JP4560164B2 - Microbial production of glycine - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for microbiological production of glycine. More specifically, glycinonitrile obtained by the reaction of formaldehyde, hydrocyanic acid and ammonia is subjected to a hydrolysis reaction by the action of microorganisms under closed reaction conditions without adjusting the pH, and the resulting glycine and ammonia are recovered from the aqueous solution. The present invention relates to a microbiological method for producing glycine. 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 used for industrially producing useful glycine efficiently.
[0002]
[Prior art]
Conventionally, glycine was once synthesized from formaldehyde, hydrocyanic acid and ammonia by the Strecker method, hydrolyzed with alkali such as caustic soda, converted to glycine soda and ammonia, and then added with acid such as sulfuric acid. Manufactured with the sum. At this time, when ammonia is hydrolyzed with an alkali, it is recovered by evaporation, and glycine is neutralized with an acid and recovered by a crystallization method (Japanese Patent Laid-Open Nos. 43-29929 and 51-19719). JP-A 49-14420, JP-A 49-35329). As described above, the conventional method has the disadvantage that a large amount of waste is generated and the environmental load is large because a large amount of salts are by-produced in the neutralization step in addition to the disadvantage of using a large amount of alkali and acid. Furthermore, since the salt formed as a neutralization by-product is very similar in solubility to glycine, in order to purify and recover glycine, complicated operations such as repeated crystallization operations and circulation of mother liquor are required. (Japanese Patent Laid-Open No. 51-34113).
[0003]
On the other hand, a method of enzymatically hydrolyzing glycinonitrile to obtain glycine is also known. Japanese Patent Publication No. 58-15120 discloses a method of suspending Brevibacterium R312 strain in a reaction solution adjusted to pH 8 with caustic potash or the like and using it for the hydrolysis reaction. A method is disclosed in which -774 strain is suspended in a reaction solution adjusted to pH 7.7 with a phosphate buffer and used for the reaction. However, according to the examples, in order to obtain glycine, it is necessary to use a large amount of cells corresponding to 1 to 10 times the weight of glycine, and these methods adjust the pH of the reaction solution. There was a problem in that a buffer solution was used. That is, since a large amount of microbial cells are used, there is a disadvantage that a large amount of medium and microbial cells are wasted, and since a buffer solution is used as a pH adjuster, there is a disadvantage that discarding of the buffer solution cannot be avoided. Further, the buffer solution becomes an ammonia salt, and there is a problem that hinders the recovery of ammonia. In Japanese Patent Laid-Open No. 3-280889 using microorganisms of the genus Rhodococcus, Arthrobacter, Caseobacter, Pseudomonas, Enterobacter, Acinetobacter, Alkaligenes, Corynebacteria, or Streptomyces Is improved to about 1/20 of the weight of glycine produced, but has a disadvantage that the reaction time is as long as about 40 hours. Further, the problem of using a buffer for the reaction has not been solved.
[0004]
As described above, the conventional method for producing glycine from glycinonitrile using microorganisms has a low activity per cell and per unit time, and a waste of a large amount of medium and cells. Disposal of the buffer solution used for adjusting the pH is unavoidable, and further, there is a problem that the buffer solution prevents the recovery of ammonia, which cannot be industrially implemented. Moreover, in the method using the conventional microorganism, the device which collect | recovers ammonia was not disclosed.
[0005]
[Problems to be solved by the invention]
In producing glycine from glycinonitrile using microorganisms, the present invention has a high activity per cell and per unit time, and does not discard a large amount of medium and cells, and adjusts the pH of the reaction solution. It is an object of the present invention to provide a method capable of recovering glycine and ammonia quantitatively and easily without using a buffer solution.
[0006]
[Means for Solving the Problems]
In order to solve the above problems, the present inventor has high activity per cell and per unit time, does not decompose or consume glycine and ammonia produced in the reaction system, quantitatively separates glycine and ammonia, and In order to construct a reaction system that can be easily recovered, suitable microorganisms were searched. As a result, glycine and ammonium are produced under closed reaction conditions where the pH is not adjusted, and a microorganism in which the reaction proceeds even when the pH of the reaction solution exceeds 10 can be found. As a result of diligent research on reaction methods and the like, the present invention has been completed.
[0007]
That is, the present invention is a method for producing glycine characterized in that a microorganism is allowed to act on an aqueous solution of glycinonitrile under a closed reaction condition in which the pH is not adjusted, and the present invention is a closed method in which the pH is not adjusted. A process for producing glycine characterized in that a microorganism is allowed to act on an aqueous solution of glycinonitrile under a mechanical reaction condition to recover glycine and ammonia produced by an enzymatic reaction.
As a microorganism used in the present invention, for example, a microorganism belonging to the genus Rhodococcus has been newly found to be suitable, but is not limited thereto.
[0008]
Rhodococcus maris BP-479-9 selected as a microorganism suitable for the present invention was originally deposited on November 2, 1993 at the Institute for Microbial Industrial Technology, and deposited internationally on September 1, 1995. And has been assigned the FERM BP-5219 accession number, and the microbiological properties are as follows.
(A) Shape and growth status 1. Shape of cell Presence or absence of cell polymorphism Yes 3. Existence of motility None 4. Presence or absence of spores None 5. Gram staining positive 6 Good growth, off-white, unknown, in broth agar plate culture
Colony state Smooth, convex, glossy [0009]
(B) Physiological properties Catalase +
2. Oxidase −
3. OF test not implemented 4. Cell wall diamino acid meso-diaminomimelic acid
(C) Other physiological properties Reduction of nitrate +
2. Pyrazine amidase +
3. Cysteine allylamidase −
4). Valine allylamidase +
5). Pyrrolidonyl allylamidase −
6). Alkaline phosphatase +
7). β-glucuronidase −
8). β-galactosidase −
9. α-Glucosidase +
10. Urease −
11. Gelatin degradation −
12 Hemolytic −
13. Adenine degradation ＋
14 Tyrosine degradation −
Growth in the presence of 15.5% NaCl. Acid generation from ribose +
[0011]
(D) Growth on each single carbon source Inositol −
2. Maltose −
3. Mannitol −
4). Rhamnose −
5). Sorbitol −
6). m-Hydroxybenzoic acid +
7). Sodium adipate −
8). Sodium citrate −
9. Sodium glutarate +
10. L-tyrosine-
11. Glycerol −
12 Trehalose −
13. Acetamide −
14 D-galactose-
When identifying strains, classify according to Bergy's Manual of Determinative Bacteriolog Volume 2 (1986) and The Prokaryotes 2nd Edition (1992). did.
[0012]
Next, general embodiments of the present invention will be described.
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. As nitrogen sources, ammonia and its salts, urea, nitrates and organics are used. Nitrogen 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 5-9, preferably pH 6-8, temperature 20-37 ° C, preferably 27-32 ° C. In the culture of the microorganism of the present invention, an enzyme inducer may be added to the medium. For example, lactam compounds (γ-lactam, δ-lactam, ε-caprolactam, etc.), nitrile compounds, amide compounds and the like may be used.
[0013]
The microorganism of the present invention can be used industrially as it is. However, a mutant strain improved by a method of inducing mutation with an appropriate mutant agent or a genetic engineering technique, for example, a mutant strain that constitutively produces an enzyme is used. You can also. The microbial cell of the present invention means a microbial cell or a processed microbial product collected from a culture solution (a crushed microbial cell, an enzyme separated from the crushed microbial cell, and an enzyme separated and extracted from the microbial cell or microbial cell. Processed product). Collection of bacterial cells from the culture solution can be performed by a known method.
[0014]
The glycinonitrile used in the present invention can be synthesized by a known method. For example, it is synthesized by a method obtained from formaldehyde, hydrocyanic acid and ammonia, or a method obtained by reacting formaldehyde with hydrocyanic acid to synthesize glycolonitrile once and subsequently reacting with ammonia. Both are collectively referred to as the Strecker method.
[0015]
In the present invention, the microbial cell and the microbial cell product separated by the above-described method are suspended in a glycinonitrile aqueous solution under a closed reaction condition in which the pH is not adjusted, so that the hydrolysis reaction proceeds rapidly, and glycine And can be converted to ammonia. That is, normally, the generated ammonia does not dissipate in an aqueous suspension that does not adjust the pH, for example, containing 0.01 to 5% by weight of the microbial cell or treated product and 1 to 30% by weight of glycinonitrile. The reaction time may be allowed to react for 1 to 24 hours, for example, using a condition of 0 to 50 ° C. as the temperature. In this case, glycinonitrile may be added at a low concentration and added over time, or the reaction temperature may be changed over time.
[0016]
Thus, glycine can be converted to glycine and ammonia in a molar yield of almost 100% and produced and accumulated as a high concentration aqueous solution of glycine ammonium salt. 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 reaction solution of glycine ammonium salt, for example, after removing bacterial cells from the reaction solution by centrifugal filtration, membrane separation, etc., glycine is then separated by crystallization, ion exchange or fractional precipitation with a poor solvent. Ammonia can be recovered by distillation or extraction after evaporation together with some water.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited by this Example.
[Example 1]
(1) Synthesis of glycinonitrile An equal amount of hydrocyanic acid was allowed to act on formalin, and an excessive amount of aqueous ammonia solution was added to the once produced aqueous glycolonitrile solution to obtain a 30% by weight aqueous glycinonitrile solution.
[0018]
(2) Culture of bacterial cells Rhodococcus maris BP-479-9 was cultured under the following conditions.
(1) Medium glycerin 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
[0019]
(2) Culture conditions 30 ℃ / 1 day
(3) The glycinonitrile hydrolyzing cells were collected from the obtained culture broth by centrifugation and washed with distilled water or the like for use in the reaction. In a sealed 100 ml glass autoclave, 62 mg of dry cell mass and 3 ml of 30% by weight glycinonitrile aqueous solution of the substrate were prepared in 17 ml of distilled water, and the reaction was started at 20 ° C. Two hours after the start of the reaction, the pH was 10. This reaction solution was analyzed by a liquid chromatography method. As a result, glycinonitrile disappeared and glycine was quantitatively produced. Therefore, the reaction temperature was raised by 5 ° C. every 2 hours, 3 ml of 30% by weight glycinonitrile aqueous solution of the substrate was 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.
[0020]
Using 2 g of the obtained 32 g of the reaction solution, the produced ammonia was quantified by the Nessler method, the raw material glycinonitrile and the produced glycine were analyzed by liquid chromatography, the glycinonitrile disappeared, glycine and ammonia Was quantitatively generated. The production amount of glycine per dry cell was 97 g / g dry cell, and the production activity of glycine was 9.7 g / g · Hr. The remaining 30 g was subjected to centrifugal filtration to remove the cells, and then concentrated to 1/10 under boiling to recover 4.9 g of glycine by crystallization. On the other hand, ammonia in the evaporated aqueous solution recovered by cooling was 1.25 g.
[0021]
[Comparative Example 1]
The same culture operation as in Example 1 was carried out using Alcaligenes faecalis (ATCC 8750), which is known for the synthesis activity of glycine, instead of Rhodococcus maris BP-479-9, and acetonitrile instead of ε-caprolactam. It was. Bacteria were collected from the obtained culture broth by centrifugation, washed with distilled water, etc., and then sealed in a 100 ml glass autoclave. The dry cell mass was 50 mg and 3 ml of a 30 wt% aqueous glycinonitrile substrate solution was added to 17 ml of distilled water. And the reaction was started at 20 ° C. Two hours after the start of the reaction, the pH was 8.5.
[0022]
When this reaction solution was analyzed by a liquid chromatography method, glycinonitrile remained and glycine was produced at 16% of the charged amount. Therefore, the reaction time was further extended for 2 hours, but the amount of glycine did not change. This suggests that the ammonia generated in the closed reactor accumulates and the pH rises, and in the case of normal cells such as Alcaligenes faecalis cultured in acetonitrile, the hydrolysis activity of nitrile is significantly inhibited. .
[0023]
【The invention's effect】
By using the microbiological production method of glycine of the present invention, a glycinonitrile obtained by reaction with formaldehyde, hydrocyanic acid and ammonia under a closed reaction condition without adjusting pH, a microorganism having hydrolytic activity at pH 10 or higher, Preferably, it is subjected to a hydrolysis reaction by the action of microorganisms belonging to the genus Rhodococcus, and glycine and ammonia are separately collected from the aqueous ammonium solution of glycine to be produced, so that a large amount of alkali, acid, and buffer solution are consumed and discarded. Thus, glycine can be produced with high activity per dry cell and per unit time without consuming the produced glycine and ammonia under the reaction conditions.
[0024]
In general, hydrolysis does not occur with good reproducibility even if glycinonitrile is hydrolyzed without adjusting the pH using cells having hydrolytic activity. In addition, when acid, alkali, or buffer solution is used for pH adjustment, it is necessary to consume and discard them in large quantities, and the recovery of the generated ammonia and glycine becomes complicated. Therefore, glycine can be produced with high activity per dry cell and per unit time by using a cell that is active even at high pH and that maintains activity even when the pH changes greatly. By not using an alkali, acid, or buffer, it was possible to eliminate such salts and to easily separate glycine and ammonia quantitatively.

Claims (1)

Without using a buffer, in a closed reaction conditions that will not adjust the pH, by the action of microorganisms in an aqueous solution of glycidyl Sino nitriles, to a process for the preparation of a glycine to recover glycine and ammonia produced by the enzyme reaction, as the microorganism A method for producing glycine, comprising using Rhodococcus maris BP-479-9 (FERMBP-5219) .