JPS58216690A - Method for obtaining l-threonine - Google Patents

Abstract

PURPOSE:To prepare L-threonine with a high efficiency, by reacting a cluture of a microorganism in which only an enzyme hydrolyzing L-threnonie contained therein is slectively inactivated with an isomeric mixture of the threonine. CONSTITUTION:A culture, microbial cell or extract therefrom of a microorganism, e.g. Psedomonas DK-2 or Bacillus DL-315, having the ability to produce D- threonine aldolase and/or L-allothreonine aldolase in kept at such a temperature as to inactivate only an enzyme hydrolyzing L-threonine selectively without deteriorating the activity of the above-mentioned enzyme for a given time, e.g. 30- 75 deg.C, preferably 35-55 deg.C, for 5min-48hr to inactivate only the enzyme hydrolyzing the L-threonine, and then the above-mentioned culture, microbial cell or extract therefrom is reacted with an isomeric mixture of the threonine.

Description

[Detailed Description of the Invention] By culturing microorganisms containing I,-allothreonine aldolase and heat-treating the culture, bacterial cells, or bacterial cell extracts, only the L-threonine degrading enzyme contained therein is selected. After being inactivated, D-threonine and DL-allothreonine are selectively decomposed without decomposing L-threonine by acting on a mixture of isomers of threonine to obtain mono-threonine. Regarding the law.

L-threonine is one of the essential amino acids for humans and animals, but because its content in various animal and plant proteins is relatively low, demand for it as a food and feed additive is expected to increase.

The chemical synthesis method for L-threonine had the disadvantage that three other isomers were generated as by-products, making it difficult to separate them.However, the present inventors have developed a new enzyme, D-
By allowing a mixture of threonine aldolase and L-allothreonine aldolase to act on a mixture of threonine isomers, three types of isomers other than L-threonine, namely D-threonine, D-allothreonine and L-allothreonine, are decomposed, He discovered a method for isolating L-threonine from a mixture of isomers and has already filed a patent application in 1982-2099.
It was disclosed as No. 85. D-threonine aldolase degrades D-threonine and D-70 threonine, but
It has no effect on L-threonine and L-allothreonine.

In this method, isomers other than L-threonine are stereospecifically decomposed to produce glycine and acetaldehyde. However, if a mixture of threonine isomers is reacted as a raw material, L-threonine can be easily converted into a single monomer. This is an excellent method in which these decomposition products can be reused as raw materials for synthesis, especially when combined with a threonine synthesis method using glycine or glycine and acetaldehyde as starting materials.

However, L-threonine is a metabolic intermediate;
Microorganisms also have an enzyme that decomposes L-threonine. Therefore, D-threonine aldolase or L-
Culture of microorganism containing allothreonine aldolase1.
When bacterial cells or bacterial cell extracts are allowed to directly act on a threonine isomer mixture, L-threonine will also be decomposed at the same time. Therefore, in order to obtain L-threonine in high yield by asymmetric decomposition using these microorganisms,
It is necessary to delete the activity of L-threonine degrading enzyme contained in the bacterial cells. If purified D-threonine aldolase or L-allothreonine aldolase from which L-threonine degrading enzyme has been removed is used, it is possible to specifically decompose only other corresponding isomers without decomposing L-threonine. Generally, extracting and purifying a specific enzyme from microorganisms is complicated, and the enzyme activity may decrease during the purification process, which poses some problems in industrial implementation.

The present inventors discovered that D-threonine aldolase and/or L.
- Only the existing L-threonine degrading enzymes can be removed without impairing the activities of D-threonine aldolase and L-allothreonine aldolase contained in the culture, bacterial cells, or bacterial cell extract of microorganisms that produce allothreonine aldolase. As a result of intensive research into the activation method, we found that L-threonine degrading enzyme is sensitive to heat and is inactivated by keeping it at 30-75°C, whereas D-threonine aldolase and 7+
, - It was found that allothreonine aldolase is relatively resistant to heat. Therefore, depending on the type of microorganism, by carefully selecting temperature, pH, and treatment time,
The present invention was completed based on the discovery that it is possible to completely inactivate L-threonine aldolase and not reduce the activities of D-threonine aldolase and L-allothreonine aldolase, but even activate them.

If microbial cultures, cells, or cell extracts are treated under these conditions, only one-threonine aldolase can be inactivated and one-threonine aldolase can be inactivated from a mixture of threonine isomers without careful isolation of specific enzymes. can be separated very easily and the other isomers recycled as useful glycine and acetaldehyde.

In the present invention, the temperature at which the microbial culture, bacterial cells, or bacterial cell extract is heat-treated is 30 to 75°C, preferably 30°C.
5° to 55°C, more preferably 40° to 50°
It is C. If the heating temperature is too low, it will not be possible to sufficiently inactivate 17-threonine degrading enzyme, while if it is too high, D-threonine aldolase, 1:H-L-
Allothreonine aldolase is also inactivated, which is not preferable. In summer, even room temperature can reach 30°C or higher, but the conditions are harsh, not only to inactivate L-threonine degrading enzyme but also to increase the activity of D-threonine aldolase and L-allothreonine aldolase. Simply leaving it under relatively high temperature conditions will never yield satisfactory results. These conditions include the type of bacteria, temperature, time,
It is relatively determined by pH etc. and cannot be specified.

In the method of the present invention, the heat treatment of the microorganism culture, cells, or cell extract is carried out in an aqueous medium, but if the pH of the solution becomes lower than 4 or exceeds 10, L-
Not only threonine degrading enzyme but also D-threonine aldolase and L-allothreonine aldolase are inactivated. pl (4 to 10
However, a particularly suitable range is from pl+5 to
It is 8. In this case, pl+ is adjusted and maintained by adding acid or alkali, and various buffers such as phosphate buffer, acetate buffer, ammonia buffer, etc. can be used for the purpose of stably maintaining the pH.

The heating time varies depending on the properties of D-threonine aldolase, L-allothreonine aldolase and L-threonine degrading enzyme, the pH and temperature during heating, etc. - Although it cannot be determined generally, it usually ranges from 5 minutes to 48 hours. Preferably, a range of 10 minutes to 24 hours is appropriate.

In the method of the present invention, pyridoxal-5'-phosphate is used as a coenzyme of D-threonine aldolase and L-allothreonine aldolase, and pyridoxal-5'-phosphate is used as a stabilizer during the heat treatment of microorganism cultures, cells, or cell extracts. It is also desirable to coexist mercaptoethanol, dithiothreitol, glutathione, cysteine, citric acid, and the like.

Furthermore, although the method of the present invention is applied to microbial cultures, microbial cells, or microbial cell extracts, it can also be effectively applied to those immobilized by means of detection. For example, any method such as an aggregation method using a flocculant such as chitosan, an entrapping method using acrylamide or the like, a crosslinking method using curcuraldehyde or the like, or a carrier bonding method using an ion exchange resin or the like can be used. Immobilization can be carried out prior to heat treatment, but
The immobilization and heat treatment may be performed at the same time, or the fixation may be performed after the heat treatment.

The microorganism used in the method of the present invention can be summarized as follows: 1) D-threonine aldolase or L-allothreonine, which stereospecifically asymmetrically decomposes -threonine and D-allothreonine or L-allothreonine to produce glycylate/acetaldehyde; Any microorganism that produces one or both of the aldolases may be used (e.g., Pseudomonas DK).
-2 Microtechnical Research Institute No. 6200 and Bacillus DK-3is
Microtechnical Research Institute No. 6202 and the like have the ability to produce the above-mentioned threonine aldolase.

In order to cause the above-mentioned threonine aldolase to act on the threonine isomer mixture, in short, a culture, bacterial cells, or these bacteria containing the above-mentioned threonine aldolase whose L-threonine degrading activity has been deleted by the method of the present invention is added to the threonine isomer mixture. All you have to do is use body extracts. The concentration of the threonine isomer mixture may be at a level that does not significantly inhibit enzyme activity, but is preferably about 0.1 to 2 molar. The solvent for the reaction system is, in principle, water, but an organic solvent or the like may be included as long as it does not inhibit the enzymatic reaction.

The pH during the reaction varies depending on the threonine aldolase produced by the microorganism used, but in the case of the enzyme produced by the exemplified microorganism, the pH is preferably around 7 to 10. The reaction temperature also depends on the enzyme used, but in the case of enzymes produced by the exemplified microorganisms, a range of 30 to 50°C is suitable. In the case of the above-mentioned threonine aldolase, the enzymatic reaction can be promoted by coexisting with about 10-3 to 0 M of pyridoxal-5-furic acid as a coenzyme.

In addition, surfactants and the like may be added depending on the purpose as long as they do not inhibit the enzymatic reaction. The reaction may be carried out batchwise or continuously. Thus,
The reaction is completed in about 5 to 100 hours.

After the reaction is complete, remove suspended solids and water-soluble polymeric substances by centrifugation, ultrafiltration, r-filtration, etc. as necessary, and then decolorize with activated carbon, etc. to remove glycine produced by the enzymatic reaction. After separation, L-threonine crystals can be obtained in pure form by crystallization. Glycine and acetaldehyde accumulate in the reaction system as decomposition products of the enzymatic reaction, but
Glycine can be easily separated and recovered, for example, by chromatography using an ion exchange resin. Acetaldehyde is preferably recovered by a method such as distillation during the reaction.

The present invention will be specifically described below with reference to Examples. Note that all percentages are by weight.

Examples] and Comparative Example 1 Polypeptone 05%, yeast extract 0.2%, Kl-Jz
p consisting of PO4o, 1%, MgSO40,05%, L-glutamic acid 0,1% and D-threonine 02%
Bacillus DK in a test tube containing 5 ml of medium + 8.0
-: SIS (Feikoken Bacteria No. 6202) was inoculated and cultured with shaking at a temperature of 30°C for 20 hours. 41 containing 5 m/+ of the obtained seed culture solution containing 100 mg of a medium of the same composition.
After inoculating into a 00ml Erlenmeyer flask and cultivating with shaking at 30°C for 20 hours, the bacterial cells were centrifuged from the culture solution and 0.9%
After washing with saline, the cells were freeze-dried to obtain dried bacterial cells.

0.1 g of dried bacterial cells was added to 10 ml of 0.1 M phosphate buffer with pH 8.0! 20 m+r+o
l e 4 Noshiichi' allothreonine 20771 mo
le/l L-threonine and Q, l, 71(I
p containing +101 e/d of pidxal 5'-phosphate
After adding H8,0 o, tM phosphate buffer go, s ml and reacting at 30°C for 60 minutes, the amount of glycine produced and the amount of remaining L-threonine in the reaction solution was quantified.

Separately, as Comparative Example 1, the amount of glycine produced and the remaining L
- Quantitate the amount of threonine and put these results together in a table - ■
It was shown to.

In the Examples and Comparative Examples, glycine and threonine were quantified by using an amino acid analyzer (model JLC-6AI, manufactured by JEOL Ltd.) in combination with paper chromatography. Quantification by paper chromatography uses a mixture of tert-butyl alcohol: methyl ethyl ketone: aqueous ammonia in the ratio of 4:3:2:1 as a developing agent, develops color with ninhydrin, and cuts out glycine and threonine spots. Copper nitrate 0.
It was extracted with methanol containing 0.005% and subjected to colorimetric determination. Furthermore, acetaldehyde was quantified by gas chromatography using a PEG600006m column.

Table 1 Example 2 and Comparative Example 2 Same as Example 1 except that Eudomonas DK-2 (Feikoken Bibori No. 6200) was used as the seed and D-threonine was used instead of L-allothreonine as the substrate. I conducted an experiment. The results are also shown in Table-1.

Separately, as Comparative Example 2, a test was conducted in the same manner as in Example 2 except that the 1 hour heat treatment operation was omitted, and the results are also shown in Table 1.

Example 3 An experiment was conducted in the same manner as in Example 1 except that the heating time in the heat treatment was different, and the obtained results are shown in Table 2.

Table 2 Example 4 and Comparative Example 3 Bacillus D K-315 was prepared in the same manner as in Example 1.
Freeze-dried bacterial cells were obtained. 01g of dried bacterial cells to 10ml
Suspended in 9% saline o, s m11! Take
After centrifuging to remove the kinetic fluid, 0.5 ml of a buffer solution of a predetermined pH was added and heated at 40°C for 6 hours.

After the heat treatment, it was washed once with 0.1 M phosphate buffer at pH 8.0, and the supernatant was removed by centrifugation. 5mm
o Ie/l of L-allothreonine and 5 mmol
e/H) L-threonine and 0.1771 mole/4
of pyridoxal 5'-phosphate at pH 8.0.
Add 1.0 ml of 1 M phosphate buffer and incubate at 30°C for 6 hours.
After reacting for 0 minutes, the amount of glycine produced and the amount of L-threonine remaining in the reaction solution were quantified.

Separately, as Comparative Example 3, the amount of glycine produced and the amount of remaining threonine were determined in the same manner as in Example 4, except that the operation of heating the bacterial cell suspension at 40°C for 6 hours was omitted, and these results were combined. It is shown in Table-3.

Table 3 Example 4 Pseudomonas DK-z (Feikoken Bokuyori No. 6200) was cultured in the same manner as in Example 1 to obtain dried bacterial cells.

Dry toy cells were suspended in 100 +++ liters of 0.1 M IJ acid buffer at pH 8.0, heated at 50°C for 2 hours, centrifuged, and washed with pure water. I) 100 ml of an aqueous solution containing 2.38 g of L-threonine and 27 mg of pyridoxal 5'-phosphoric acid was added to the treated bacterial cells thus obtained.
1 was added thereto, and the resulting acetaldehyde was removed and recovered from the reaction solution under reduced pressure, while the reaction was carried out at 40° C. for 24 hours. 0 during the reaction. Adjust the pH of the reaction solution to 7.5 with IN and NaOH.
It was held at 8.5.

After the reaction was completed, the reaction solution was centrifuged to remove bacterial cells, decolorized with activated carbon, and then treated with Dowex 50 W X8 with increased Fl.
After washing with water, the threonine-containing fraction was eluted with 0.05M aqueous ammonia, and the eluate was dried under reduced pressure. Add 2 ml of pure water to this and dissolve it.
1. Separate the precipitated crystals by gradually adding methanol J and oml. ．． 02 g (dry weight) of white crystals were obtained.

It was confirmed by paper chromatography that the crystals were threonine. Also, the specific optical rotation is [α几7=
-28,7(1-120)teari, which corresponded to that of L-threonine.

0.05 M ammonia water was further passed through to elute the glycine-containing fraction, and the eluate was dried under reduced pressure.

Add 2 ml of pure water to this, dissolve, and 10 ml of methanol.
was gradually added and the precipitated crystals were separated to obtain 065 g (dry weight) of white crystals. It was confirmed by paper chromatography that this crystal consisted only of glycine.

On the other hand, the amount of acetaldehyde recovered under reduced pressure during the reaction was 038 g.

Example 5 Bacillus DK-315 (Feikoken Bacillus No. 6202) was cultured in the same manner as in Example 1 to obtain dried bacterial cells.

A suspension of 1.0 g of dried bacterial cells in 100 ml of 0.1 M phosphate buffer with a pl+ of 8.0 was heated at 35° C. for 12 hours, centrifuged, and washed with pure water. The treated bacterial cells thus obtained were given L-threonine (1,19,9) and L-allothreonine (119) instead of DL-threonine.
The bacterial cell reaction and the separation operation after the reaction were performed in the same manner as in Example 1 except that the mixture in g) was allowed to react for 28 hours. As a result, 1.04 g of white crystals of L-threonine were obtained.
g, 064 g of white crystals of glycine and 0.39 g of acetaldehyde were obtained. The specific optical rotation of the threonine crystal was [α]'D'--28.7 (I20), which coincided with that of I-northreonine.

Example 6 Pseudomonas DK-2 (Feikoken Bacillus No. 6200) and Bacillus DK-315 (Feikoken Bacillus No. 6202)
were cultured in the same manner as in Example 1 to obtain dried bacterial cells.

The dried bacterial cells of DK-2 and DK-:315 were heated in the same manner as in Examples 4 and 5, mixed, centrifuged, and washed with pure water. To the thus obtained treated bacterial cells, I) DL-threonine (1.19 g) instead of L-threonine and I) L-allothreonine (1.
, 1, 99) was reacted for 38 hours in the same manner as in Example 4, and the bacterial cell reaction and separation operation after the reaction were performed. As a result, 0.51 g of white crystals of L-threonine and 0.51 g of white crystals of glycine were obtained. White crystal 097I and acetaldehyde 0.5! I got J g. In addition, the specific optical rotation of the threonine crystal is [α]2□=-28,7(LhO)
and was consistent with that of L-threonine.

Patent Applicant Denki Kagaku Kogyo Co., Ltd. Agent Patent Attorney Sadako Suzuki Procedural Sleeve Letter filed in August 1980 by Kazuo Wakasugi, Commissioner of the Japanese Patent Office1, Indication of the Case Showa 5 Fi1 Patent Application No. 985212, Invention Name Method of obtaining L-threonine 3 Relationship with the person making the amendment 41 cases Patent applicant 4, Agent 〒150 6, Number of inventions increased by amendment None 7, Subject of amendment Scope of claims in the specification and (1) Patent Wr: The scope of claims is amended as shown in the attached sheet.

(2) Specification I, line 19, page 4, line 2, page 4, line 4, page 4, line 17, last line of page 5, page 6, line 5, page 6, line 15, page 6, lines 19 to 20, and page 7. Replace “degrading enzyme” in line 8 with “degrading enzymes”
Correct.

(3) In the specification, page 3, line 14, "...degrading enzymes" is corrected to "...degrading enzymes."

(4) In the specification, "L-threonine aldolase" in line 4 of page 5 and line 11 of page 5 is referred to as "L-threonine decomposition: bald species."
Correct the appearance.

(Above) Revised Patent Claims At least one of the culture, bacterial cells, and bacterial cell extract of a microorganism that produces D-threonine aldolase and/or L-allothreonine aldolase, and the above-mentioned D-threonine aldolase and/or or L-threonine sea% 11. without impairing the activity of L-allothreonine aldolase. ＃＋
9. (A method for obtaining monothreonine, which is characterized by maintaining the temperature at which threonine is inactivated for a predetermined period of time, and then acting on a mixture of isomers of threonine.

Claims (1)

[Scope of Claims] [1] At least one of the culture, bacterial cells, and bacterial cell extract of a microorganism that produces -threonine aldolase and/or L-allothreonine aldolase as described above in 1) - threonine aldolase and/or L-allothreonine aldolase. A method for removing and removing L-threonine, which is characterized by maintaining the temperature for a predetermined period at a temperature that inactivates L-allothreonine aldolase and then allowing it to act on a mixture of isomers of threonine. .