JPH022597B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing D-β-hydroxyamino acids using a novel enzyme. As various D-amino acids have been discovered in recent years, their physiological significance is being clarified, and D-
Many amino acids are useful as raw materials for the synthesis of various medicines such as antibiotics and enzyme inhibitors, agricultural chemicals, and other physiologically active substances. Some of these D-amino acids are produced by optically resolving DL-amino acids produced by synthetic methods, but most of them are produced by optically resolving L-amino acids after racemizing them. In that case, D-amino acids are manufactured through a long process, which requires a lot of effort.
Moreover, the yield was also extremely low. The present inventors have developed a new technique for producing specific D-amino acids all at once from glycine and aldehydes, which are produced at low cost and in large quantities. That is, they developed a method for producing the corresponding D-β-hydroxyamino acid from glycine and aldehyde all at once using D-threonine aldolase, a novel enzyme. Until now, L-
L-threonine aldolase (EC4.1.2.5), which breaks down threonine into glycine and acetaldehyde
From glycine and aldehydes using
It is known that -β-hydroxy amino acids can be produced. However, regarding D-β-hydroxyamino acids, D-
The existence of threonine aldolase itself is completely unknown. The present inventors happened to know that a specific microorganism can produce this D-threonine aldolase, and as a result of further research, we found that using this enzyme, we can convert glycine and aldehydes into corresponding D-
The present invention was completed based on the discovery that β-hydroxyamino acids can be produced all at once. That is, the present invention provides glycine and the general formula R-
A general formula characterized by reacting an aldehyde compound represented by CHO (wherein R represents hydrogen or a saturated alkyl group) in the presence of D-threonine aldolase (However, R represents hydrogen or a saturated alkyl group.) This relates to a method for producing a D-β-hydroxyamino acid represented by the following formula. D-threonine aldolase is an enzyme that acts on D-threonine and decomposes it into glycine and aldehyde.
No., and Arthrobacter
DK-19 Microtech Research Institute No. 6201 has the ability to produce this D-threonine aldolase. Pseudomonas DK-2 FEK No. 6200 and Arylobacter DK-19 FEK No. 6201
The mycological properties of this issue are shown below. (a) Form

【table】 (b) Growth status in each medium

【table】 (c) Physiological properties

【table】

[Table] Based on the above mycological properties, the classification is based on the ``Bergey's Manual of Determinative Bacteriology, 8th edition (1974)''.
DK-2 bacterium is a Gram-negative bacillus with polar flagella.
It was identified as belonging to the genus Pseudomonas because it was positive for oxidase and denitrification. On the other hand, the DK-19 bacterium was identified as belonging to the genus Arilobacter because it is a bacillus with weak Gram staining, is pleomorphic, has pericytium, and cannot assimilate sugars. D-threonine aldolase can be produced, for example, by culturing these microorganisms in a nutrient medium. The nutrient medium may be a normal one for culturing bacteria, and the carbon source may be glycose, xylose,
Sugars such as glycerol and molasses, or organic acids such as acetic acid and malic acid; nitrogen sources such as ammonium sulfate, ammonium chloride, and urea; organic nutritional sources such as yeast extract, peptone, meat extract, and cornstarch liquor; and inorganic sources. Ions containing magnesium, iron, manganese, potassium, phosphate, etc. are used. The culture method can be carried out according to the conventional method for culturing bacteria, and the pH of the medium should be set at 4 to 10, and after inoculating the bacteria,
It may be cultured aerobically at 60°C for 1 to 3 days. In this way, D-threonine aldolase is mainly produced and accumulated within the bacterial body, but the bacterial body itself may also be used as the enzyme source for the reaction.
In addition, D-threonine aldolase was isolated from bacterial cells.
It may be used at any stage of the purification process. When D-threonine aldolase is isolated from a culture solution, the bacterial cells are first disrupted by a known method such as a mechanical method, an enzyme treatment method, or an autolytic method to obtain a crude extract. Then, this crude extract is subjected to ammonium sulfate precipitation, solvent precipitation with acetone or ethanol, DEAE-Sepharose, DEAE
- Highly pure enzyme preparations can be obtained by purification using appropriate combinations of chromatography using various ion exchangers and adsorbents such as Cephadex and calcium phosphate gel. The activity of this enzyme requires pyridoxal-5'-phosphate as an auxiliary enzyme, so it is normally used during the reaction.
It is present at 10 ^-3 to ^{10 -5} M. Next, the results of measuring the physicochemical properties of the enzyme preparation obtained in Enzyme Production Example 1 will be described. Action and Substrate Specificity The enzyme decomposes D-threonine and D-allothreonine to produce glycine and acetaldehyde. On the other hand, L-threonine and L-
It has no effect on allothreonine. Optimal PH: 30 at each PH using D-threonine as a substrate
When the reaction was carried out at ℃ for 10 minutes and the aldehyde produced was quantified, the optimum pH of this enzyme was between 7 and 9. The buffer used was 0.1M for pH 4 to 7.5.
Phosphate buffer, 0.1M Tris for pH 7-9
HCl buffer and pH 9-11 are 0.1M sodium carbonate buffer. 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 6 to 9. The buffers used were 0.1M phosphate buffer for pH 4 to 7.5;
For pH 7 to 9, use 0.1M Tris-HCl buffer and
For pH 9 to 11, 0.1M sodium carbonate buffer is used. Measurement method for titer: Transfer 0.1 ml of enzyme-containing solution to 0.1 M Tris-HCl buffer with pH 8.0 containing 100 μmol of D-threonine.
0.9ml and heated at 30℃ for 10 minutes to generate acetaldehyde using the Paz method [Arch.Biochem.
Biophys., Vol. 109, p548 (1965)]. In addition, the enzyme activity that decomposes 1 μmole of D-threonine per minute was defined as 1 U. Suitable temperature range for action Using D-threonine as a substrate in 0.1M Tris-HCl buffer with a pH of 8.0, the reaction was carried out for 10 minutes at each temperature and the acetaldehyde produced was measured, and the optimum temperature for this enzyme was found to be 50-50. It was ℃. Thermostability After heating an enzyme solution dissolved in 0.1M Tris-HCl buffer at pH 8.0 for 1 hour at each temperature, the residual activity in the solution was measured, and the stable temperature of this enzyme was found to be
The temperature was below 40℃. Conditions for inactivation due to PH, temperature, etc. This enzyme is PH5 or lower, PH11 or higher, and temperature 70.
At temperatures above ℃, it becomes inactive within 1 hour. Inhibition, Activation and Stabilization The enzyme is activated and stabilized by mercaptoethanol, sodium sulfite, sodium bisulfite, dithiothreitol, Mn ²⁺ , Co ²⁺ , Fe ²⁺ , Mg ²⁺ . On the other hand, Ag ¹⁺ , Cu ²⁺ ,
Hg ²⁺ , Zn ²⁺ , Pd ²⁺ , hydroxylamine, p
- Chlormercury is inhibited by benzoic acid. Coenzyme The coenzyme of this enzyme is pyridoxal-5'-phosphate. The enzyme obtained in Enzyme Production Example 1 has the above-mentioned physical and chemical properties, but all conventionally known threonine aldolases decompose L-threonine, but not D-threonine. Since it is completely unknown, this enzyme is a novel enzyme with a completely new action. The enzyme used for the reaction of glycine and an aldehyde compound may be any enzyme as long as it can decompose D-threonine to generate glycine and acetaldehyde. Furthermore, this enzyme is not limited to a form that can exhibit enzymatic activity, and is not limited to an isolated form. Therefore, semi-refined products may be used, crude extracts,
Furthermore, it may be a culture, live bacterial cells, freeze-dried bacterial cells, acetone-dried bacterial cells, or a ground product of these bacterial cells. Furthermore, the enzyme itself or the bacterial cells may be immobilized by known means and used. D-
Threonine aldolase is not limited to those derived from microorganisms as mentioned above, but may be derived from other animals or plants. The aldehyde compound is R in the general formula R-CHO.
is hydrogen or a saturated alkyl group. The number of carbon atoms is preferably 20 or less, and suitable examples include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and laurylaldehyde. The reaction can be carried out by mixing D-threonine aldolase, glycine, and an aldehyde compound.
The order of addition does not matter. The aldehyde compound may be used as long as it does not significantly inhibit enzyme activity.
The amount is preferably about 0.05 to 0.2 mol/degree. The amount of glycine may be about equimolar to that of the aldehyde compound, but in order to increase the reaction yield of glycine, it is preferable to use less than the amount of the aldehyde compound. The reaction temperature may be about 10 to 70°C, but preferably about 10 to 40°C. PH during reaction
is preferably maintained at about 6 to 9.5, preferably 7 to 8. When pyridoxal-5'-phosphate is added to the reaction system as a coenzyme, the enzyme activity can be increased and the reaction can be accelerated. The reaction may be carried out in batch mode or in continuous mode. Thus, the reaction is completed in about 5 to 50 hours. After the reaction is complete, if necessary, remove suspended matter by centrifugation, filtration, etc., then purify by ion exchange resin treatment, crystallization, etc., decolorize with activated carbon, etc., and concentrate this decolorized liquid. D-β-hydroxy amino acids can be isolated. Next, an example of enzyme production will be shown. Note that all percentages are by weight. Enzyme production example 1 Polypeptone 0.5%, yeast extract 0.5%,
PH consisting of _{KH2PO4 0.1} %, _MgSO4 0.05%, L-glutamic acid 0.1%, and D-threonine 0.1%
Prepare the culture medium of 7.5 and place it in a 5 volume culture tank.
was added and heat sterilized at 120°C for 15 minutes. In this medium, Arylobacter DK-19
No. was inoculated and cultured at 30°C for 20 hours with aeration and stirring while maintaining the pH at 7.5. After culturing, centrifuge the cells from culture solution 1, wash once with physiological saline, and remove the wet cells at 0.1
The suspension was suspended in 100 ml of 0.1 M Tris-HCl buffer, pH 7.5, containing mM pyridoxal-5'-phosphate and 10 mM mercaptoethanol. This bacterial suspension
The bacterial cells were destroyed by ultrasonication at 20 KHz for 10 minutes, and then centrifuged and decanted to obtain 105 ml of crude enzyme extract. Add ammonium sulfate to the obtained crude enzyme extract to give a concentration of 0.3 to 0.5
The saturated fraction was separated and this fraction was dialyzed overnight against the above buffer. DEAE Cephadex A-50
Fill the column with 100 ml and equilibrate it with the above buffer before passing the dialysis residue through the column to adsorb the enzyme, then pass through the column with varying concentrations of sodium chloride solution from 0.1 to 0.4M. Then, the D-threonine aldolase activity fraction of each fraction of the solution was collected. This active category has a concentration of sodium chloride.
It was around 0.3M. The collected active fraction was passed through a column packed with 200 ml of Cephadex G-200 for gel filtration, and the D-threonine aldolase active fraction was collected and concentrated using a membrane filter to obtain 15 ml of an enzyme concentrate. The protein content in this enzyme solution was 2.4 mg/mg, the specific activity for D-threonine was 1.24 U/mg, and the specific activity for D-allothreonine was 3.33 U/mg. on the other hand,
It showed no activity against L-threonine and L-allothreonine. Enzyme Production Example 2 Using Pseudomonas DK-2 Microtechnical Research Institute No. 6201 and Alcaligenes haecalis IFO12669, both were cultured in the same medium as in Enzyme Production Example 1, and when the enzyme was separated from the culture solution, Pseudomonas DK- In the case of 2 bacteria, the protein concentration is 2.3
12 ml of enzyme solution of mg/ml and Alcaligenes
In the case of S. falciparum, the protein concentration is 2.1
11 ml of mg/ml enzyme solution was obtained. When the enzyme activity was measured, the former had a specific activity of 1.01 U/mg for D-threonine and 2.96 U/mg for D-allothreonine. On the other hand, the latter has a specific activity toward D-threonine.
The specific activity for D-allothreonine was 2.95 U/mg. In addition, none of them showed any activity against L-threonine and L-allothreonine. Examples are shown below. The quantitative determination of the product and the threo isomer/allo isomer ratio are as follows: t-butanol: methyl ethyl ketone: 25% ammonia water ratio: 4:3:
Paper chromatography was performed using the mixture of No. 1 as a developing solvent, color was developed with ninhydrin, a spot was cut out, extracted with methanol containing 0.005% copper nitrate, and determined by colorimetry. Example 1 Alcaligenes flycharis IFO12669, Pseudomonas DK-2 F.K. No. 6200, and Arylobacter DK-19 F.K. No. 6201 obtained by culturing in the same manner as in Enzyme Production Examples 1 and 2. Centrifuge 1 ml of each culture solution to collect bacterial cells.
The operation of adding 0.9% saline and washing was repeated twice. A substrate solution consisting of 200 μmoles of glycine, 200 μmoles of acetaldehyde, and 1 ml of 0.1M Tris-HCl buffer with pH 8.0 was added to each washed bacterial cell and allowed to react at 30° C. for 20 hours. After the reaction, D-threonine and D
- Allothreonine was quantified and the results shown in the table below were obtained.

[Table] It was confirmed that the threonine produced was D-form by reacting each bacteria on a single scale. That is, the reaction solution was passed through a column packed with 500 ml of H ⁺ type Dowex 50WX8, washed with water, and then eluted with 0.2N ammonia to separate the threonine fraction and the glycine fraction. After concentrating the threonine fraction, it was decolorized with activated carbon, and ethanol was added to the decolorized solution to obtain crystals. NMR, infrared absorption spectrum, elemental analysis, and specific rotation of this crystal were measured, and it was found that this crystal was D
-Confirmed to be threonine. On the other hand, each reaction solution was measured by a bioassay method using Streptococcus flycallis IFO3181, and it was confirmed that the reaction solution did not contain any L-form. Example 2 Using the same culture solution as in Example 1 of Arylobacter DK-19 FEK No. 6201, the bacterial cells were centrifuged, washed, and freeze-dried. 50 mmole of each aldehyde shown in the table below, glycine
50 mmole and 500 ml of 0.1 M Tris-HCl buffer with pH 8.0.
g each, and reacted at 30°C for 40 hours. After the reaction was completed, the amount of D-β-hydroxyamino acid in the solution was determined and the results are shown in the table below. The ratio of threo isomer to allo isomer was approximately 1.6 in each solution.

[Table] Example 3 Add 200 μmole of glycine, 200 μmole of acetaldehyde, and mercaptoethanol to 2 ml of the D-threonine aldolase-containing solution obtained in Enzyme Production Example 1.
A substrate solution consisting of 100 μmole and 5 ml of 0.1M Tris-HCl buffer with pH 7.5 was added and reacted at 30°C for 10 hours. After the completion of the reaction, D-threonine and D-allothreonine were quantified and found that D-threonine
The amount of D-allothreonine was 85 μmole and 53 μmole of D-allothreonine.

Claims (1)

[Claims] 1. A method characterized by reacting glycine with an aldehyde compound represented by the general formula R-CHO (where R represents hydrogen or a saturated alkyl group) in the presence of D-threonine aldolase. general formula (However, R represents hydrogen or a saturated alkyl group.) A method for producing a D-β-hydroxyamino acid represented by the following.