JPS60244286A - Preparation of oxalic acid oxidase - Google Patents

Abstract

PURPOSE:To obtain the titled enzyme with high efficiency, by cultivating a microorganism having the ability to produce oxalic acid oxidase belonging to the genus Pseudomonas in producing the oxalic acid oxidase with the microorganism, and collecting the above-mentioned enzyme from the resultant culture. CONSTITUTION:A microorganism, e.g. Pseudomonas maltophilia OX-54, belonging to the genus Pseudomonas, and having the ability to produce oxalic acid oxidase is cultivated in a culture medium containing a nitrogen source, carbon source, inorganic substance, vitamins, etc. After completing the cultivation, microbial cells are separated to collect the aimed oxalic acid oxidase from the microbial cells.

Description

[Detailed description of the invention] The present invention relates to a method for producing oxalate oxidase.

Conventionally, as oxalate oxidase, for example, mold (mo
ld) (Jay, Hoget and A.

Mayer (J, Houget and A, Mayer
), :! 7pt.

Rend, (Compt, Rend,), / (S
'j, 3 o4LJj (/ri 27 years), Y, Suzuki & J, D, Me S- (y, 5uz
uki and J, 11. Meesue), Plant Cell Ph.
ysiol, ), Otsu.

2l pages (2013)], barley seedlings [Jay, Chiripoga (
J, Chiriboga), 7th, Biochem,
Hi-Ophis, (Arch, BioChem, Bi
ophys, ), / /Otsu.

617, p.
, Leek and V, S, Butt), Broc, Biochem, G.I., (Proc, Bioche
m, J,), /, II.

! r7 page (/'173)], fungi (E, H.

Baisei & Bui, H, Chelderin (E.

B. Vaisey and V. H. Sheld
eline), 7chi, Biochem, Biophys, (Arch, BioChem, Bio-phy
s, , rit, gg page (15 i'g/year)], etc. are known.

Therefore, apart from the above, the present inventors conducted a wide search for microorganisms capable of producing oxalate oxidase from the natural world, and as a result, newly isolated Pseudomonas (Peudomonas
The present invention was completed based on the knowledge that a strain belonging to the genus Domon, as) efficiently produces the enzyme within the bacterial body.

That is, the present invention is a method for producing oxalate oxidase, which comprises culturing a microorganism belonging to the genus Pseudomonas and having the ability to produce oxalate oxidase in a medium, and then collecting the enzyme from the culture.

The present invention will be explained in detail below.

First, the bacteria used in the present invention are Pseudomonas (
Any microorganism may be used as long as it belongs to the genus Psθuaomonas and produces oxalate oxidase, and p< may be a mutant strain of these microorganisms.

Specific examples of such microorganisms include Pseudomonas and Maltophilia.
to-philia) OX-j 41 and the like.

The above Pseudomonas maltophilia ox-! r≠ is
This strain was newly found in soil, and its mycological properties are as shown below.

Mycological properties of Pseudomonas maltophilia ox, 5-hiro (a) Morphology Microscopic observation (30℃ on broth agar slant medium,
/6 hour culture) ■Cell shape and size (nearly 0.5 μm)<7 to 2 μm, short rods.

■Presence or absence of cell pleomorphism; Not observed.

③ Motile or not. It has two to several polar flagella and is motile.

■Presence or absence of spores: Not formed.

■Durham staining: Negative.

■Anti-acidity: Negative.

(b) Growth status in each medium ■ Juicy agar plate culture: Yellow colonies were obtained by static culture at a temperature of 30°C and a waiting time of ≠g. Does not produce diffusible pigments.

■Meat juice agar slant culture: ■Same as described for gravy agar plate culture.

■ Broth liquid culture: Good growth.

■Meat juice gelatin puncture culture: Gelatin is not liquefied.

■ Lidmus milk: No change observed.

(C) Physiological properties ■Reduction of nitrate: No reduction.

■Denitrification reaction: Negative.

■MR test: Negative.

■VP test: Negative.

■Indole production: Negative.

■Generation of hydrogen sulfide: Negative.

■Starch hydrolysis: Negative.

■Use of citric acid: Christensen (Chris-t)
Koser (Kos)
θr) medium, its utility is unknown due to poor growth.

■ Utilization of inorganic nitrogen: Both ammonia and nitrate are utilized in small amounts.

[Phase] Pigment formation: Negative.

■Urease: Negative.

■Oxidase: Negative.

0 catalase: positive. “ ■Growth range: Optimal temperature: 20-37℃ Optimal pH: j~ri [Phase] Attitude towards oxygen: Aerobic.

[Co., Ltd.] O-F test: Oxidizing property.

Production of acid and gas from O-saccharides Acid production Gas production (IIL-arabinose + - (2) D-xylose + - (3) D-glucose 10- (4) D-Mannose 10- (5) D-Fructose - (6) D-galactose + - (7) Maltose 10- (8) Sugar 10- (9) Lactose -- (10) Trehalose + - (11) D-Sorvit - - (12D-Mannit +- (131 Inojitsu - - α4 glycerin 10- (19 starch -- (d) Other properties ■Lipase: Negative.

■Auxotrophic requirement: Methionine.

The mycological properties of the above Pseudomonas maltophilia ox-jll were analyzed using the Purge's Manual of Determinative Bacteriology (Bergey's Manual of Determinative Bacteriology).
s Manual Of Determinative
Bacterio-1ogy) Rth edition (/ri7≠
In contrast to the classification described in 2010, this bacterium is negative for Durham staining, is an aerobic, nonsporing bacillus, has flagella, is motile, and
It is recognized that it belongs to the genus Pseudomonas because it is catalase positive, and it belongs to Pseudomonas maltophilia because the colony is yellow, it requires methionine as a growth factor, and it is oxidase negative. It was classified as a thing.

However, it was determined that it is a new strain that belongs to Pseudomonas maltophila and is different from the conventional strain that belongs to Pseudomonas maltophila in terms of its isolation source and ice-melting ability of gelatin. It was named ≠.

In addition, the above Pseudomonas maltophilia Ox-≠
is the 7th microbiological research institute at the Institute of Microbial Technology, Agency of Industrial Science and Technology.
It has been deposited as No. 6O1 (°FERM P-7tO/).

Next, the medium used for oxalate oxidase production in the present invention may be any synthetic or natural medium as long as it contains appropriate amounts of selected substances such as nitrogen sources, carbon sources, inorganic substances, vitamins, etc. It can also be used.

Examples of the nitrogen source include yeast extract, peptone,
Examples of carbon sources include meat extract, soybean, amino acids, ammonium sulfate, potassium nitrate, etc.
Examples include carbohydrates such as glucose, sucrose, and xylose, and organic acids such as oxalic acid.

Further, as the inorganic substance, for example, potassium phosphate, sodium phosphate, magnesium sulfate, ferrous sulfate, sodium chloride, etc. are used.

The bacteria used in the present invention may be cultured by a solid culture method, but it is usually advantageous to use a liquid culture method. Specifically, aerobic culture using shaking culture, stirring culture, aeration culture, etc. Culture.

The culture temperature is, for example, usually 36°C, preferably 30°C, and the pH during culture is! -♂, preferably pH!
It is.

Under such culture conditions, the enzyme is produced and accumulated in the culture by culturing for about 2 to 2 hours, although the culture time varies depending on the culture form.

After completion of the culture, oxalate oxidase can be collected from the culture using a conventional enzyme collection method.

However, since this enzyme exists within the bacterial body,
After culturing, for example, add a surfactant such as Triton I can do it.

In addition, it is preferable to collect the enzyme from the culture after separating the bacterial cells by an operation such as filtration and centrifugation; in this case, the bacterial cells may be used as they are. However, it is possible to destroy the bacterial cells using various destructive means such as an ultrasonic destroyer, French press, or Dynomill, to dissolve the bacterial cell wall using a cell wall-dissolving enzyme such as lysozyme, or to use an abrasion agent. A method of grinding the bacterial cells or a method of applying osmotic shock is used.

The above-mentioned bacterial cells or those destroyed in this way,
Alternatively, a bacterial cell extract can be obtained by removing solid matter from the cell wall lysed product through an appropriate processing operation such as filtration or centrifugation, or by extraction with water, a buffer solution, or an appropriate solvent, This can be obtained as it is as a crude enzyme, or if necessary, cyprotamine sulfate, polyethyleneimine, etc. can be added to the extract to remove nucleic acids, or further, freeze-drying, alcohol precipitation, acetone precipitation, etc. Crude enzyme powder is obtained by appropriately selecting and carrying out the procedure.

In order to obtain a further purified enzyme preparation from the above crude enzyme solution or crude enzyme powder, for example, gel filtration method using Sephadex G-200 etc., adsorption elution method using ion exchanger, hydroxyapatite etc., sucrose density Purified specimens of this enzyme can be obtained by appropriately selecting and combining sedimentation methods such as gradient centrifugation, Affinity chromatography, fractionation methods using molecular sieve membranes or hollow fiber membranes, etc. I can do it.

Furthermore, the present enzyme can be obtained by treating the culture filtrate obtained by autolysis after long-term culture in the same manner.

The physicochemical properties of the enzyme thus obtained are as follows.

(1) Action Oxalic acid is oxidized in the presence of oxygen to produce 2 molecules of carbon dioxide and 7 molecules of hydrogen peroxide.

This enzyme ↓ (C00H)2+ 02 →2 CO24-Hz 02
(2) Substrate specificity It has extremely high substrate specificity for oxalic acid, and has a slight effect on glyoxylic acid.

(3) Optimum pH and stable pH 9. ,'Q,/M The optimum pH in citrate buffer was 0 for oxalic acid.

Further, the stable pH range was 4'l- to 7.

Note that this range includes buffer solutions of each pH (pH 2,0 to b,
.

So, 0. /'M citrate buffer, also pH 6, θ~
In 9.5, 0.1M potassium phosphate buffer was used, respectively. ) was stored at a temperature of 2J°C for 2 days, and then the enzyme activity was measured.

(4) Potency measurement method M potassium phosphate buffer (pHj:O) 0.3rtt
l, M potassium oxalate o, i punishment, o, oozcs t-aminoantipyrine, 0.02% phenol or N, N
-Dimethylaniline, peroxidase ≠ unit and this enzyme solution! Oμt (diluted or concentrated to an appropriate amount) was added to bring the total amount to 3-, and after reacting at a temperature of 37°C for 20 minutes, the visible absorption (jJ-[n
m) was measured, and the amount of hydrogen peroxide produced was calculated based on the absorbance value of the known amount of hydrogen peroxide measured in advance to obtain the enzyme unit.

In addition, a method of measuring oxygen absorption using an oxygen electrode was also used as necessary.

Note that the enzyme activity/unit is the amount of enzyme that produces/micromole of hydrogen peroxide in 7 minutes at a temperature of 37°C.

(5) Range of suitable temperature for action As shown in Figure 2, the range of suitable temperature for action is 3! ~jO℃
The temperature range was determined by adding the present enzyme to a reaction solution using oxalic acid as a substrate at each temperature and measuring the enzyme activity when the enzyme was allowed to react for 10 minutes.

(6) 1) Conditions for deactivation due to H, temperature, etc. As shown in Figure 3, deactivation starts from a temperature of 70°C.
It becomes almost inactive.

Note that the conditions are 0. This was done by measuring the remaining enzyme activity when this enzyme was treated for 70 minutes in a potassium phosphate buffer (CpH 70).

'palAs is inactivated on the acidic side; on the other hand, at pH'70
It is also deactivated in highly alkaline conditions.

(7) Inhibition, activation and stabilization respectively/mM Hg, Pb 1CHaCOO- and C1
- containing solution C0,0! ;M citrate buffer, pH left O
) The enzyme activity after reacting at a medium temperature of 30°C for /θ minutes is:
When no treatment was set as /0.0, the values were 23.2 and ≠/, respectively.

(8) Purification method The separation and purification of this enzyme can be carried out by conventional methods.
For example, purification methods such as fractional precipitation with ammonium sulfate, adsorption and elution with DEAFi-cellulose, and gel filtration with Biogel A-QJm can be used in appropriate combinations.

(9) Contains SDS with a molecular weight of 0.7%! The result of measurement by polyacrylamide electrophoresis was 37,000.

(XO) Acrylamide disk gel electrophoresis As shown in Figure 4, an acrylamide gel with a pore size of 7J% (
pI! As a result of performing diacrylamide disc gel electrophoresis in a conventional manner using 4t), a single band was confirmed.

As a result of Uυ isoelectric focusing disk gel focusing electrophoresis measurement, lA
It was 7.

As described above, according to the present invention, bacteria of the genus Pseudomonas have not been known to produce oxalate oxidase, and compared to molds, barley seedlings, etc., which are known to have oxalate oxidase, the production of oxalate oxidase is extremely short. The present invention is industrially extremely meaningful because the present enzyme can be obtained efficiently within a short period of time.

Furthermore, this enzyme is extremely useful as an enzyme for quantifying oxalic acid in oxalic acid-containing substances such as body fluids and foods and drinks.

The present invention will be specifically described below with reference to Examples.

Example phosphoric acid/potassium 0. /%, dipotassium phosphate 0. /
H, magnesium sulfate 0.2/%, ferrous sulfate 0.00
10 ml of a solution (pHj:
After dispensing the mixture into Pseudomonas maltophilia ○X-J-≠ (F
KRMP-7OtsuO/) was inoculated and cultured with shaking at a temperature of 30°C for 22 hours to obtain a culture.

Next, in addition to the above medium composition, 0.0% potassium oxalate was added.
2 tf solution with addition of 2 j % and 0.3 % glycine
After dispensing into a mini jar fermenter (capacity: 2 t), this was heat sterilized for 70 minutes at multiple temperatures/2θ°C in a conventional manner to obtain a medium, and the culture obtained as described above was added to this. Things end O
ml was inoculated and cultured with rotational agitation at a temperature of 30° C. for 72 hours to obtain a culture.

Triton x-io was added to the culture thus obtained.

(manufactured by Wako Tsumugi Pharmaceutical ■) at a temperature of 2! After incubating at ℃ for 20 hours,
10% polyethyleneimine solution (pH 7O) ljrrt
l and ammonium sulfate! ≠1 was added, and insoluble matter was filtered off to obtain a crude enzyme solution.

Next, the crude enzyme solution was diluted with 0.0/M') acid buffer (p
H70) diluted 3 times with the phosphate buffer ('
into a -DEAE-cellulose column (3,j r, m x 30 crn) equilibrated with pH'7:O),
After adsorbing the enzyme, 0.07M phosphate buffer CpH
7,0) Contains C0,2tM ammonium sulfate. ) to elute the adsorbed enzyme and obtain the active fraction.

Then, the active fraction was converted into ammonium sulfate! θ~zO%
Fractionate at saturation, 0.0/M') acid buffer (pH 70')
) was diluted 3 times with 0.0/) A') acid buffer (1) H7 (1)) and passed through a DEAE-Sepharose CL, 4B column to adsorb the enzyme. After that, the adsorbed enzyme was eluted using 0.0/M phosphate buffer (containing 0./4M ammonium sulfate).
After concentrating the obtained active fraction using a collodion bag, biogel A, Q, j m (2, j
The purified oxalate oxidase preparation ysrrq (yield/
3.2%, specific activity 1A♂ units/■ protein) was obtained.

[Brief explanation of drawings]

Figure 1 shows the optimum pH of this enzyme, Figure 2 shows the range of action and optimum temperature, Figure 3 shows the conditions for inactivation due to temperature, and Figure 4 shows acrylamide disc gel electrophoresis. . Patent applicant: Noda Institute of Industrial Science Foundation

Claims (1)

[Claims] (1) A method for producing oxalate oxidase, which comprises culturing a microorganism belonging to the genus Pseudomonas and having the ability to produce oxalate oxidase in a medium, and collecting the enzyme from the culture.