KR101782205B1 - Process for preparing medium chain fatty acids from long chain fatty acids by bioconversion and chemical reaction - Google Patents
Process for preparing medium chain fatty acids from long chain fatty acids by bioconversion and chemical reaction Download PDFInfo
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Abstract
The present invention relates to a process for producing an ester compound from a long-chain fatty acid through a bioconversion process using a microorganism capable of expressing an alcohol dehydrogenase and BVMO (Baeyer-Villiger monooxygenase) Chain fatty acid from a long chain fatty acid, which comprises the step of producing a medium chain fatty acid from an ester compound. According to the method for producing a heavy chain fatty acid according to the present invention, it is possible to partially reduce the manufacturing cost by partially using the conventional biological conversion process, and to partially maximize the final production concentration and productivity of the heavy chain fatty acid using a chemical process , Which can be used to produce more economical heavy chain fatty acids.
Description
The present invention relates to a method for producing a heavy chain fatty acid from a long chain fatty acid using bioconversion and chemical conversion. More specifically, the present invention relates to a method for producing a heavy chain fatty acid from a long chain fatty acid, A step of obtaining an ester compound from a long chain fatty acid having 16 to 20 carbon atoms through a bioconversion process using a microorganism capable of reacting with an ester compound obtained from the ester compound obtained through chemical hydrolysis and oxidation, Chain fatty acids comprising the step of producing an omega-hydroxy fatty acid or an alpha, omega-dicarboxylic acid.
Medium chain alpha, omega-dicarboxylic acids and omega-aminocarboxylic acids are widely used in plastics such as polyamides and polyesters, pharmaceuticals, plasticizers, lubricating oils, And as building blocks and / or building blocks for the production of such products. For example, omega-aminoundecanoic acid is used for the synthesis of polyamide 11. It offers a wide range of flexibility and outstanding chemical, thermal and impact resistance compared to other high performance and engineering plastics. Generally, heavy chain alpha, omega-dicarboxylic acids and omega-aminocarboxylic acids are used under high temperature and pressure conditions such as strong acids (eg sulfuric acid, nitric acid; H2SO4, HNO3), strong oxidizing agents (eg ozone) For example, omega-aminandecdexinic acid can be produced from ricinoleic acid by the addition of hydrogen bromide after ammonia and nucleophilic substitution at high and high pressure. .
Recently, it has been reported that a recombinant microorganism such as recombinant Escherichia coli is used to synthesize a heavy chain fatty acid such as omega-amino undecylic acid and alpha, omega-undecanoic anoxic acid from long chain fatty acids (Korean Patent Publication No. 2013-0132254) . As one example, the C12-hydroxyl group of ricinoleic acid is oxidized to the ester compound via 12-ketooleic acid by treatment with an alcohol dehydrogenase and Baeyer-Villiger monooxygenase, The compound is hydrolyzed by esterase to produce n-heptanoic acid and omega-hydroxy undec-9-ioic acid. The resulting omega -hydroxy undec-9-ioic acid is converted to alpha, omega-undecanoic acid by treatment with a reducing enzyme and an alcohol dehydrogenase, or by treatment with an alcohol dehydrogenase and an omega- - < / RTI > amino undecanoic acid. The production of heavy chain fatty acids by using microorganisms has the advantage of reducing the cost and environmental pollution compared with the chemical process. However, due to the high toxicity of the produced heavy chain fatty acid to the microbial catalyst and the complicated bioconversion pathway It has been reported that it is difficult to improve the final concentration and productivity of the target product (Process Biochemistry 49 (2014) 617-622, Advanced Synthesis & Catalysis, 356 (2014) 1782-1786). Studies to increase the productivity of heavy chain fatty acids have been actively conducted to overcome these disadvantages, but they have not achieved much yet.
Under these circumstances, the present inventors have made intensive researches to develop a method for improving the productivity when producing a medium chain fatty acid using microorganisms. As a result, they have found that the biotransformation process using microorganisms does not show cytotoxicity from long chain fatty acids, It has been confirmed that the productivity of the medium chain fatty acid can be improved when the low ester compound is produced and the produced ester compound is subjected to a chemical hydrolysis reaction and an oxidation reaction to produce a medium chain fatty acid, Respectively.
It is an object of the present invention to provide a method for producing a heavy chain fatty acid from a long chain fatty acid through a biological conversion process, a chemical hydrolysis reaction and an oxidation reaction.
The present inventors have developed a method for overcoming the disadvantage that the productivity of the medium chain fatty acid is lowered due to the cytotoxicity of the produced heavy chain fatty acid and the complex bioconversion pathway in the bioconversion step of producing the medium chain fatty acid from the long chain fatty acid using microorganisms During the course of the research, we have developed a method to combine the biological conversion process with the chemical method. That is, in the bioconversion process, the ester compounds having low cytotoxicity from microorganisms are produced by a conventional bioconversion process, and the produced ester compounds are applied to a chemical hydrolysis reaction and an oxidation reaction to produce a final product, The ester compound having a low cytotoxicity can be produced with high concentration and high productivity and the ester compound can be converted into a medium chain fatty acid through a relatively simple hydrolysis and oxidation reaction, The results are summarized as follows.
For example, the production of hydroxyundec-9-Ioic acid from ricinoleic acid using a recombinant E. coli catalyzer has been difficult due to the toxicity of the reaction products, resulting in the production of hydroxyendec-9-ioic acid at 20 mM or higher , Productivity was also not as high as 3.2 mM / h (Process Biochemistry 49 (2014) 617-622). However, using the method provided in the present invention, it was possible to produce ester having low toxicity by using recombinant Escherichia coli and to stably produce the final concentration up to 54 mM, and the production rate was 6.6 mM / h in Process Biochemistry (49 (2014) 617 -622) (Examples 1 and 2). ≪ tb >< TABLE >
In order to accomplish the above object, the present invention provides a method for producing a heavy chain fatty acid from a long chain fatty acid through a biological conversion process, a chemical hydrolysis reaction and an oxidation reaction.
Specifically, the present invention provides a method for producing a heavy chain fatty acid from a long chain fatty acid, comprising the steps of: (a) culturing a transformant capable of expressing an alcohol dehydrogenase and BVMO (Baeyer-Villiger monooxygenase) Further producing an ester compound; And (b) subjecting the resulting ester compound to a chemical reaction to produce a heavy chain fatty acid.
Hereinafter, the method for producing the heavy chain fatty acid from the long chain fatty acid provided by the present invention will be described in detail with reference to the steps.
(a) Bioconversion process
The first step of the method for producing the heavy chain fatty acid from the long chain fatty acid provided by the present invention is that the transformant capable of expressing the alcohol dehydrogenase and BVMO (Baeyer-Villiger monooxygenase) is cultured and then reacted with the long chain fatty acid, It is a bioconversion process that produces compounds.
The term " bioconversion "of 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 biotransformation step is a step of converting a long-chain fatty acid into an ester compound by treating the long-chain fatty acid with an alcohol dehydrogenase to remove hydrogen to generate a keto acid derivative from the long chain fatty acid and treating the resulting keto acid derivative with BVMO As shown in FIG.
The term " alcohol dehydrogenase " of the present invention means an enzyme that catalyzes the reaction of removing hydrogen from alcohol to generate aldehyde or ketone.
In the present invention, the alcohol dehydrogenase may be used for the purpose of generating a keto acid derivative by removing hydrogen from the long chain fatty acid. The alcohol dehydrogenase may be, for example, one derived from a microorganism such as a micrococcus strain or a Pseudomonas sp. Strain.
The term " Baeyer-Villiger monooxygenase (BVMO) "of the present invention means a kind of monooxygenase which can catalyze various oxidation reactions including Baeyer-Villiger oxidation reaction in which a ketone is oxidized to produce a lactone or an ester compound . For the purpose of the present invention, an enzyme catalyzing a reaction for producing a fatty acid derivative (for example, 10-octyloxy-10-oxodecanoic acid from 10-chitostearic acid) expressed in a transformant and having an ester group introduced into the chain from chitosan fatty acid As long as it exhibits activity, the BVMO is not particularly limited, but preferably Pseudomonas sp., Rhodococcus sp. Brevibacterium sp., Comanonas sp., Acinetobacter sp., Arthrobacter sp., And Brkimonas sp. Strains. The strains of Brevibacterium sp., Comanonas sp., Acinetobacter sp. And BVMO derived from microorganisms such as Brachymonas sp., And more preferably Pseudomonas sp. fluorescens), Pseudomonas footage is (Pseudomonas putida , Pseudomonas veronii , Rhodococcus jostii ) or BVMO derived from Pseudomonas sp. strain HI-70, and most preferably BVMO derived from Pseudomonas putida. The nucleotide sequence of the gene encoding BVMO can be obtained from a known database such as NCBI's GenBank. A gene represented by CAFK01000010, a gene obtainable from an expression vector pJOE-KT2440BVMO (Biotechnol. Lett., 29: 1393-1398, 2007) prepared to express the BVMO gene, and the like.
The term "transformant " of the present invention means a cell or microorganism that has been transformed into a polynucleotide encoding a target protein by introducing the polynucleotide into a host using a vector and then expressing the target protein. At this time, the polynucleotide introduced into the host cell may be in any form as long as it can be introduced into the host cell and expressed.
The transformant provided in the present invention can be produced by introducing an expression vector containing a gene encoding each enzyme into a host cell so as to express a known alcohol dehydrogenase and BVMO. In this case, the host cell that can be used is not particularly limited. As an example, a culturable single-celled prokaryotic or eukaryotic cell suitable for application to the bioconversion process may be used, and E. coli, yeast, etc., As another example, Escherichia coli BL21 (DE3) cells can be used.
The long chain fatty acid used in the bioconversion process may be a fatty acid composed of 16 to 20 carbon atoms and the final heavy chain fatty acid to be produced may be a fatty acid composed of 6 to 14 carbon atoms, May also be used. In the case where the long chain fatty acid does not have a hydroxyl group, a gene encoding a hydratase may be further introduced to form a hydroxy fatty acid from the fatty acid, which can then be used as a substrate.
The term "cultivation" of the present invention means a process of growing microorganisms under an appropriately artificially controlled environmental condition.
In the present invention, the culturing can be understood as a process for growing the transformant to carry out a biotransformation process. Specifically, the culturing can be performed in a batch or continuous manner through a batch process, an implantation batch process, .
The culture medium used for the above cultivation should meet the requirements of a specific strain in an appropriate manner while controlling the temperature, pH and the like under aerobic conditions in an ordinary medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, and the like. Carbon sources that may be used include sugars, fats, fatty acids and glycerol such as glucose, sucrose, and lactose. 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 phosphate; Amino acids such as glutamic acid, and organic peptides such as peptone, meat extract, yeast extract, malt extract, corn steep liquor, and casein hydrolyzate. These nitrogen sources may be used alone or in combination.
The method may further comprise the step of injecting oxygen or an oxygen-containing gas (e.g., air) into the culture to maintain the aerobic state during the culture, and the temperature of the culture may be in the range of 27 to 37 Lt; 0 > C, and the incubation time can be 10 to 100 hours.
(b) chemical processes
The second step of the method for producing the heavy chain fatty acid from the long chain fatty acid provided by the present invention is a chemical process for producing the heavy chain fatty acid by performing a chemical reaction on the ester compound produced in the first biological conversion step.
As an example, the chemical reaction performed on the ester compound may be a hydrolysis reaction.
When the hydrolysis reaction is performed, H 2 O is added to the ester group to be cleaved to produce a medium chain fatty acid and a carboxylic acid in the form of an omega -hydroxy fatty acid.
In addition, when the oxidation reaction is further performed on the produced omega -hydroxy fatty acid, a dicarboxylic acid type heavy chain fatty acid can be produced.
As another example, a chemical reaction performed on the ester compound may be a methanol decomposition reaction.
When the methanol decomposition reaction is performed, methanol is added to the ester group to be cleaved to produce an omega hydroxy fatty acid type heavy chain fatty acid and methyl carboxylate.
According to an embodiment of the present invention, there is provided a process for producing a (Z) -11- (heptanoyloxy) undec-9-enoic acid which is an unsaturated ester compound by applying ricinoleic acid, The unsaturated ester compound was hydrogenated to produce a saturated ester compound, 11- (heptanoyloxy) undecanoic acid. The saturated ester compound was hydrolyzed to obtain an omega-hydroxy fatty acid, 11-hydroxyindecanoic acid, and a carboxylic acid, n- Heptanoic acid was obtained, and the omega-hydroxy fatty acid was oxidized to prepare dicarboxylic acid, undecanediiodic acid. Further, the ester compound (Z) -11- (heptanoyloxy) undec-9-enoic acid was subjected to methanolysis to obtain (Z) -11-hydroxyindec- Methylheptanoate was prepared.
According to another embodiment of the present invention, 10-hydroxystearic acid, a kind of long-chain fatty acid, is applied to a bioconversion process to produce a saturated ester compound, 9- (nonanoyloxy) nonanoic acid, The compound was hydrolyzed to obtain an omega -hydroxy fatty acid, 9-hydroxynonanoic acid, and the obtained omega-hydroxy fatty acid was oxidized to prepare dicarboxylic acid, azelaic acid. In addition, the above saturated ester compound, 9- (nonanoyloxy) nonanoic acid, was decomposed by methanol to prepare omega-hydroxy fatty acid and methylnonanoate as methyl carboxylate.
According to the method for producing a heavy chain fatty acid according to the present invention, it is possible to partially reduce the manufacturing cost by partially using the conventional biological conversion process, and to partially maximize the final production concentration and productivity of the heavy chain fatty acid using a chemical process , Which can be used to produce more economical heavy chain fatty acids.
1 is a schematic view showing a progress of a bioconversion process for producing an omega -hydroxy fatty acid, which is a high-value heavy chain fatty acid, from a long chain fatty acid, ricinoleic acid, by a bioconversion step, wherein 1 represents ricinoleic acid, Represents a keto acid derivative, 3 represents an ester compound, 4 represents an omega -hydroxy fatty acid, and 5 represents a carboxylic acid.
2 is a schematic view showing the progress of a step of producing a dicarboxylic acid from an ester compound by a chemical reaction, wherein 3 is an ester compound, 5 is n-heptanoic acid, 6 is a saturated ester compound, 7 represents omega-hydroxy fatty acid, and 8 represents dicarboxylic acid.
3 is a schematic diagram showing the progress of a process for producing methyl carboxylate and omega -hydroxy fatty acid from an ester compound by a chemical reaction, wherein 3 represents an ester compound, 4 represents an omega -hydroxy fatty acid, 9 Represents methyl carboxylate.
FIG. 4 is a graph showing the results of comparing concentrations of ricinoleic acid, a keto acid derivative, and an ester compound with time in a biological conversion process when an ester compound is produced from ricinoleic acid through a biological conversion process.
FIG. 5A is a graph showing the results of culturing a secondary transformant at a cell concentration of 10 g per unit volume of culture medium (l), and then treating the culture with 30 mM ricinoleic acid and performing a biotransformation process for 8 hours .
FIG. 5B is a graph showing the results of culturing a second transformant at a cell concentration of 20 g per unit volume of culture medium (l), treating 63 mM ricinoleic acid in the culture medium, and performing a biotransformation process for 8 hours .
6a shows the result of culturing a second transformant at a cell concentration of 3 g per unit volume (L) of the culture medium, treating the culture with 5 mM of 10-hydroxystearic acid, and performing a biotransformation process for 12 hours FIG.
6B shows the result of culturing the second transformant at a cell concentration of 10 g per unit volume of culture medium (l), treating 30 mM 10-hydroxystearic acid in the culture medium, and performing a biotransformation process for 15 hours FIG.
FIG. 7 is a graph showing changes in the content of the reaction product with time in the bioconversion process for producing 9- (nonanoyloxy) nonanoic acid from oleic acid by the bioconversion step. FIG.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
Example
One:
From ricinoleic acid
n-heptanoic acid (n-
heptanoic
acid and (Z) -11-hydroxy undec-9-enoic acid ((Z) -11-
hydroxyundec
-9-
enoic
acid production
When the long-chain fatty acid is treated in the culture medium after the cultivation of Escherichia coli transformed to express alcohol dehydrogenase, BVMO (Baeyer-Villiger monooxygenase) and estrase, the long-chain fatty acid is absorbed and the long chain fatty acid is absorbed by the alcohol dehydrogenase The resulting keto-acid derivative is converted to an ester compound by BVMO, and the ester compound is cleaved by esterase, resulting in a bioconversion process in which an omega-hydroxy fatty acid is produced. (Korean Patent Publication No. 2013-0132254) (Fig. 1). 1 is a schematic view showing a progress of a bioconversion process for producing an omega -hydroxy fatty acid, which is a high-value heavy chain fatty acid, from a long chain fatty acid, ricinoleic acid, by a bioconversion step, wherein 1 represents ricinoleic acid, Represents a keto acid derivative, 3 represents an ester compound, 4 represents an omega -hydroxy fatty acid, and 5 represents a carboxylic acid.
However, since the omega -hydroxy fatty acids and carboxylic acids are toxic to the transformed E. coli, the production of omega -hydroxy fatty acids and the productivity are lowered (Process Biochemistry 49 (2014) 617-622) The present inventors have found that a method for producing an ester compound at a maximum concentration by a biological conversion process and converting the produced ester compound into an omega -hydroxy fatty acid by a chemical process, thereby improving the production concentration and productivity of the omega -hydroxy fatty acid .
In addition, the produced ester compound was chemically reacted to produce a dicarboxylic acid, a carboxylic acid, or an omega-hydroxy fatty acid (FIGS. 2 and 3).
2 is a schematic view showing the progress of a step of producing a dicarboxylic acid from an ester compound by a chemical reaction, wherein 3 is an ester compound, 5 is n-heptanoic acid, 6 is a saturated ester compound, 7 represents omega-hydroxy fatty acid, and 8 represents dicarboxylic acid.
3 is a schematic diagram showing the progress of a process for producing methyl carboxylate and omega -hydroxy fatty acid from an ester compound by a chemical reaction, wherein 3 represents an ester compound, 4 represents an omega -hydroxy fatty acid, 9 Represents methyl carboxylate.
As an example of the above-described method, the present inventors have produced (Z) -11-hydroxy undec-9-enoic acid which is an omega-hydroxy fatty acid and dicarboxylic acid, dicarboxylic acid, from ricinoleic acid, Was performed as follows.
Example 1-1: Production of ester compounds using bioconversion
According to a known method (Korean Patent Laid-Open Publication No. 2013-0132254), pACYC-ADH, an expression vector containing an alcohol dehydrogenase gene, was introduced into Escherichia coli BL21 (DE3) to perform primary transformation , And pJOE-BVMO, which is an expression vector containing the BVMO gene, was introduced into the primary transformant thus prepared to prepare a secondary transformant capable of expressing the alcohol dehydrogenase and BVMO.
The thus-prepared secondary transformants were cultured in LB medium (5 g / l yeast extract, 10 g / l tryptone and 10 g / l NaCl) containing ampicillin and chloramphenicol to obtain cultures. Cells were obtained from the cultures and then cultured in a regenberg medium (4 g / l (NH 4 ) 2 HPO 4 , 13.5 g / l KH 2 PO 4 containing 10 g / l glucose and antibiotics (ampicillin and chloramphenicol) , 1.7 g / ℓ of citric acid, 1.4 g / ℓ MgSO 4 and 10 mL / ℓ metal solution (10 g / ℓ FeSO 4, 2.25 g /
Then, 15 mM ricinoleic acid and 0.1
After the biotransformation was performed, the ester compound (3 in Fig. 1) was recovered from the cultured secondary transformant. Specifically, the culture transformed with the biotransformation was centrifuged to obtain cells, and the process of extracting the cells with ethylacetate was repeated to obtain an ethyl acetate extract. The ethyl acetate extract was mixed with an aqueous solution of sodium chloride, and then an organic solvent layer was obtained. Sodium sulfate was added to the obtained organic solvent layer, which was filtered and the solvent was evaporated to recover the ester compound.
After the completion of the culture, the concentration of the long chain fatty acid (ricinoleic acid) remaining in the culture medium and the concentration of the keto acid derivative (12-keto octadec-9-enoic acid) and the ester compound ((Z ) -11- (heptanoyloxy) undec-9-enoic acid) was measured by gas chromatography / mass spectrometry (GC / MS).
Approximately, each TMS derivative is obtained by adding TMS (N-methyl-N- (trimethylsilyl) trifluoroacetamide) to the ricinoleic acid, the intermediate compound or the ester compound contained in the culture medium or the ethyl acetate extract, The TMS derivatives were analyzed using an ion trap mass detector (Thermo ITQ1100GC-ion Trap MS, Thermo Scientific) and Thermo Ultra equipped with a non-polar capillary column (30 m length, 0.25 μm film thickness, HP-5MS, Agilent Technologies Palo Alto, Calif. GC system and then measured at a linear temperature gradient (degree of change) of 5 DEG C / min and an injection port temperature of 230 DEG C in the range of 90 to 280 DEG C, and the measured scan spectrum was measured at a range of 100-600 m / z (Fig. 4).
FIG. 4 is a graph showing the results of comparing concentrations of ricinoleic acid, a keto acid derivative, and an ester compound with time in a biological conversion process when an ester compound is produced from ricinoleic acid through a biological conversion process. As shown in FIG. 4, the biotransformation process was performed for 12 hours. As a result, it was confirmed that an ester compound of 11 mM was generated from 15 mM ricinoleic acid to show a bioconversion yield of about 73%. It was confirmed that the bioconversion process was successfully performed.
Example
1-2: Transformants
The cell concentration
Effect of ester compound on final product concentration and productivity
At the time of culturing the secondary E. coli transformant, the cells were cultured at a cell concentration of 10 g or 20 g in dry weight per unit volume (ℓ) of the final cultured cells, and 30 mM or 63 mM of ricinoleic acid, respectively, And the biotransformation process was performed for 8 hours, the respective ester compounds were produced and compared (Fig. 5 (a) and (b)) using the same method as in Example 1-1.
FIG. 5A is a graph showing the results of culturing a secondary transformant at a cell concentration of 10 g per unit volume of culture medium (l), treating 30 mM rhizinoleic acid in a culture medium, and performing a biotransformation process for 8 hours , FIG. 5B is a graph showing the results of culturing the second transformant at a cell concentration of 20 g per unit volume (L) of the culture medium, treating 63 mM of ricinoleic acid in the culture medium, and performing a biotransformation process for 8 hours to be.
As shown in FIG. 5A, when a secondary transformant cultured at a cell concentration of 10 g / L was used, 27 mM of an ester compound was generated from 30 mM ricinoleic acid, and a bioconversion yield of about 90% h, and as shown in FIG. 5b, when a secondary transformant cultured at a cell concentration of 20 g / L was used, 54 mM of the ester compound was generated from 63 mM ricinoleic acid and about 86% It was confirmed that the conversion yield was 6.6 mM / h.
Therefore, it was found that as the cell concentration of the transformant was increased, the production concentration and productivity of the ester compound could be increased.
Example
1-3: Dicarboxylic acids and omega-hydroxides from ester compounds by chemical processes
Shiji
produce
Example
1-3-1: Purification of ester compounds
First, (Z) -11- (heptanoyloxy) undec-9-enoic acid (Z-11- (Heptanoyloxy) undec-9-enoic acid) which was an ester compound recovered in Example 1-1 was dissolved in silica gel , And purified by elution using a mixed solvent of 20% ethyl acetate and hexane.
The NMR analysis results of the purified ester compound are as follows.
(M, 2H), 1.65 (d, J = 6.0 Hz, 2H) (75 MHz, CDCl 3): δ 178.2, 172.0, 133.4, 121.6, 58.3, 32.5, 32.2, 29.5, 27.4 (m, 4H) , 27.2, 27.1, 27.0, 26.9, 25.6, 23.0, 22.7, 20.6, 12.1.
Example 1-3-2: Production of dicarboxylic acid from ester compound through multistage reaction
Ester compounds were subjected to hydrogenation, hydrolysis and oxidation in order to produce dicarboxylic acids.
Example 1-3-2-1: Hydrogenation reaction
A solution obtained by dissolving 1.0 g of the ester compound purified in Example 1-3-1 in 10 ml of methanol was obtained. The solution was added to the filter paper and 0.4 g of hydrated Raney-Ni was added thereto. Subsequently, hydrogen was added to the ester compound by adding 1 atm of hydrogen for 8 hours to convert it to a saturated ester compound, and the reaction mixture was filtered through celite and washed with methanol. The filtrate was evaporated to give 11- (heptanoyloxy) undecanoic acid (0.95 g, 95%) as a saturated, ester compound showing a semi-solid yellow color.
The results of NMR analysis of the purified saturated ester compound are as follows.
1 H NMR (300 MHz, CDCl 3)? 4.05 (t, J = 6.6 Hz, 2H), 2.31-2.26 (m, 4H), 1.61-1.59 (m, 6H), 1.28-1.25 t, J = 6.0 Hz, 3H); 13C NMR (75 MHz, CDCl3) 灌 174.09 (2C), 64.38, 34.41, 31.45, 29.71, 29.40, 29.31, 29.18, 29.03, 28.81, 28.61, 25.89, 24.97, 24.65, 22.47, 14.01
Example
1-3-2-2: Hydrolysis reaction
First, a reaction solution in which 1 N NaOH was dissolved in a mixed solvent containing methanol and distilled water at a ratio of 4: 1 (v / v) was obtained. To 25 ml of the reaction solution obtained above, , 950 mg of the purified saturated ester compound was added to conduct hydrolysis reaction to obtain a hydrolysis product, 11-hydroxyundecanoic acid, which is an omega-hydroxy fatty acid, and n-heptanoic acid, ). After the reaction was completed, the reaction solution was cooled, 6N HCl was slowly added thereto to adjust the pH to 2, and the solvent was evaporated to adjust the volume of the reaction solution to 5 ml. Then, NaCl was added to the reaction solution to saturate, and the mixture was extracted three times with 30 ml of ethyl acetate. The extract was washed with 20 ml of NaCl solution and 20 ml of distilled water, dried by adding anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain a sample containing the heavy chain fatty acid. The obtained sample was applied to column chromatography using silica gel and eluted using a mixed solvent of 50% ethyl acetate and hexane to obtain 11-hydroxyindecanoic acid (550 mg, 90%) as an omega -hydroxy fatty acid, .
NMR analysis of the purified omega-hydroxy fatty acid is as follows.
2H), 1.64-1.51 (m, 4H), 1.35-1.25 (m, 12H), 2.32 (t, J = 7.5 Hz, 2H) ; 13C NMR (75 MHz, CDCl3) 灌 179.47, 62.96, 34.01, 32.60, 29.41, 29.28, 29.25, 29.12, 28.96, 25.63, 24.63
Example
1-3-2-3: Oxidation reaction
The omega -hydroxy fatty acid solution was obtained by dissolving 470 mg of the omega-hydroxy fatty acid purified in Example 1-3-2-2 in a mixed solvent containing acetonitrile and distilled water at a ratio of 3: 1 (v / v) 13 ml of H 5 IO 6 / CrO 3 solution (2.5 equivalents of H 5 IO 6 and 0.01 equivalent of CrO 3 ) was added to the obtained solution, and the oxidation reaction was carried out at 5 ° C. for 30 minutes. After completion of the reaction, the reaction was allowed to stand at 0 ° C for 45 minutes, and an aqueous Na 2 HPO 4 solution was added to terminate the reaction. 15 ml of NaCl solution, 15 ml of 5% NaHSO 3 solution and 15 ml of NaCl solution were sequentially added to the obtained extract, followed by washing Respectively. Subsequently, anhydrous sodium sulfate was added and dried, followed by concentration to obtain a sample containing a dicarboxylic acid. The sample was recrystallized in a mixed solvent of ethyl acetate and hexane to give undecanedioic acid 450 (dicarboxylic acid) mg, 90%) was purified
NMR analysis results of the purified dicarboxylic acid are as follows.
1H NMR (300 MHz, DMSO-d6)? 2.17 (t, J = 6.6 Hz, 4H), 1.49-1.44 (m, 4H), 1.23 (m, 10H); 13C NMR (75 MHz, DMSO-d6)? 174.90 (2C), 34.09 (2C), 29.23, 29.14 (2C), 28.98 (2C), 24.92 (2C).
Example 1-3-3: Production of dicarboxylic acid from ester compound by simplified process
Since the reaction process described in Example 1-3-2 is carried out by a purification process using column chromatography every time, there is a problem in that it takes a lot of time and cost for the production of dicarboxylic acid, so that it is suitable for industrial production Did not do it.
Thus, it was attempted to produce dicarboxylic acid from the ester compound more easily by simplifying the process.
That is, the ester compound obtained in Example 1-1 was applied to the hydrogenation reaction of Example 1-3-2-1, and the reaction solvent, methanol, was evaporated to reduce the volume of the reaction solution . Subsequently, 1 N aqueous sodium hydroxide solution was added to the reaction solution, and the hydrolysis reaction was carried out at 60 DEG C for 2.5 hours. After completion of the hydrolysis reaction, hydrochloric acid was added to the reaction solution to titrate the reaction mixture to
When the multistage process of Example 1-3-3 is performed, the dicarboxylic acid can be produced at a yield of about 80%, while when the process is simplified, the yield of dicarboxylic acid Since the acid can be produced, the multistage process is effective in terms of the yield rate of the final product, while the process simplification method which can reduce the cost and time required for the process produces the dicarboxylic acid It was analyzed that the process simplification method could be used for industrial production.
Example
1-3-4: Omega-Hydroxyl from ester compound
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0.35 g of the ester compounds obtained in Examples 1-1 and 1-2 were dissolved in 8 ml of methanol, 28 mg of dibutyltin oxide was added, and the mixture was heated at reflux for 12 hours Methanol decomposition reaction was carried out. After the completion of the reaction, the reaction product obtained from the reaction and a saturated solution of sodium hydrogencarbonate were mixed to obtain a mixture, and the mixture was extracted with ethyl acetate. The extract was filtered with celite to obtain a dibutyltin compound, which was then dried by adding sodium sulfate, and then filtered again. The solvent was then removed from the filtrate under vacuum and methyl heptanoate (136.5 mg, 85%) was obtained via fractional distillation. Omega-hydroxy fatty acid (Z) -11-hydroxyundec-9-enoic acid (206 mg, 92%) was purified.
NMR analysis results of the methylheptanoate and omega-hydroxy fatty acid are as follows.
Methylheptanoate
2H), 1.30-1.17 (m, 6H), 0.82 (t, 3H), 2.24 (t, J = 7.5 Hz, 2H) J = 6.0 Hz, 3H); ≪ 13 > C NMR (75 MHz, CDCl3) [delta] 174.16, 51.28, 34.01, 31.39, 28.76, 24.85, 22.41, 13.89.
(Z) -11-hydroxy unedec-9-enoic acid
(M, 2H), 4.19 (d, J = 6 Hz, 2H), 2.33 (t, J = 7.5 Hz, 2H) , 2.09-2.02 (m, 2H), 1.65-1.57 (m, 2H), 1.38-1.28 (m, 8H); 13 C NMR (75 MHz, CDCl 3)? 179.38, 133.07, 128.14, 58.40, 34.04, 29.42, 28.97, 28.91, 28.88, 27.31, 24.63.
Example
2: Omega-
Hydroxynonanoic acid
(Omega-Hydroxynonanoic Acid) and alpha, omega-
Norenadeioi
The acid (?,? -
Nonanedioic
Acid) production
As another example of the method described in Example 1, the inventors of the present invention have found that, from oleic acid, which is a kind of long-chain fatty acid, or 10-hydroxystearic acid derived therefrom, azelaic acid, a dicarboxylic acid, and 9-hydroxynonanoic acid, an omega- The process for producing the liquor acid was carried out as follows.
Example
2-1: Production of ester compounds using bioconversion
According to the method of Example 1-1, the resulting alcohol dehydrogenase and a secondary transformant expressing BVMO were inoculated into a Regenberg medium and cultured. To the resulting culture, 5 mM 10-hydroxystearic acid ( 10-hydroxystearic acid) and 0.5 g / l of Tween 80 were added to the reaction mixture to conduct a bioconversion step. The ester compound 9- (nonanoyloxy) nonanoyloxyacetic acid (9- nonanoic acid. At this time, it was confirmed that 10-keto stearic acid was produced as a keto acid derivative.
Meanwhile, as in Example 1-2, in order to ascertain whether increasing the cell concentration of the transformant can increase the bioconversion yield of the ester compound, it was found that when the secondary transformant was cultured, The cultured cells were cultured at a cell concentration of 3 g or 10 g in terms of dry weight per unit volume (ℓ) of the culture medium, and 10 mM of hydroxystearic acid of 5 mM or 30 mM, respectively, The respective ester compounds were produced and compared using the same method as in Example 1-1 except that the conversion process was performed (Figs. 6A and 6B).
6a shows the result of culturing a second transformant at a cell concentration of 3 g per unit volume (L) of the culture medium, treating the culture with 5 mM of 10-hydroxystearic acid, and performing a biotransformation process for 12 hours FIG. 6B is a graph showing the results of cultivation of a secondary transformant at a cell concentration of 10 g per unit volume of medium (l), followed by treatment with 30 mM 10-hydroxystearic acid in a culture medium and biotransformation step for 15 hours And FIG.
As shown in FIG. 6A, when a secondary transformant cultured at a cell concentration of 3 g / L was used, 4.1 mM of 9- (nonanoyloxy) nonanoic acid from 5 mM 10-hydroxystearic acid As shown in FIG. 6B, when a secondary transformant cultured at a cell concentration of 10 g / L was used, it was found that 30 mM of 10-hydroxystearic acid was converted into 21 mM 9- (nonanoyloxy) nonanoic acid was produced and it was confirmed that the yield of bioconversion was about 70%.
Example 2-2: Production of ester compound using oleic acid
The rate of bioconversion of 10-hydroxystearic acid in Example 2-1 was significantly lower. One of the reasons for this is that 10-hydroxystearic acid is present as a solid powder and is very low in water solubility and is low in the rate of migration into cells. Therefore, bioconversion was performed using oleic acid, which is an unsaturated fatty acid present in a liquid phase, as a substrate in order to increase the rate of biological conversion. Hydroxy fatty acid, 10-hydroxystearic acid, can be produced by treating the oleic acid with a hydrating enzyme. Thus, the expression vector pET-OhyA containing the gene encoding the secondary transformant and the oleosin hydrolase obtained in Example 1-1 was introduced to transform the transformed Escherichia coli capable of expressing the oleosin hydrolase 10-hydroxystearic acid (10-HSA), 10-keto-stearic acid (10-hydroxystearic acid) and the like were added to the culture, (10-KSA) and 9- (nonanoyloxy) nonanoic acid ester were measured (Fig. 7).
FIG. 7 is a graph showing changes in the content of the reaction product with time in the bioconversion process for producing 9- (nonanoyloxy) nonanoic acid from oleic acid by the bioconversion step. FIG. As shown in FIG. 7, oleic acid was consumed at the end of 3 hours, and the content of 10-hydroxystearic acid rapidly increased until 3 hours had elapsed. However, the content of 10-hydroxystearic acid was steadily decreased after that, Acids were detected at the end of 3 hours, but were measured in trace amounts. On the other hand, the content of 9- (nonanoyloxy) nonanoic acid increased steadily after the initiation of the bioconversion process, and it was confirmed that the content was maintained at a constant level after 9 hours had elapsed.
Compared with the result shown in FIG. 6B, when the medium was treated with the same amount of oleic acid or 10-hydroxystearic acid, the production rate of the ester compound was remarkably high when oleic acid was treated Could know.
Example
2-3: Dicarboxylic acids and omega-hydroxides from ester compounds by chemical processes
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Example
2-3-1: Purification of ester compounds
First, 9- (nonanoyloxy) nonanoic acid, an ester compound obtained in Example 2-1, was purified by the method of Example 1-3-1.
The NMR analysis results of the purified ester compound are as follows.
2H), 2.40-2.26 (m, 4H), 1.63-1.59 (m, 6H), 1.32-1.26 (m, 18H), 0.88 (m, t, J = 6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 179.17, 174.09, 64.34, 34.41, 33.85, 31.85, 31.81, 29.23, 29.14, 29.11, 29.05, 29.03, 28.95, 28.61, 25.86, 25.02, 22.65, 14.10.
Example 2-3-2: Production of dicarboxylic acid from ester compound through multistage reaction
Ester compounds were hydrolyzed and oxidized sequentially to produce dicarboxylic acid.
Example
2-3-2-1: Hydrolysis reaction
First, a reaction solution in which 1 N NaOH was dissolved in a mixed solvent containing methanol and distilled water at a ratio of 4: 1 (v / v) was obtained. To 10 ml of the reaction solution obtained above, Hydroxynonanoic acid (9-hydroxynonanoic acid) and carboxylic acid (n-butyl ester) as hydrolysis products at 60 ° C for 3 hours, Nonanoic acid was obtained. After completion of the reaction, the reaction solution was cooled, 6N HCl was slowly added thereto to adjust the pH to 2, and the solvent was evaporated to adjust the volume of the reaction solution to 2 ml. Then, 5 ml of distilled water was mixed with the reaction solution and diluted. Then, NaCl was added to saturate the reaction solution, and 15 ml of ethyl acetate was added thereto for extraction three times. The extract was washed with 10 ml of NaCl solution and 10 ml of distilled water, dried by adding anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain a sample containing the omega -hydroxy fatty acid. The obtained sample was applied to column chromatography using silica gel and eluted using a mixed solvent of 50% ethyl acetate and hexane to obtain 9-hydroxy nano-nano acids (59 mg, 85%) as an omega -hydroxy fatty acid, .
NMR analysis of the purified omega-hydroxy fatty acid is as follows.
J = 7.5 Hz, 2H), 1.66-1.52 (m, 4H), 1.38-1.32 (m, 8H) ; ≪ 13 > C NMR (75 MHz, CDCl3) [delta] 178.85, 63.03, 33.83, 32.68, 29.14 (2C), 28.93, 25.61, 24.63.
Example
2-3-2-2: Oxidation reaction
100 mg of the omega -hydroxy fatty acid purified in Example 1-3-2-2 was dissolved in a mixed solvent containing acetonitrile and distilled water at a ratio of 3: 1 (v / v) to obtain an omega -hydroxy
NMR analysis results of the purified dicarboxylic acid are as follows.
1 H NMR (300 MHz, DMSO-d 6)? 2.17 (t, J = 7.5 Hz, 4H), 1.47 (m, 4H), 1.24 (m, 6H); 13 C NMR (75 MHz, DMSO-d 6)? 174.95 (2C), 34.07 (2C), 28.90, 28.86 (2C), 24.89 (2C).
Example 2-3-3: Production of dicarboxylic acid from ester compound by simplified process
The process for producing dicarboxylic acid from the ester compound was made easier by simplifying the process as in the case of Example 1-3-3.
That is, the ester compound was recovered after the bioconversion reaction as in the case of Example 1-1 or 2-1, and was added to a methanol / distilled
Example
2-3-4: Omega-Hydroxyl from ester compound
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150 mg of the ester compound obtained in Example 2-1 was dissolved in 5 ml of methanol, 20 mg of dibutyltin oxide was added, and the reaction solution was heated at reflux for 12 hours to carry out methanolysis . After the completion of the reaction, the reaction product obtained from the reaction and a saturated solution of sodium hydrogencarbonate were mixed to obtain a mixture, and the mixture was extracted with ethyl acetate. The extract was filtered with celite to obtain a dibutyltin compound, which was then dried by adding sodium sulfate, and then filtered again. The solvent was then removed from the filtrate using vacuum conditions and methylnonanoate (110 mg, 80%) and 9-hydroxynonanoic acid were obtained via reduced pressure distillation.
The results of NMR analysis of the 9-hydroxynonanoic acid are the same as those described in Example 2-3-2-1, and NMR analysis results of methylnonanoate are as follows.
Methylnonanoate
2H), 1.60-1.56 (m, 2H), 1.29-1.20 (m, 10H), 0.84 (t, J = 6.0 Hz, 3H); 13 C NMR (75 MHz, CDCl 3)? 173.84, 51.03, 33.83, 31.68, 29.10, 29.02, 29.00, 24.79, 22.49, 13.82.
Claims (15)
(b) a step of subjecting the resulting ester compound to a chemical reaction to produce a heavy chain fatty acid,
Wherein the chemical reaction is a hydrogenation reaction, an oxidation reaction, or a methanolysis reaction, and the long chain fatty acid is lysinolic acid.
A method for producing a medium chain fatty acid from a long chain fatty acid.
wherein the alcohol dehydrogenase in step (a) is derived from a strain of the genus Micrococcus or a strain belonging to the genus Pseudomonas.
wherein said BVMO of step (a) is selected from the group consisting of Pseudomonas sp., Rhodococcus sp. Wherein the strain is derived from a strain of the genus Brevibacterium, a strain of the genus Comany, a genus of the genus Ashtona, a genus of the genus Arthrobacter, or a genus of the genus of the genus Brcky.
wherein the transformant of step (a) further comprises a gene selected from the group consisting of a gene encoding lipase, a gene encoding dehydratase, and combinations thereof.
Wherein said heavy chain fatty acid is a fatty acid having 6 to 14 carbon atoms.
Wherein said heavy chain fatty acid is an omega-hydroxy fatty acid.
Wherein the omega -hydroxy fatty acid is (Z) -11-hydroxy undec-9-enoic acid.
Wherein a methyl carboxylate is additionally produced in addition to the heavy chain fatty acid.
Wherein said methyl carboxylate is methyl heptenoate.
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