JP2001275692A - Method for microbiologically producing glycine with no occurrence of discoloration of glycine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce glycine from glycinonitrile by using a microorganism that can quantitatively form glycine and ammonia with high activity based on the dried cell bodies of the microorganism and per unit time without accompaniment of decomposition and discoloration during the cultivation, and can form glycine and ammonia quantitatively without a large volume of waste disposal of the cell bodies and culture medium and the addition or exhaustion of acids, alkalis of buffering solutions for adjusting the pH of the reaction mixture and can recover the glycine and the ammonia separately. SOLUTION: In the presence of a reductive biochemical compound, a microorganism or its treated product is allowed to act on glycinonitrile, preferably under the reaction condition in no need of addition of acid, alkali or buffer for adjusting the pH, or under the closed-system of the reaction condition or under reaction condition where the formed ammonia is exhausted out of the reaction system, as it is formed, a microorganism or its treated product is allowed to act on glycinonitrile to recover the formed glycine and the ammonia separately.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a microbiological production method in which glycine is prevented from being colored. More specifically,
The present invention relates to a microbiological production method for preventing the coloration of glycine, wherein a microorganism or a processed 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 synthesizing medical and agricultural chemicals. The production method of the present invention can be used to produce useful glycine efficiently and industrially.

[0002]

2. Description of the Related Art There is known a method for obtaining glycine by hydrolyzing glycinonitrile with a weak alkaline aqueous solution using a microorganism. Japanese Patent Publication No. 58-15120 discloses a method of using Brevibacterium R312 strain at a pH of 8, and Japanese Patent Application Laid-Open No. 3-62391 discloses a method wherein Corynebacterium strain N-774 is added to a reaction solution adjusted to a pH of 7.2. And JP-A-3-280889 discloses that a reaction solution having a pH adjusted to about 7.7 contains Rhodococcus, Arthrobacter, Caseobacter, Pseudomonas, Enterobacter, Acinetobacter and Alcaligenes. , Corynebacteria or Streptomyces are disclosed.

[0003] It is known that glycinonitrile is unstable in such a weak alkaline aqueous solution. For example, pH
Is 2.5 or more, the stability is poor, and it is disclosed that the higher the pH, the higher the temperature, and the longer the elapsed time, the more easily the metabolism such as decomposition or coloration is easily caused (JP-A-49-14).
No. 420, JP-A-54-47720, JP-A-54-4
6721). Such decomposition or denaturation not only lowers the yield of glycine, but also requires a complicated treatment using activated carbon or a special ion-exchange resin for decolorization (JP-A-3-190851, JP-A-4-226949). Specification). Further, in the conventional method, since an equal amount of ammonia accumulates in the aqueous solvent with the production of glycine, the pH becomes further higher and the alkali becomes more alkaline, so that there is a problem that glycinonitrile is inevitably colored or denatured. As described above, the conventional method using a microorganism requires a complicated operation for lowering the glycine yield and decoloring, and cannot be carried out industrially.

[0004]

SUMMARY OF THE INVENTION The present invention relates to the production of glycine from glycinonitrile using a microorganism.
Addition of acid, alkali or buffer solution to adjust the pH of the reaction solution without decomposing or coloring reaction, high activity per dry cell and per unit time, without large amount of cells and medium wasted It is an object of the present invention to provide a method for producing glycine, in which glycine and ammonia are quantitatively generated without accompanying or discarding the glycine and ammonia separately without decomposing and consuming them.

[0005]

In order to solve such industrial problems, the inventor of the present invention does not involve decomposition or coloring reaction,
Construct a reaction system that has high activity per cell and per unit time, does not decompose or consume glycine and ammonia generated in the reaction system, and can collect glycine and ammonia separately, quantitatively and easily. Investigation was carried out earnestly. Surprisingly, they have found that a specific compound does not impair the activity of such microorganisms, suppresses the coloring of the reaction solution, and obtains a high glycine yield, and has completed the present invention.

That is, according to the present invention, a microorganism or a processed product thereof is allowed to act on glycinonitrile in the presence of a reducing biochemical compound, preferably by adding an acid, alkali or buffer for adjusting pH. Under reaction conditions
In addition, by causing the microorganism or the treated product to act on glycinonitrile in the presence of a sulfite compound under the reaction conditions of a closed system or a reaction condition in which generated ammonia is separated out of the system at the same time as the reaction, decomposition and coloring reactions are caused. Acid, for adjusting the pH of the reaction solution without drying the cells and the medium, and having a high activity per unit time and a high amount of activity per unit time.
A method for producing glycine is provided, wherein glycine and ammonia are quantitatively produced without adding or discarding an alkali or a buffer, and without separately decomposing and consuming glycine and ammonia.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below. The glycinonitrile used in the present invention uses not only pure glycinonitrile but also a reactant capable of forming glycinonitrile under reaction conditions such as a reactant of formaldehyde, hydrocyanic acid and ammonia, or a reactant of glycolonitrile and ammonia. You can do it.

[0008] In the present invention, the reducing biochemical compound coexisting in the reaction solution is not limited. For example, ascorbic acid esters such as L-ascorbic acid and L-ascorbic acid stearic acid ester, and sodium L-ascorbate and the like. Cysteine salts such as salts, glutathione, L-cysteine, L-cysteine hydrochloride monohydrate, cysteine esters such as L-cysteine ethyl ester hydrochloride and L-cysteine methyl ester hydrochloride, or N-acetyl-L- N-substituted cysteines such as cysteine are used. Preferably, L-ascorbic acid is used. The amount of the reducing biochemical compound to be added is 0.001 mol% to 5 mol%, preferably 0.01 mol% to glycinonitrile.
It may be from 2 mol% to 2 mol%.

[0009] Examples of the microorganism used in the present invention include the genus Acinetobacter, the genus Corynebacterium, and the genus Alkaline.
aligenes) have been newly found to be suitable, but not limited thereto. Acinetobacter sp.A selected as a microorganism suitable for the present invention
K226 strain (A.sp.AK226) (hereinafter abbreviated as AK226) and Acinetobacter sp.AK227 strain (A.sp.AK227) (hereinafter AK227)
Was abbreviated to the Institute of Microorganisms and Industrial Technology Research Institute on May 28, 1985.
No. and the accession number of Microtechnological Laboratory No. 8272 are given, and the microbiological properties are as shown in Table 1 below.

[0010]

[Table 1]

[0011] The strains were identified according to the Bergy's Manual of Determinative Bacteriolog, 8th edition (1974). Further, Corynebacterium sp. C5 selected as a microorganism suitable for the present invention.
The strain (hereinafter abbreviated as C5) has been deposited with the Research Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology, and has been accorded the accession number of No. 8931 of Microbiological Research Institute, whose microbiological properties are described in JP-A-63-1299.
No. 88, as shown.

Also, Corynebacterium nitriophilus ATCC21 selected as a microorganism suitable for the present invention.
419 strains, Alcaligenes faecalis ATCC87
For culturing the microorganism used in the present invention, which can also use 50 strains, commonly used carbon sources, for example, glucose, glycerin, organic acids, dextrin, maltose, etc. are used, and ammonia and its salts,
Urea, nitrates and organic nitrogen sources, such as yeast extract,
Malt extract, peptone, meat extract and the like are used.

[0013] In addition, inorganic nutrients such as phosphate, sodium, potassium, iron, magnesium, cobalt, manganese and zinc are appropriately added to the medium. The cultivation is carried out at pH 5 to 9, preferably pH 6 to 8, at a temperature of 20 to 37 ° C, preferably at 2
Performed aerobically at 7 to 32 ° C. In culturing the microorganism of the present invention, an enzyme inducer may be added to the above-mentioned medium, for example, lactam compounds (γ-lactam, δ-lactam, ε
-Caprolactam and the like), nitrile compounds, amide compounds and the like. Although the microorganism of the present invention can be used industrially as it is, it is necessary to grow and use a mutant strain improved by a method for inducing mutation with a suitable mutagen or a genetic engineering technique, for example, a mutant strain that produces an enzyme constitutively. Can also. The cells of the present invention are cells immobilized with cells collected from a culture solution or treated cells (crushed cells, enzymes separated from the cells, and enzymes separated or extracted from the cells). Processed material). Collection of the cells from the culture solution can be performed by a known method.

In the present invention, the cells isolated by the above-mentioned method and the treated cells are suspended in an aqueous glycinonitrile solution, whereby the hydrolysis reaction proceeds rapidly to produce glycine. That is, usually, the microbial cells or the processed cells are, for example, 0.01 to 5% by weight, the substrate glycinonitrile is 1 to 30% by weight, and the glycinonitrile is 0.001 mol% to 5 mol%. An aqueous suspension containing preferably 0.01 mol% to 0.2 mol% of the reducing biochemical compound is charged into the reactor at a temperature of, for example, 0 to 60 ° C., preferably 10 to 50 ° C., and the reaction time is, for example, The reaction may be performed for 1 hour to 24 hours, preferably 3 hours to 8 hours.

In this case, glycinonitrile may be added at a low concentration and added over time, or the reaction temperature may be changed over time. Also, in reducing waste after the reaction,
It is preferable not to add a buffer for adjusting H, an acid or an alkali to the reaction solution. In addition, in order to recover the generated ammonia, the generated ammonia may be temporarily accumulated in the reaction vessel using a closed reaction vessel, but the reaction in which the generated ammonia is separated simultaneously with the reaction in order to suppress a rise in pH is performed. Preferably, a separating device is provided. Such a method of reacting and separating ammonia can be carried out by a reactive distillation method of ammonia or a flow method of an inert gas. When performing reactive distillation, a single-tube tower with a cooler for cooling and recovering water accompanying ammonia, a shelf tower,
Alternatively, it is preferable to provide a packed tower and continuously or intermittently perform reduced-pressure reactive distillation under a pressure condition of not less than the boiling pressure of the reaction aqueous solution, for example, 20.0 kPa or more at 60 ° C. to 0.6 kPa or more at 0 ° C.

[0016] More preferably, from 12.6 kPa to 1.
Vacuum reactive distillation can be performed under a pressure condition of 3 kPa. When flowing an inert gas, the system is provided with a nozzle for blowing the inert gas and a cooling trap for recovering ammonia and accompanying water from the inert gas. Can be entrained in an inert gas and separated from the reaction solution. Further, in order to promote the separation of ammonia, the reduced pressure reactive distillation can be performed under an inert gas flow condition. The reaction system can be carried out by a batch type system, a flow type reaction system, or a combination thereof.

Thus, glycinonitrile is almost 100
% Of the resulting ammonia can be once produced and accumulated in a closed reaction vessel as a high-concentration aqueous solution of glycine containing an ammonium salt of glycine. Further, all or most of the produced ammonia is separated from the reaction solution by a reactive distillation method or an inert gas flow method at the same time as the reaction, and is cooled and recovered. If glycinamide remains, it can be completely converted to glycine and ammonia by additionally adding cells or enzymes having glycinamide hydrolysis activity. Glycine is recovered from a high-concentration aqueous solution of glycine containing an ammonium salt of glycine, for example, after removing cells by centrifugal filtration or membrane separation from the reaction solution, glycine is crystallized,
It can be recovered by an ion exchange method or a fractional precipitation method using a poor solvent, and ammonia can be recovered by distillation or extraction after evaporating together with a part of water. The present invention will be described based on examples, but the present invention is not limited to these examples.

[0018]

Example 1 In order to prevent the incorporation of oxygen, all reaction operations were performed under a nitrogen atmosphere, and all aqueous solutions used for the reaction were about 5
The operation of cooling to ° C., pressurizing once with nitrogen gas, and returning to normal pressure again was repeated several times to replace the air. (1) Synthesis of glycinonitrile An excess amount of aqueous ammonia solution was added to an aqueous solution of glycolonitrile once produced by reacting an equal amount of hydrocyanic acid to formalin under a nitrogen atmosphere, and reacted for 2 hours. And excess water were removed under reduced pressure to obtain a 30% by weight glycinonitrile aqueous solution. The absorbance of the aqueous solution measured at a wavelength of 380 nm was 1 mol of glycinonitrile and 0.08 per 10 mm quartz cell.

(2) Culture of cells The 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 ・ 7 hydrate 0.02 Ammonium chloride 0.1 Ferric sulfate ・ 7 hydrate 0.003 Manganese chloride ・ 4 water Salt 0.002 Cobalt chloride hexahydrate 0.002 pH 7.5 (2) Culture conditions 30 ° C / 1 day

(3) Hydrolysis of glycinonitrile The cells were collected from the obtained culture by centrifugation, washed with distilled water, replaced with nitrogen gas, and used for the reaction. A 100 ml glass autoclave purged with nitrogen gas contains 49 mg of dry cells and 6.3 mg of L-ascorbic acid.
The reaction was started at 20 ° C. by mixing 3 ml of a 30% by weight aqueous glycinonitrile solution with 17 ml of distilled water. After the reaction starts 2
After hours, the pH had reached 10. The reaction mixture was analyzed by liquid chromatography, and glycinonitrile was lost and glycine was quantitatively generated. Then, the reaction temperature was raised by 5 ° C. every 2 hours, and 3 ml of the above 30% by weight glycinonitrile aqueous solution was additionally added, 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 reaction solution of 32 g,
The produced ammonia was quantified by the Nessler method, and the raw material glycinonitrile and the produced glycine were analyzed by liquid chromatography. The glycinonitrile disappeared, and glycine and ammonia were produced quantitatively. The amount of glycine produced per dry cell is 120 g / g dry cells,
The production activity of glycine was 12 g / g · Hr. 2 ml of the reaction solution was centrifuged at 10500 rpm for 15 minutes to separate the cells, and the ultraviolet-visible absorption spectrum of the supernatant was measured. Absorbance at a wavelength of 380 nm is 1 mol 1 of glycine
It was 0.11 per cm.

[0022]

Comparative Example 1 The same reaction as in Example 1 was carried out without adding L-ascorbic acid. The amount of glycine produced per dry cell was unchanged, 120 g / g dry cells, and the glycine production activity was 12 g / g · Hr. The absorbance of the supernatant after centrifugal filtration was 0.7 g / mol glycine / cm.
Nine.

[0023]

Examples 2 to 4 The same culturing operation and reaction as in Example 1 were carried out by changing the cells and reducing biochemical compounds as shown in Table 2. The results are shown in Table 2 together with Example 1 and Comparative Example 1.

[0024]

[Table 2]

[0025]

Example 5 The reaction was carried out using the 30% by weight aqueous glycinonitrile solution synthesized in Example 1 and changing the reaction system. The cells were collected from the resulting culture by centrifugation, washed with distilled water, replaced with nitrogen gas, and used for the reaction. Sampling connected to a single-tube distillation column connected to a vacuum pump through a dry ice trap, a pressure sensor, a thermometer, and a liquid feed pump in a 1000 ml thermostatic jacket tank type three-neck separable flask with a stirrer. With a tube.

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 was dried.
And 170 ml of distilled water. The pressure inside 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, the reaction solution was analyzed by liquid chromatography, and it was found that glycinonitrile had disappeared and glycine had been generated quantitatively. Therefore, 30 ml of a 30% by weight aqueous solution of glycinonitrile as a substrate was additionally added. This operation was repeated three more times every hour, and the reaction was carried out for a total of 5 hours.
20 g of solid was recovered in the dry ice trap.

When the solid was dissolved in 50 ml of water and quantified by the Nessler method, 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 glycinonitrile as a raw material and the produced glycine were analyzed by liquid chromatography. Glycinonitrile disappeared, glycine was quantitatively formed, and a trace amount of ammonia remained. The amount of glycine produced per dry cell is 8
1 g / g dry cells and glycine producing activity of 16 g / g
g · Hr. 2 ml of the reaction solution was centrifuged at 10500 rpm for 15 minutes to separate the cells, and the ultraviolet-visible absorption spectrum of the supernatant was measured. The absorbance at a wavelength of 380 nm was 0.09 per 1 cm1 of glycine.

[0028]

According to 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 reducing biochemical compound, and preferably an acid, alkali or buffer solution for adjusting pH is prepared. Under reaction conditions without addition,
Decomposition and coloration of glycinonitrile in the presence of reducing biochemical compounds by the action of microorganisms or their processed products under closed-system reaction conditions or reaction conditions in which generated ammonia is separated out of the system simultaneously with the reaction Addition or disposal of acid, alkali or buffer to adjust the pH of the reaction solution without reaction, high activity per dried cell and per unit time, without accompanying large amount of cells and medium. Without this, glycine and ammonia are quantitatively generated, and there is an effect that glycine and ammonia can be separately recovered without accompanying their decomposition and consumption.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:15) C12R 1:15) (C12P 13/04 (C12P 13/04 C12R 1:05) C12R 1 : 05) (C12N 1/20 (C12N 1/20 A C12R 1:01) C12R 1:01) (C12N 1/20 (C12N 1/20 A C12R 1:15) C12R 1:15) (C12N 1/20 (C12N 1/20 A C12R 1:05) C12R 1:05)

Claims (8)

[Claims] 1. A microbiological production method in which glycine is prevented from being colored, wherein a microorganism or a processed product thereof is allowed to act on an aqueous glycinonitrile solution in the presence of a reducing biochemical compound. 2. The method according to claim 1, wherein the reducing biochemical compound is ascorbic acid. 3. The method according to claim 1, wherein the reducing biochemical compound is glutathione. 4. The method according to claim 1, wherein the reducing biochemical compound is cysteine. 5. Glycinonitrile is formaldehyde,
The method according to any one of claims 1 to 4, wherein the method is obtained by a reaction between hydrocyanic acid and ammonia. 6. The method according to claim 1, wherein the microorganism is Accinetobact.
The method according to any one of claims 1 to 5, wherein the microorganism is a microorganism belonging to the genus er), the genus Corynebacterium, or the genus Alcaligenes. 7. The method according to claim 1, wherein the conditions under which the microorganisms or the processed product thereof act are reaction conditions in which no acid, alkali or buffer for adjusting pH is added. The described method. 8. The method according to claim 7, wherein the reaction conditions are such that ammonia generated in the reaction solution is separated from the reaction solution.