg -1 protein min -1 ) Inactivation of oleic acid hydrolase (umol / enzyme -1 min -1 ) Oleic acid 10-Hydroxystearic acid 5.36 + 0.07 3.69 ± 0.0023 Palmitoleic acid 10-hydroxypalmitoleic acid
(10-hydoroxypalmitic acid) 2.21 ± 0.03 1.95 ± 0.0018 Linoleic acid 10-Hydroxyoctadec-12-enoic acid 1.67 ± 0.19 0.91 + 0.0005 Alpha-linolenic acid 10-hydroxyoctadec-12,15-dienostric acid
(10-hydroxyoctadec-12,15-dienoic acid) 1.05 + 0.09 0.54 + - 0.0004 Gamma-linolenic acid 10-Hydroxyoctadec-6,12-dienostric acid
(10-hydroxyoctadec-6, 12-dienoic acid) 0.78 + 0.04 0.33 ± 0.00008

As shown in Table 1, the hydration reaction activity of oleic acid hydrolase 2 was measured using various unsaturated fatty acids as a substrate. As a result, it was found that the most suitable unsaturated fatty acid as a substrate was oleic acid, and then palmitoleic acid, linoleic acid, Gamma-linolenic acid.

In addition, as shown in Table 1, the activity of the oleosin hydrolase 2 (the amount of the product according to the amount of the enzyme in the reaction time) of the oleosin hydrolase 2 can be measured by using oleic acid hydration Were found to be 45%, 10%, 80%, 90%, and 130% higher than those of the enzyme substrate, respectively, for oleic acid, palmitoleic acid, linoleic acid, alpha-linolenic acid or gamma-linolenic acid.

therefore. Reaction activity and production rate of oleic acid hydrolase 2 were highest when oleic acid was used as a substrate, and when the above substrate was used, it was found to be higher than conventional oleic acid hydrolase.

Experimental Example  1.5: Concentration of oleic acid hydrolase 2

In order to investigate the concentration of oleic acid hydrolase 2 capable of optimizing the reaction activity of the oleosan hydrolase 2, oleic acid hydration using oleic acid as a substrate was carried out under various enzyme concentration conditions, and the reaction product of 10-hydroxystear The yield of the acid was compared.

L of oleic acid hydrolase 2 ranging from 0.5 g / L to 3.0 g / L was added to a 50 mM citrate / phosphate buffer (pH 6.0) containing 10 g / L oleic acid and 5% dimethyl sulfoxide, Roxystearic acid was measured. The reaction was carried out in 1 mL of a 15 mL plastic container at 35 ° C for 3 hours, and 1 mL of ethyl acetate was added to terminate the reaction.

As a result of the experiment, it was confirmed that 10-hydroxystearic acid production was increased as the concentration was increased until the concentration of oleic acid hydrolase 2 was 2.5 g / L, but it was not increased after 2.5 g / L (FIG. 3) .

Therefore, when the concentration of oleic acid hydrolase 2 is 2.5 g / L, oleic acid is maximally converted to 10-hydroxystearic acid, so that the reaction activity is the highest at this concentration.

Experimental Example 2. Production of 10-hydroxystearic acid using oleic acid hydrolase 2

In order to confirm the production rate of 10-hydroxystearic acid by using oleic acid hydrolase 2, the production amount of the reaction product at the time of bioconversion was measured by using 10 g / L of oleic acid as a substrate at pH 6.0 and 35 ° C, Of oleic acid hydrolase.

As a result, oleic acid hydrolase 2 produced 10 g / L of 10-hydroxystearic acid and showed a productivity of 3.4 g / L per hour and a conversion yield of 90% or more with respect to the reduced substrate. In addition, it was confirmed that oleic acid hydrolase 2 exhibited a productivity of about 20% or higher as compared with the productivity of 10-hydroxystearic acid using a conventional oleic acid hydrolase (FIG. 4).

Therefore, it was found from the above results that oleic acid hydrolase 2 is an oleic acid hydrolase superior to oleic acid hydrolase derived from the same strain and 10-hydroxystearic acid can be produced with high yield using the enzyme.

Experimental Example  3. Recombinant E. coli containing oleic acid hydrolase 2, 10- Of hydroxy fatty acids  production

Experimental Example  3.1: Synthesis of 10- < RTI ID = 0.0 > Hi Production of 10-hydroxyoctadec-12-enoic acid

In order to confirm the production rate of 10-hydroxyoctadec-12-enoic acid using the recombinant E. coli of Example 1, the production amount of the reaction product over time during the bioconversion was measured and compared with the conventional oleic acid hydrolase.

The recombinant Escherichia coli was cultured in a regenberg culture medium, and linolenic acid was added to the culture solution at 30 mM (8.4 g / L) for 6 hours.

As a result, the recombinant Escherichia coli expressing oleosin hydrolase 2 produced 20 mM 10-hydroxyoctadec-12-enoic acid, and the conversion yield of the reduced substrate was 90% or more (FIG. 5). Further, as a result of comparing the production rate of 10-hydroxyoctadec-12-enoic acid of the recombinant E. coli expressing the conventional oleosin hydrolase, the recombinant E. coli expressing oleosin hydrolase 2 exhibited a production rate higher than 5 times Respectively.

Thus, the above results show that oleic acid hydrolase 2 is an oleic acid hydrolase superior to oleic acid hydrolase derived from the same strain, and 10-hydroxyoctadec-12-enoic acid can be produced with high yield using the enzyme Could know.

Experimental Example  3.2: Recombinant E. coli containing oleic acid hydrolase 2 and 10- Hydroxyoctadec -12- Enoch  Acid and 10-hydroxystearic acid of  production

The production rate of 10-hydroxyoctadec-12-enoic acid and 10-hydroxystearic acid was confirmed by biotransformation from soybean oil using the recombinant E. coli of Example 1 and TLL (Genoporac, Daejon) which is a fatty acid hydrolase In order to investigate the effect of the E. coli and TLL on the biosynthesis of the reaction product, the yield of the reaction product was measured and compared with the conventional oleic acid hydrolase.

As a result, 7 mM 10-hydroxyoctadec-12-enoic acid and 5 mM 10-hydroxystearic acid were produced using recombinant E. coli expressing oleosin hydrolase 2, About 80% or more (Fig. 6). As a result of comparison with conventional recombinant E. coli expressing oleic acid hydrolase, it was found that the production rate of 10-hydroxyoctadec-12-enoic acid was lowered when recombinant E. coli expressing oleosin hydrolase 2 was used And more than four times higher.

Therefore, it was found from the above results that oleic acid hydrolase 2 was an oleic acid hydrolase superior to the same strain-derived oleic acid hydrolase, and in particular, it was confirmed that the 10-hydroxyoctadec-12- And the yield can be obtained.

Experimental Example  4. Recombinant E. coli containing oleic acid hydrolase 2, Alcohol  Dehydrogenase dehydrogenase ) And Bayer- Billy  Oxidase ( Bayer - Villiger  monooxygenase, BVMO ) And Escherichia coli containing 9- < RTI ID = 0.0 > Hi Production of deoxynonanoic acid

Recombinant Escherichia coli expressing oleic acid hydrolase 2 of Example 1, recombinant Escherichia coli expressing ADH and BVMO (Green Chemistry, 2015) and TLH (fatty acid hydrolyzing enzyme TLL (Genoporac, Daejeon) In order to confirm the production rate of 9-hydroxynonanoic acid, the production yield of 9-hydroxynonanoic acid was measured at the time of bioconversion using the recombinant E. coli and TLL of Example 1.

Recombinant E. coli were cultured in a regenberg culture medium. To the culture medium of recombinant E. coli expressing ADH and BVMO, 5 g / L of recombinant Escherichia coli expressing oleic acid hydrolase 2 and 5 g / L of soybean oil were added and reacted for 12 hours.

As a result of the experiment, it was confirmed that about 8 mM of 9-hydroxynnonanoic acid was produced using the recombinant Escherichia coli expressing the oleic acid hydrolase 2 of Example 1, and the conversion yield of the reduced substrate was about 70% or more 7).

Thus, it was found from the above results that oleic acid hydrolase 2 can also be used for the production of 9-hydroxynonanoic acid from soybean oil.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> Ewha University - Industry Collaboration Foundation          Konkuk University Industrial Cooperation Corp <120> Novel oleate hydratase 2 and uses thereof <130> KPA151378-KR <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1986 <212> DNA <213> Artificial Sequence <220> <223> Stenotrophomonas maltophilia, oleate hydratase 2 <400> 1 atgagccagc ccaccgcacc gggacgcaac gcaggggcca cgccggcctt cgagcacgag 60 ccggacagca ccggcggcta ctggtccaac cggccggaga acacactgcc accgccggac 120 atgatggggg cctacatgcg caaccggccg ctgccgccgg aggacgtggc gcagcgcaag 180 gcctacatca tcggcaccgg catcgccggg ttggcggcgg cgttctacct gatccgcgac 240 ggcggcatgc cgccggccaa catcacgctg ctggacagcc tggagatcga aggcggttcg 300 ctggatggcg cgggggatgc cgagcagggc tacctgatcc gcggcggccg cgagatgaac 360 tggaactacg acaatttctg ggacctgttc caggatgtgc cggcactcga actgccggcc 420 ggcttcagcg tgctcgacga gtaccgcgcc gtcaacgaca acgatccgaa ctggtccaag 480 gcgcggctgc tgcaccagca gggcaaggtc aaggatttcg ccacgttcgg gttgagccgc 540 ggccagcaat gggagctggt caagctgctg ctcaagcgca aggaagacct ggacgacgtc 600 accatcgagg actacttcag cgaaggcttt ctgcagagca acttctggtt cttctggcgc 660 tcgatgttcg ccttcgagaa ctggcagagc ctgctcgaga tgaagctgta catgcatcgg 720 ttcctggatg ccatcgacgg tctgaacgac atgtccgcgc tggtgtttcc caagtacaac 780 caatacgaga gttttgtggt gccgctgtcg cggatgctgc gcgcgcaggg cgtcaacgtg 840 cagttcgata cccgcgtcca cgacctggag atggcggtgg acgggcagtc acgcaccgtc 900 accgcgctgc gctgccgggt ggccggcaac gagaccacgc tgccggttgc ggcgggcgac 960 ctggtgttcg cgctcaccgg ttcgatgacc gaaggcacgg cgtacggcga catggacacc 1020 gtgccgccgc tggcgcgcga ccgccgggac ccgggcgagg acagtgactg ggcgctgtgg 1080 cgcaatctcg cgcggcagtc gccgatcttc ggcaagccgg agaagttcta cggcgacgtg 1140 gaccgctcga tgtgggagtc ggccacgttg acctgccgcc cctcgccgct ggtggacaag 1200 atccgcacgt tgtcggtcaa cgatccgtac tccgggcgca ccgtgaccgg tggggtcatc 1260 accatcaccg attccaactg ggtgctcagc ttcaccgtca accgccagcc gcatttcgtg 1320 gaccagccca aggacgtgct ggtggtatgg gtctatgccc tgttgatgga tcaggacggc 1380 aacacatca aaaagccgat gccggcgtgt accggacgcg aggtgctggc cgaactgtgc 1440 caccacctgg gcatcggcga ccagatcgat gcggtggccg ccgcgaccag ggtgcggctg 1500 gcgttgatgc cgtacatcac cgcgcagttc atgccgcgtg ccgctggcga ccgtccgcac 1560 gtggtaccgg ccggctgcac caacctgggc ctgctcggcc agttcgtgga aacgcgcaac 1620 gatgtgatct tcacgatgga aagctcgatc cgcacggcgc gtgtggcggt gtacaccctg 1680 ctggggctgc gcaagcaggt accggacctg agcccgaccc agtacgacat ccgcaatctg 1740 atcaaagcgg cacgggcgtt gaacaacaac gcgccgttcc ccggcgaacg gctgctgcac 1800 cgtctgctcg gcaacagtta ttacgcccac atcctgccgc cgctgccaca gcctgaaaag 1860 ggccgggagg ccttccttga agaggagctg tcgtggttgt caggcaaggg cagcgtggtg 1920 ctgaaggacc tgtctgcacg gctggatcgg cttggcgaaa cgctgggcag gcggcgcgcg 1980 ccctag 1986 <210> 2 <211> 661 <212> PRT <213> Artificial Sequence <220> <223> Stenotrophomonas maltophilia, oleate hydratase 2 <400> 2 Met Ser Gln Pro Thr Ala Pro Gly Arg Asn Ala Gly Ala Thr Pro Ala   1 5 10 15 Phe Glu His Glu Pro Asp Ser Thr Gly Gly Tyr Trp Ser Asn Arg Pro              20 25 30 Glu Asn Thr Leu Pro Pro Pro Asp Met Met Gly Ala Tyr Met Arg Asn          35 40 45 Arg Pro Leu Pro Pro Glu Asp Val Ala Gln Arg Lys Ala Tyr Ile Ile      50 55 60 Gly Thr Gly Ile Ala Gly Leu Ala Ala Phe Tyr Leu Ile Arg Asp  65 70 75 80 Gly Gly Met Pro Pro Ala Asn Ile Thr Leu Leu Asp Ser Leu Glu Ile                  85 90 95 Glu Gly Ser Leu Asp Gly Ala Gly Asp Ala Glu Gln Gly Tyr Leu             100 105 110 Ile Arg Gly Gly Arg Glu Met Asn Trp Asn Tyr Asp Asn Phe Trp Asp         115 120 125 Leu Phe Gln Asp Val Ala Leu Glu Leu Pro Ala Gly Phe Ser Val     130 135 140 Leu Asp Glu Tyr Arg Ala Val Asn Asp Asn Asp Pro Asn Trp Ser Lys 145 150 155 160 Ala Arg Leu Leu His Gln Gln Gly Lys Val Lys Asp Phe Ala Thr Phe                 165 170 175 Gly Leu Ser Arg Gly Gln Gln Trp Glu Leu Val Lys Leu Leu Leu Lys             180 185 190 Arg Lys Glu Asp Leu Asp Asp Val Thr Ile Glu Asp Tyr Phe Ser Glu         195 200 205 Gly Phe Leu Gln Ser Asn Phe Trp Phe Phe Trp Arg Ser Met Phe Ala     210 215 220 Phe Glu Asn Trp Gln Ser Leu Leu Glu Met Lys Leu Tyr Met His Arg 225 230 235 240 Phe Leu Asp Ala Ile Asp Gly Leu Asn Asp Met Ser Ala Leu Val Phe                 245 250 255 Pro Lys Tyr Asn Gln Tyr Glu Ser Phe Val Val Pro Leu Ser Arg Met             260 265 270 Leu Arg Ala Gln Gly Val Asn Val Gln Phe Asp Thr Arg Val His Asp         275 280 285 Leu Glu Met Ala Val Asp Gly Gln Ser Arg Thr Val Thr Ala Leu Arg     290 295 300 Cys Arg Val Ala Gly Asn Glu Thr Thr Leu Pro Val Ala Ala Gly Asp 305 310 315 320 Leu Val Phe Ala Leu Thr Gly Ser Met Thr Glu Gly Thr Ala Tyr Gly                 325 330 335 Asp Met Asp Thr Val Pro Pro Leu Ala Arg Asp Arg Arg Asp Pro Gly             340 345 350 Glu Asp Ser Asp Trp Ala Leu Trp Arg Asn Leu Ala Arg Gln Ser Pro         355 360 365 Ile Phe Gly Lys Pro Glu Lys Phe Tyr Gly Asp Val Asp Arg Ser Met     370 375 380 Trp Glu Ser Ala Thr Leu Thr Cys Arg Pro Ser Pro Leu Val Asp Lys 385 390 395 400 Ile Arg Thr Leu Ser Val Asn Asp Pro Tyr Ser Gly Arg Thr Val Thr                 405 410 415 Gly Gly Val Ile Thr Ile Thr Asp Ser Asn Trp Val Leu Ser Phe Thr             420 425 430 Val Asn Arg Gln Pro His Phe Val Asp Gln Pro Lys Asp Val Leu Val         435 440 445 Val Trp Val Tyr Ala Leu Leu Met Asp Gln Asp Gly Asn His Ile Lys     450 455 460 Lys Pro Met Pro Ala Cys Thr Gly Arg Glu Val Leu Ala Glu Leu Cys 465 470 475 480 His His Leu Gly Ile Gly Asp Gly Ile Asp Ala Val Ala Ala Ala Thr                 485 490 495 Arg Val Arg Leu Ala Leu Met Pro Tyr Ile Thr Ala Gln Phe Met Pro             500 505 510 Arg Ala Gly Asp Arg Pro His Val Val Pro Ala Gly Cys Thr Asn         515 520 525 Leu Gly Leu Leu Gly Gln Phe Val Glu Thr Arg Asn Asp Val Ile Phe     530 535 540 Thr Met Glu Ser Ser Ile Arg Thr Ala Arg Val Ala Val Tyr Thr Leu 545 550 555 560 Leu Gly Leu Arg Lys Gln Val Pro Asp Leu Ser Pro Thr Gln Tyr Asp                 565 570 575 Ile Arg Asn Leu Ile Lys Ala Ala Arg Ala Leu Asn Asn Asn Ala Pro             580 585 590 Phe Pro Gly Glu Arg Leu Leu His Arg Leu Leu Gly Asn Ser Tyr Tyr         595 600 605 Ala His Ile Leu Pro Pro Leu Pro Gln Pro Glu Lys Gly Arg Glu Ala     610 615 620 Phe Leu Glu Glu Glu Leu Ser Trp Leu Ser Gly Lys Gly Ser Val Val 625 630 635 640 Leu Lys Asp Leu Ser Ala Arg Leu Asp Arg Leu Gly Glu Thr Leu Gly                 645 650 655 Arg Arg Arg Ala Pro             660 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 catatgagcc agcccaccgc ac 22 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 ctcgagtcag ggcgcgcgcc gcctg 25

Claims (17)

delete delete delete 1 &lt; / RTI &gt; that encodes the oleate hydrolase 2, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:
An expression vector comprising the polynucleotide of claim 4.
A transformant into which the expression vector of claim 5 is introduced.
7. The transformant according to claim 6, wherein the transformant is recombinant E. coli. Transformants.
(a) culturing the transformant of claim 6 to obtain a culture; And
(b) recovering the oleoresinase 2 from the culture.
A process for producing a hydroxy fatty acid, which comprises the step of treating the transformant of claim 6 with an unsaturated fatty acid to cause a hydration reaction.
10. The method of claim 9, wherein the unsaturated fatty acid is selected from the group consisting of oleic acid, palmitoleic acid, linoleic acid, alpha-linolenic acid, gamma-linolenic acid, And an unsaturated fatty acid selected from the group consisting of combinations thereof.
10. The method of claim 9, wherein the unsaturated fatty acid is a fatty acid degradation product obtained by treating fatty acid hydrolyzate (TLL) in soybean oil.
10. The composition of claim 9, wherein the hydroxy fatty acid is selected from the group consisting of 10-hydroxystearic acid, 10-hydroxypalmitic acid, 10-hydroxyoctadec-12-enoic acid 10-hydroxyoctadec-12-enoic acid, 10-hydroxyoctadec-6,12-dienoic acid, 10-hydroxyoctadec-12,15-di Hydroxyoctadec-12,15-dienoic acid or 9-hydroxy fatty acid.
13. The method of claim 12, wherein the 9-hydroxy fatty acid is 9-hydroxynonanoic acid. delete delete delete delete
KR1020160025454A 2016-01-07 2016-03-03 Novel oleate hydratase 2 and uses thereof KR101856308B1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
KR1020160025454A KR101856308B1 (en) 2016-03-03 2016-03-03 Novel oleate hydratase 2 and uses thereof
PCT/KR2016/011224 WO2017119576A1 (en) 2016-01-07 2016-10-07 Bioconversion process using oleic acid hydratase 2

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020160025454A KR101856308B1 (en) 2016-03-03 2016-03-03 Novel oleate hydratase 2 and uses thereof

Publications (2)

Publication Number Publication Date
KR20170104015A KR20170104015A (en) 2017-09-14
KR101856308B1 true KR101856308B1 (en) 2018-05-10

Family

ID=59926721

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020160025454A KR101856308B1 (en) 2016-01-07 2016-03-03 Novel oleate hydratase 2 and uses thereof

Country Status (1)

Country Link
KR (1) KR101856308B1 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102527521B1 (en) * 2020-10-12 2023-05-03 주식회사 그린위치 Novel Stenotrophomonas sp. strain and method for manufacturing hydroxy fatty acid by using the same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GenBank Accession No. WP_017356052.1

Also Published As

Publication number Publication date
KR20170104015A (en) 2017-09-14

Similar Documents

Publication Publication Date Title
KR101730035B1 (en) Variant Microorganism Producing L-Aspartic acid Derivative and Method for Preparing L-Aspartic acid derivatives Using the Same
CN101617038A (en) Utilize coryneform bacteria from the carbon source that contains glycerine, to produce the method for leavened prod
KR101642583B1 (en) Method for the production of 13-hydroxylinoleic acid by lipoxygenase from Burkholderia thailandensis and composition therefor
KR101934900B1 (en) Composition for producing hydroxyfatty acid, hepoxilin b or trioxilin b
KR101087760B1 (en) Novel recombinant yeasts and methods of simultaneously producing ethanols and target proteins using them
KR101749429B1 (en) Oleate Hydratase Gene, Expression Vector Containing the Gene, and Transformant Transformed by the Vector
KR101856308B1 (en) Novel oleate hydratase 2 and uses thereof
US20120149074A1 (en) Transformant Comprising Gene Coding for ws/dgat and Method of Producing Fatty Acid Ethyl Esters Using the Same
KR102170228B1 (en) A mutant lipase having enhanced transesterification activity and its application for biodiesel production
KR101725454B1 (en) Gene encoding lysine decarboxylase derived from H. alvei, recombinant vector, host cell and method for producing cadaverine using the same
KR101460228B1 (en) Mutant lipase with advanced activity and process for production bio-diesel using the same
KR101775226B1 (en) Fatty acid double bond hydratases acting in periplasm and process for producing hydroxy fatty acids using thereof
KR101741873B1 (en) Novel esterase fusion protein with improved activity
KR101500827B1 (en) Process for preparing medium chain fatty acids and ω-hydroxyfatty acids from long chain fatty acids
KR102028161B1 (en) Process for preparing 2,3-butanediol using transformant
KR102424603B1 (en) A Mutants of Alcohol Dehydrogenase, and A Method for Preparing Aldehyde Compounds Using The Same
JP5057727B2 (en) Transformant containing hydroxynitrile lyase gene and nitrilase gene, and method for producing α-hydroxycarboxylic acid using the same
KR101897189B1 (en) Lipase derived from Penicillium chrysogenum and preparation method of lipase using the same
KR101563307B1 (en) A METHOD FOR THE PRODUCTION OF 13-HYDROXY-FATTY ACID BY USING LACTOBACILLUS ACIDOPHILUS 13-LINOLEATE HYDRATASE AND δ-DECALACTONE FROM THE HYDROXY FATTY ACID BY USING WALTOMYCES LIPOFER AND A COMPOSITION THEREFOR
KR20160042254A (en) Process for preparing lipase derived from Candida antarctica using Pichia pastoris
CN113265343B (en) Construction method and application of recombinant hansenula polymorpha
KR102398203B1 (en) Novel oxygenase from fungi Podospora anserina S mat+ and method for preparing thereof
KR101543297B1 (en) Method for the production of 5,8-dihydroxylinoleic acid by diol synthase from Aspergillus nidulans and composition therefor
KR102693953B1 (en) Recombinant Corynebacterium glutamicum producing aromatic aldehydes and method for producing aromatic aldehydes using the same
KR101945925B1 (en) Protein for isobutylene synthesis and isobutylene preparation method using the same

Legal Events

Date Code Title Description
A201 Request for examination
E902 Notification of reason for refusal
E90F Notification of reason for final refusal
E701 Decision to grant or registration of patent right
GRNT Written decision to grant