JPS58116680A - D-threonine aldolase and its preparation - Google Patents

Abstract

PURPOSE:To prepare the titled novel enzyme capable of decomposing D-threonine into glycine and acetaldehyde, by culturing specific microbial strain belonging to Arthrobacter genus, Pseudomonas genus or Alcaligenes genus. CONSTITUTION:Microorganisms belonging to Arthrobacter genus, Pseudomonas genus or Alcaligenes genus and capable of producing D-threonine aldolase, e.g. Alclaigenes faecalis IFO 12669, Pseudomonas DK-2 (FERM-P NO.6200) or Arthrobactoer DK-19 (FERM-P NO.6201), are cultured in a nutrient medium, and the novel enzyme D-threonine aldolase is separated from the cultured product.

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.
L-threonine aldolase (L C, 4, 1, 2, 5), which specifically acts on threonine and L-allothreonine and decomposes it into glycine and acetaldehyde, is used not only in animals and plants, but also in bacteria, yeast, actinomycetes, etc. It is also known to exist in microorganisms. However, the existence of D-threonine aldolase, which decomposes D-threonine into glycine and acetaldehyde, was completely unknown.

The present inventors focused on a method of optically resolving DL-threonine as a method for producing L-threonine, which has a large potential market as food additives, feed additives, etc. As a result of extensive searches for enzymes that degrade fC, certain microorganisms belonging to the genus Arilobacter, the genus Pseudomonas, and alkaline earth metals produce a novel enzyme, D-threonine aldolase, which degrades D-threonine into total glycine and acetaldehyde. Based on this discovery, the present invention has been completed.

That is, in the present invention, microorganisms capable of producing D-threonine aldolase, which is a novel enzyme, and D-threonine aldolase belonging to the genus Arilobacter, Pseudomonas, or an alkaline earth metal genus are cultured in a nutrient medium, and the culture is The present invention relates to a method for producing D-threonine aldolase, which is characterized by collecting D-threonine aldolase from.

The microorganisms used in the present invention are Arylobacter, Pseudomonas, or alkaline earth metal species.
- has the ability to produce threonine aldolase,
Examples include Alcaligenes
gene8 faecalis) Engineering FO12669, Pseudomonas (Pseud, omonas) DK-2 Microtechnical Research Institute No. 62o0, Engineering Arislobacta = (Ar
throbacter) DK-+ 9Feikoken Bacterial Serial No. 62o1.

Pseudomonas DK-2 Microtechnological Laboratory No. 62o.

No. and Arylobacter DK-+ 9 Microtechnical Laboratory No. 6
The mycological properties of No. 2o1 are shown below.

(b) Growth status in each medium (C) Based on mycological properties that are more than physiological properties [Virginie's Manual of Determinative Bacteriology Vol.
Classifying with reference to version (+974) J, the DK-2 bacterium is a Durham-negative bacillus with polar flagella. have, oxidase positive,
It was identified as belonging to the genus Pseudomonas because of the positive denitrification reaction. On the other hand, the DK-49 bacterium is a bacillus with weak Durham staining, and is pleomorphic and pericytous. It was identified as belonging to the genus Arilobacter because it was unable to assimilate sugars. It is sufficient for the microorganism of the present invention to belong to each of the above-mentioned genera and have the ability to produce D-threonine aldolase. Needless to say, this includes L-threonine aldolase-deficient strains obtained by mutating this strain. There are no particular difficulties in culturing such microorganisms to produce D-threonine aldolase, and usually It can be carried out according to the culture of bacteria.

That is, regarding the culture medium, carbon sources include sugars such as glucose, glycerol, and molasses, or organic acids such as acetic acid and malic acid, nitrogen sources include W77monium sulfate, ammonium chloride, and urea sudo, and organic nutrients include yeast extract, Peptone, meat extract, cornstarch liquor, etc., and inorganic ions such as magnesium, iron, mankan, potassium, and phosphate are used. Addition of about 0.05 to 0.05% D-threonine ligation to the medium may increase the amount of enzyme production.

The culture method may be any conventional method, for example, the culture medium has a pH of 14 to
After inoculating the bacteria as No. 10, the cells may be cultured aerobically at 20 to 60°C for 1 to 3 days.

The D-threonine aldolase obtained in this way is mainly produced and accumulated within the bacterial body. Therefore, when using D-threonine aldolase as an enzyme source, the culture or the bacterial cells isolated from it may be used directly or It may be used dried, but if it is necessary to use D-threonine aldolase in a more purified form, the bacterial cells must first be destroyed by a known method such as mechanical methods, enzymatic treatment, or autolytic methods. to obtain a crude extract. Then, this crude extract is subjected to ammonium sulfate precipitation, solvent precipitation with acetone or ethanol, DKlli-Sepharose, DllI
l:A Fi - Chromatography using various ion exchangers and adsorbents such as Sephadex and calcium phosphate gel? A highly pure enzyme preparation can be obtained by appropriately combining and purifying the enzymes. To express the activity of this enzyme, all of the pyridoxal-5'-IJ acid is required as a cofermentation process, so the reaction usually takes place between 10"-1l' and 10"-10
-Exist at 5M.

Next, the results of measuring the physicochemical properties of the enzyme preparation obtained in 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. 0 ■ 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, the optimum pH of this enzyme was found to be between 7 and 9. The buffer used was 0.1M phosphate buffer from pH 4 to Z5, and pH 7.
~9 ViO, iM) Lys-HC1 buffer and p
H9 to H11 are 01M sodium carbonate buffers.

■ 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. pH4-7.5
(up to 0.1 M IJ non-acid buffer, up to pH 7-9 0.1 M) IJ Su-He/: buffer and pH 9-9
Items up to 11 are α1M sodium carbonate buffer.

■Measurement method of titer Enzyme-containing solution 0.1 ml f 100 μmol D
PaZ method (Arch, Bio
chem, Biophys, vOl-j 09
.

p548 (1965)). still,
The enzyme activity that decomposes 1 μmole of D-threonine per minute was defined as 1 U.

■ Suitable temperature range for action: 0 and 1 M at pH 8.0 as D-Threoninke substrate.
Using a 17-hydrochloric acid buffer solution, the reaction was carried out for 10 minutes at each temperature, and the amount of acetaldehyde produced was measured, and the optimum temperature for this enzyme was found to be 40 to 50°C.

■ Thermostable The fermentation bath containing pHao dissolved in cL1M Tris-HCl buffer was heated at various temperatures for 1 hour, and the residual activity in the solution was measured, and the stable temperature of this enzyme was 40°C or lower. .

Conditions for inactivation such as pH 5 temperature, etc. This enzyme is at pH 5 or lower, pH 11 or higher, and temperature 70°C.
In this case, it is deactivated at V11 hour.

■ Inhibition, activation and stability This enzyme contains mercaptoethanol, sodium sulfite, sodium bisulfite, dithiothreitol, Mn2+,
Activated and stabilized by C02+, Fe2+, and Mg2+. On the other hand, Ag1+, Cu total, Hg2+, z
Inhibited by nz+, Pd2+, hydroxylamine, p-chloromercury-benzoic acid.

(2) Coenzyme The coenzyme of this enzyme is pyridoxal-5-I monoacid.

The enzyme obtained in the example has the above-mentioned physicochemical properties, but all conventionally known threonine aldolases degrade L-threonine, and like this enzyme, D-threonine aldolase degrades D-threonine. This enzyme is clearly a new enzyme with a completely new action, as nothing is known about what it does.

This enzyme can simplify the optical resolution process if used, for example, in a method for producing L-threonine ligation using DL-threonine as a raw material, 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 05, yeast extract 0.5%, KH2PO
pHa consisting of 40.1%, MgSO40.05%, L-glutamic acid 0.1%, and D-threonine α1%
A culture medium of 57-f was prepared, and 57-f was added to a culture tank with a volume of 5 and sterilized by heating at 120° C. for 15 minutes. This medium was inoculated with Arylobacter DK-19 Microtechnical Research Institute No. 6201, and cultured at 30° C. for 20 hours with aeration and stirring while maintaining the pH at 5.

After culturing, the cells were centrifuged from 1 ton of the culture solution, washed once with physiological saline, and the wet cells were washed with 0.1M Tris, pH 7.5, containing α1mM pyridoxal-5-phosphate and 10mM mercaptoethanol. -Suspended in 100ml of hydrochloric acid buffer. This bacterial suspension 220 KH2 is 10
Ultrasonic treatment for 12,000 r to destroy bacterial cells
-p,m for 60 minutes and decantation to obtain a crude enzyme extract of 105g.

Ammonium sulfate was added to the obtained crude enzyme extract to separate the 03-α5 saturated fraction, and this fraction was dialyzed against the above buffer solution overnight. ] JAFi Sephadex A-50+00m
The dialysis residue was added to a column packed with 1 and equilibrated with the above buffer solution. After the solution was passed through the column to adsorb the enzyme, a sodium chloride solution was passed through the column at varying concentrations from 0.1 to 0.4M, and the D-threonine aldolase activity portion of each fraction of the eluate was collected. This active zone was located near a concentration of 0.5 M sodium chloride. All the collected active fractions were passed through a column packed with 200 mg'i of Sephadex G-200 and subjected to gel filtration, and the D-threonine aldolase active fraction was collected and concentrated with a Membram filter to obtain 15 grams of enzyme concentrate. Ta. The protein content in this enzyme solution was 2.4 get/-, the specific activity for D-threonine was 1.24 U/.9, and the specific activity for D-allothreonine was A 55 U/mf. On the other hand, it showed no activity against L-threonine and L-allothreonine.

Example 2 Using Pseudomonas DK-2 Microtechnical Laboratory No. 6200 and Alcaligenes fly sue FO+2669i,
Both were cultured in the same manner as in Example 1, and the enzyme was separated from the culture solution. In the case of Pseudomonas DK-2, enzyme solution 1 with a protein concentration of 2.5 my/fnt
In the case of 2-, Alcaligenes flies, an enzyme solution 11 with a protein concentration of 2.1 mW/- was obtained.

When all of these enzyme activities were measured, the specific activity of the former against D-threonine was 1.01 U/m? The specific activity for D-allothreonine was 2.96 U/IF. On the other hand, the latter had a specific activity of 0.86 U/mf for 1dD-threonine and 2.95 U/mf for D-allothreonine. In addition, none of them showed any activity against L-threonine and L-allothreonine.

Both enzymes were eluted at the same NaC concentration as the enzyme of Example 1, and had similar physicochemical properties such as substrate specificity, optimum temperature, temperature stability, optimum pH, p)1 stability, and molecular weight. It was determined that they were the same enzyme.

Patent Applicant Denki Kagaku Kogyo Co., Ltd. Agent Mitsuben Nakamoto Continued from page 1 @ Inventor Masahisa Ikemi Inside Denki Kagaku Kogyo Co., Ltd. Central Research Laboratory, 3-5-1 Asahi-cho, Machida City (inventor) Inventor: Haruo Gomi, 3-5-1 Asahicho, Machida City, Denki Kagaku Kogyo Co., Ltd. Central Research Laboratory Inventor: Yoshiaki Ishimatsu, 3-5-1 Asahicho, Machida City, Denki Kagaku Kogyo Co., Ltd. Central Research Laboratory Inventor Noriaki Koizumi 2-11-8 Sengen, Sakuramura, Niiharu-gun, Ibaraki Prefecture Shinouchi Hai No. 102 Procedural Amendment (voluntary amendment) April 19, 1980 Commissioner of the Patent Office Shima 1) Haruki Tono 1, Indication of the case 1988 Patent Application No. 209981 2, Invention Name: D-threonine aldolase and its manufacturing method 6. Relationship with the case concerning amendments Patent Applicant Address: 1-4-1 Yurakucho, Chiyoda-ku, Tokyo Name (
329) Denki Kagaku Kogyo Co., Ltd. Representative Shinohara
Akira Address: 302 Nishi-Shinbashi Chuo Building, 6-15-8 Nishi-Shinbashi, Minato-ku, Tokyo Telephone: (437)-346'7 Name: Patent Attorney (7850) Hiroshi Nakamoto Address: Same
Name Patent Attorney (8702) Inoue
Date of the 1980 amendment order Voluntary amendment 6, Detailed explanation of the invention column 7 of the specification subject to amendment 7 Contents of the amendment The description in the Detailed explanation of the invention column of the specification is amended as follows.

(1) Add the following statement after line 8 on page 13 of the specification.

As a result of gel filtration using Sephadex G-200, the molecular weight was determined to be 100,000 to iso, ooo.

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

C: 51.7% F] Near, 8% 1st: 157chi　　　　　　　　　　

Claims (2)

[Claims] (1) D-threonine al'r'lase. (2) A microorganism belonging to the genus Arilobacter, the genus Pseudomonas, or alkaline earth metals and capable of producing D-threonine aldolase is cultured in a nutrient medium, and D-threonine aldolase is collected from the culture. Method for producing D-threonine aldolase〇