JPH02154686A - Production of sucrose phosphorylase - Google Patents

Abstract

PURPOSE:To produce the subject enzyme in extremely high efficiency without adjusting the pH of the medium by culturing a microbial strain in a medium containing at least one kind of salt selected from carbonate, acetate, hydrochloride and nitrate. CONSTITUTION:A microbial strain belonging to genus Leuconostoc [e.g. Leuconostoc mesenteroides (ATCC 12291)] is cultured in a liquid culture medium containing a carbon source (e.g. sucrose), a nitrogen source (e.g. trypton), an inorganic salt (e.g. monopotassium phosphate), a vitamin (e.g. thiamine) and at least one kind of salts selected from inorganic salt such as carbonate (e.g. calcium carbonate), acetate (e.g. sodium acetate), hydrochloride (e.g. sodium chloride) and nitrate (e.g. sodium nitrate) by conventional cultivation process. The cultured microbial cells are disintegrated and extracted and the obtained extract liquid of sucrose phosphorylase is purified and, as necessary, concentrated to obtain purified enzyme. An enzyme useful as an enzymatic agent for quantitative determination or diagnosis can be produced with simple operation in high efficiency by this process.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing sucrose phosphorylase, and its purpose is to efficiently obtain sucrose phosphorylase with high enzymatic activity.

Sucrose phosphorylase is an enzyme that acts on sucrose in the presence of inorganic phosphate to produce glucose monophosphate and fructose. Methods for quantifying phosphoric acid (e.g., JP-A-63-146800, JP-A-63-491)
(see Publication No. 00) or quantitative analysis of sea-close [
Analytical Biochemistry 142, 3
56-561 (1984)), etc.
Its development as a diagnostic enzyme agent is expected in the future.

[Conventional technology]

Conventionally, a method is known in which bacteria belonging to the genus Leuconostoc, Neudomonas (1, Acetobacter, etc.) and having the ability to produce sucrose phosphorylase are cultured, and sucrose phosphorylase is collected from the culture.

[Problem to be solved by the invention]

However, the conventional culture method has the disadvantage that the culture pH drops to 5 or less due to the production of organic acids during culture, resulting in a significant decrease in the yield of NAKES phosphorylase. In order to prevent the pH from decreasing, the pH of the culture solution is adjusted by adding alkali such as ammonia or sodium hydroxide. This method has disadvantages in that it requires a large culture device and the culture operation is complicated.

[Means to solve the problem]

The present inventors have conducted intensive studies to solve the various drawbacks of conventional culturing methods for sucrose phosphorylase-producing bacteria, and as a result, the present inventors have found that the present invention contains at least one salt selected from carbonates, acetates, hydrochlorides, and nitrates. If sucrose phosphorylase-producing bacteria are inoculated and cultured in a medium containing
The present invention was completed based on the knowledge that sucrose phosphorylase can be obtained extremely efficiently without H adjustment.

That is, the present invention involves culturing a microorganism capable of producing sucrose phosphorylase in a medium containing at least one salt selected from carbonate, acetate, hydrochloride, and nitrate, and collecting sucrose phosphorylase from the culture. This is a method for producing sucrose phosphorylase.

The present invention will be explained in detail below.

The microorganisms capable of producing sea-clase phosphorylase used in the present invention may be of any origin, but bacteria belonging to the genus Leuphustock, Pseudomonas, Acetobacter, etc. are particularly preferred.

Specific examples of the above-mentioned sucrose phosphorylase-producing bacteria include Metsucentiloides (ATCC 12291), which belongs to the genus Leifstock, Satsulophila (ATCC 15946), which belongs to the genus Pseudomonas, and Xylinum (J, Natl., which belongs to the genus Acetobacter).
Sci, Covmc, Sri Lanka
to (2), 80-169 (1982)].

Next, a method for culturing a microorganism having the above-mentioned sucrose phosphorylase-producing microorganism will be described.

First of all, the culture medium used in the present invention includes a carbon source such as /nykose and molasses, a nitrogen source such as tryptone, yeast extract, meat extract, soybean flour, and corn staple liquor, monopotassium phosphate, and dipotassium phosphate. , a liquid culture medium containing inorganic salts such as magnesium sulfate and iron sulfate, and vitamins such as thiamine and ascorbic acid (medium pH is 6.0~
8.0 degree) is used.

Next, add carbonate, acetate, hydrochloride and 1i1 to the above medium.
At least one salt selected from 'l salts is added.

Examples of the above-mentioned carbonates include calcium carbonate, potassium carbonate, sodium carbonate, and ammonium carbonate; examples of acetates include sodium acetate, ammonium acetate, potassium acetate, and acetate; and examples of hydrochlorides include sodium chloride, ammonium chloride, and ammonium chloride. Nitrates include sodium nitrate, potassium nitrate, potassium chloride, etc.
Ammonium nitrate, calcium nitrate, etc. are preferably used.

The amount of at least one salt selected from carbonate, acetate, hydrochloride, and nitrate added in the above-mentioned medium is such that the pH of the culture solution is 5.0 or more, preferably in the range of 5.5 to 8.0. It is recommended to add 0.05% (W/V) or more to the medium, preferably about 0.1 to 5.0 (W/V), to maintain the quality of the culture. However, it is preferable to add it when preparing the medium.

In addition, the culture method in the present invention includes ordinary shaking culture,
Using an appropriate liquid culture method such as agitation culture, aeration culture, or static culture, the culture time is 5 at a culture pH in the range of 5.0 to 8.0.
Culture for about 25 hours.

In order to culture the above-mentioned sucrose phosphorylase-producing bacteria and collect sucrose phosphorylase from the culture, for example, the cultured cells obtained by culturing the sucrose phosphorylase-producing bacteria are crushed and extracted by a conventional method to produce the enzyme. Obtain an extract, remove nucleic acids with polyethyleneimine, manganese sulfate, protamine sulfate, etc., and perform adsorption and elution using an ion exchanger, gel filtration, adsorption and elution using hydroxyapatite, hydrophobic chromatography, etc. as appropriate. Refined by selecting and combining,
A purified enzyme can be obtained by concentrating this if necessary.

〔Effect of the invention〕

According to the present invention, microorganisms capable of producing sucrose phosphorylase are cultured in a medium containing at least one kind of salt selected from carbonate, acetate, hydrochloride, and nitrate. It is possible to obtain sucrose phosphorylase that is useful as a quantitative enzyme agent or a diagnostic enzyme agent, and the present invention is extremely significant industrially.

Hereinafter, the present invention will be specifically illustrated by examples.

〔Example〕

Example 1 Shake sugar 2.0% (W/V), sodium acetate 2.0% (W/V), yeast extract 1.0% (W/V)
), trypto/(manufactured by Difco) 1.0% (W/v),
Put 100 ml of a medium (pH 7.0) consisting of 1.5% (W/V) dipotassium phosphate and water into a 200 ml Erlenmeyer flask, sterilize it at 120°C for 115 minutes, and add the vitamin/myrrhal solution [ Thiamine salt a salt 0.
1% (W/V), ascorbic acid 0.5% <W/V),
Magnesium sulfate 4.0% (W/V), ferrous sulfate 0.
1% (W/V) and manganese sulfate 2.0% (W/V)
Leuconostoc metsucentiloides (ATCC
Three platinum loops were inoculated from the broth agar slant culture medium of No. 12291), and this was cultured stationary at 30"C for 14 hours in an incubator.

The culture solution 4 me thus obtained was centrifuged to collect the bacterial cells, and the resulting bacterial cells were mixed with 0.05 MKH2PO, t
-The cells were suspended in NaOH buffer (pH 6,5) 4 yJ and centrifuged to prepare washed bacterial cells.

Next, the washed bacterial cells were washed with 0.0% lysozyme (manufactured by Seikagaku Kogyo Co., Ltd.).
5% (W/V), Triton X-1000,
1% (V/V), to mM ethylenediamine 4
0,03 M KH2PO containing disodium acetate
The mixture was suspended in 4 ml of NaOH buffer (pH 6,5) and 4 trtt. Then, boil it at 37°C for 30 minutes to lyse the bacteria, and then add ethyleneimine polymer (manufactured by Tokyo Kasei Kogyo Co., Ltd.).
was added to a final concentration of 0.005% (V/V) to remove nucleic acids, and then centrifuged to collect the supernatant (crude enzyme solution).

As a result of measuring the enzyme activity of the supernatant, the Nyuk p-phosphorylase activity per #It of culture solution was 1.69 units.

As a control, when cultured in exactly the same manner as above in a medium with only sodium acetate removed from the above medium composition, the pH during the culture was lowered to below 5.0, and the resulting crude enzyme solution The di-close phosphorylase activity was as low as 0.53 units/me.

Note that the enzyme activity was measured as follows.

Substrate s-GIX was added to 0.05 M KH2P.
The substrate concentration was adjusted to 160 mM by dissolving it in O4-NaOH buffer (pH 6,8). This substrate solution 3.0xr
l of a concentration of 0.01 M EDT dissolved in the above buffer.
A-2 Sodium solution 0.03 d, 1% (W/V
) NADP (manufactured by Oriental Yeast Co., Ltd.) solution 0.1 shoulder/, 0.01% (W/V) glucose-1,6-2
Phosphoric acid (manufactured by Peringarman Im Co.) solution 0.1
ml, l M chloride-2gnesium m solution 0.05 ml,
Phosphoglucomutase (manufactured by Boehringer Mannheim A>
Add o, ot scraps 11 glucose 6-phosphate dehydrogenase (manufactured by Oriental Yeast Co., Ltd.) Q, Q1 shoulder/, 25
Prewarm at °C for approximately 5 minutes. Add 0.02 d of enzyme solution and measure the change in absorbance per minute at a wavelength of 340 nm.

As a control, instead of 0.02 m/ml of this enzyme solution, 0.01
M HEPES-NaOH buffer (pH 7) was added to 0.
02ml was added.

The produced glucose 1-phosphate was quantified from the difference in absorbance change per minute between the enzyme reaction solution and the control at 340 nm.

The titer was expressed at 25''C, with 1 unit being the amount of enzyme that produced 1 micromole of glucose monophosphate per minute.

Example 2 Sucrose 2.0% (W/V), carbonate cal/rum 0
．． 3% (W/V), yeast extract 1.0% (W/V)
, Triphton 1.0% (W/V
), dipotassium phosphate 1.5% (W/V)
and 100 ml of a medium (pH 6,9) consisting of water!
', ff Put into a 200 ml Erlenmeyer flask and sterilize at 20°C for 115 minutes, then add vitamin/myrrhal solution [thiami/hydrochloride 0.1% (W/v),
Ascorbic acid 0.5% (W/V), magnesium sulfate 4.0% (W/v), ferrous sulfate 0.1% (W/V)
) and mangaf sulfate 2.0% (W/V) dissolved in water and sterilized and filtered with a membrane filter] to 1.0%
% (V/V), inoculated with 3 platinum loops of Leuconostoc metsucentiloides (ATCC 12291) from a broth agar slant, and placed in a thermostat at 30°C for 12 hours. The mixture was allowed to stand for a period of time to perform intoxication culture.

Next, Nyucrose 2.0% (W/V), Cal/Rum Carbonate 0.5% (W/V), Yeast M Extract 1.0%
(W/V), )! j-% ton t,
o% <W/V), dipotassium phosphate 1.5%
(W/V) and water (pH 6,9) 2
1 was placed in the culture tank of a stirring type small culture device (manufactured by Iwashiya Co., Ltd.) and sterilized at 120"C for 20 minutes, and then a vitamin/myrrhal solution [thiamine hydrochloride 0, t% (W)
/V), ascorbic acid 0.5% (W/V), magnesium sulfate 4.0% (W/V), ferrous sulfate 0.1% (
W/V) and manganese sulfate 2.0% (W/V) dissolved in water and sterilized through a membrane filter] were added to L, 0% (V/V). , 2% (V/V) of the seed culture solution obtained by the above seed culture was inoculated, and stirred submerged culture was performed at 30°C for 14 hours without aeration.

The culture solution obtained in this way was centrifuged to collect the bacterial cells, and the resulting bacterial cells were concentrated at 0.05 M (PC) 4Na.
After suspending in OH buffer (pH 6,5) 4 me,
Washed bacterial cells were prepared by centrifugation.

Next, the washed bacterial cells were washed with 0.0% lysozyme (manufactured by Seikagaku Kogyo Co., Ltd.).
5% (W/V), Triton X-1000,1
% (V/V), 0.05 M KH2PO4 containing 10 mM “disodium ethylenediaminetetraacetate
-Suspended in NaOH buffer (pH 6,5) for 4 d. Then, the cells were heated at 37°C for 30 minutes to lyse the bacteria, and then ethyleneimide/polymer (manufactured by Tokyo Kasei Kogyo Co., Ltd.) was added to a final concentration of 0.005% (V/V) to remove nucleic acids. Afterwards, the supernatant (crude enzyme solution) was collected by centrifugation.

As a result of determining the enzyme activity of the supernatant, the culture solution 1 at
The sucrose phosphorylase activity per sample was 2.5 units.

As a control, when culturing was performed in exactly the same manner as above using a medium with only carbonate from the above medium composition, the aph of the culture decreased to 5.0 or less, and the sucrose phosphorylase in the crude enzyme solution obtained decreased. Activity is 0.51 unit/d
Met.

Claims (1)

[Claims] A microorganism capable of producing sucrose phosphorylase is cultured in a medium containing at least one salt selected from carbonate, acetate, hydrochloride, and nitrate, and sucrose phosphorylase is collected from the culture. A method for producing sucrose phosphorylase.