JPS6411279B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel enzyme, D-threonine aldolase, and a method for producing this enzyme using microorganisms. Threonine aldolase is an enzyme that decomposes threonine into glycine and acetaldehyde. Until now, L-threonine aldolase (EC4.1.2 .5) In addition to animals and plants, bacteria, yeast,
It is also known to exist in microorganisms such as actinomycetes. However, the existence of D-threonine aldolase, which decomposes D-threonine into glycine and acetaldehyde, was completely unknown. The present inventors have developed a method for producing L-threonine, which has a large potential market for food additives, feed additives, etc.
-Focusing on the method of optically resolving threonine, D-
As a result of a wide search for enzymes that degrade threonine,
The inventors happened to discover that certain microorganisms belonging to the genus Arilobacter, Pseudomonas, and Alcaligenes produce a novel enzyme, D-threonine aldolase, which decomposes D-threonine into glycine and acetaldehyde, and based on this finding, they completed the present invention. . That is, the present invention cultivates a microorganism capable of producing D-threonine aldolase, which is a novel enzyme, and D-threonine aldolase belonging to the genus Arilobacter, Pseudomonas, or Alcaligenes in a nutrient medium, and extracts D-threonine from the culture. - A method for producing D-threonine aldolase, which comprises collecting threonine aldolase. The microorganism used in the present invention belongs to the genus Arilobacter, Pseudomonas, or Alcaligenes and has the ability to produce D-threonine aldolase, and an example is Alcaligenes faecalis.
IFO12669, Pseudomonas
DK-2 Microtechnical Laboratory No. 6200, and Arthrobacter DK-19 Microtechnical Laboratory No. 6200
No. 6201 can be mentioned. 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, DK-2 bacteria is a Gram-negative bacillus when classified with reference to the 8th edition of Virgies Manual of Determinative Bacteriology (1974). It was identified as belonging to the genus Pseudomonas because it had polar flagella and was positive for oxidase and denitrification reactions. On the other hand, the DK-19 bacterium is a bacillus with weak Gram staining, is pleomorphic, has perichomes, and cannot assimilate sugars, so it was identified as belonging to the genus Arilobacter. The microorganisms of the present invention belong to each of the above-mentioned genera.
It is sufficient to have the ability to produce threonine aldolase,
For example, it goes without saying that strains containing both D-threonine aldolase and L-threonine aldolase in their cells are included in the microorganisms of the present invention, as well as L-threonine aldolase-deficient strains obtained by mutationally treating this strain. Also included. There is no particular difficulty in culturing such microorganisms to produce D-threonine aldolase.
This may be carried out in accordance with the usual culture of bacteria. That is, for the medium, glucose is used as the carbon source,
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 ions. As such, substances containing magnesium, iron, manganese, potassium, phosphate, etc. are used. Addition of about 0.05 to 0.5% D-threonine to the medium may increase the amount of enzyme produced. The culture method may be any conventional method, for example, by adjusting the pH of the medium.
After inoculating the bacteria at a temperature of 4 to 10, the bacteria may be cultured aerobically at 20 to 60°C for 1 to 3 days. The D-threonine aldolase thus obtained is mainly produced and accumulated within the bacterial cells. Therefore,
When using D-threonine aldolase as an enzyme source, the culture or bacterial cells isolated from it may be used as is or dried, but it is necessary to use D-threonine aldolase in a more purified form. In some cases, the bacterial cells are first destroyed by a known method such as a mechanical method, an enzyme treatment method, or an autolysis method to obtain a crude extract. Then, this crude extract is purified by appropriate combinations of 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. The physicochemical properties of D-threonine aldolase of the present invention were measured using the enzyme preparations obtained in Examples 1 and 2 below, and the following results were obtained. a Action and Substrate Specificity This 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. b Optimum 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 the enzyme was found to be between 7 and 9. The buffer used was 0.1M from PH4 to 7.5.
Phosphate buffer, 0.1M Tris for pH 7-9
HCl buffer and pH 9-11 are 0.1M sodium carbonate buffer. c PH Stability 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, with 100% activity remaining. In addition, the buffer used has a pH of 4 to 7.5.
0.1M phosphate buffer, 0.1M Tris-HCl buffer for pH 7-9, and 0.1M sodium carbonate buffer for pH 9-11. d Optimum temperature 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, and the optimum temperature for this enzyme was found to be 40-50. It was ℃. e 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℃. f Inhibition, activation and stability This 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. g Coenzyme Pyridoxal-5'-phosphate is required as a coenzyme for the activity of this enzyme to be expressed. h Molecular Weight As a result of gel filtration using Cephadex G-200, the molecular weight was determined to be 100,000 to 150,000. i Inactivation conditions: This enzyme has a pH of 5 or lower, a pH of 11 or higher, and a temperature of 70
At temperatures above ℃, it becomes inactive within 1 hour. j Elemental Analysis Values The elemental analysis values of each enzyme obtained in Examples 1 and 2 show similar values, but are listed according to the genus from which they are derived. C; 51.7%, H; derived from Arilobacter genus;
7.8%, N; 15.7%, O; 24.8% C from Pseudomonas genus; 50.5%, H; 7.1
%, N; 14.8%, O; 27.6% Alcaligenes C; 52.7%, H; 8.1
%, N; 16.4%, O; 22.8% The enzyme titer measurement method described above is
0.1ml contains 100μmol of D-threonine
Add to 0.9 ml of 0.1 M Tris-HCl buffer with pH 8.0,
The acetaldehyde generated by heating at 30℃ for 10 minutes was processed 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. The enzyme obtained in the example has the above-mentioned physicochemical properties, but all conventionally known threonine aldolases decompose L-threonine, and like this enzyme, D-threonine decomposes. Since it is completely unknown what can be decomposed,
This enzyme is clearly a new enzyme with a completely new action. For example, if this enzyme is used in a method for producing L-threonine using DL-threonine as a raw material, the optical resolution process can be simplified, but it is also effective for quantitative analysis of D-threonine. Examples are shown below. Note that all percentages are by weight. 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 8.0 medium and add 3 to 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 by 0.1 m.
The suspension was suspended in 100 ml of 0.1 M Tris-HCl buffer, pH 7.5, containing M pyridoxal-5'-phosphate and 10 mM mercaptoethanol. This bacterial suspension
The bacterial cells were destroyed by ultrasonic treatment at 20 KHz for 10 minutes, and then centrifuged and decanted at 12,000 rpm for 60 minutes 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/Sephadex A-50
The residual dialysis solution was passed through the column filled with 100 ml and equilibrated with the above buffer solution to adsorb the enzyme, and then the sodium chloride solution was passed through the column at varying concentrations from 0.1 to 0.4M. The D-threonine aldolase activity fraction was collected from each fraction of the eluate. In this active category, the concentration of sodium chloride 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/ml, 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. Example 2 Pseudomonas DK-2 microorganisms No. 6200 and Alcaligenes haecalis IFO12669 were cultured in the same medium as in Example 1, and the enzyme was separated from the culture solution. In the case of , the protein concentration is 2.3mg/
ml of enzyme solution is equivalent to 12 ml of enzyme solution with a protein concentration of 2.1 mg/ml in the case of Alcaligenes flycharis bacteria.
11 ml was obtained. When this enzyme activity was measured, the former had a specific activity against D-threonine.
The specific activity for D-allothreonine was 2.96 U/mg. On the other hand, the latter has a specific activity of 0.86 U/mg for D-threonine and a specific activity for D-allothreonine.
It was 2.95U/mg. In addition, none of them showed any activity against L-threonine and L-allothreonine. Both enzymes are eluted at the same NaCl concentration as the enzyme of Example 1, and have similar physicochemical properties such as substrate specificity, optimum temperature, temperature stability, optimum PH, PH stability, and molecular weight. It was determined that they were the same enzyme.

Claims (1)

[Claims] 1. D-threonine aldolase having the following physicochemical properties. a Action and substrate specificity; D-threonine and D
- Decomposes allothreonine to produce glycine and acetaldehyde. On the other hand, it does not act strongly on L-threonine and L-allothreonine. b Optimal PH; pH was between 7 and 9. c PH stability: PH6-9 after heating at 30℃ for 1 hour
Almost 100% activity remained within this range. d Optimum temperature: 40°C to 50°C at PH8.0. e Thermal stability: Stable up to 40°C after heating at PH8.0 for 1 hour. f inhibitors, stabilizers and activators; Ag ¹⁺ , Cu ²⁺ ,
Hg ²⁺ , Zn ²⁺ , Pd ²⁺ , hydroxylamine, p
- Chlormercury is inhibited by benzoic acid. On the other hand, it is stabilized and/or activated by mercaptoethanol, sodium sulfite, sodium bisulfite, dithiothreitol, Mn ²⁺ , Co ²⁺ , Fe ²⁺ , Mg ²⁺ . g Coenzyme: Requires pyridoxal-5'-phosphate as a coenzyme for activity expression. h Molecular weight: As a result of gel filtration using Cephadex G-200, the molecular weight was determined to be 100,000 to 150,000. i Conditions for inactivation: Inactivation occurs if left at PH5 or lower, PH11 or higher, or 70°C or higher for 1 hour. j Elemental analysis value; C derived from Arilobacter genus; 51.7%, H;
7.8%, N; 15.7%, O; 24.8% C from Pseudomonas genus; 50.5%, H; 7.1
%, N; 14.8%, O; 27.6% Alcaligenes C; 52.7%, H; 8.1
%, N: 16.4%, O: 22.8% 2 A microorganism that belongs to the genus Arilobacter, Pseudomonas, or Alcaligenes and can produce D-threonine aldolase is cultured in a nutrient medium, and D-threonine aldolase is extracted from the culture. A method for producing D-threonine aldolase, which comprises collecting D-threonine aldolase.