JP2001269190A - Microbiological method for production of glycine prevented from discoloration - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing glycine from glycinonitrile using a microorganism, wherein the process is not accompanied by decomposition or discoloration, is efficient per dry cell mass per unit time, is not accompanied by the disposal of a large amount of cell or spent broth, is not accompanied by the addition or disposal of acid, alkali or buffer for adjusting pH of the reaction liquid, permits stoichiometrically producing glycine and ammonia, is not accompanied by any decomposition or consumption of these, and permits separately collecting these. SOLUTION: This method comprises such a process that glycinonitrile is subjected to a microorganism or product therefrom in the presence of a sulfite compound, preferably under a reaction condition that does not require adding acid, alkali or buffer for adjusting pH, in a closed system or system that permits simultaneous separation of formed ammonia outside the system, thus separately collecting glycine and ammonia.

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 in which glycine is prevented from being colored, wherein a microorganism or a treated product thereof is allowed to act on an aqueous glycinonitrile solution in the presence of a sulfite. 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, by reacting a microorganism or a processed product thereof with glycinonitrile in the presence of a sulfite compound, a reaction is preferably performed without adding an acid, alkali or buffer for adjusting pH. Decomposition and coloration of glycinonitrile in the presence of a sulfite compound by the action of microorganisms or their processed products under the conditions of a closed system or under the reaction conditions of separating generated ammonia out of the system simultaneously with the reaction Without a reaction, per dry cell,
It has high activity per unit time and does not involve large amounts of cells and medium, and does not require the addition or disposal of acids, alkalis or buffers to adjust the pH of the reaction solution, and glycine and ammonia can be quantitatively determined. A process for the production of glycine is provided, wherein the glycine and the ammonia are produced separately, without the decomposition and consumption thereof, and glycine and ammonia are separately recovered.

[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.

In the present invention, the sulfite compound coexisting in the reaction solution is not limited. For example, sulfurous acid gas, sulfurous acid, disodium sulfite, sodium hydrogen sulfite, dipotassium sulfite, potassium hydrogen sulfite, diammonium sulfite and the like are used. Preferably, diammonium sulfite is used as the ammonium salt of sulfite. The addition amount of the sulfite compound is 0.001 mol% based on glycinonitrile.
~ 5mol%, preferably 0.01mol% to 2mo
1% may be sufficient.

[0009] Examples of the microorganisms used in the present invention include the genus Acinetobacter, the genus Rhodococcus, and the genus Corynebacteria.
Microorganisms belonging to the genus acterium or the genus Alcaligenes have been newly found to be suitable, but are not limited thereto. Acinetobacter sp. AK226 strain (A. sp. AK22) selected as a microorganism suitable for the present invention
6) (hereinafter abbreviated as AK226), or Acinetobacter sp.A
The K227 strain (A.sp. AK227) (hereinafter abbreviated as AK227) has 19 strains.
Deposited on May 28, 1985, with the Institute of Microbial Industry and Technology, the National Institute of Advanced Industrial Science and Technology
No. 272 has been assigned an accession number, 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). The Rhodococcus maris BP-479-9 strain selected as a microorganism suitable for the present invention was originally deposited on November 2, 1993 at the Institute of Microbial Industry and Technology, and on September 1, 1995, It was transferred to the deposit and given a deposit number of FERM BP-5219, and the microbiological properties are as shown in Table 2 below.

[0012]

[Table 2]

[0013] In identifying the strain, the Berg's Manual of Determinative Bacteriolog (Bergy's Manual of Systematic Bacteriolog)
Vol. (1986) and The Prokaryote
s) Classified according to the second edition (1992). In addition, Corynebacterium s selected as a microorganism suitable for the present invention
p. strain C5 (hereinafter abbreviated as C5) was deposited with the National Institute of Microbial Industry and Technology, and given the accession number of Microtechnical Research Bacteria No. 8931. −
As described in JP-A-129988.

In addition, 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.

[0015] 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, a mutant strain improved by a method of inducing a mutation with a suitable mutagen or a genetic engineering technique, for example, a mutant strain that produces an enzyme constitutively, is grown. Can also be used. 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 a sulfite compound is charged into a reactor at a temperature of, for example, 0 to 60 ° C., preferably 10 to 50 ° C., and the reaction time is, for example, 1 hour to The reaction may be performed for 24 hours, preferably for 3 to 8 hours.

In this case, glycinonitrile may be charged 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 the reactive distillation, the hydrolysis reaction apparatus is provided with a single-tube tower, a tray tower, or a packed tower equipped with a cooler for cooling and recovering water accompanying ammonia. For example, it is preferable to continuously or intermittently perform the reduced pressure distillation under a pressure condition of 20.0 kPa or more at 60 ° C. to 0.6 kPa or more at 0 ° C. More preferably, the distillation under reduced pressure can be performed under a pressure condition of 12.6 kPa to 1.3 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 by, for example, removing cells from the reaction solution by centrifugal filtration, membrane separation, etc., and then glycine is crystallized, ion-exchanged or poor solvent. And ammonia can be recovered by distillation or extraction after evaporating together with some of the water. The present invention will be described based on examples, but the present invention is not limited to these examples.

[0022]

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. In a 100 ml glass autoclave purged with nitrogen gas, 49 mg of dry cells and 2.4 g of diammonium sulfite monohydrate were added.
3 ml of 30% by weight glycinonitrile aqueous solution containing 17 mg
The reaction was started at 20 ° C. prepared in distilled water. Two hours after the start of the reaction, 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 two hours,
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 performed for a total of 10 hours. 3 obtained
Using 2 g of the 2 g reaction solution, the generated ammonia was quantified by the Nessler method, and the raw material glycinonitrile and the generated glycine were analyzed by liquid chromatography,
Glycinonitrile disappeared, and glycine and ammonia were produced quantitatively. The amount of glycine produced per dried cell was 120 g / g dried cells, and the activity of producing glycine was 12 g / g · Hr. 2 ml of the reaction solution was rpm 105
The cells were separated by centrifugal filtration at 00 for 15 minutes, and the ultraviolet-visible absorption spectrum of the supernatant was measured. Wavelength 380nm
Was 0.12 per 1 cm 1 of glycine.

[0026]

Comparative Example 1 The same reaction as in Example 1 was carried out without adding diammonium sulfite monohydrate. The amount of glycine produced per dry cell is 120 g / g dry cells unchanged,
The production activity of glycine was 12 g / g · Hr. The absorbance of the supernatant after centrifugal filtration was 0.79 per 1 cm1 of glycine.

[0027]

Examples 2 to 5 The same culturing operation and reaction as in Example 1 were carried out by changing the cells and sulfite compounds as shown in Table 3. The results are shown in Table 3 together with Example 1 and Comparative Example 1.

[0028]

[Table 3]

[0029]

Example 6 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 the separable flask was replaced with nitrogen gas, 30 ml of a 30% by weight aqueous glycinonitrile solution containing 650 mg of dry cells and 24 mg of diammonium sulfite monohydrate and 170 ml of distilled water were prepared. 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 of the substrate was additionally added. Perform this operation 3 more times every hour.
The reaction was repeated 5 times for a total of 5 hours. 20 g of solid was recovered in the dry ice trap. The solid was dissolved in 50 ml of water and quantified by the Nessler method.
g had been 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 was 88 g / g dry cells, and the activity of producing glycine was 18 g / g · Hr. 2 ml of reaction solution
Was centrifuged 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 0.08 per 1 cm1 of glycine.

[0032]

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 sulfite compound, and preferably no acid, alkali or buffer for adjusting pH is added. The reaction of microorganisms or a treated product thereof with glycinonitrile in the presence of a sulfite compound under reaction conditions and under closed reaction conditions or under reaction conditions in which generated ammonia is separated out of the system at the same time as the reaction, causes degradation or degradation. Without coloring reaction, per dry cell,
It has high activity per unit time and does not involve large amounts of cells and medium, and does not require the addition or disposal of acids, alkalis or buffers to adjust the pH of the reaction solution, and glycine and ammonia can be quantitatively determined. Glycine and ammonia can be separately recovered without producing and decomposing and consuming them.

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Claims (10)

[Claims] (1) A microbiological production method in which coloring of glycine is prevented, wherein a microorganism or a processed product thereof is allowed to act on an aqueous glycinonitrile solution in the presence of a sulfite compound. 2. The method according to claim 1, wherein the sulfite compound is an ammonium sulfite. 3. The process according to claim 1, wherein the glycinonitrile is obtained by the reaction of formaldehyde, hydrocyanic acid and ammonia. 4. The method according to claim 1, wherein the microorganism is Synechobacter (Accinetobacte).
r) genera, Rhodococcus, Corynebacterium and Alcalig
4. The method according to claim 1, wherein the microorganism is a microorganism belonging to the genus enes). 5. 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 the pH is added. The described method. 6. The method according to claim 5, wherein the reaction conditions are closed reaction conditions. 7. The method according to claim 5, wherein the reaction conditions are such that ammonia formed in the reaction solution is separated from the reaction solution. 8. The method according to claim 7, wherein the method for separating ammonia from the reaction solution is reactive distillation. 9. The method according to claim 8, wherein the reactive distillation is performed under reduced pressure. 10. The process according to claim 9, wherein the reactive distillation is carried out in the presence of an inert gas.