JP5165393B2 - Method for producing glycolic acid or ammonium glycolate - Google Patents

Description

  The present invention provides a practical industrial method for producing glycolic acid or ammonium glycolate from glycolonitrile using a biocatalyst having nitrilase.

  The method of synthesizing a target compound using a biocatalyst having enzyme activity can simplify the reaction process because the reaction conditions are mild, or can obtain a high-purity reaction product with few byproducts. Due to its advantages, it has recently been used in the production of various compounds. Among them, hydrolyzing enzymes such as nitrilase, which has the activity of converting nitrile compounds into carboxylic acids or ammonium carboxylates, and nitrile hydratase, which has the activity of converting nitrile compounds into carboxylic acid amides, have various reaction behaviors. Studies for use in the production of carboxylic acid or ammonium carboxylate or carboxylic acid amide have been made.

  Among them, many methods for producing glycolic acid or ammonium glycolate, in which glycolonitrile is converted into glycolic acid or ammonium glycolate using a biocatalyst having nitrilase activity, have been studied. For example, a method for producing glycolic acid or ammonium glycolate using Corynebacterium genus (Patent Document 1), a method for producing glycolic acid or ammonium glycolate using Rhodococcus genus or Gordona genus (Patent Document 2), or Acidovorax genus Glycolic acid or ammonium glycolate using a method (Patent Document 3, Patent Document 4), or using a biocatalyst in which a nitrilase derived from the genus Acidovorax or a modified enzyme thereof is expressed in a host such as Escherichia coli, Methods for producing ammonium glycolate (Patent Document 5, Non-Patent Document 1) and the like are disclosed.

  However, these conventional techniques have not been industrially satisfactory production methods, and further improvements have been demanded. That is, in producing glycolic acid or ammonium glycolate using glycolonitrile as a raw material by hydrolysis reaction using such a biocatalyst, 1) sufficient average production rate, 2) sufficient It has been desired to achieve three of ammonium glycolate accumulation concentration and 3) sufficient catalyst productivity at the same time.

  For example, Patent Document 1 discloses an example of synthesizing glycolic acid or ammonium glycolate using Corynebacterium genus, but there is no description regarding the average production rate. Incidentally, it is described that the initial activity is 29.3 [mmol-ammonium glycolate / g-dried cells / Hr], and it is not expected to achieve a very sufficient average production rate. In Patent Document 2, Rhodococcus genus is used to achieve an ammonium glycolate accumulation concentration of 48.2% by weight. The average production rate at that time is 47.3 [mmol-ammonium glycolate / g-dried cells / Hr ] (20 ° C x 24Hr), which is also not enough to achieve a sufficient average production rate. Moreover, in the said patent document 3 and the patent document 4, using the Acidovorax genus, the average production rate of ammonium glycolate is a low value of 0.42 [mmol-ammonium glycolate / g-dry microbial cell / Hr] (25 ° C. × 40 Hr). It cannot be said that this also achieves a sufficient average production rate. On the other hand, in Patent Document 5, Patent Document 6 and Non-Patent Document 1, the initial specific activity is 266 [mmol-mmol] by using a biocatalyst in which a modified enzyme of Nitrilase derived from the genus Acidovorax is expressed in a host such as Escherichia coli. Ammonium glycolate / g-dried cells / Hr] (25 ° C) has been achieved, and the E. coli is immobilized with carrageenan and repeated reactions, and the reaction temperature is kept low at 25 ° C. As a result, 1225 [g-ammonium glycolate / g-dried cells] and very high catalyst productivity were achieved, but the ammonium glycolate accumulation concentration was 30% by weight or less, and a sufficient accumulation concentration was achieved. It can not be said.

Japanese Patent Publication No. 3-38836 JP-A-9-28390 JP 2005-504506 A US6416980 A1 WO2006069110 A1 WO2006069114 A1 Adv. Synthe. Catal. 349,1462-1474 (2007)

 The object of the present invention is to produce glycolic acid or ammonium glycolate using glycolonitrile as a raw material by hydrolysis reaction using nitrilase: 1) sufficient average production rate, 2) sufficient ammonium glycolate accumulation concentration, 3) To provide a process for producing glycolic acid or ammonium glycolate from glycolonitrile, which is industrially advantageous, and can achieve three of sufficient catalyst productivity at the same time.

  In the production of glycolic acid or ammonium glycolate from glycolonitrile by a hydrolysis reaction using nitrilase, the present inventors have 1) sufficient average production rate, 2) sufficient ammonium glycolate accumulation concentration, 3) As a result of intensive studies on a process for producing glycolic acid or ammonium glycolate from glycolonitrile, which is industrially advantageous and can achieve three of the sufficient catalyst productivity at the same time, It was discovered that the catalyst productivity can be drastically improved by setting the methanol concentration in the raw material containing ronitrile to 2% by weight or less, and the present invention has been completed. In other words, in order to improve catalyst productivity, the biocatalyst is immobilized with carrageenan or the like, and / or sufficient average production rate is secured without using a method that keeps the reaction temperature low, and sufficient ammonium glycolate accumulation As a method that can achieve sufficient catalyst productivity while achieving concentration, pay attention to methanol, which is an impurity contained in raw materials containing glycolonitrile, and reduce this in advance using a method such as stripping. It has become possible to simultaneously achieve 1) a sufficient average production rate, 2) a sufficient ammonium glycolate accumulation concentration, and 3) a sufficient catalyst productivity. Incidentally, when methanol is contained in a raw material containing glycolonitrile, when the glycolonitrile is synthesized from hydrocyanic acid and formalin, the stabilizer of the formalin, that is, what is present in formalin as a polymerization inhibitor remains in the raw material as it is. It is a substance that must be mixed in the conventional method for producing glycolonitrile. Thus, by paying attention to the influence of methanol in the raw material, a novel method for improving catalyst productivity can be found, and the present invention has been completed.

That is, the present invention has a configuration as described below.
(1) In a method for producing glycolic acid or ammonium glycolate, which comprises reacting nitrilase with glycolonitrile, the concentration of methanol in the raw material containing glycolonitrile is 2% by weight or less, or A method for producing ammonium glycolate.
(2) The method for producing glycolic acid or ammonium glycolate according to (1), wherein the concentration of methanol in the raw material is adjusted to 2% by weight or less by stripping a raw material containing glycolonitrile.
(3) The nitrilase is any one or more biocatalysts selected from microbial cells or treated products thereof, or immobilized nitrilases or suspensions of microbial cells (1) or (2) A process for producing glycolic acid or ammonium glycolate as described in 1.
(4) The nitrilase is a nitrilase derived from Acinetobacter sp. AK226 (accession number FERM BP-08590) or a protein enzyme encoded by the nitrilase gene of Acinetobacter sp. AK226 (accession number FERM BP-08590), (1) To a method for producing glycolic acid or ammonium glycolate according to any one of (3).
(5) The glycolic acid according to any one of (1) to (4), wherein the nitrilase is a biocatalyst selected from an immobilized product and / or a suspension of a gram-negative bacterium and / or a processed product thereof A method for producing ammonium glycolate.
(6) The glycolic acid according to any one of (1) to (5), wherein the nitrilase is a biocatalyst selected from an immobilized product and / or a suspension of cells of the genus Acinetobacter and / or a processed product thereof Or the manufacturing method of ammonium glycolate.
(7) From (1), wherein the nitrilase is a biocatalyst selected from an immobilized product and / or suspension of a microbial cell of Acinetobacter sp. AK226 (Accession No. FERM BP-08590) and / or its treated product ( 6) The method for producing glycolic acid or ammonium glycolate according to any one of the above.

  The present invention uses glycolonitrile synthesized substantially from hydrocyanic acid and formalin as raw materials to produce glycolic acid or ammonium glycolate using nitrilase, 1) sufficient average production rate, 2) sufficient glycol It is possible to provide an industrially advantageous production method capable of simultaneously achieving three acid ammonium accumulation concentrations and 3) sufficient catalyst productivity.

  The notation “glycolic acid or ammonium glycolate” in the present invention will be described first. When glycolonitrile is hydrolyzed using nitrilase, N in glycolonitrile is converted to ammonia. Usually, under the hydrolysis reaction conditions using nitrilase, the ammonia is instantly produced together with glycolic acid that is produced at the same time. Since a salt is formed, ammonium glycolate is finally produced. However, since the process of synthesizing glycolic acid has passed through the reaction mechanism, the present invention adopts the notation of glycolic acid or ammonium glycolate as a method meaning both glycolic acid or ammonium glycolate.

  The average production rate defined in the present invention represents a value calculated as an average value from the initial stage to the end of the reaction weight of ammonium glycolate per hour per dry biocatalyst weight used, and the unit is [mmol- Ammonium glycolate / g-dry biocatalyst weight / Hr]. The dry biocatalyst weight represents the dry weight of the microbial cell when microbial cells are used, the dry weight of nitrilase when nitrilase is used, and the fixed weight when the immobilized microbial cell or nitrilase is used. This represents the dry weight of microbial cells or nitrilase in the chemical. As the average production rate defined here is higher, glycolic acid or ammonium glycolate having a target concentration can be produced in a shorter time, which becomes very important as an indicator of practicality.

  The ammonium glycolate accumulation concentration defined in the present invention represents the weight concentration of ammonium glycolate in the reaction solution at the end of the reaction, and the unit is wt%. The high concentration of ammonium glycolate indicates that the biocatalyst having the nitrilase used is less susceptible to product (ammonium glycolate) inhibition and the activity of the nitrilase enzyme is maintained even at high salt concentrations. The above-mentioned high average production rate and high concentration of ammonium glycolate at the same time are very important as an index indicating the practicality of glycolic acid or ammonium glycolate as a production method.

The catalyst productivity defined in the present invention represents the weight of ammonium glycolate produced per reaction weight per dry biocatalyst used, and the unit is [g-ammonium glycolate / g-dry biocatalyst weight]. . Here, the reaction end time indicates a time when the substantial catalyst activity approaches zero as much as possible, and indicates a time when it is determined that the catalyst productivity hardly increases even if the reaction time is further extended. The high productivity of the catalyst means that the biocatalyst having the nitrilase used is less susceptible to product (ammonium glycolate) inhibition, as in the above-mentioned ammonium glycolate accumulation concentration. Indicates that activity is maintained. The high productivity of the catalyst is very important as an indicator of practicality as a method for producing glycolic acid or ammonium glycolate.

The glycolonitrile used as a raw material in the present invention can be synthesized by a generally known technique. The method for producing glycolonitrile is not particularly limited, but as a general synthesis method, a method of synthesizing from hydrocyanic acid and formaldehyde in an aqueous medium with an alkali catalyst is known. (Japanese Patent Publication No. 7-30004, JP-A-6-135923) Of these, formaldehyde is usually used in the form of an aqueous solution because of its easy handling. When handling formalin, from the viewpoint of preventing polymerization. The addition of the stabilizer methanol is known to those skilled in the art. The concentration of formaldehyde in formalin varies depending on the product, but generally it is about 35 to 40% by weight. The concentration of methanol in formalin also varies depending on the product, but is generally about 5 to 8% by weight. ing. Glycolonitrile in an aqueous solution of glycolonitrile synthesized by using such formalin as a raw material and reacting with hydrocyanic acid usually has a concentration of 52 to 57% by weight. Since methanol is mixed as it is, the methanol concentration is about 3 to 6% by weight.

  Impurity methanol in this reaction has little influence on the specific activity of nitrilase, but has a significant effect on catalyst productivity. The lower the methanol concentration, the higher the catalyst productivity. To produce glycolic acid or ammonium glycolate economically, the higher the productivity of the catalyst, the more advantageous. Therefore, in order to reduce the methanol concentration during the reaction, the methanol concentration in the raw glycolonitrile should be reduced. Is important. The methanol concentration in the raw material glycolonitrile in the present invention is preferably 2% by weight or less, more preferably 1% by weight or less, still more preferably 0.5% by weight or less.

  Various methods for reducing the methanol concentration in the present invention are conceivable. For example, it can be reduced by stripping operation of an aqueous solution of glycolonitrile. This stripping operation may be performed under any conditions as long as the thermal stability of glycolonitrile is sufficiently maintained, but glycolonitrile tends to become unstable at high temperatures and high pH. It causes the formation of formaldehyde and hydrocyanic acid by the decomposition reaction and the subsequent polymerization reaction of hydrocyanic acid, or the oligomerization and polymerization reaction of glycolonitrile, and the product is an alkaline substance such as an amine. In the worst case, it can cause an explosion. Therefore, the stripping operation of the glycolonitrile aqueous solution is preferably performed at a low temperature and a low pH as much as possible. On the other hand, since it is necessary to entrain some water to perform stripping of methanol, it is performed near the boiling point of water. Since the boiling point of water can be lowered by changing the operating pressure from atmospheric pressure to reduced pressure, it is better to set the reduced pressure condition to an industrially possible range when aiming at a low temperature condition. As a method for lowering the pH, it is conceivable to use an inorganic acid or an organic acid as a stabilizer for glycolonitrile. Commonly used are inorganic acids such as sulfuric acid and phosphoric acid, or organic acids such as acetic acid. Glycolic acid may be used for quality reasons. The pH range to be set is usually an acidic range below 7. However, in order to ensure stability, the pH is preferably 5 or less, more preferably 4 or less, still more preferably 3 or less, and most preferably 2 or less. Is good. The lower limit of the pH is not particularly limited as long as hydrolysis by acid does not occur, but it is usually preferably set to 1 or more. In addition, as a method for reducing the methanol concentration in the aqueous solution of glycolonitrile, it may be possible to remove methanol in advance by stripping operation from formalin used immediately before the synthesis of glycolonitrile.

  The nitrilase in the present invention may be of any kind as long as it has the ability to hydrolyze glycolonitrile and synthesize glycolic acid or ammonium glycolate. Examples of organisms from which the nitrilase enzyme is derived include microorganisms, animal and plant cells, and the like, but it is preferable to use a microbial nitrilase from the viewpoint of the amount of enzyme expression per weight and ease of handling.

  Many microbial species are known as the above microorganism species. Examples of those having high nitrilase activity include Rhodococcus genus, Acinetobacter genus, Alcaligenes genus, Psudomonas genus, Corynebacterium 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 described in JP-A No. 2001-299378, JP-A No. 11-180971, JP-A No. 06-303991, JP-A No. 63-209592 and JP-B No. 63-2596.

  Another nitrilase enzyme that is a natural or artificially improved nitrilase gene expressed by a microorganism incorporating a nitrilase gene having high conversion activity from glycolonitrile to glycolic acid or ammonium glycolate by genetic engineering techniques. However, in order to prepare nitrilase economically advantageously, it is desirable to use a microorganism that highly expresses nitrilase as much as possible.

  As the form of the biocatalyst in the present invention, microorganisms / animal / plant cells or the like may be used as they are, or the microorganisms / animal / plant cells etc. themselves, or those treated by crushing, or the microorganisms / animal / plant cells, etc. A product obtained by removing a necessary nitrilase enzyme and immobilized by a general entrapment method, a crosslinking method, a carrier binding method or the like may be used. 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 using microorganisms, animal and plant cells as they are, they may be suspended only in water (distilled water and / or ion-exchanged water), or suspended in an inorganic salt or organic acid salt buffer solution due to osmotic pressure. May be. Moreover, when using what was fix | immobilized, it is usually good to use suspending in a buffer solution from the relation of osmotic pressure. The buffer solution concentration 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 and activity of the enzyme, it is usually less than 0.1 M, preferably 0. 01 to 0.08M, more preferably 0.02 to 0.06M. Further, it is one method to use glycolate in order to reduce impurities.

  As for the conditions for the hydrolysis reaction of glycolonitrile, the pH is preferably 6 to 8, and preferably 6.5 to 7. As described above, since glycolonitrile as a raw material is a very unstable substance, it usually contains an acid component such as sulfuric acid, phosphoric acid or acetic acid as a stabilizer. Therefore, it is essential to add an alkali to the reaction system in order to adjust the pH in the reaction system. In this case, the alkali used is not particularly limited as long as it does not affect the reaction, but it is desirable to use ammonia as one of the products. The form of ammonia may be gas or ammonia water, but ammonia water is usually desirable because of easy handling. As for the reaction temperature, if the reaction temperature is too low, the reaction activity becomes low, and more reaction time is required when producing a high concentration of ammonium glycolate. On the other hand, if the reaction temperature is too high, thermal degradation of the nitrilase enzyme will make it difficult to reach that concentration if the final ammonium glycolate concentration is high, resulting in the addition of a biocatalyst with a new nitrilase enzyme. Therefore, the cost of the catalyst becomes high. Moreover, when the temperature is too high, the decomposition of the substrate glycolonitrile into hydrocyanic acid and formaldehyde is promoted, and the reaction activity is further reduced, such as reaction inhibition and deactivation. Therefore, the reaction temperature is usually from 30 to 60 ° C, preferably from 40 to 50 ° C.

  The reaction method for producing glycolic acid or ammonium glycolate 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 half-batch reaction. When is used, 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 having nitrilase may be used in a single batch or repeatedly. 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 steady concentration of glycolonitrile as a reaction substrate is preferably 2% by weight or less, more preferably 0.1 to 1.5% by weight, still more preferably 0.1 to 1.0% by weight, most preferably 0. It is better to control to 2 to 0.5% by weight. If the concentration of glycolonitrile is too high, the effects of product inhibition and / or deactivation, or substrate inhibition and / or deactivation that become noticeable only at high product accumulation concentrations rapidly increase, and the reaction has been progressing until then. May stop. On the other hand, if the concentration of glycolonitrile is too low, the reaction rate is lowered, which is disadvantageous because glycolic acid or ammonium glycolate cannot be produced efficiently. For the above reasons, it is very important to control the steady concentration of glycolonitrile during the reaction.

  The weight ratio of the dry biocatalyst used to the produced ammonium glycolate is preferably 1/100 or less, preferably 1/100 to 1/200, more preferably 1/200 to 1/300, and even more preferably 1/300 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.

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

  The method for measuring the weight of the dried biocatalyst in the biocatalyst suspension was performed as follows. First, an appropriate amount of a biocatalyst suspension having an appropriate concentration was taken, cooled to −80 ° C., completely dried using a freeze dryer, and the concentration of the biocatalyst suspension was calculated from the weight value. For the immobilized product, the dry biocatalyst weight was calculated from the amount of the biocatalyst suspension that was known at the time of immobilization and the amount of the crosslinking agent and the immobilized carrier.

The analysis of the reaction solution and the treatment solution was performed as follows. The substrate, glycolonitrile, and the product, glycolic acid or ammonium glycolate, and the by-product, glycolamide, 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)
In addition, analysis of methanol in the raw material and the reaction solution was performed by gas chromatography. The main body of the gas chromatograph is Shimadzu GC-14B, the column is a strong polarity column (TC-FFAP manufactured by GL Sciences), the injection temperature is 250 ° C, the column temperature is 50 ° C x 5 min, the temperature is raised 20 ° C / min, 220 ° C x 5 min, the detector Temperature: 250 ° C., detector: FID, sample injection volume: 2 μL

[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 250 ml of a culture solution containing 0.005% by weight of the Japanese product was charged into an Erlenmeyer flask, adjusted with sodium hydroxide to a pH of 7, sterilized at 121 ° C. for 20 minutes, and then 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%, calcium chloride dihydrate 0.01%, iron sulfate heptahydrate 0.003%, zinc sulfate heptahydrate 0.002%, copper sulfate pentahydrate 0.002% A 5 L jar fermenter was charged with 3 L of a broth containing 2% by weight and 2% soybean oil, sterilized at 121 ° C. for 20 minutes, inoculated with the pre-culture solution, 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 10% by weight) suspended in phosphate buffer was obtained.

[Preparation of raw material glycolonitrile]
For the preparation of the raw material glycolonitrile, an aqueous glycolonitrile solution (52 wt% glycolonitrile) manufactured by Tokyo Chemical Industry was used. As a result of gas chromatography analysis of an aqueous solution of glycolonitrile manufactured by Tokyo Chemical Industry, it was found to contain 0.90% by weight of methanol. The raw material having a higher methanol concentration was prepared by adding commercially available methanol (manufactured by Wako Pure Chemical Industries). In addition, the raw material with a low methanol concentration is Tokyo Kasei's glycolonitrile, which is stripped under the conditions of 50 ° C x 40 mmHg to distill off the methanol together with water, and the concentrated glycolonitrile concentration is the original 52 wt. It was prepared by adding distilled water to return to%.

[Examples 1 and 2, Comparative Example 1]
Using the Acinetobacter sp. AK226 suspension (10 wt%) obtained as described above and distilled water, a 16.1 g cell suspension (dry cell 0.16 g) was charged into a 200 mL four-necked flask. 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 raw material 52 wt% glycolonitrile aqueous solution was fed at 3.6 g / Hr using a liquid chromatography pump. In order to neutralize sulfuric acid contained as a stabilizer in the raw material glycolonitrile, 1.5 wt% aqueous ammonia was fed by a tube pump. The ammonia water feed pump was set so that the pH of the internal solution was 6.9 ± 0.1 by control with a pH meter. Sampling was periodically performed during the reaction, and the concentrations of glycolonitrile and ammonium glycolate were measured by high performance liquid chromatography, and the amount of raw material added was adjusted so that the steady glycolonitrile concentration was 2% by weight or less. The methanol concentration of the aqueous glycolonitrile solution used and the results of the reaction are shown in Table 1, FIG. 1, FIG. 2, and FIG.

  According to the present invention, 1) sufficient average production rate, 2) sufficient ammonium glycolate accumulation concentration, 3) sufficient to produce glycolic acid or ammonium glycolate using glycolonitrile as a raw material using nitrilase It is possible to provide an industrially advantageous production method that can simultaneously achieve three of the catalyst productivity.

FIG. 1 shows the change over time in the concentration of ammonium glycolate accumulated when a hydrolysis reaction is performed in the presence of methanol at various concentrations using a glycolonitrile aqueous solution as a raw material and using a biocatalyst. FIG. 2 shows the production amount of ammonium glycolate accumulated when a hydrolysis reaction is performed in the presence of methanol at various concentrations using a glycolonitrile aqueous solution as a raw material and using a biocatalyst. FIG. 3 shows the relationship between methanol concentration and catalyst productivity when a hydrolysis reaction is carried out using a glycolonitrile aqueous solution as a raw material and using a biocatalyst in the presence of each concentration of methanol.

Claims (7)

In a process for producing glycolic acid or ammonium glycolate, which comprises allowing nitrilase to act on glycolonitrile synthesized from formalin and methanol containing methanol as a stabilizer, the concentration of methanol in the raw material containing glycolonitrile is 0.5 wt. % Or less, a method for producing glycolic acid or ammonium glycolate. The glycolic acid or ammonium glycolate according to claim 1, wherein methanol in the raw material is removed by stripping a raw material containing glycolonitrile, and the methanol concentration is 0.5 wt% or less. Production method. The glycolic acid according to claim 1 or 2, wherein the nitrilase is any one or more biocatalysts selected from microbial cells or treated products thereof, or immobilized nitrilase or suspension derived from microbial cells. Or the manufacturing method of ammonium glycolate. Nitrilase is Acinetobacter sp. (Acinetobacter sp.) Nitrilase derived from AK226 (Accession No. FERM BP-08590) or Acinetobacter sp. (Acinetobacter sp.) The method for producing glycolic acid or ammonium glycolate according to any one of claims 1 to 3, which is a protein enzyme encoded by the nitrilase gene of AK226 (accession number FERM BP-08590). The production of glycolic acid or ammonium glycolate according to any one of claims 1 to 4, wherein the nitrilase is a biocatalyst selected from an immobilized product and / or a suspension of a gram-negative bacterium and / or a processed product thereof. Method. The glycolic acid or glycol according to any one of claims 1 to 5, wherein the nitrilase is a biocatalyst selected from an immobilized product and / or a suspension of cells of the genus Acinetobacter and / or a processed product thereof. Method for producing ammonium acid. Nitrilase is Acinetobacter sp. (Acinetobacter sp.) Any one of claims 1 to 6, which is a biocatalyst selected from an immobilized product and / or a suspension of a bacterial cell of AK226 (Accession No. FERM BP-08590) and / or a processed product thereof. A process for producing glycolic acid or ammonium glycolate as described in 1.

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