JPS58116692A - Separation of l-threonine from threonine mixture - Google Patents

Abstract

PURPOSE:To obtain L-threonine at a low cost, by reacting a solution containing DL-threonine and DL-allothreonine with D-threonine aldolase and L-allothreonine aldolase. CONSTITUTION:Microorganism capable of producing L-allothreonine aldolase [e.g. Bacillus DK-315 (FERM-P No.6202)]is inoculated in a conventional nutrient medium containing carbon source, nitrogen source, organic nutrient, inoganic ion, etc., and cultured aerobically at 4-10pH and 20-60 deg.C for 1-3 days to obtain L-allothreonine aldolase. D-threonine aldolase and L-allothreonine aldolase are added to an aqueous solution containing DL-threonine and DL-allothreonine, and made to react with each other at 7-10 pH and 30-45 deg.C for 5- 100hr. If necessary. The suspended material is removed and the solution is purified by ion exchange treatment, crystallization, etc., decolored, and concentrated to obtain L-threonine crystal.

Description

Detailed Description of the Invention The present invention produces isomers other than L-threonine by treating a mixture of DL-threonine and DL-allothreonine with allothreonine aldolase and D-threonine aldolase, a novel enzyme. The present invention relates to a method for decomposing D-threonine, D-allothreonine, and L-allothreonine, and obtaining only L-threonine in a pure form.

L-threonine is an essential amino acid for humans and animals, and because its content in various animal and plant proteins is relatively low, it has great potential demand as a food and feed additive. Conventionally, L-threonine could be produced by extraction from natural products or fermentation, but since it was relatively expensive, an inexpensive production method was long awaited. On the other hand, if a synthetic method is used, it can be easily synthesized using glycine etc. as a raw material (7), but D-threonine is produced as a by-product in the same amount as Sono-L, and D-allothreonine and J-L-allothreonine (their DL form) are produced as by-products. Since DL-threonine is also produced as a by-product, the separation and purification process is very complex, and the yield is therefore low.This makes monothreonine, which is produced by synthetic methods, expensive.In other words, DL-threonine Examples of optical resolution methods include Bull, SacChim, .
Vot, 2[1, p903 (1953) and Vot.
t.

23, p. 447 (1956), etc., but this optical resolution method is very complicated and has a low yield. There are also methods for separating allothreonine, including a method using bis(acetaldehyde)threonine steel (Japanese Patent Publication No. 36-19562), and a method using sodium ethylate in ethanol to form a sodium salt and utilizing the difference in solubility (US Patent No. 36-19562). No. 2461847) and other complicated and inefficient methods (/C).

-t(7) Moreover, D-threonine and allothreonine have the disadvantage that there is almost no demand and racemization is not easy.

The present inventors solved the above problems and used a synthetic method to
- As a result of various studies to develop a method for supplying threonine at low cost, we have found that - 1) - A new enzyme that acts on threonine and D-allothreonine to decompose it into glycine and acetaldehyde - i.e. D -Threonine aldolase is discovered, and if this enzyme is combined with L-allothreonine aldolase and applied to a mixture of synthetic reaction products DL-threonine and OL-allothreonine, only -threonine remains, On the other hand, since the decomposition products become useful and become lysine and acetaldehyde,
It was discovered that L-threonine can be easily obtained and the by-products can be easily utilized, and that all of the above problems can be solved12, and based on this, the present invention has been completed. Ta. -1 That is, according to the present invention, in a solution containing DL-threonine and DL-allothreonine, D-threonine aldolase,
and L-allothreonine aldolase.

D-threonine aldolase is an enzyme that acts on D-threonine and decomposes it into lysine and acetaldehyde, and also acts on D-allothreonine and decomposes it into glycine and acetaldehyde. Examples are Alca, Alcaligenes fae.
ca], is) IFO12669, Pseudomonas (
Pseudomonas) DK-2 Bichoken Bacteria No. 6200, and Arthrobacter
Enzymes produced by DK-19 (acter) DK-19, which has both D-threonine aldolase activity and D-allothreonine aldolase activity, can be used in the present invention7. The mycological properties of Pseudomonas DK-2 No. 6200 and Arylobacter DK-19 No. 6201 are shown below.

(a) Morphology (b) Growth status in each culture medium (c) Physiological properties Based on the above mycological chambers, "Purge Single's Manual of Determinative Bacteriology, 8th Edition" (1974) J, 1) K-2 bacteria are crumb-negative rods with polar flagella, oxidase-positive,
It was identified as belonging to the genus Pseudomonas because of the positive denitrification reaction. On the other hand, the DK-19 bacterium is a bacillus with weak crumb staining, has pleomorphic hairs, and is able to assimilate sugars.
Because there was no such thing, it was identified as belonging to Arilobacter m.

An enzyme having both D-threonine aldolase activity and D-allothreonine aldolase activity can be produced, for example, by culturing the microorganism of Tono 1 et al. in a nutrient medium. The nutrient medium may be a normal one for culturing bacteria, and carbon sources include sugars such as glucose, glycerol, and molasses, or organic acids such as acetic acid and malic acid, and nitrogen sources include ammonium sulfate, ammonium chloride, urea, etc.
Yeast extract, peptone, meat extract, cornstarch liquor, etc. are used as organic nutritional sources, and inorganic ions such as magnesium, iron, manganese, potassium, and phosphate are used.

The culture method can be carried out according to the conventional method for culturing bacteria.
The pH of the culture medium should be adjusted to 4 to 10, and after inoculating the bacteria, the culture should be aerobically cultured at 20 to 60°C for 1 to 6 days.

The thus obtained enzyme having both D-threonine aldolase activity and D-allothreonine aldolase activity is mainly produced and accumulated within the bacterial cells. Therefore, when this enzyme is isolated from a culture solution, the bacterial cells are first destroyed by known methods such as mechanical methods, enzyme treatment methods, and autolytic methods to obtain a crude extract. Then, this crude extract is purified by an appropriate combination of methods such as total ammonium sulfate precipitation, solvent precipitation with acetone or ethanol, and chromatography using various ion exchangers and adsorbents such as DEAE-Sepharose, DEAE-Sephadex, and calcium phosphate gel. By doing so, a highly pure enzyme preparation can be obtained. Since pyridoxal-5'-phosphate is required as a coenzyme for the expression of activity of this enzyme, it is usually present at 10-3 to 10-5 M during the reaction.

Next, the results of measuring the physical and chemical properties of the enzyme preparation obtained in Enzyme Production Example 1 below will be described.

(2) Action and substrate specificity This enzyme decomposes D-threonine and D-allothreonine to produce glycine and acetaldehyde. on the other hand,
It has no effect on L-threonine and L-allothreonine.

■ Optimum pH 1 at 30°C at each pH using D-threonine as a substrate.
When the reaction was carried out for 0 minutes and the aldehyde produced was quantified, it was found to be at the optimum pH of the enzyme, 7 to 9. The buffer used was pF (0.1M phosphate buffer from 4 to 7.5, pF
For H7-9n, 0.1M+-Lis-H (l buffer and pH
9 to 11 are 0.1M sodium carbonate buffers.

(2) Stable pH range After heating the enzyme solution at 30° C. for 1 hour at each pH, the residual activity in the solution was measured, and the stable pH range of this enzyme was found to be 6 to 9. The buffer used had a pH of 4 to 7.5.
Until 0. IMIJ acid buffer, p) (0.1M Tris-HCt buffer and pH 9-111 from 7 to 9)
This is a 0.1M sodium carbonate buffer.

■Measurement method of titer 0.1 ml of enzyme-containing solution was mixed with 0.4 ml of p[(8,0 0.1M) Lis-HCl buffer containing 100 p mole of D-threonine! In addition, the acetaldehyde produced by heating at 30°C for 10 minutes was subjected to the Paz method (Arc
h, Biochem, Biophys,
Vom 109°p54B (1965)').

In addition, the enzyme activity that decomposes 1 μmole of D-threonine per minute was defined as 1 U.

■ Range of suitable temperature for action Using D-threonine as a substrate and 0.1M Tris-HCl buffer at pH 8.0, the reaction was carried out for 10 minutes at each temperature, and the acetaldehyde produced was measured. The temperature was 40-50°C.

■ Thermostability After heating an enzyme solution dissolved in 0.1M (pH 8.0) Lis-HCl buffer at each temperature for 1 hour, the residual activity in the solution was measured, and the stable temperature of this enzyme was 40℃ or lower. Met.

■ Conditions for inactivation by pH 1 temperature, etc.
At temperatures above ℃, it becomes inactive within 1 hour.

■ Inhibition, activation and stabilization This enzyme contains silcabutoethanol, sodium sulfite, sodium bisulfite, dithiothreitol, Mn'', C
It is activated and stabilized by oz+, FeZ+, and Mg2+. On the other hand, Ag + +, Cu2+, )(
g2+, zn2+, Pd2+, hydroxylamine,
It is inhibited by p-remercury benzoic acid.

(2) Coenzyme The coenzyme of this enzyme is pyridoxal-5'-phosphate.

The enzyme obtained in Enzyme Production Example 1 has the above-mentioned physicochemical properties, but the conventionally known threonine aldolase and allothreonine aldolase both [
This enzyme is a novel enzyme with a completely new action, as there is no known enzyme that decomposes D-1 and D-1.

The O-threonine aldolase used in the present invention may essentially be one that can decompose D-threonine and D-allothreonine to generate glycine and acetaldehyde.

L-Aerothreonine aldolase is an enzyme that decomposes L-allothreonine into glycine and acetaldehyde, and its existence is known in sheep liver, corn germination, etc., but it is completely unknown that microorganisms produce it. Not yet.

The present inventors happened to find that Bacillus spp., Pseudomonas spp.
He discovered that bacteria belonging to the genus Arylobacter and the alkaline earth genus produced this enzyme, paving the way for industrial mass production of this enzyme.

Regarding the genus Bacillus, Bacillus DK-315 microorganism No. 6202 isolated from soil can be mentioned as an example of a microorganism having the ability to produce L-allothreonine aldolase. For the genus Pseudomonas, the above-mentioned Pseudomonas DK-2 Microtech Research Institute No. 6200, for the genus Arilobacter, the above-mentioned Arilobacter DK-19 Microtech Research Institute No. 621] No. 1, and for the genus Alcaliges. Alcaligenes flycalis (A1.cal, igenes
faecaNS) IFo No. 12669.

The dental properties of Bacillus OK-315 FEI No. 6202 are shown below.

(a) Morphology■ Cell shape and size; rod, 0.8X2. Gμ ■ Polymorphism: None ■ Motility: Ayu, hairy ■ Spores: Yes, circular and located at a distance from the center ■ Crumb staining: Positive ■ Anti-acidity: None b) Each Growth status in the medium■ Meat juice agar plate culture: Good growth, circular ■ Meat juice agar slant culture: Good growth, translucent, glossy ■ Meat liquid culture: Good growth■ Meat juice seratin puncture culture: Thread-like, grows well on the surface■ Lidomus milk; decolorizes and liquefies (c) Physiological properties ■ Nitrate reduction; + ■ Denitrification reaction; +■ MR
Test ;+(weak) ■ vp Te
St: ~■ Generation of indole
- ■ Production of hydrogen sulfide; ■ Hydrolysis of starch; +■ Koser medium using citric acid; - Christensen medium; - ■ Use of inorganic nitrogen sources Nitrate; - Ammonium salts: Production of ten pigments; - ■ Urease; -0 Oxidase; 10 Catalase; + (weak)
■Growth range pH: p85-12 Temperature: 5-40℃ [Phase] Attitude toward oxygen: Aerobic Q O-F test (Fuugh Leifson method); Generation of acid and gas from FO sugars (2) D-xylose - (3) D-glucose + ten (4) D-
Mannose + ten (5) D-fructose + + (6) D-galactose
+ 10 (7) maltose (8)
−(9) Lactose −
0□ trehalose + ell) D-nruvit - α埠 D-mannit + - (to) inosit q4 Glycerin + + θ9 Starch + [phase] Salt tolerance; Does not grow on NaC15 0 Gelatin degradability; 10@ DNAase: 10■ Vitamin requirement: None Based on the above dental properties [Referring to Virginie's Manual of Determinative Bacteriology, 8th edition (1974) J, DK-315 bacterium This bacterium was identified as belonging to the genus Bacillus because it is a clam-positive bacillus, moves with periflagella, and has the ability to form spores.

Allothreonine aldolase can be produced, for example, by culturing various of the above-mentioned microorganisms in a nutrient medium.

The nutrient medium may be one commonly used for culturing bacteria; carbon sources include sugars such as glucose, glycerol, and molasses; organic acids such as acetic acid and malic acid; nitrogen sources include ammonium imilate, ammonium chloride, urea, etc. Organic nutritional sources include yeast extract, peptone, meat extract, cornstarch liquor, etc., and inorganic ions include magnesium, iron, manganese, potassium, phosphate, etc.

The culture method can be carried out according to the conventional method for culturing bacteria.
After inoculating the bacteria, set the p[( of the medium to 4 to 10) to
The cells may be cultured aerobically for 1 to 3 days.

Alloslemenine aldolase, which cannot be obtained in this way, is mainly produced and accumulated within the bacterial body.

Therefore, when this enzyme is to be isolated from a culture solution, a crude extract is obtained by destroying the bacterial cells by a known method such as a mechanical method, an enzyme treatment method, or an autolytic method. Then, the crude extracted solution was subjected to ammonium sulfate precipitation, solvent precipitation with acetone or ethanol, DEAF-Sepharose, DE
Highly pure enzyme preparations can be obtained by purification using appropriate combinations of chromatography using various ion exchangers and adsorbents such as AE-Sephatex and calcium phosphate gel. Simple physical property values are shown below.

1. Optimum pH pH 8-9 2. Optimum temperature 60-70℃ 3. Conditions for deactivation pF (5-11 or p
Inactivated in 1 hour at 50°C or higher in H84 Inhibitor: Inhibited by Cu2+, HF2+, Ag I+5, Stabilizer: Mercaptoethanol, dithiothreitol, sodium sulfite6, Coenzyme: Pyridoxal-57-phosphate Activity of the main enzyme Since expression requires pyridoxal-5'-phosphate as a coenzyme, 10-3 to 10-
Let 5M exist.

It goes without saying that the allothreonine aldolase used in the present invention is not limited to those derived from the above-mentioned microorganisms, as long as it decomposes L-allothreonine to produce chrysin and acetaldehyde.

Furthermore, the enzyme used in the present invention may be either D-threonine aldolase or L-allothreonine aldolase in a form that can develop enzymatic activity;
Nothing 1 is limited to isolated form. Therefore, raw rice products, crude extracts, cultures, raw cocoons, freeze-dried bacterial cells, acetone-dried bacterial cells, crushed bacterial cells, crushed sheep liver, etc. may be used. . Furthermore, the enzyme itself or bacterial cells may be immobilized using a known immobilization stage. A wide variety of immobilization methods such as carrier binding, crosslinking, entrapping, and aggregation methods can be used.1 The mixture of DL-threonine and DL-allothreonine may be obtained by any known method. For example, α-amino-
A method of synthesizing β-hydroxybutyric acid ester, reacting it with thionyl chloride, and hydrolyzing it under heat to form an oxazoline ester (T, Am, Chem, S+
nc,, Vat, 71°pH01 (1949)),
Using vinyl acetate as a raw material, α-acetoxybrobionaltehyde is synthesized by hydroformylation using the oxo method, and α-amine-
A method may be used in which β-hydroxybutyronitrile is obtained and hydrolyzed to form an oxazolidone derivative by the action of phosgene (Japanese Patent Publication No. 11608/1983). However, in the method of the present invention, the target substance is L-threonine, and the by-products are chrysin and acetaldehyde, so the synthesis reaction produces less allo-isomer by-products, and it is convenient to use mataglycine and acetaldehyde as raw materials. be. In this respect, the method of treating chrysin with a metal salt to form a glycine metal complex and condensing acetaldehyde therewith (Japanese Patent Publication No. 56-19562, Japanese Patent Publication No. 47-39093, etc.) is a method of D L in the present invention.
- Suitable as a method for producing a mixture of threonine and DL-allothreonine. The solution containing this mixture may be obtained once in the form of crystals and dissolved in water, but it may also be a synthesis reaction solution or various intermediate purification process solutions from which the above-mentioned mixture crystals are obtained. . In other words, in the present invention, a solution containing OL-threonine and DL-allothreonine may be dissolved as long as it contains these substances, and the ratio of D-integral to integral, the ratio of threo-to-allo-isomer, etc. does not matter. By the way, it's not. Moreover, it goes without saying that it may also contain various impurities in addition to these. In that case, remove any enzyme reaction inhibitors in advance. For example, if the metal ions used in the synthesis reaction are harmful, they are removed in advance using a cation exchange resin or the like.

A solution containing D-threonine and D-allothreonine is added with D-threonine aldolase and - There is no particular difficulty in making allothreonine aldolase act; the key is to have these enzymes present in this solution.

The concentration of DL-threonine and DL-allothreonine should be at a level that does not significantly inhibit enzyme activity, but the total amount of threonine and allothreonine is preferably about 0.1 to 2 mol/. In principle, the solvent for the reaction system is water, but
It may also contain an organic solvent that does not inhibit the enzymatic reaction. The pF at the time of reaction varies depending on the enzyme used, but in the case of the enzyme obtained in the enzyme production example, pi (around 7 to 10 is preferable.The reaction temperature also depends on the enzyme used, but In the case of enzymes, a temperature range of 30 to 45°C is suitable.In addition, in the case of the enzyme of this production example, about 10-3 to 10-5M of pyridoxal is added as a coenzyme.
When 5-phosphoric acid is present, the enzymatic reaction can be promoted. Addition of surfactants etc. for various other purposes7
It's okay. The reaction may be carried out batchwise or continuously. Furthermore, both enzymes may be added together and the reaction may proceed simultaneously, or may be reacted individually.

The reaction time for each enzyme may be about 5 to 100 hours.

After the reaction is completed, if necessary, remove suspended matter by centrifugation, filtration, etc., and then purify by ion exchange resin treatment, crystallization, etc.
[-6-threonia bond A can be obtained in a pure form by decolorizing with activated carbon or the like and concentrating the decolorizing solution f. The reaction products of any enzyme are glycine and acetaldehyde, and glycine can be easily separated and recovered, for example, by chromatography using an ion exchange resin. Acetaldehyde condenses with glycine, which is produced as a by-product during the enzymatic reaction, or undergoes a reverse reaction with each enzyme and recombines with glycine.1. Therefore, it is preferable to recover acetaldehyde by distillation or the like during the reaction.

By using the method of the present invention, D-threonine and allothreonine, which were previously difficult to separate, can be easily separated from L-threonine and allothreonine.
It can be removed from threonine. Therefore, the method for separating the above-mentioned isomers, which had been a major problem when producing L-threonine by the conventional synthetic method, was solved all at once, and a way was opened to provide L-threonine at a low price by the synthetic method. There are some things. In addition, the by-products glycine and acetaldehyde also have high market value, and it is particularly preferable to combine this method with a method for synthesizing threonine using glycine or glycine and acetaldehyde as raw materials, since these by-products can be reused as raw materials for synthesis.

Next, an example of enzyme production will be shown. In addition, all ts are it%, D-allothreonine aldolase activity and 1. −
Allothreonine aldolase activity was measured in the same manner as in the method for measuring the titer of D-threonine aldolase described above, except that the substrate was changed according to each enzyme. I agree, the titer is also 1 μmol of the substrate allothreonine per minute.
The activity to decompose e was defined as 1U.

Enzyme production example 1 Polypeptone 0.5%, yeast extract 0.2%, KH2
Prepare a pH 7.5 medium consisting of PO40.1%, M, gsO40.05, L-glutamic acid 0.1%, and D-threonine 0.5, and put 3 tons of it into a culture tank with a capacity of 5. and heat sterilized at 120°C for 10 minutes. In this medium, Arylobacter 0K-19
Inoculate No. 1 from 1 to 20 at 30℃ while maintaining pFJ 8.
The cells were cultured for hours with aeration and stirring.

After culturing, centrifuge the bacterial cells from the culture solution 1~, o, q%
After washing with saline, the wet bacterial cells were soaked in 100 me of 0.1M phosphate buffer, pH 7.5, containing 10mM mercaptoethanol and 0.1mM pyridoxal-5-phosphate.
dispersed inside. This bacterial cell dispersion was treated with 20 KHz ultrasound five times for 5 minutes, then centrifuged, and the supernatant liquid was collected.
Next, protamine sulfate was added to the supernatant to remove nucleic acids, and the supernatant was further fractionated with ammonium sulfate to collect fractions containing D-threonine aldolase activity.

Both parts were combined with 10.71M mercaptoethanol and Q
, pH 7,5 containing 1mM pyridoxalu-5 to phosphoric acid.
After dialysis with 0.01MIJ acid buffer, the solution was passed through a column packed with DEAE-Sephadex 8-5 and 100-, and eluted using the KC1 salt concentration gradient method, and the D-threonine aldolase activity fraction was collected. . This fraction was concentrated by ultrafiltration and [.

- A purified D-threonine aldolase solution 10- containing no threonine deaminase was obtained. The D-threonine aldolase activity of this solution was 5 U/-1 and the D-allothreonine aldolase activity was 12.5 U/-.

Enzyme Production Example 2 Using Pseudomonas DK-2 Microtechnological Laboratory No. 6200 and Alcaligenes flycharis TFO12669, both were cultured in the same medium as in Enzyme Production Example 1, and the separation and purification operations from the culture solution were performed in the same manner. Ten purified D-threonine aldolase solutions were obtained. The enzyme activity of the obtained solution was D-threonine aldolase activity of 4.5 U/m/! in the case of Pseudomonas 0K-2. In the case of Alcaligenes no. there were.

Enzyme Production Example 3 Using Bacillus DK-315 FEI No. 6202,
A standing culture was carried out in the same medium as in Enzyme Production Example 1, and the separation and purification operation was carried out in the same manner from the culture solution to obtain L-allothreonine aldolase solution 10me manufactured by Cff. The allothreonine aldolase activity of this solution was 4.5 U/-, and D-allothreonine and Oo-threonine had no activity at all. There wasn't.

Examples are shown below. In addition, the ratio of threonine, allothreonine, and glycine Iri, tert, -phthyl alcohol dimethyl ethyl ketone: water: concentrated ammonia is 4.
Perform paper chromatography using a mixed solution of :3:2:j as a developing solvent, develop the color with ninhydrin, cut out spots of glycine, threonine, and allothreonine, and extract with methanol containing 0.005% steel nitrate. It was determined colorimetrically. In addition, acetaldehyde PEG
It was quantified by gas chromatography using a 6000 x 6m column.

Next, the present invention will be explained with reference to Examples, but the present invention (r) is not limited thereto. Also, DL-threonine used in the following Examples is synthesized by the following synthesis method. According to

Synthesis example of OL-Threonine Glycine 75? was heated and melted under 3.8 tons of water pressure, and basic carbonated steel was gradually added to react by heating. After the Fj reaction is completed, excess basic copper carbonate is removed from the hot solution, and the p solution is cooled under reduced pressure to obtain crystalline glycine steel 110F.

Next, 0.8t of methanol in which 61% of potassium hydroxide was dissolved
crystalline glycine copper 587. Acetaltecht 502 was added and reacted at 50-60°C for 1.5 hours with stirring.

After the reaction, take the solution, remove a small amount of insoluble matter, and add acetic acid 6 to the P solution.
．． 42 was added and the solvent was distilled off under reduced pressure. After thoroughly washing the remaining crude threonine copper compound with water, the crystals were dissolved in 0.5 t of 6N ammonia water, and this solution was diluted 6 times, followed by chelate resin TOWEX A-1 (NH4 type).
Pass through a column packed with 2t to remove copper and collect the threonine effluent fraction. This solution was heated to 100 m under reduced pressure.
The mixture was condensed to a volume of 400 g, and the concentrate was added to VC methanol 4007 and cooled. The precipitated crystals were recrystallized from water-containing methanol to obtain DL-threonine crystals 382. The allothreonine content of this product was 10%. Example 1 DL-allothreonine obtained by the above synthesis example 10%.
DL-threonine crystal 2.3El' containing
Purified D containing D-allothreonine aldolase activity obtained in Enzyme Production Example 1 was added to 20 ml of an aqueous solution containing 10-6 moles of pyridoxal 5-phosphate, 10-4 moles of mercaptoethanol, and 10-3 moles of manganese chloride.
-10ml of threonine aldolase solution! and 10 m of the purified A T-arthreonine aldolase solution obtained in Enzyme Production Example 6 (!) was added and distilled under reduced pressure. Reaction was carried out for 24 hours.During the reaction, 0.I NN
The pH of the reaction solution was maintained at 7.5-8.5 with aOH.

After the reaction is completed, the reaction solution is neutralized with #hydrochloric acid (~), then filled with H+ type Dowex 50 WX 8 100-, left in the column, washed with water, and adsorbed threonine, glycine, etc. with 0.2N ammonia. The eluate was concentrated under reduced pressure to dryness, redissolved in water, diluted with dilute hydrochloric acid, and made into an acidic aqueous solution.
Pass liquid I through the column packed with 50WX B 100-
-ta. After washing with water, 0.05N ammonia was passed through to elute the threonine-containing fraction, and the eluate was dried under reduced pressure. To this dried product was added 10 me of water to dissolve it, and 10 me of ethanol was gradually added to separate the precipitated crystals. The dry weight of the obtained crystals was 0.9 t, and it was confirmed by paper chromatography that they were pure threonine. In addition, the specific optical rotation is also [α] -2
8.7 (H2O), which was consistent with that of pure L-threonine.

0.05N ammonia was further passed through to elute the glycine-containing fraction (7. The eluate was dried under reduced pressure.

4 ml of water was added to the dried product to dissolve it, and 4 ml of ethanol was gradually added to separate the precipitated crystals.

The obtained crystal had a dry weight of 0.65 f, and analysis by paper chromatography revealed that it contained only glycine.

On the other hand, the amount of acetaldehyde recovered by vacuum distillation during the reaction was 0.34 t.

Example 2 Pseudomonas OK, using enzyme production example 2 instead of the D-threonine aldolase solution made by n obtained in enzyme production example 1.
-2 Fine Technology Research Institute No. 6200 Using 10 ml of the obtained purified D-threonine aldolase solution, the reaction was carried out in the same manner as in Example 1, and the separation operation was performed. As expected, it did not contain any allothreonine. Threonine crystal 0.91
F, 0.66f of glycine crystals, and 0.34f of acetaldehyde were obtained. The specific rotation of the threonine crystal is [α)27 = -28,7(H2O), and L
- It was only threonine.

Example 3 The same procedure as in Example 1 was carried out except that 10 ml of the purified D-threonine aldolase solution obtained from Alcaligenes hygrophilis IFO12669 in Enzyme Production Example 2 was used instead of the purified D-threonine aldolase solution obtained in Enzyme Production Example 1. After reaction and separation, we found 0.91 F crystals of threonine containing no allothreonine, 0.66 F crystals of glycine, and 0.3 F crystals of acetaldehyde.
I got 41. In addition, the specific optical rotation of threonine crystal is [
α)z, t = −28,7(H2O), and only L-threonine was present.

Example 4 The reaction and separation operations were carried out in the same manner as in Example 1, except that the vacuum distillation operation was not performed during the reaction. As a result, threonine crystals containing 10% allothreonine, 0.8F,
Glycine crystal 0.5f, and acetaldehyde 0.0
5f was obtained.

Example 5 Shutomonas DK-2 microtechnical research group No. 6200 was purified according to Enzyme Production Example 1 (-D-threonine aldolase activity class 10- and L-allothreonine aldolase class 10- were collected). Each solution was condensed by ultrafiltration.
- Two types of enzyme solutions containing no threonine deaminase were obtained.

A mixture of this enzyme solution, 2v of L-threonine, and 1 part of L-allothreonine was operated in the same manner as in Example 7 except that the reaction temperature was 55°C. Crystal 0.85f', [α
]q: = -28,6(H2O) Glycine 0.6
4f, and 0.3Or of acetaldehyde 1. Patent applicant Denki Kagaku Kogyo Co., Ltd. Agent Hiroshi Nakamoto Agent I-hi Akira Continued from page 1 Inventor Noriaki Koizumi Kangen, Sakuramura, Niiharu District, Ibaraki Prefecture 2-11-8 Shinouchi High No. 102 No. 42 Author: Hideaki Yamada No. 1, No. 19, Matsugasakimoto, Sakyo Ward, Kyoto City Procedural amendment (voluntary amendment) April 19, 1980 Commissioner of the Japan Patent Office Shima 1) Haruki Indication of Tono 1 case 1982 Patent Application No. 209985 2
Name of the invention Method for obtaining L-threonine from threonine admixtures 6@Relationship with the person who makes a correction Patent applicant address 1-4-1 Yurakucho, Chiyoda-ku, Tokyo Name
(329) Denki Kagaku Kogyo Co., Ltd. Representative Shino
Akira Hara Address: 3F12, Nishi-Shinbashi Chuo Building, 3-15-8 Nishi-Shimbashi, Minato-ku, Tokyo Telephone: (437)-3467 Name: Patent Attorney (7850) Yasu Nakamoto Address: Same
The upper elemental analysis values are as follows.

6. Subject of amendment Detailed explanation of the invention column of the specification Z Contents of amendment The description in the column of the detailed explanation of the invention of the specification is amended as shown in σ) below.

(b) Add the following statement in its entirety next to page 15, line 12 of the specification.

60 Molecular Weight As a result of gel pox filtration with Sephadex G-200F, the molecular weight was determined to be 1 nO,000 to 150,000.

■　Elemental analysis value The elemental analysis values are as follows.

U: 51.7% ■(: 18% N: 15.7% (b) Add the following statement next to page 22, line 9.

As a result of gel filtration with 7 molecular weight Sephadex G-200 (F), the molecular weight was determined to be 100,000 to 150,000.

8. Elemental analysis value (21H near, 5% N: 15.2% (c) Page 26, lines 13-14 "Reaction time may be as long as 0" The entire statement is corrected to the following.

The reaction time is selected appropriately depending on the intended use of the -threonine.For example, if you want to obtain an I, -threonine that allows the mixture of undecomposed isomers, the enzymatic decomposition reaction is completed. All you have to do is stop the reaction before
``Even at L, the reaction time for each enzyme is about 5 to 100 hours.''

Claims (1)

[Claims] A combination of DL-threonine and HDL-allothreonine characterized by acting on a solution containing DL-threonine and DL-allothreonine with D-threonine aldolase and -allothreonine aldolase. How to obtain Monokara L-su L/Onin.