KR101275855B1 - Oleate hydratase and method for the production of 10-hydroxystearic acid by the same - Google Patents

Abstract

The present invention relates to a production method of 10-hydroxystearic acid (10-Hydroxystearic acid), more specifically Lysinibacillus fusiformis ) is a method of producing 10-hydroxystearic acid in high yield by optimizing the reaction conditions of oleate hydratase derived from bacteria and by reacting oleate under the optimized conditions.
According to the present invention, 10-hydroxystearic acid, which is a raw material of an industrial product, can be produced in high yield with a high specificity and an environmentally friendly method, and the 10-hydroxystearic acid thus produced is useful as a starting material for the synthesis of lactone. Can be used.

Description

Oleate hydratase and method for the production of 10-hydroxystearic acid by the same}

The present invention relates to a method for preparing 10-hydroxystearic acid using oleate hydratase, and more particularly, to an enzyme for converting from oleic acid to 10-hydroxystearic acid. And a method for producing 10-hydroxystearic acid in high yield using the same.

Hydroxy fatty acids are naturally occurring substances found in triglycerols, waxes, cerebrosides and other lipids present in plants, insects and microorganisms. Hydroxy fatty acids, in which microorganisms and enzymes produce unsaturated fatty acids through biological conversion, are of three types: monohydroxy, dihydroxy and trihydroxy fatty acids. Hydroxy fatty acids are important industrial materials as precursors of lactones that make up fragrances and are used as starting materials for resins, waxes, nylons, plastics, lubricants and coatings because they are more reactive than non-hydroxy fatty acids. It is also used as a cosmetic ingredient.

10-hydroxystearic acid is an important substance that produces sebacic acid, which is used as a lubricant and used to synthesize plasticizers and resins. Hydroxy fatty acids, such as 10-hydroxystearic acid, are converted to gamma-decalactone by yeast. Gamma-dekaralactone is characterized by a peach or apricot flavor and is essential for food processing and is increasingly used in the food industry. However, since these substances are present in a small amount in some fruits, they are synthesized by organic synthesis.However, due to the increase in national income and the demand of natural foods and fragrances, and the high quality of consumer goods, natural substances are stable and highly There is an absolute need for methods using biotransformation as the preferred trend in.

Microorganisms producing 10-hydroxystearic acid include Flavobacterium (sp. NRRL B-14859), Nocardia cholesterolicum , and runimal bacteria. bacteria ) and enzymes, such as hydratase, but these microorganisms and enzymes produce 10-hydroxystearic acid in low yields, and by-products such as 10-kedostearic acid are also uneconomical for industrialization. In addition, the case of using a recombinant microorganism containing enzymes or related enzymes to increase the productivity has not been reported so far.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned problems and the need for the above-described problem, and an object of the present invention is to provide a manufacturing method for producing high-efficiency 10-hydroxystearic acid.

It is an object of the present invention to provide a composition for producing high efficiency 10-hydroxystearic acid.

In order to achieve the above object, the present invention provides a composition for preparing 10-hydroxystearic acid (10-hydroxystearic acid) comprising an oleic acid hydratase having an amino acid sequence of SEQ ID NO: 1 and an organic solvent.

In one embodiment of the present invention, the enzyme is preferably derived from Lysinibacillus fusiformis , but is not limited thereto.

In another aspect, the present invention provides a composition for preparing 10-hydroxystearic acid (10-hydroxystearic acid) comprising a gene and an organic solvent encoding the oleic acid hydrase having a nucleotide sequence of SEQ ID NO: 2 as an active ingredient.

The present invention also provides a method for producing 10-hydroxystearic acid by bioconversion using the enzyme and substrate of claim 1 to produce hydroxy fatty acids from unsaturated fatty acids.

In one embodiment of the invention, the substrate is preferably, but not limited to oleic acid (oleic acid).

In a preferred embodiment of the present invention, the method is preferably carried out in the presence of ethanol as an organic solvent, but is not limited thereto.

In another preferred embodiment of the present invention, the method is preferably performed at a pH of 6.0 to 7.0 range, a temperature of 25 ℃ to 35 ℃ range, but is not limited thereto.

Hereinafter, the present invention will be described.

The present invention makes it possible to prepare 10-hydroxystearic acid using oleate as a substrate. Lysinibacillus fusiformis ) provides oleic acid hydratase derived from the strain.

The present invention also provides a recombinant expression vector comprising oleic acid hydratase.

Specifically, the present invention Lysinibacillus fusiformis ) provides a recombinant expression vector pET 28 (+) a / oleate hydratase derived from the strain and comprising the oleic acid hydratase gene.

The present invention also provides a microorganism transformed with a recombinant expression vector comprising oleic acid hydratase. Lysinibacillus fusiformis ) provides E. coli ER 2566 pET 28 (+) a / oleate hydratase transformed with a recombinant expression vector comprising an oleic acid hydratase gene derived from a strain.

In the present invention, as long as the recombinant expression vector can be used for the production of 10-hydroxy stearic acid, any vector used in the gene recombination method including the expression vector pET-28a (+) can be used. As a microorganism to be transformed with E. coli ER 2566 is preferably used, any strain that can be transformed with a recombinant expression vector to overexpress the desired gene and produce an active enzyme protein.

In addition, the present invention (1) to produce a recombinant expression vector comprising the oleic acid hydrase gene; (2) culturing the microorganism transformed with the recombinant expression vector; (3) inducing the expression of the oleic acid hydratase gene (4) provides a recombinant protein expressed and a method for producing the enzyme including the same.

In the present invention, as long as the recombinant expression vector can be used for the production of 10-hydroxystearic acid, any vector used in the gene recombination method including the expression vector pET-28a (+) can be used. E. coli ER 2566 is preferably used as the microorganism to be transformed. However, any strain can be used as long as it can be transformed with a recombinant expression vector to overexpress the desired gene and produce an active enzyme protein.

Provided is a production method for obtaining 10-hydroxystearic acid in high yield using cultured oleic acid hydratase.

Specifically, the present invention provides a method for producing 10-hydroxystearic acid using the oleic acid hydratase capable of selectively synthesizing only 10-hydroxystearic acid without producing by-products.

The oleic acid hydrase is preferably used to induce the expression of the recombinant enzyme gene by culturing E. coli transformed with a recombinant expression vector containing the oleic acid hydrase gene as described above, and then recover the expressed protein.

It is also preferable to use fatty acids as the substrate, more preferably oleate. In addition, the concentration of the substrate is preferably in the range of 10 to 50 g / l, more preferably in the range of 30 g / l to 40 g / l.

In addition, the enzyme reaction is preferably made in the range of pH 5.0 to 8.0, more preferably in the range of pH 6.5.

In addition, the enzymatic reaction is preferably made in the temperature range of 20 to 40 ℃, more preferably in the temperature range of about 30 ℃.

In addition, the time of the said enzyme reaction can be suitably adjusted according to a conventional method.

Hereinafter, the present invention will be described.

The present inventors have tried to develop a method for producing 10-hydroxystearic acid more effectively using oleic acid hydratase, Lysinibacillus fusiformis ) and a large amount of oleic acid hydrase and recombinant strains derived from, and confirmed that it can produce 10-hydroxystearic acid in high yield without the production of by-products in this environmentally friendly process, and completed the present invention.

The present inventors investigated enzymes capable of converting oleic acid, which is abundant unsaturated fatty acids of plants into hydroxy fatty acids, and selected enzymes capable of producing 10-hydroxystearic acid in high yield without by-products.

Lysinibacillus fusiformis ) was used to construct the oleic acid hydrase gene and to use it to prepare a large amount of oleic hydrase. It confirmed and completed this invention.

The present invention has the effect of providing a production method for obtaining a high yield of 10-hydroxystearic acid using a recombinant expression vector, an expressed protein containing an oleic acid hydrase gene.

The oleic acid hydrase derived from Lysinibacillus fusiformis of the present invention can produce 10-hydroxystearic acid, a raw material of industrial products, in high yield in a high specificity and environmentally friendly method. -Hydroxystearic acid can be usefully used as a starting material for synthesis of various lactones.

Figure 1a shows the effect of the pH of the oleic acid hydrolase of the present invention on the 10-hydroxystearic acid production activity (●: MES buffer, ▲: PIPES buffer), Figure 1b is the temperature of the oleic acid hydrolase 10-hydroxy It shows the effect on the oxystearic acid production activity.
Figure 2a shows the type of the organic solvent in the production of 10-hydroxystearic acid of the oleic acid hydrase of the present invention, Figure 2b shows the results according to the concentration of the organic solvent.
Figure 3 shows the optimal enzyme concentration in producing 10-hydroxystearic acid of oleic acid hydratase of the present invention (●: 10-Hydroxystearic acid, ○: oleic acid), Figure 4 is the concentration of the substrate (●: 10-Hydroxystearic acid, ○: conversion rate).
Figure 5 shows the hourly production of 10-hydroxystearic acid using oleic acid hydrating enzyme derived from Lysinibacillus fusiformis. KCTC 3454 of the present invention. (●: 10-Hydroxystearic acid, ○: oleic acid)

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art of fatty acids that the scope of the present invention is not to be construed as being limited by these examples.

Example  1. Preparation of recombinant expression vector and protein containing oleic acid hydratase gene

In order to prepare the oleic acid hydratase gene of the present invention, The oleic acid hydratase gene derived from Lysinibacillus fusiformis strain was first isolated.

Specifically, the gene sequence and amino acid sequence is already specified Lysinibacillus fusiformis KCTC 3454 strain (obtained from Korea Research Institute of Bioscience and Biotechnology) (KCTC) was screened and primers based on the DNA sequence of oleic acid hydrase derived therefrom were selected. Forward primer): 5'- GCTAGC ATGTATTACAGTAATGGTAACTATGAA-3 '(SEQ ID NO: 3); Reverse (reverse primer): 5'-GG CTCGAG CTATATTAGTTTACTTTCTTTCA-3' (SEQ ID NO: 4)] And amplified the nucleotide sequence of the gene.

The oleic acid hydratase genes obtained in large quantities were inserted into the plasmid vector pET 28 (+) (manufactured by Novagen) using restriction enzymes Nhe and Xho to prepare pET 28 (+) a / oleic acid hydrase.

The recombinant expression vector obtained as described above was transformed into E. coli ER 2566 strain (obtained from Korea Biotechnology Research Institute (KCTC)) by a conventional transformation method, the transformed microorganism is 20% glycerin (glycerine) The solution was added and stored frozen before incubation for production of 10-hydroxystearic acid.

Example  2. Preparation and Recovery of Oleic Acid Hydrolase

Recombinant E. coli for protein expression of the enzyme was incubated at 37 ° C. under aeration conditions of 200 rev / min in a flask containing 500 ml of LB (Difco, Sparks, MD, USA) medium and 50 g / ml kanamycin. When the absorbance of the bacteria reached 0.6 at 600 nm, IPTG was added at a final concentration of 0.1 mM to induce oleic hydratase expression, and the culture was incubated with stirring at 200 rev / min at 37 ° C. for 6 hours.

E. coli cells containing cultured oleic acid digestive enzymes were collected and used. In addition, the oleic acid hydrase produced by overexpression as described above, the culture medium of the transformed strain was washed twice with 0.85% sodium chloride (NaCl) by centrifugation at 6,000xg for 30 minutes at 4 ℃, 50mM phosphate The cell solution was disrupted with a sonicator by adding sodium, 300 mM sodium chloride, 10 mM immidazole, 0.1 mM protease inhibitor (phenylmethylsulfonyl fluoride). The cell lysate was again centrifuged at 13,000 × g for 20 minutes at 4 ° C., cell pellets were removed, and only cell supernatant was obtained for fast protein liquid chromatography (Bio-Rad Laboratories, Hercules, CA, USA). It was equipped with a Hisrap HP adsorption column using a His-tag, and separated as an enzyme liquid used for the production of 10-hydroxystearic acid.

Example  3.Oleic Acid Hydrolase pH Go 10- Hydroxy stearic acid  Effect on Acid Production Activity

In order to investigate the pH effect on the production of 10-hydroxystearic acid of the oleic acid hydrase, pH 5.5 to 7.5 range in 50mM citrate / phosphate buffer solution for 10 g / L oleic acid as a substrate. Enzyme reaction was performed. As a result, as shown in FIG. 1A, the optimum pH was found to be 6.5.

Example  4. The temperature of oleic hydrase is 10- Hydroxy stearic acid  Effect on Acid Production Activity

In order to investigate the temperature effect on the production of 10-hydroxystearic acid of the oleic acid hydrolase, a temperature of 50mM citrate / phosphate buffer pH 6.5 for 10 g / L oleic acid was increased to 20-45 ° C. The reaction was carried out for 10 minutes each.

 As a result, as shown in FIG. 1B, the optimum temperature was found to be 30 ° C.

Example  5. Investigation of Bioconversion Activity According to Organic Solvent Type and Concentration of Oleic Acid Hydrolase

In order to investigate the activity of producing 10-hydroxystearic acid from oleic acid using oleic acid hydratase derived from Ricinibacillus fusiformis, an enzyme was prepared in the same manner as in Example 2.

To investigate the effects of organic solvent species, the reaction was selected from ethanol, methanol, isopropanol, acetone, ethyl acetate, butanol, toluene, and 50 mM citrate / phosphate buffer containing 10 g / L oleic acid and 0.1 mg / mL enzyme. The solution was reacted at pH 6.5 for 10 minutes, at which time 6N HCl was added to terminate the reaction, and then the yield of 10-hydroxystearic acid was measured. The relative results are shown in FIG. It was confirmed that.

To investigate the effects of ethanol concentration, 50 mM citrate / phosphate buffer containing 10 g / L oleic acid and 0.1 mg / mL enzyme in the range of 0% to 6% was reacted at pH 6.5 for 10 minutes each. After completion of the reaction using 6N HCl, the production of 10-hydroxystearic acid was measured, and the relative results are shown in FIG. 2B, and ethanol 4% was the most optimal.

Example 6 Investigation of Bioconversion Activity in the Changes of Oleic Acid Hydrolase Concentration and Oleic Acid Concentration

In the present invention, an enzyme was prepared in the same manner as in Example 2 to investigate the amount of 10-hydroxystearic acid from oleic acid using oleic acid hydratase derived from Ricinibacillus fusiformis.

In the present invention, in order to investigate the conditions showing the high production rate of 10-hydroxystearic acid from oleic acid using oleic acid hydratase derived from Ricinibacillus fusiformis, the enzyme and substrate are reacted at various enzyme amounts and substrate concentration conditions. The yield of 10-hydroxystearic acid was compared.

First, in order to investigate the effect of the concentration of oleic acid hydratase derived from Ricinibacillus fusiformis, from 0.5 mg / ml to 30 g / L oleic acid and 4% ethanol and pH 6.5, 50 mM citrate / phosphate buffer solution from 9 to 9 The reaction was carried out at an enzyme concentration in the range of mg / ml. The reaction amount was 1ml in a 1.5ml plastic container. At this time, reaction temperature was 30 degreeC and reaction time was adjusted to 5 hours. Then, the reaction was terminated by adding 6N HCl, and the amount of 10-hydroxystearic acid produced was measured. The results are shown in FIG. 3 and showed the maximum conversion at an enzyme concentration of 7 mg / ml.

In order to investigate the effect of oleic acid concentration, the reaction was carried out with a pH of 50 mM citrate / phosphate buffer containing 7 mg / ml enzyme, 4% ethanol at a concentration of 10 g / l to 60 g / l of oleic acid. The reaction was carried out under 6.5 conditions, at which time 6N HCl was added to terminate the reaction, and then 10-hydroxystearic acid production was measured to confirm 100% conversion to 30 g / l.

Figure 4 shows the production by confirming the production of 10-hydroxystearic acid according to the oleic acid concentration using the oleic acid hydrase derived from Ricinibacillus Fushiformis under the conditions of pH 6.5 and 30 ℃ of the present invention.

Example  7. 10- Using Oleic Acid Hydrolase Hydroxy stearic acid  Production of acid

In order to confirm the productivity of 10-hydroxystearic acid using the oleic acid hydratase derived from Ricinibacillus fusiformis of the present invention, 10- Hourly production of hydroxystearic acid was measured.

As a result, 38 g / l of 10-hydroxystearic acid was produced, yielding a productivity of 7.6 g / l per hour and a conversion yield of 95% (Fig. 5).

As described above, the present invention relates to a method for mass-producing hydroxy fatty acids, which are intermediates of flavor lactone production and used as lubricants, using oleic acid hydratase derived from Ricinibacillus fusiformis. Specifically, since the substrate can be produced using only oleic acid and oleic acid hydratase derived from Ricinibacillus fusiformis, by controlling enzyme concentration, temperature and pH, only 10-hydroxystearic acid can be produced. Compared with environmentally friendly conditions, the use of unsaturated fatty acids contained in plants as raw materials can be used to maximize production efficiency while effectively reducing production costs even in bioconversion processes, specifically 10-hydroxystearic acid. Bay can be obtained with high yield and high productivity.

As described above, specific portions of the contents of the present invention have been described in detail, and for those skilled in the art of fatty acids, these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (10)

A composition for preparing 10-hydroxystearic acid (10-hydroxystearic acid) comprising an oleic acid hydratase having an amino acid sequence of SEQ ID NO: 1 and an organic solvent. The composition for preparing 10-hydroxystearic acid according to claim 1, wherein the enzyme is derived from Lysinibacillus fusiformis . The composition for preparing 10-hydroxystearic acid according to claim 1, wherein the organic solvent is ethanol. A composition for preparing 10-hydroxystearic acid (10-hydroxystearic acid) comprising a gene encoding an oleic acid hydrase having a nucleotide sequence of SEQ ID NO: 2 and an organic solvent as an active ingredient. The composition for preparing 10-hydroxystearic acid according to claim 4, wherein the organic solvent is ethanol. A method for preparing 10-hydroxystearic acid by bioconversion in the presence of 4% ethanol using oleic acid hydrase and oleic acid having the amino acid sequence of SEQ ID NO: 1. delete delete The method of claim 6, wherein the method is performed at a pH of 6.0 to 7.0. The method of claim 6, wherein the method is performed at a temperature of 25 ° C. to 35 ° C. 7.

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