JP4901293B2 - Decolorization method of bio-method glycolic acid aqueous solution or ammonium glycolate aqueous solution - Google Patents

Description

  The present invention relates to a method for decolorizing an aqueous glycolic acid solution or an aqueous ammonium glycolate solution produced using a biocatalyst having nitrile hydrolysis ability.

  Glycolic acid has been conventionally used as an important component for cosmetics, hair dyes, shampoos, treatments, cleaning agents (household cleaning agents, industrial cleaning agents, etc.), metal treatment agents, and tanning agents. In particular, when used for cosmetics and the like, not only is it highly pure, but also the coloring caused by trace coloring impurities is required to be reduced in terms of product quality.

  On the other hand, as a method for producing glycolic acid by a bio method, a method of producing glycolic acid from glycolonitrile using a microorganism belonging to the genus Corynebacterium (Patent Document 1), a nitrilase derived from a microorganism belonging to the genus Acidborax, And a method for producing glycolic acid from glycolonitrile (Patent Document 2), a method for producing glycolic acid from glycolonitrile using a microorganism belonging to the genus Rhodococcus or Gordona (Patent Document 3), and the like. None of the above references describes the coloring of the hydrolysis reaction solution.

  A number of prior literatures have also been disclosed regarding the desalting of various organic acid salts using electrodialysis, and among them, electrodialysis of a solution obtained by saponifying chloroacetic acid with an excess antkari metal hydroxide. A representative example is a method for producing high-purity glycolic acid (Patent Document 4). However, there is no description in the above-mentioned document regarding coloring of the hydrolysis reaction solution and decolorization by electrodialysis.

  In addition, as a decolorization method by purification of glycolic acid, particularly removal of colored substances, for example, a method of decoloring by subjecting α-hydroxy acid to at least two crystallization stages (Patent Document 5), extraction of α-hydroxy acid , Method of concentrating and crystallizing (Patent Document 6), method of subjecting α-hydroxy acid to distillation step after crystallization step (Patent Document 7), method of decolorizing crude hydroxy acid using granular activated carbon (Patent Document 7) Reference 8) and the like, but all are not examples of using ammonium glycolate electrodialysate produced from a biocatalyst with glycolonitrile as a raw material and having a nitrile hydrolysis ability, but simple and sufficient decolorization It's not a method.

That is, there is no simple and sufficient decolorization method as a practical industrial method for decolorizing an aqueous glycolic acid solution or an aqueous ammonium glycolate solution produced using a biocatalyst having nitrile hydrolysis ability from glycolonitrile. It's real.
JP-A 61-56086 JP 2005-504506 A JP-A-9-28390 JP 9-216848 A Japanese translation of PCT publication No. 2004-509092 Japanese translation of PCT publication No. 2004-509091 JP-T-2004-509090 Japanese Patent Laid-Open No. 49-1224025 JP 2001-299378 A JP-A-11-180971 Japanese Patent Laid-Open No. 06-303991 Japanese Patent Laid-Open No. 63-209592 Japanese Examined Patent Publication No. 63-2596

  The object of the present invention is to easily and sufficiently decolorize glycolic acid aqueous solution or ammonium glycolate aqueous solution produced using a biocatalyst having nitrile hydrolysis ability from glycolonitrile as a raw material. An object of the present invention is to provide a method for decolorizing a glycolic acid aqueous solution or an ammonium glycolate aqueous solution that can obtain a sufficiently high purity glycolic acid aqueous solution or ammonium glycolate.

  The inventors of the present invention have described that an aqueous solution of ammonium glycolate produced using glycolonitrile as a raw material using a biocatalyst having a nitrile hydrolyzing ability contains a trace amount of a colored substance exhibiting specific absorption in the UV to visible light region. In view of the above, the colored substance was not sufficiently decolorized even after the deammonium operation by electrodialysis, and therefore it was necessary to decolorize the deammonium aqueous solution of the reaction solution separately. As a result, it was surprisingly found that the aqueous solution was decolorized simultaneously with a cation exchange operation using a cation resin for the purpose of removing residual ammonium salts that could not be removed by electrodialysis. The invention has been completed.

That is, the present invention has a configuration as described below.
[1] Remove colored substances from glycolic acid aqueous solution or ammonium glycolate aqueous solution produced using a biocatalyst having nitrilase or nitrile hydratase and amidase and having nitrile hydrolyzing ability using glycolonitrile as a raw material A glycolic acid aqueous solution or a glycolic acid aqueous solution or a glycolic acid aqueous solution, which is brought into contact with a strongly acidic cation exchange resin that has been regenerated into a proton (H ⁺ ) type in advance. Decolorization method of ammonium glycolate aqueous solution.
[2] The method for decolorizing an aqueous glycolic acid solution or an aqueous ammonium glycolate solution according to claim 1, wherein the aqueous glycolic acid solution is an aqueous glycolic acid solution obtained by desalting an aqueous ammonium glycolate solution by electrodialysis .

  In the present invention, when decolorizing an aqueous glycolic acid solution or an aqueous ammonium glycolate solution produced using a biocatalyst having a nitrile hydrolyzing ability from glycolonitrile as a raw material, it can be easily and sufficiently decolored. A decolorization method of a glycolic acid aqueous solution or an ammonium glycolate aqueous solution capable of obtaining a high-purity glycolic acid aqueous solution or ammonium glycolate can be provided.

  The nitrile hydrolyzing ability referred to in the present invention is broadly divided into 1) the ability to directly convert a nitrile group into a corresponding carboxylic acid (ammonium) group (nitrilase), and 2) the nitrile group into a corresponding acid amide group. There are two, the ability (nitrile hydratase) and the ability to simultaneously convert the acid amide group to the corresponding carboxylic acid (ammonium) group (amidase). In the present invention, the biocatalyst having any one or both of these two abilities may be in any form.

  The biocatalyst referred to in the present invention may be any organism as long as it is derived from the organism having nitrile hydrolysis activity or from the organism. Specific examples include an enzyme itself, a microorganism containing the enzyme, animal and plant cells, and the like, but any form may be used as long as the activity of the enzyme is basically exhibited. Usually, it is preferable to use microbial cells from the viewpoint of enzyme expression per weight and ease of handling.

Many microorganism species are known, but there is no particular limitation as long as it has nitrilase activity or nitrile hydratase activity and amidase activity. For example, microorganisms having nitrilase activity include Rhodococcus, Acinetobacter, Alcaligenes, Psudomonas, Corynebacterium, Caseobacter, Brevibacterium, Nocardia, Gordona, Arthrobacter, Bacillus, Aureobacterium, Enterobacter, Escherichia , Micrococcus genus, Streptomyces genus, Flavobacterium genus, Aeromonas genus, Mycoplana genus, Cellulomonas genus, Erwinia genus, Candida genus, Bacteridium genus, Aspergillus genus, Penicillium genus, Cochliobolus genus, Fusarium genus, Rhodopseudomonas genus and the like. Among these, the genus Acinetobacter and Alcaligenes, which are Gram-negative bacteria, are particularly preferable in the present invention, and more preferably the genus Acinetobacter.
Specifically, Acinetobacter sp. AK226 (FERM BP-08590) and Acinetobacter sp. AK227 (FERM BP-08591). These strains are disclosed in prior patents. (Patent Documents 9 to 13)

  On the other hand, examples of microorganisms having nitrile hydratase and amidase activity include Rhodococcus, Corynebactgerium, Pseudomonas, Artrobacter, Alcaligenes, Batillus, Bacteridium, Micrococcus, Brevibacterium, and Nocardia.

  Examples of the biocatalyst in the present invention include, for example, a natural or artificially improved nitrilase gene and / or a microorganism incorporating a nitrile hydratase and amidase gene by genetic engineering techniques, or a nitrilase enzyme extracted from the microorganism. Alternatively, a nitrile hydratase and amidase enzyme may be used, but a carboxylic acid (ammonium) can be produced using a small amount of a microorganism having a low expression amount of the nitrile degrading enzyme or a microorganism expressing an enzyme having a low nitrile hydrolysis activity. Requires a longer reaction time, so use microorganisms that express nitrile hydrolase as high as possible and / or microorganisms that express nitrile hydrolase with high conversion activity, or nitrile hydrolase extracted from them. It is desirable.

  As the form of the biocatalyst, microorganisms / animal / plant cells or the like may be used as they are, or the microorganisms / animal / plant cells, etc. themselves, those obtained by crushing the microorganism / animal / plant cells etc., or the microorganisms / animal / plants A nitrile hydrolase extracted from cells or the like may be immobilized by a general entrapment method, a crosslinking method, a carrier binding method, or the like. Examples of the immobilization carrier used for immobilization include, but are not limited to, glass beads, silica gel, polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, alginic acid, and photocrosslinking resin.

  When microorganisms, animal and plant cells are used as they are, they may be suspended only in water (distilled water and / or ion-exchanged water), but are usually suspended in an inorganic salt buffer solution due to osmotic pressure. . Moreover, also when using what was fix | immobilized, it is normally suspended and used for a buffer solution from the relationship of osmotic pressure. The concentration of the buffer solution at this time is preferably as low as possible from the viewpoint of reducing impurities in the reaction solution. However, from the viewpoint of maintaining the stability of the biocatalyst and specific activity, it is usually less than 0.1M, preferably It is 0.01-0.08M, More preferably, it is 0.02-0.06M.

  The concentration of the glycolic acid aqueous solution or ammonium glycolate aqueous solution produced using the above biocatalyst is usually 20% by weight or more, but for economic reasons, a higher concentration is better, preferably 30% by weight or more. Preferably it is 35 weight% or more, More preferably, it is 40 weight% or more.

  The reaction method for producing the glycolic acid aqueous solution or the ammonium glycolate aqueous solution may be any of a fixed bed, a moving bed, a fluidized bed, a stirring tank, etc., and may be a continuous reaction or a semi-batch reaction. When using bacterial cells, a half-batch reaction using a stirring tank is preferable because of the ease of reaction. In that case, it is good to perform appropriate stirring from the viewpoint of reaction efficiency.

  In addition, when a half-batch reaction is performed, the biocatalyst may be disposable for one batch or may be repeatedly performed. However, when the reaction is repeated, the biocatalyst is rapidly changed from a high concentration of ammonium glycolate to a low concentration, so that the specific activity may decrease due to the influence of osmotic pressure or the like, so care must be taken.

  The dry biocatalyst weight used for the ammonium glycolate produced is preferably 1/50 or less, preferably 1/100 to 1/1000, more preferably 1/200 to 1/500. If the weight of the dry biocatalyst used relative to the ammonium glycolate produced is too large, a large amount of impurities from the biocatalyst suspension is entrained in the reaction solution, which increases the purification cost and lowers the product quality. Conversely, if the weight of dry biocatalyst used for the ammonium glycolate produced is too small, the productivity per reactor volume will be reduced, and a large reactor size will be required, which will be economically disadvantageous.

  If the reaction temperature is too low, the reaction activity becomes low, and more reaction time is required to produce a high concentration of ammonium glycolate. On the other hand, if the reaction temperature is too high, the biocatalyst is deactivated by heat, and if the target ammonium glycolate concentration is high, it is difficult to reach the concentration, resulting in the addition of a new biocatalyst, etc. Therefore, the cost of the catalyst becomes high. Therefore, the reaction temperature is usually from the freezing point to 70 ° C, preferably from 10 to 60 ° C, more preferably from 20 to 50 ° C.

  The reaction charging solution for suspending microorganisms at the initial stage of the reaction is not particularly limited as long as it is an aqueous solution, but water is usually used. Further, a buffer solution may be used from the viewpoint of increasing the stability of the microorganism due to the osmotic pressure. Moreover, you may mix a polar organic solvent in arbitrary ratios, if it is a range which does not reduce activity.

  The pH of the reaction solution is preferably adjusted to the optimum pH for the nitrile hydrolysis activity of the biocatalyst to be used. Usually, the reaction solution pH is preferably 6 to 13, and more preferably 9 to 11. However, for the convenience of the product to be obtained, this is not limited as long as the required pH is defined, and it can be arbitrarily selected within a range in which the decrease in reaction activity does not appear extremely.

  In cation exchange using a cation exchange resin in the present invention, an aqueous ammonium glycolate solution produced under the above conditions can be used as it is, or glycolic acid obtained by subjecting to desalination treatment by electrodialysis can be used. Although an aqueous solution can be used, the aqueous solution after the desalting treatment by electrodialysis is preferable in order to increase the efficiency of decolorization.

  As the cation exchange resin used in the present invention, a weak acid cation exchange resin or a strong acid cation exchange resin can be used, and a strong acid cation exchange resin is preferable. Specifically, for example, Diaion SK1B, SK104, SK110, SK112, SK116, PK208, PK212, PK216, PK220, PK228, UBK530, UBK550, UBK535, UBK555 ( (Mitsubishi Chemical Corporation), Lebatit S100, S109, SP112, STV40, MSD1368 (above Bayer), Amberlite IR120B, 120BN, IR124, 1006F, 200CT, 252 (above organo) ), Dowex Monosphere 650C, Marathon C, HCR-S, Marathon MSC (manufactured by Dow Chemical Company) and the like, but are not necessarily limited thereto. These cation exchange resins are used after being regenerated into a proton (H +) type by a conventional method.

  As a method of using the cation exchange resin in the present invention, a usual method is adopted. That is, a batch in which a predetermined amount of a cation exchange resin is added to a glycolic acid aqueous solution or an ammonium glycolate aqueous solution produced using the above biocatalyst, which contains a trace amount of a coloring substance exhibiting specific absorption in the UV to visible light region. Alternatively, a column method in which the cation exchange resin is filled in a resin tower and the aqueous glycolic acid solution or the aqueous ammonium glycolate solution is passed can be employed. In the case of the batch type, after stirring for a sufficient time to reach the equilibrium adsorption of the coloring substance to the cation exchange resin, if the supernatant is recovered, a sufficiently decolorized glycolic acid aqueous solution or ammonium glycolate aqueous solution can be obtained. Obtainable. In the case of the column method, the resin passing solution is a sufficiently decolorized glycolic acid aqueous solution or ammonium glycolate aqueous solution.

  The amount of cation exchange resin used is different between the case of treating an aqueous ammonium glycolate solution and the case of treating an aqueous glycolic acid solution that is a desalting solution by electrodialysis. In the case of an ammonium glycolate aqueous solution, the amount of the resin corresponding to the total exchange capacity of the resin equal to or more than that of ammonium ions is essential, and in order to remove the coloring component more reliably, the resin is usually 1.2 times equivalent or more. It is good to use. In the case of an aqueous glycolic acid solution, the total exchange capacity of the resin is possible as long as the amount of the resin is equivalent to an equivalent amount or more of the cation component that is slightly contained, but in this case as well, it is better to use the resin in excess. preferable. Further, in the case of the column method, in order to increase the time until resin breakthrough and resin regeneration, it is common practice to use more excess resin.

  The temperature during the resin treatment may be room temperature, but if necessary, the resin may be heated within a range in which the heat resistance of the resin is guaranteed. Usually, it is performed at 70 ° C. or lower. In the case of the column method, the liquid flow rate is in the range of 1 to 20, preferably 2 to 10, in terms of space velocity (L / L-resin / Hr).

  In the case of the column method, the point where mixing of colored substances is confirmed in the resin passage liquid is used as a breakthrough point, and reverse regeneration is performed using an appropriate reverse regeneration agent (e.g., alkaline liquid such as sodium hydroxide aqueous solution) from there. Alternatively, the resin can be used repeatedly if it is regenerated using a suitable regenerant (eg, mineral acid such as dilute hydrochloric acid or dilute sulfuric acid).

The electrodialysis in the present invention is a general method known to those skilled in the art and is not particularly limited. For example, a method of obtaining a glycolic acid aqueous solution by combining a bipolar membrane and a cation membrane and passing the cation membrane through ammonium ions and bringing it into a separate chamber, or combining a bipolar membrane and an anion membrane to make the anion membrane an glycolate anion To obtain a glycolic acid aqueous solution by passing it through a separate chamber, or combining a bipolar membrane, a cation exchange membrane, and an anion exchange membrane to pass ammonium ions and glycolate anions, respectively, and bring them into a separate chamber. And a method for obtaining an aqueous solution of glycolic acid and aqueous ammonia.
<Example>

  Hereinafter, the present invention will be described in more detail with reference to examples. In addition, this invention is not limited by these Examples, A various change and modification are possible unless it exceeds the summary.

The reaction solution was analyzed as follows.
The substrate α-hydroxynitrile and the product α-hydroxy acid aqueous solution or ammonium glycolate were measured by high performance liquid chromatography. The column is an ion exclusion column (Shimadzu Shim-pack SCR-101H), the column temperature is 40 ° C., the mobile phase is phosphoric acid aqueous solution (pH = 2.3), the detector is UV (Shimadzu SPD-10AV vp, 210 nm) and RI (Shimadzu RID-6A).

Evaluation of decolorization of the cation exchange treatment solution was performed as follows.
The evaluation of the degree of coloring (change in hue) was carried out using a chromaticity meter. The apparatus used was an air bath method trace melting point measuring apparatus manufactured by Yanagimoto Seisakusho. The evaluation results are represented by three values of L value, a value, and b value according to the color difference display method (JIS Z 8730). The L value represents the degree of black and white with lightness. When L is 100, it is completely white, and when L is 0, it is completely black. Further, the redness becomes darker as the value a becomes larger, and the greenness becomes darker as the value a becomes smaller. Moreover, the b value increases as the b value increases, and the blue color increases as the b value decreases.

[Preparation of biocatalyst]
Sodium chloride 0.1 wt%, potassium dihydrogen phosphate 0.1 wt%, magnesium sulfate heptahydrate 0.05 wt%, ferrous sulfate heptahydrate 0.005 wt%, ammonium sulfate 0.1 wt% %, Potassium nitrate 0.1 wt% Manganese sulfate pentahydrate 250ml culture solution 250ml was placed in an Erlenmeyer flask, adjusted to pH 7 with sodium hydroxide and sterilized at 121 ° C for 20 minutes After that, 0.5% by weight of acetonitrile was added. This was inoculated with Acinetobacter sp. AK226 and cultured with shaking at 30 ° C. (preculture). Mist powder 0.3% by weight, sodium glutamate 0.5% by weight, ammonium sulfate 0.5% by weight, dipotassium hydrogen phosphate 0.2% by weight, potassium dihydrogen phosphate 0.15% by weight, sodium chloride 0. 1% by weight, magnesium sulfate heptahydrate 0.18% by weight, manganese chloride tetrahydrate 0.02% by weight, calcium chloride dihydrate 0.01% by weight, iron sulfate heptahydrate 0.003% by weight 3L of culture broth containing 0.002% by weight of zinc sulfate heptahydrate, 0.002% by weight of copper sulfate pentahydrate and 2% by weight of soybean oil was charged into a 5L jar fermenter and sterilized at 121 ° C for 20 minutes. After that, the pre-cultured solution was inoculated and aerated and stirred at 30 ° C. Soybean oil feed was started 10 hours after the start of the culture. The pH was controlled with phosphoric acid and aqueous ammonia so that the pH was 7, and finally a suspension of about 5% by weight of Acinetobacter sp. AK226 was obtained. Furthermore, it was washed twice with 0.06M phosphate buffer, and finally Acinetobacter sp. AK226 suspension (dry cell concentration 5 to 10% by weight) suspended in phosphate buffer was obtained.

[Preparation of bio-method ammonium glycolate aqueous solution]
Using the Acinetobacter sp. AK226 suspension obtained above, an ammonium glycolate accumulation experiment by hydrolysis of glycolonitrile was conducted. A suspension of Acinetobacter sp. AK226 having a known bacterial cell concentration was suspended in 316 ml of distilled water previously charged in a 2 L four-necked flask so that the concentration was 1.02% by weight. A pH meter and a thermometer were installed in the flask so that the pH and temperature of the reaction solution could be monitored. The flask was placed in a 50 ° C. constant temperature water bath and stirred with a stirrer, and held for a while until the internal temperature reached 50 ° C. Next, a 55 wt% aqueous solution of glycolonitrile (manufactured by Tokyo Chemical Industry) is continuously added using a metering pump, and at the same time, the pH is maintained at 6.5 to 7 with a 5 wt% KOH aqueous solution metering pump. Added. Sampling was periodically performed during the reaction, the concentration of glycolonitrile and ammonium glycolate was measured by high performance liquid chromatography, and the feed pump feed rate of the raw material was adjusted so that the steady glycolonitrile concentration was 2% by weight or less. The final ammonium glycolate accumulation concentration was 56.7% by weight. The obtained ammonium glycolate was filtered under reduced pressure with a membrane filter (1 micron) to remove AK226 cells and debris and proteins derived therefrom, to obtain 852 g of a biomethod ammonium glycolate aqueous solution.

[Preparation of aqueous bioglycolic acid solution]
Using a part of the ammonium glycolate aqueous solution obtained above, 500 g of a 50.2 wt% ammonium glycolate aqueous solution was prepared, and deammonium was removed using a bipolar membrane electrodialyzer. The apparatus used was ACILIZER EX3B (manufactured by Tokuyama Corporation), with 10 rooms and a membrane area of 550 cm2. It consists of a combination of a bipolar membrane and a cation exchange membrane. 500 g of the ammonium glycolate is used in the raw material chamber, 500 g of 1.0 wt% ammonia water is used in the alkali chamber, and 0.5 N sodium hydroxide aqueous solution is used in the electrode chamber. It was. At the beginning of desalting, a constant voltage operation of 30 V was performed, and the operation was switched to a constant current operation of 4 A in about 80 minutes from the start of operation. The liquid temperature during operation is kept constant at 40 ° C by the cooling operation, and the transition of operation is monitored by the pH and conductivity of the raw material chamber, the end point is determined, and finally 37.8 wt% glycolic acid aqueous solution is obtained. It was.

[Decolorization test]
300 g of the aqueous glycolic acid solution obtained above was passed through a resin tower packed with 40 ml of a strongly acidic cation exchange resin Amberlite IR120B (trade name Organo Co., Ltd.) that had been regenerated into a proton (H ⁺ ) type in advance. Liquid was collected and collected with a fraction collector. The resin tower had an inner diameter of 14 mm, a feed rate of 2.66 ml / min (space velocity = 4), a fraction condition of 5.3 ml / tube, and a treatment temperature of room temperature. When the raw material liquid disappeared, the extrusion operation was performed by feeding distilled water under the same conditions as the raw material liquid. The pH of each fraction was measured, and those having a pH range of 1.69 to 0.71 were collected and mixed to obtain a treatment liquid. The hue change before and after the treatment was as shown in Table 1.

Resin tower packed with 100 ml of a strongly acidic cation exchange resin Amberlite IR120B (trade name Organo Co., Ltd.), in which a portion of 20 g of the ammonium glycolate aqueous solution obtained in Example 1 was previously regenerated into a proton (H ⁺ ) type. The solution was passed through a down flow and collected with a fraction collector. The resin tower was 14 mm in inner diameter, the feed rate was 3.33 ml / min (space velocity = 2), the fraction conditions were 1 ml / tube, and the treatment temperature was room temperature. When the raw material liquid disappeared, the extrusion operation was performed by feeding distilled water under the same conditions as the raw material liquid. The pH of each fraction was measured, and those having a pH range of 1.72 to 0.73 were collected and mixed to obtain a treatment liquid. The hue change before and after the treatment was as shown in Table 2.

  According to the present invention, since a sufficiently high purity glycolic acid aqueous solution or ammonium glycolate aqueous solution can be produced in terms of product quality, the obtained high purity glycolic acid aqueous solution or ammonium glycolate aqueous solution can be used in various applications. In particular, it can be suitably used for cosmetic applications.

Claims (2)

Decolorization by removing colored substances from glycolic acid aqueous solution or ammonium glycolate aqueous solution produced using biocatalyst having nitrilase or nitrile hydratase and amidase and having nitrile hydrolyzing ability from glycolonitrile as raw material A glycolic acid aqueous solution or an ammonium glycolate characterized in that an aqueous glycolic acid solution or an aqueous ammonium glycolate solution is brought into contact with a strongly acidic cation exchange resin that has been regenerated into a proton (H ⁺ ) type in advance. Decolorization method of aqueous solution. The method for decoloring an aqueous glycolic acid solution or an aqueous ammonium glycolate solution according to claim 1, wherein the aqueous glycolic acid solution is an aqueous glycolic acid solution obtained by desalting an aqueous ammonium glycolate solution by electrodialysis .