JP4647059B2 - Microbiological production method that prevents coloring of glycine - Google Patents

Description

【００３２】
【発明の効果】
本発明の製造方法は、蟻酸化合物の存在下グリシノニトリルに微生物またはその処理物を作用させることで、分解や着色反応を伴わず、乾燥菌体当たり、且つ単位時間当たり高活性であって菌体や培地の多量廃棄を伴わず、反応液のpHを調整するための酸、アルカリまたは緩衝液の添加や廃棄を伴わず、グリシンとアンモニアが定量的に生成し、これらの分解および消費を伴わなず、グリシンとアンモニアを別々に回収出来る効果を有する。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a microbiological production method in which coloring of glycine is prevented. More specifically, the present invention relates to a microbiological production method for preventing glycine coloring, characterized in that a microorganism or a treated product thereof is allowed to act on an aqueous glycinonitrile solution in the presence of a reducing biochemical compound. The obtained glycine is useful as a food additive, a detergent, and a raw material for synthetic medicine and agricultural chemicals. The production method of the present invention can be utilized for efficiently industrially producing useful glycine.
[0002]
[Prior art]
A method for obtaining glycine by hydrolyzing glycinonitrile with a weakly alkaline aqueous solution using a microorganism is known. Japanese Patent Publication No. 58-15120 describes a method using Brevibacterium R312 strain maintained at pH 8, Japanese Patent Application Laid-Open No. 3-62391 discloses Corynebacterium N-774 strain in a reaction solution adjusted to pH 7.2. In addition, in JP-A-3-280889, a reaction solution whose pH is adjusted to around 7.7 is added to the genus Rhodococcus, Arthrobacter, Caseobacter, Pseudomonas, Enterobacter, Acinetobacter, Alkagenes. , A method using a microorganism belonging to the genus Corynebacteria or Streptomyces is disclosed.
[0003]
It is known that glycinonitrile is unstable in such weak alkaline aqueous solution. For example, it is disclosed that when pH is 2.5 or more, the stability is poor, and that the higher the pH, the higher the temperature, and the longer the elapsed time, the easier it is to undergo degradation, coloring, etc. 14420, JP 54-46720, JP 54-46721). Such decomposition and modification not only reduce the yield of glycine, but also require complicated treatment using activated carbon or a special ion exchange resin for decolorization (Japanese Patent Application Laid-Open Nos. 3-190851 and 4-226949). Issue description).
[0004]
Furthermore, in the conventional method, since an equal amount of ammonia accumulates in the aqueous solvent as glycine is produced, the pH becomes higher and the alkali becomes strongly alkaline. Therefore, there is a problem that coloring and modification of glycinonitrile cannot be avoided. As described above, the conventional method using microorganisms requires a troublesome operation because of a decrease in glycine yield and decolorization, and it has not been industrially feasible.
[0005]
[Problems to be solved by the invention]
In producing glycine from glycinonitrile using microorganisms, the present invention is not accompanied by decomposition or coloring reaction, is highly active per dry cell and per unit time, and does not involve a large amount of waste of cells or medium. Glycine and ammonia are quantitatively produced without the addition or disposal of acids, alkalis or buffers to adjust the pH of the reaction solution, and glycine and ammonia are recovered separately without their decomposition and consumption. An object of the present invention is to provide a method for producing glycine.
[0006]
[Means for Solving the Problems]
In order to solve such industrial problems, the present inventor has no activity of decomposition or coloring reaction, has high activity per cell and per unit time, and decomposes or consumes glycine and ammonia generated in the reaction system. First, the inventors intensively studied to construct a reaction system that can easily and quantitatively collect glycine and ammonia separately. Surprisingly, when producing glycine by reacting a microorganism or its treated product with an aqueous glycinonitrile solution, it was found that coloring can be prevented by reacting in the presence of a formic acid compound, and the present invention is completed. It came. That is, the present invention is a microbiological production method for preventing coloring of glycine, characterized in that a microorganism or a treated product thereof is allowed to act on an aqueous glycinonitrile solution in the presence of a formic acid compound.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be specifically described below. The glycinonitrile used in the present invention is not only pure glycinonitrile but also a reactant capable of forming glycinonitrile under reaction conditions such as a reaction product of formaldehyde, hydrocyanic acid and ammonia, a reaction product of glycolonitrile and ammonia. I can do it.
Although there is no restriction | limiting in the formic acid compound made to coexist in a reaction liquid by this invention, Formic acid esters, such as formates, such as formic acid and ammonium formate, methyl formate, and ethyl formate, are used, for example. Preferably, formic acid or ammonium formate is used. The addition amount of the formic acid compound may be 0.002 mol% to 8 mol%, preferably 0.02 mol% to 4 mol% with respect to glycinonitrile.
[0008]
The microorganisms used in the present invention, for example, Acinetobacter (Accinetobacter) genus Rhodococcus (Rhodococcus) genus, it is newly discovered that Corynebacterium (Corynebacterium) genus and Arukarigene scan (Alcaligenes) microorganism belonging to the genus is suitable However, the present invention is not limited to this. Specifically, Acinetobacter Espi-AK226 (FERM BP-2451), Acinetobacter Espi-AK227 (Mikken Kenki No. 8272), Rhodococcus mariis BP-479-9 (FERMBP-5219), Corynebacterium sp. C5 ( Microtechnical Laboratories No. 8931), Corynebacterium nitrilophilus ATCC 21419, Alkaligenes faecalis ATCC 8750. These strains are described in JP-A-2-84198, JP-A-7-30396, and JP-A-63-129988.
[0009]
For culturing the microorganisms used in the present invention, commonly used carbon sources such as glucose, glycerin, organic acids, dextrin, maltose and the like are used, and ammonia and its salts, urea, nitrate and organic nitrogen are used as nitrogen sources. Sources such as yeast extract, malt extract, peptone, meat extract and the like are used. In addition, inorganic nutrient sources such as phosphate, sodium, potassium, iron, magnesium, cobalt, manganese, and zinc are appropriately added to the medium. Culturing is carried out aerobically at pH 5 to 9, preferably pH 6 to 8, temperature 20 to 37 ° C, preferably 27 to 32 ° C. In the culture of the microorganism of the present invention, an enzyme inducer may be added to the above medium, for example, lactam compounds (γ-lactam, δ-lactam, ε-caprolactam, etc.), nitrile compounds, amide compounds, etc. may be used. Good.
[0010]
The microorganism of the present invention can be used industrially as it is, but a mutant strain improved by a method of inducing a mutation with an appropriate mutant agent or a genetic engineering technique, for example, a mutant strain that constitutively produces an enzyme should be bred and used. You can also. The microbial cells of the present invention are immobilized microbial cells or treated microbial cells collected from the culture solution (broken microbial cells, enzymes separated from crushed microbial cells, and enzymes separated and extracted from microbial cells or microbial cells) Processed product). Collection of bacterial cells from the culture solution can be performed by a known method.
[0011]
In the present invention, the bacterial cells and the treated bacterial cells separated by the above-described method can be temporarily suspended and stored in distilled water or a buffer solution. In this case, it is preferable to use distilled water in order to reduce waste after the reaction. In addition, a stabilizer such as glycine can be added to the storage solution for storage stabilization. Also in this case, it is preferable to use glycine in order to reduce waste after the reaction.
[0012]
Add glycinonitrile to the suspension of the bacterial cells and treated cells thus obtained, or directly add the suspension of the obtained bacterial cells and treated cells, or the treated cells and cells. By adding to an aqueous glycinonitrile solution, the hydrolysis reaction proceeds rapidly, and glycine can be produced. That is, usually, the microbial cell or the processed microbial cell, for example, 0.01 to 5% by weight in terms of dry cell, 1 to 30% by weight of the substrate glycinonitrile, is charged into the reaction apparatus as a temperature, for example The reaction may be performed at 0 to 60 ° C., preferably 10 to 50 ° C., for example, for 1 to 24 hours, preferably 3 to 8 hours.
[0013]
In this case, glycinonitrile may be added at a low concentration and added over time, or the reaction temperature may be changed over time. In this way, glycinonitrile is hydrolyzed and ammonia is generated at the same time as glycine, and the pH of the reaction solution is increased after the reaction compared to before the reaction. Thus, in order to suppress the increase in pH with the progress of the reaction, a buffer solution can be added before the reaction, or an acid or an alkali can be added during the reaction. However, in order to reduce waste after the reaction, it is preferable not to add such a buffer solution, acid or alkali to the reaction solution.
[0014]
Although the reaction can be carried out in an open reactor, the ammonia produced under closed reaction conditions using a closed reaction vessel in order to prevent environmental pollution due to the scattering of the produced ammonia and to collect valuable ammonia Is preferably temporarily stored in the reaction container. In this case, it is more preferable to attach a reaction separation device that separates ammonia generated at the same time as the reaction in order to suppress an increase in pH. Such a reaction separation method of ammonia can be carried out by a reaction distillation method of ammonia or an inert gas flow method.
[0015]
In the case of performing reactive distillation, the hydrolysis reaction apparatus is provided with a single tube tower, a shelf tower, or a packed tower with a cooler that cools and collects water accompanying ammonia, and is above the boiling pressure of the aqueous reaction solution, for example, 60 ° C. It is preferable to carry out reduced pressure reactive distillation continuously or intermittently under pressure conditions of 20.0 kPa or more to 0 ° C. or more and 0.6 kPa or more. More preferably, the reaction distillation under reduced pressure can be performed under a pressure condition of 12.6 kPa to 1.3 kPa.
[0016]
When circulating an inert gas, it is equipped with an inert gas blowing nozzle and a cooling trap that recovers ammonia and accompanying water from the inert gas. Can be separated from the reaction solution along with an inert gas. In addition, reduced pressure reactive distillation can be performed under inert gas flow conditions to facilitate ammonia separation. The reaction method can be performed by a batch method, a flow reaction method, or a method combining these.
[0017]
Thus, glycinonitrile is hydrolyzed with a molar yield of almost 100%, and all of the ammonia produced can be produced and accumulated as a high-concentration aqueous solution of glycine containing the ammonium salt of glycine once in a sealed reaction vessel. . Also, all or most of the ammonia produced is separated from the reaction solution by the reactive distillation method or inert gas flow method simultaneously with the reaction, and is cooled and recovered.
[0018]
If glycinamide remains, it can be completely converted to glycine and ammonia by adding a cell or enzyme having hydrolytic activity of glycinamide. Recovery of glycine from a high-concentration aqueous solution of glycine containing ammonium salt of glycine, for example, after removing cells from the reaction solution by centrifugal filtration, membrane separation, etc., glycine can be crystallized, ion-exchanged or poor solvent The ammonia can be recovered by distillation or extraction after evaporation together with some water.
The present invention will be described based on examples, but the present invention is not limited to these examples.
[0019]
[Example 1]
In order to prevent oxygen contamination, all reaction operations are performed in a nitrogen atmosphere. All aqueous solutions used for the reaction are cooled to about 5 ° C, pressurized with nitrogen gas, and then returned to normal pressure several times. Was replaced.
[0020]
(1) Synthesis of glycinonitrile An excess amount of an aqueous ammonia solution was added to an aqueous solution of glycolonitrile once produced by allowing an equal amount of hydrocyanic acid to act on formalin in a nitrogen atmosphere and reacted for 2 hours, followed by unreacted ammonia. And excess water was removed under reduced pressure to obtain a 30 wt% aqueous glycinonitrile solution. When the absorbance of the aqueous solution was measured at a wavelength of 380 nm, it was 0.08 per 1 mol of glycinonitrile and 10 mm quartz cell.
[0021]
(2) Culture of bacterial cells Acinetobacter AK226 strain was cultured under the following conditions.
(1) Medium fumaric acid 1.0% by weight
Meat extract 1.0
Peptone 1.0
Salt 0.1
ε-Caprolactam 0.3
Potassium phosphate 0.2
Magnesium sulfate heptahydrate 0.02
Ammonium chloride 0.1
Ferric sulfate, heptahydrate 0.003
Manganese chloride tetrahydrate 0.002
Cobalt chloride hexahydrate 0.002
pH 7.5
(2) Culture conditions 30 ° C / 1 day [0022]
(3) The glycinonitrile hydrolyzed bacterial cells were collected from the obtained culture broth by centrifugation, washed with distilled water, then replaced with nitrogen gas and used for the reaction. The reaction was started at 30 ° C. in a 100 ml glass autoclave purged with nitrogen gas and 3 ml of a 30 wt% glycinonitrile aqueous solution containing 58 mg of dry cell mass and 3.3 mg of formic acid in 17 ml of distilled water. Two hours after the start of the reaction, the pH was 10. This reaction solution was analyzed by liquid chromatography, and glycinonitrile disappeared and glycine was quantitatively produced.
[0023]
Therefore, 3 ml of the above 30% by weight glycinonitrile aqueous solution was added every 2 hours, and the reaction solution was analyzed by liquid chromatography. This operation was repeated four times and the reaction was carried out for a total of 10 hours. Using 2 g of the obtained 32 g reaction solution, the produced ammonia was quantified by the Nessler method, the raw material glycinonitrile and the produced glycine were analyzed by a liquid chromatography method, glycinonitrile disappeared, and glycine and ammonia were It was generated quantitatively.
[0024]
The production amount of glycine per dry cell was 104 g / g dry cell, and the production activity of glycine was 10 g / g · Hr. 2 ml of the reaction solution was subjected to centrifugal filtration at 10500 rpm for 15 minutes to separate the cells, and the UV-visible absorption spectrum of the supernatant was measured. The absorbance at a wavelength of 380 nm was 0.12 per 1 mol / cm of glycine.
[0025]
[Comparative Example 1]
The same reaction as in Example 1 was performed without adding formic acid. The amount of glycine produced per dry cell remained unchanged at 120 g / g dry cell, and the production activity of glycine was 12 g / g · Hr. The absorbance of the supernatant after centrifugal filtration was 0.79 per 1 mol / cm of glycine.
[0026]
[Examples 2 to 5 ]
The same culture operation and reaction as in Example 1 were performed, and the type of bacterial cells and the type of formic acid compound were changed. Table 1 shows the types of cells and the types of formic acid compounds. Table 1 also shows the results of Example 1 and Comparative Example 1.
[0027]
[Reference example]
The reaction was carried out using the 30% by weight glycinonitrile aqueous solution synthesized in Example 1 and the bacterial cells. The cells were collected from the obtained culture broth by centrifugation, washed with distilled water, and then replaced with nitrogen gas for use in the reaction. Sampling connected to a single-tube distillation column, pressure sensor, thermometer, and liquid feed pump connected to a vacuum pump via a dry ice strap in a 1000 ml constant temperature jacketed tank type 3 neck separable flask with a stirrer Equipped with a tube.
[0028]
After replacing the separable flask with nitrogen gas, 30 ml of a 30% by weight glycinonitrile aqueous solution containing 740 mg of dry cells and 63 mg of L-ascorbic acid and 170 ml of distilled water were prepared. The pressure in the flask was adjusted to 10 kPa with a vacuum pump, and the reaction was started at 30 ° C. One hour after the start of the reaction, this reaction solution was analyzed by liquid chromatography. As a result, glycinonitrile disappeared and glycine was quantitatively produced. Therefore, 30 ml of a 30% by weight aqueous glycinonitrile solution was additionally added. This operation was repeated three more times every hour for a total of 5 hours.
[0029]
20 g of solid was recovered in the dry eye strap. The solid was dissolved in 50 ml of water and quantified by the Nessler method, and 14 g of ammonia was recovered. 300 g of the reaction solution was recovered. Using 2 g of the reaction solution, the produced ammonia was quantified by the Nessler method, and the raw material glycinonitrile and the produced glycine were analyzed by a liquid chromatography method. The glycinonitrile disappeared, and glycine was quantitatively produced, and a trace amount of ammonia remained.
[0030]
The production amount of glycine per dry cell was 81 g / g dry cell, and the production activity of glycine was 16 g / g · Hr. 2 ml of the reaction solution was subjected to centrifugal filtration at 10500 rpm for 15 minutes to separate the cells, and the UV-visible absorption spectrum of the supernatant was measured. Absorbance at a wavelength of 380 nm was 0.09 per 1 mol / cm of glycine.
[0031]
[Table 1]

[0032]
【The invention's effect】
In the production method of the present invention, a microorganism or a processed product thereof is allowed to act on glycinonitrile in the presence of a formic acid compound, so that it is free from decomposition or coloring reaction, has high activity per dry cell and per unit time, and has a high activity. Glycine and ammonia are quantitatively produced without the waste of body and medium, and without the addition or disposal of acids, alkalis or buffers to adjust the pH of the reaction solution. However, it has the effect of collecting glycine and ammonia separately.

Claims (7)

Microbiology that prevents glycine from coloring, characterized by allowing microorganisms or their processed products to act on glycinonitrile aqueous solution in the presence of formic acid compound without adding acid, alkali or buffer to adjust pH Manufacturing method.   The method according to claim 1, wherein the formic acid compound is formic acid.   The method according to claim 1, wherein the formic acid compound is ammonium formate.   4. The process according to claim 1, wherein the glycinonitrile is obtained by a reaction of formaldehyde, hydrocyanic acid and ammonia.   5. The method according to claim 1, wherein the microorganism belongs to the genus Accinetobacter, Rhodococcus, Corynebacterium, or Alcaligenes. . The method according to any one of claims 1 to 5 , wherein the reaction condition for causing the microorganism to act is a reaction condition for separating ammonia produced in the reaction solution from the reaction solution. 7. The method according to claim 6 , wherein the method for separating ammonia from the reaction solution is reactive distillation.