KR20080085377A

KR20080085377A - Method for preparing l-ornithine salts

Info

Publication number: KR20080085377A
Application number: KR1020070026801A
Authority: KR
Inventors: 김민홍
Original assignee: 주식회사 엠에이치투 바이오케미칼
Priority date: 2007-03-19
Filing date: 2007-03-19
Publication date: 2008-09-24

Abstract

A method for preparing L-ornithine salts is provided to eliminate a conventional purification process using ion exchange resin, significantly reduce generation of waste water, and improve productivity and yield of L-ornithine salts by using solubility difference between ornithine salt and urea in alcohol aqueous solution. A method for preparing L-ornithine salts comprises the steps of: (a) reacting L-arginine with arginase to form L-ornithine and urea; (b) adding an acid selected from an organic acid or an inorganic acid into the enzyme reaction solution formed in the step (a); and (c) adding alcohol into the mixture at 40-65 deg. C to selectively crystallize L-ornithine salts, and further comprises a step of decolorizing, filtering and concentrating the produced mixture of L-ornithine salts and urea after the step (b). Further, the inorganic acid is one or more selected from hydrochloric acid, phosphoric acid and sulphuric acid.

Description

Method for preparing L-ornithine salts {Method for preparing L-Ornithine salts}

Figure 1 shows a manufacturing process of the L- ornithine salt according to the present invention.

The present invention relates to a method for preparing L-ornithine salt, and more specifically, to the L-ornithine salt and urea obtained by acid-treating the reaction solution produced by the enzymatic reaction, only L-ornithine salt is added. The present invention relates to a method for preparing L-ornithine salt to be selectively crystallized.

Ornithine (ornithine) is one of the amino acids present in the urea cycle in vivo, relatively contained in plasma, liver, skin and the like. High levels of ammonia in the blood cause liver damage. Ornithine and citrulline bind to ammonia in the blood and become citrulline and arginine, respectively, to reduce the level of ammonia in the blood.

As shown in Scheme 1, L-ornithine reacts with hydrochloric acid, L-aspartic acid, alpha ketoglutaric acid, malic acid, and the like to form a salt (conjugate), and L-ornithine is difficult to crystallize industrially. Prefers L-ornithine salts.

Scheme 1

L-ornithine + acid ------> L-ornithine salt (conjugate)

L-ornithine salt is used as a health food material in the form of capsules, tablets, etc., or added to foods. Recently, the market has been gradually expanding due to fatigue recovery, growth hormone secretion and wrinkle improvement.

Until now, in order to industrially manufacture L-ornithine salts, a fermentation method using L-arginine nutrient constitutive strains or a method of chemically or enzymatically converting arginine has been used.

The fermentation method uses glucose as a raw material, so the raw material price is relatively low, but the fermentation equipment is expensive, the productivity of the equipment is low, and the refining burden is high. On the other hand, the chemical conversion method isomer is produced by isomerization reaction, There was a problem in that side reactions such as citrulline were produced.

In general, the enzyme can be used to selectively obtain L-ornithine, and has the advantage of higher productivity than the fermentation method. Despite the advantages of the enzyme method as described above, so far, L- ornithine The reason why ornithine production was difficult to industrialize was because the conversion rate was lower than 90% and the removal of urea, a byproduct of the enzymatic reaction, was complicated.

Ornithine is produced with urea by the action of arginase from arginine, as shown in Scheme 2 below.

Scheme 2

Arginase

L-arginine + water <----------------> L-ornithine + urea

The conversion rate is defined as the percentage of moles of ornithine produced to the moles of arginine introduced. Conventionally, ion exchange resins are mainly used to remove urea from the reaction solution. For example, at or above pH 9, ornithine has an anion and urea is neutral, so ornithine is adsorbed on the anion exchange resin and urea is It is discharged as it is.

By eluting the adsorbed ornithine with 5% hydrochloric acid, an aqueous ornithine solution from which urea is removed can be obtained. However, this method has disadvantages in that the amount of wastewater generated is excessive and the final yield is low as 65% or less.

The present invention is to solve the problems of the prior art as described above, an object of the present invention is to remove urea with dehydration liquid by using the difference in solubility in the crystallization process of L- ornithine salt, and selectively crystallize only L- ornithine salt It is thereby to provide a method for producing L-ornithine salts that can significantly reduce the generation of wastewater and significantly improve the productivity and yield of L-ornithine salts.

In preparing L-ornithine salt using enzyme, firstly, the factor that has a big influence on manufacturing cost should be obtained by applying cell enzyme to a high concentration of arginine aqueous solution to obtain 100% conversion rate. Should be removed.

The present invention for achieving the above object, (a) an enzyme reaction step of producing a ureachin and urea by reacting arginase to L-arginine; (b) adding an acid to the resulting enzyme reaction solution to produce a mixture of salts and urea of L-ornithine; And (c) it provides a method for producing L- ornithine salt comprising the step of selectively crystallizing the L- ornithine salt by adding alcohol to the mixture.

Hereinafter, the present invention will be described in detail for each process step.

(a). Enzyme reaction

Arginase used in the enzymatic reaction of the present invention is an enzyme derived from Bacillus sp. Microorganisms, which can be purchased relatively cheaply compared to animal-derived arginase, and has a relatively high temperature of enzyme reaction. Has the advantage of having.

In addition, the arginase used in the enzymatic reaction of the present invention has a characteristic of L-ornithine conversion of 95% or more, preferably 98 to 99.5%, and the crystallization of L-ornithine conversion of 95% or less. This is because L-arginine, which is a substrate in the process, is precipitated together with the L-ornithine salt crystal targeted in the present invention.

The enzyme reaction may be carried out at a reaction temperature of 45 ~ 60 ℃, preferably 50 ~ 55 ℃ and conditions of pH 7.5 ~ 12.5, when the reaction temperature is less than 35 ℃ and 50 ℃ or more there is a concern that the enzyme activity is lowered In addition, the pH conditions for the reaction are not particularly limited because they differ depending on the specific enzyme used, but it is preferable to perform the reaction in the range of pH 7.5 to 12.5 in consideration of the enzyme activity.

As in the present invention, the high reaction temperature of the enzyme can increase the concentration of arginine in the substrate solution, prevent contamination by pathogenic microorganisms, and prevent the action of other enzymes of host cells such as E. coli by genetic manipulation. Has the advantage that

However, any enzyme can be used as long as the conversion rate of L-arginine according to the enzyme reaction is 95% or more, and the type, origin, and reaction conditions of the enzyme do not limit the effectiveness of the present invention.

L-arginine provided as a substrate is provided in a high concentration in an aqueous solution, it may be provided in a liquid phase of 5 to 30% by weight, preferably 10 to 15% by weight.

(b). On the mountain By L- Ornithine salt And mixed solution of urea

As described above, a predetermined amount of acid is added to the reaction solution in which the enzymatic reaction is completed, or an acid is added to a predetermined pH to generate a mixed solution of L-ornithine salt and urea.

The acid added to the present invention can be used without limitation as long as it is an acid having a property capable of forming ornithine and a salt or ligand thereof. Specifically, an inorganic acid may be hydrochloric acid, sulfuric acid, phosphoric acid, or a mixture thereof. For example.

In addition, the organic acid is L- aspartic acid, alpha keto glutaric acid, lactic acid, citric acid, tartaric acid, fumaric acid, malic acid, itaconic acid, acetic acid, gluconic acid, 2-ketogluconic acid, 5-ketogluconic acid, kojic acid and these The mixed liquid of is mentioned as an example.

The molar ratio of the acid added to the enzyme reaction solution to L-ornithine is preferably 0.5 to 1, and the content of residual L-arginine in the enzyme reaction solution is 5% or more of L-arginine before the start of the enzyme reaction. L-arginine at% or less (95% or more conversion), preferably 2% or less (98% or more conversion) will remain.

The present invention may further comprise a step of decolorizing and filtering the mixture of L-ornithine salt and urea produced by the addition of the acid, and then concentrating.

Activated carbon and diatomaceous earth are added and stirred for a predetermined time in order to remove the cells or enzyme proteins, pigments, etc. contained in the mixed solution of L-ornithine salt and urea, and then filtered using a soap. The filtered liquid in a clear state is concentrated in vacuo in a range where crystals do not precipitate below 50 ° C.

(c). L- Ornithine Crystallization stage

The mixed solution of the L-ornithine salt and urea is transferred to a crystal tube, where the crystal tube is capable of sufficient agitation, temperature control, and a structure capable of adding alcohol.

The L-ornithine salt and the urea can be added to the mixture of alcohol to crystallize the L-ornithine salt, and the urea is discharged to the mother liquor in the dehydration process can be produced very economically L- ornithine salt.

Since alcohol has an effect of lowering the solubility of L-ornithine salt, as shown in Table 1 below, L-ornithine salt and L-ornithine salt are selectively selected from the mixture of L-ornithine salt and urea using the difference in solubility of L-ornithine salt and urea. By crystallization, the yield of crystals can be increased.

Table 1 Solubility of L-ornithine hydrochloride and urea

division L-ornithine hydrochloric acid Element water 55 g 100 g 100% methanol <1 g 16 g 95% ethanol <1 g 10 g

* 100ml solvent, room temperature

The alcohol may be used without limitation as long as the difference in solubility between the L-ornithine salt and urea is large enough, and preferably an aliphatic alcohol having 1 to 3 carbon atoms may be used. Specifically, 1 type, or 2 or more types of alcohol aqueous solution chosen from the group which consists of methanol, ethanol, and isopropyl alcohol, or is selected from the group which consists of methanol, ethanol, and isopropyl alcohol are mentioned.

Determination conditions of L-ornithine salt during the addition of the alcohol is 40 ~ 60 ℃ temperature, 45 ~ 85% (v / v) alcohol concentration in the final crystal solution, preferably 55 ~ 75% (v / v) In this condition, urea does not precipitate and L-ornithine salt can be selectively crystallized. In this case, when the concentration of alcohol is too low, the cost of alcohol is low, but there is a problem that the crystal yield of L-ornithine salt is small.

In the method for producing L-ornithine salt according to the present invention, as shown in Figure 1, after the crystallization step, after the dehydration process to discharge the urea to the mother liquor, the step of drying and screening the L- ornithine salt product It may further include.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, it should not be construed that the scope of the present invention is limited to these examples.

Example 1 Preparation of Cellulase (Arginase) and Production of L-ornithine and Urea by Arginase Enzyme Reaction

Thermophilic Bacillus Keldobelox caldovelox ) was cultured to isolate genomic DNA, and then amplified only the heat-resistant arginase gene of Bacillus Keldobelox by PCR, and then cloned into E. coli BL21 (DE3) by inserting into an expression vector. Subsequently, the recombinant E. coli BL21 (DE3) cloned with the arginase gene was cultured in an ampicillin-added medium, and the cells were recovered, wherein the recovered cells were 4500 U / g of lysate.

The production process of the cell enzyme can be variously changed to those skilled in the art, the following references for convenience of understanding (Maria C.Bewley, J. Shaun Lott, Edward N. Baker, Mark L. Patchett, FEBS Letters, Vol 386, pp 215-218 (1996)).

Example 2 Production of L-ornithine and Urea by Arginase Enzyme Reaction

The substrate used for the enzymatic reaction was dissolved in arginine in purified water at 50 ° C. to 10% (w / v) and dissolved in MnCl ₂ to 0.1 mM. At this time, the pH of the substrate aqueous solution was 10.5 ± 0.5.

The amount of enzyme used was 20 g of wet cells per kg of arginine. Within 15 hours after the initiation of the reaction, the conversion was 98% or more, and the pH after completion of the reaction was 9.5 ± 0.3. The activity of arginase 1 U was defined as the activity in which 1 micromole of L-ornithine was produced for 1 minute, and the typical composition of the solution after the enzymatic reaction was shown in Table 2 below.

TABLE 2 Composition of reaction solution before and after enzyme reaction

division Before starting reaction Termination of reaction L-arginine 10 0.1 L-ornithine 0 7.4 Element 0 3.3 Sum 10 10.8

Unit:% (w / v)

Example 3 Preparation of L-ornithine Hydrochloric Acid and Urea Removal

As in Example 2, the pH of the solution was adjusted to 5.5 by adding 35% hydrochloric acid to the solution after the enzymatic reaction was completed, and activated carbon and diatomaceous earth were adsorbed to adsorb enzymes and other coloring matters, and then using the second tablet. Filtration was performed at this time, and the transmittance (T%) of the filtrate measured at wavelength 430 nm using spectrophotometer was 97%.

The filtrate was concentrated at 45 ° C. to 50 Brix and transferred to a crystal tube. While slowly stirring in the crystal tube, the ethanol content of 95% ethanol was slowly injected to make the final ethanol content of 60% (v / v).

After cooling slowly until the temperature of the crystal solution reached 10 ° C, the solution was decanted by centrifugation, and washed twice with a washing liquid having an alcohol content of 80% (v / v). The wet cake obtained above was dried with hot air dryer at 60 ° C. for 1 hour.

The dried crystals were in the form of a thick needle, the apparent specific gravity was 0.55, the yield of dried crystals was 71%, the urea content in the dried crystals was 580 ppm.

Example 4 Preparation of L-ornithine-L-aspartic Acid and Removal of Urea

As in Example 2, the aspartic acid crystal powder was added to the finished solution of the enzyme reaction in the same molar ratio as L-ornithine produced. After stirring for 1 hour, the pH of the liquid was 6.5.

Activated carbon and diatomaceous earth were added to adsorb enzymes and other pigments, followed by filtration using a silkworm.

The filtrate was concentrated at 45 ° C. to 45 Brix and transferred to a crystal tube. Slowly injecting 99.5% methanol while stirring in a crystal tube to bring the final content of methanol to 65% (v / v).

After cooling slowly until the temperature of the crystal solution reached 10 ° C, the solution was decanted by centrifugation, and washed twice with a washing solution adjusted to 65% (v / v) of methanol. The wet cake obtained above was dried with hot air dryer at 80 ° C. for 1 hour.

The apparent specific gravity of dried crystals was 0.63, the yield of dried crystals was 75%, and the urea content in the dried crystals was 460 ppm.

Example 5 Preparation of L-ornithine-Alphaketoglutalic Acid and Urea Removal

As in Example 2, the alpha ketoglutaric acid crystal powder was added to the produced L-ornithine in a molar ratio of 2: 1 to the finished solution of the enzyme reaction, and stirred for 1 hour. 6.4.

Activated carbon and diatomaceous earth were added to adsorb enzymes and other pigments, and the filtrate was filtered. The permeability (T%) of the filtrate measured at 430 nm using spectrophotometer was 95.5%.

The filtrate was concentrated at 45 ° C. to 45 Brix and transferred to a crystal tube. While slowly stirring in the crystal tube, the ethanol content of 95% ethanol was slowly injected to obtain a final alcohol content of 60% (v / v).

After cooling slowly until the temperature of the crystal solution was 10 ℃, the solution was removed by centrifugation, and washed twice with a washing liquid adjusted to the content of alcohol to 80% (v / v). The wet cake obtained above was dried with hot air dryer at 70 ° C. for 1 hour.

The apparent specific gravity of dried crystals was 0.62, the yield of dried crystals was 70%, and the urea content in the dried crystals was 660 ppm.

According to the present invention, L-arginine is used as a substrate to generate L-ornithine and urea using arginase, and then, various acids are added to prepare urea salts (or ligands) of ornithine. It is also efficiently removed to provide a very economical method for producing L-ornithine salt.

According to the present invention, since the solubility difference between ornithine salt and urea in an aqueous alcohol solution can be omitted, a conventional purification process using an ion exchange resin can be omitted, thereby significantly reducing the generation of waste water and reducing the productivity of L-ornithine salt. Yields can be dramatically improved.

Claims

(a). An enzymatic step of reacting L-arginine with arginase to produce L-ornithine and urea;

(b). Adding an acid to the resultant enzyme reaction solution to produce a mixture of salts and urea of L-ornithine; And

(c). Method of producing an L- ornithine salt comprising the step of selectively crystallizing the L- ornithine salt by adding alcohol to the mixture.

The method of claim 1, wherein the enzymatic reaction of step (a) is characterized in that the conversion to L-ornithine is 95% or more.

The method for producing L-ornithine salt according to claim 1, wherein the acid is an inorganic acid or an organic acid.

The method for producing L-ornithine salt according to claim 3, wherein the inorganic acid is one or two or more selected from the group consisting of hydrochloric acid, phosphoric acid and sulfuric acid.

The method of claim 3, wherein the organic acid is L- aspartic acid, alpha keto glutaric acid, lactic acid, citric acid, tartaric acid, fumaric acid, malic acid, itaconic acid, acetic acid, gluconic acid, 2-ketogluconic acid, 5-ketogluconic acid and coke Method for producing L-ornithine salt, characterized in that one or two or more selected from the group consisting of acid.

The method for preparing L-ornithine salt according to claim 1, wherein the molar ratio of the acid added in step (b) to L-ornithine is 0.5 to 1.

The method for producing L-ornithine salt according to claim 1, wherein the alcohol is an aliphatic alcohol having 1 to 3 carbon atoms.

According to claim 7, wherein the aliphatic alcohol is one or two or more selected from the group consisting of methanol, ethanol and isopropyl alcohol, or one or two or more alcohols selected from the group consisting of methanol, ethanol and isopropyl alcohol Method for producing L-ornithine salt, characterized in that the aqueous solution

The method for preparing L-ornithine salt according to claim 1, wherein the concentration of alcohol in the crystal solution is 45% to 85% (v / v) after the alcohol is added in step (c).

The method for producing L-ornithine salt according to claim 1, wherein the crystal liquid temperature during the addition of alcohol in step (c) is 40 to 65 ° C.

The method of claim 1, further comprising the step of decolorizing and filtering the mixed solution of L-ornithine salt and urea produced after step (b) and concentrating the L-ornithine salt.