JPS6354359B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing L-allothreonine aldolase using microorganisms. L-allothreonine aldolase (E.
C.4.1.2.6) is an enzyme that decomposes L-allothreonine into glycine and acetaldehyde. There is relatively little research on this enzyme, but sheep liver [J.Gen.
Physiol., Vol.38, p181 (1954)], corn germination (Agricultural Chemistry 1981 Conference Abstracts, 49
Its existence is recognized in the following pages). The present inventors conducted various studies to produce this L-allothreonine aldolase using microorganisms, and as a result, it happened that bacteria belonging to the genus Bacillus, Arilobacter, Pseudomonas, and Alcaligenes produced L-allothreonine aldolase. Based on this discovery, the present invention was completed. It should be noted that since it is completely unknown that L-allothreonine aldolase is produced by microorganisms, production by microorganisms has been achieved for the first time in the present invention. The present invention relates to the genus Bacillus, the genus Arilobacter,
Belongs to the genus Pseudomonas and Alcaligenes L
- A method for producing L-allothreonine aldolase, which comprises culturing a microorganism capable of producing allothreonine aldolase in a nutrient medium, and collecting L-allothreonine aldolase from the culture. One of the microorganisms used in the present invention belongs to the genus Bacillus and has the ability to produce L-allothreonine aldolase, and an example is Bacillus DK-315 isolated from soil No. 6202. be able to. The mycological properties of Bacillus DK-315 Microtechnical Laboratory No. 6202 are shown below. (a) Morphology Cell shape and size: Bacillus, 0.8×2.0μ Pleomorphism: None Motility: Yes, pericyria Spores: Yes, circular and located off center Gram staining: Positive Acid-fastness: None (b) Growth status in each medium Meat juice agar plate culture: Good growth, circular Meat juice agar slope culture: Good growth, translucent, shiny Meat juice liquid culture: Good growth Meat juice gelatin puncture culture: Thread-like, grows well on the surface Litmus・Milk: Decolorizes and liquefies (c) Physiological properties Reduction of nitrate: + Denitrification reaction: + MR test: + (weak) VP test: - Formation of indole: - Formation of hydrogen sulfide: - Starch Hydrolysis: + Utilization of citric acid Koser medium: - Christensen medium: - Utilization of inorganic nitrogen sources Nitrate: - Ammonium salt: + Production of pigment: - Urease - Oxidase: + Catalase: + (weak) Growth range PH: PH5-12 Temperature: 5-40℃ Attitude towards oxygen: Aerobic O-F test (Hugh Leifson method): F Production of acid and gas from sugars

[Table] Salt tolerance: Does not grow in 5% NaCl Gelatin degradability: + _DNAase : + Vitamin requirement: None Based on the above bacteriological properties, "Vergey's Manual of Determinative Bacteriology" 8th Edition (1974)”, DK-
Bacterium 315 is a Gram-positive bacillus that moves with periflagella and has spore-forming ability, so it was identified as belonging to the genus Bacillus. It is sufficient that the microorganism of the present invention belongs to the genus Bacillus and has the ability to produce L-allothreonine aldolase, and it goes without saying that it includes, for example, DK-315 bacteria and artificial mutant strains of other bacteria. Regarding other microorganisms used in the present invention, microorganisms belonging to the genus Arylobacter include Arylobacter DK-19 Kaikoken Bacteria No. 6201;
A microorganism belonging to the genus Pseudomonas is Pseudomonas DK-2, and a microorganism belonging to the genus Alcaligenes is Alcaligenes faecalis.
IFO No. 12669 can be mentioned. The above-mentioned Arylobacter DK-19
No. 6201 and Pseudomonas DK-2
The mycological properties of No. 6200 are as follows. (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 bacteria are Gram-negative rods 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. The medium for culturing such microorganisms may be a conventional medium for culturing bacteria belonging to the genus Bacillus, Arilobacter, Pseudomonas, and Alcaligenes, and contains a carbon source, a nitrogen source, an organic nutrient source, an inorganic ion, etc. use something Carbon sources include sugars such as glucose, glycerol, and molasses, organic acids such as acetic acid and malic acid, nitrogen sources include ammonium sulfate, ammonium chloride, and urea, and organic nutritional sources include yeast extract, peptone, meat extract, and cornstarch. Epliquor, etc., and inorganic ions such as magnesium, iron, manganese, potassium, phosphate, etc. may be added as appropriate. The amount of enzyme produced may be increased by adding about 0.05 to 0.5% of L-threonine or L-allothreonine to the medium. 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 L-allothreonine aldolase thus obtained is mainly produced and accumulated within the bacterial cells.
Therefore, when L-allothreonine aldolase is used as an enzyme source, the culture or bacterial cells isolated from it may be used as is or dried; however, if L-allothreonine aldolase is present, purified form If it is necessary to use the bacteria, first, the bacterial cells should be treated mechanically, with enzymes,
The crude extract is obtained by destroying it by a known method such as an autolysis method. 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. Simple physical property values are shown below. 1 Optimal PH PH8-9 2 Optimum temperature 60-70℃ 3 Conditions for inactivation 30℃ and PH5-11 or PH8 and 50℃
Inactivated in 1 hour 4 Inhibitor Inhibited by Cu ²⁺ , Hg ²⁺ , Ag ¹⁺ 5 Stabilizer Mercaptoethanol, dithiothreitol, sodium sulfite 6 Coenzyme Pyridoxal-5'-phosphate 7 Molecular weight Cephadex G As a result of gel filtration using -200, the molecular weight was determined to be 100,000 to 150,000. 8 Elemental analysis values The elemental analysis values are as follows. C: 52.4% H: 7.5% N: 15.2% Since pyridoxal-5'-phosphate is required as a coenzyme for the activity of this enzyme to be expressed, it is usually present in an amount of 10 ^-3 to ^{10 -5} M during the reaction. For example, this enzyme can be extracted from L from the threonine synthesis reaction solution.
- It can be used in a method for decomposing and removing L-allothreonine in the process of separating and obtaining threonine, but it can also be used for quantitative analysis of L-allothreonine. To measure the activity of this enzyme, use an enzyme-containing solution.
Add 0.1 ml to 0.9 ml of PH8.0 Tris-HCl buffer containing 100 μmole of L-allothreonine and incubate at 30°C.
The acetaldehyde produced by heating for 10 minutes at
Paz method [Arch.Biochem.Biophys., Vol.109,
p548 (1965)].
In addition, 1U of enzyme activity was defined as the enzyme activity that decomposes 1 μmole of L-allothreonine per minute. Next, examples will be shown. It should be noted that all % represents weight %. Example 1 Polypeptone 0.5%, yeast extract 0.5%,
PH consisting of KH ₂ PO ₄ 0.1%, MgSO ₄ 0.05%, L-glutamic acid 0.1%, and L-threonine 0.1%
Three 7.5 culture media were prepared, poured into a 5-volume culture tank, and sterilized by heating at 120°C for 15 minutes. This medium was inoculated with Bacillus DK-315 FEI No. 6202,
The cells were cultured at 30°C for 20 hours with aeration and stirring while maintaining the pH at 7.5. After culturing, centrifuge the bacterial cells from culture solution 1, wash once with physiological saline, and remove the resulting wet bacterial cells.
0.1mM pyridoxal-5'-phosphate and 10mM
It was suspended in 100 ml of 0.1M Tris-HCl buffer containing mercaptoethanol and pH 8.0. This bacterial cell suspension was sonicated at 20 KHz for 10 minutes to destroy the bacterial cells, and then centrifuged and decanted at 1200 rpm for 60 minutes to obtain 103 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 Cefdex 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 L-allothreonine aldolase activity fraction was collected from each fraction of the eluate. Activity category is NaCl concentration 0.1-0.15M
It was near the. This active fraction was then passed through a column packed with 200 ml of Cephadex G-200 for gel filtration, and the L-allothreonine aldolase active fraction was collected and concentrated using a membrane filter to obtain 22 ml of an enzyme concentrate. The protein content in this enzyme solution was 1.3 mg/ml, and the specific activity against L-allothreonine was 1.14 U/mg. On the other hand, the specific activity of this purified sample against L-threonine is
The specific activity for D-threonine and D-allothreonine was 0.01 U/mg. Examples 2 to 4 Bacillus DK-315 of Example 1
In place of Arylobacter DK-19 FEK 6202, Pseudomonas DK-2 FEK 6200 and Alcaligenes haecalis
A purified sample of L-allothreonine aldolase in the form of a white powder was obtained in the same manner as in Example 1 except that IFO No. 12669 was used. The activity after treatment with Cephadex G-200 was compared as follows. Both enzymes are eluted at the same NaCl concentration as the enzyme of Example 1, and have similar and identical physicochemical properties such as substrate specificity, optimum temperature, temperature stability, optimum PH, PH stability, and molecular weight. It was determined to be an enzyme.

【table】

Claims (1)

[Claims] 1. A method of culturing L-allothreonine aldolase-producing microorganisms belonging to the genus Bacillus, Arilobacter, Pseudomonas, and Alcaligenes in a nutrient medium, and collecting L-allothreonine aldolase from the culture. ―
Method for producing allothreonine aldolase.