JPS6031470B2 - Method for producing choline dehydrogenase - Google Patents

Description

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

More specifically, the present invention relates to a method for producing choline dehydrogenase, which comprises culturing a microorganism having a choline dehydrogenase production facility in a medium containing choline, and collecting choline dehydrogenase from the culture. Choline dehydrogenase (ECI.1
．． 99.1) is an enzyme that catalyzes the reversible oxidation reaction of choline to betainaldehyde, and is known to exist in the liver and plants.

In addition, recently, Pseudomonas aeruginosa A-16
It has been reported that the production of choline dehydrogenase by fungi
Group A. Biol. Chem. , 39(7), 1513~
1514 (1975)]. Choline dehydrogenase is used to quantify choline. The present inventors conducted various studies in order to develop an inexpensive industrial method for producing choline dehydrogenase.

As a result, among the strains including Pseudomonas floretscens, Pseudomonas iodeinum, Pseudomonas melanogenum, Pseudomonas ovalis, Pseudomonas seuilkiriensis, and Pseudomonas chinkisan, Pseudomonas aeruginosa A-16
They discovered that there are strains with better choline dehydrogenase-producing ability than Pseudomonas, and that there are strains that produce choline dehydrogenase even among strains belonging to other genera than Pseudomonas. The present invention will be explained in detail below.

The choline dehydrogenase of the present invention has the following properties (as the enzyme, an enzyme preparation obtained from Pseudomonas floretscens KY4032 in Example 1 below was selected as a representative enzyme, but enzymes derived from other strains of KY4032 are also used).
032).

'1} Action Catalyzes the reversible oxidation reaction of choline to betaaldehyde.

{2} Substrate specificity The relative activity of each substrate is as follows when the activity for choline is set as 100.

Substrate Relative active choline
100 cytidine diphosphate choline 1 petaine
7 Glucosamine 5 Glycine
4 Ethanol 5 {3} Optimum pH The pH action curve of this enzyme is shown in FIG.

This figure shows that the optimum pH of this enzyme is around 9.0. {4) Optimal temperature The temperature action curve of this enzyme is shown in Figure 2.

This figure shows that the optimum temperature for this enzyme is around 4,000. The activity of choline dehydrogenase in the present invention is measured by using choline as a substrate, dehydrogenating it by acting on this enzyme, and coexisting with a dye such as nitroflutetrazolium or an ultraviolet absorbing substance as a receptor for this hydrogen. The titer of the enzyme can be determined by measuring the amount of change in visible or ultraviolet absorption within a certain period of time.

The details of this measurement method are as follows. IM choline chloride
Mix 0.1 M of '100 M nitroflutetrazolium solution with 1 M of 0.08 M phosphate buffer (pH 7.4) and 3
After leaving at 70°C for 5 minutes, 0.1% of the enzyme solution whose activity is to be measured was added, and after reacting at 37°C for 10 minutes, 0.1% of the enzyme solution whose activity was to be measured was added. Add 1 part of a hydrochloric acid solution to stop the reaction, and add 5 parts of the reaction solution.
The absorption of 3.3 m is measured using a colorimeter (absorption value of the reaction solution).

On the other hand, in the above method, without adding enzyme solution, 3
70. After leaving for 10 minutes, 0. Add 1M hydrochloric acid solution,
0.1 of the enzyme solution was added to this, and the absorption at 53 cm was measured, and this was taken as a blank value. The difference between the absorption value of the reaction solution and the blank value is defined as ΔE, and the amount of enzyme is determined by the following formula. Amount of enzyme in culture solution 1 (U/[) = △E In the display, 1 unit (U) is the amount of enzyme that converts 1 mole of choline into betaine aldehyde per minute.

Bacterial strains used in the present invention include Pseudomonas floretscens, Pseudomonas iodeinum,
Pseudomonas melanogenum, Pseudomonas ovalis, Pseudomonas seuilkii 1'ensis, Pseudomonas sinquisanta or Agrobacterium, Arthrobacter, Bacillus, Brevibacterium, Herwinia, Microbacterium, Paracolobacterium Examples include strains belonging to the genus Salmonella, Sarcina, Asbergillus, or Mucor and having the ability to produce choline dehydrogenase.

In culturing the microorganisms used in the present invention, a medium containing choline or a choline-containing substance is used in addition to a normal nutrient medium.

In this case, as the choline, choline acid salts such as choline chloride, choline sulfate, free choline, etc. are used. As for the timing of adding choline to the soil, it may be present in the medium from the beginning, or it may be added to the medium during cultivation, and the amount used is 0.5 to 3.0 t/d' (choline ) is preferred. As the carbon source used in the medium, glucose, fructose, starch, recommended sugar, molasses, etc. are used alone or in combination.

Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used.

Nitrogen sources include inorganic and organic nitrogen sources such as ammonia, ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, ammonium acetate, and natural nutritional sources such as beptone, meat extract, yeast extract, and corn steep.・Liquor etc. are used alone or in combination.

In addition, inorganic salts such as common salt, dipotassium phosphate, and magnesium sulfate are added as necessary. In addition, substances necessary for the growth of microorganisms such as biotin, substances that promote the production of choline dehydrogenase, or natural products containing such substances are added. Culture is usually carried out by shaking culture or aeration loss culture.

The culture temperature is preferably 20 to 4,000, and the pH of the medium is preferably in the range of 6.0 to 8.0. Usually 1~
When cultured for 4 days, choline dehydrogenase is produced and accumulated in the bacterial cells and the culture solution. When the presence of choline dehydrogenase is recognized, preferably when oxygen activity reaches the maximum, the culture is stopped and the bacterial cells are separated by centrifugation or the like. The bacterial cells are disrupted by an appropriate means, and a supernatant is obtained from the disrupted fluid by centrifugation or the like, and this is used as an enzyme solution. The solid matter is removed from the culture solution, and this (culture supernatant) is used as the enzyme solution. Purification and isolation of this enzyme from an enzyme solution can be carried out using methods normally used for enzyme purification, such as salting out, organic solvent precipitation, dialysis, isosilk spot precipitation, column chromatography using Sephadex, etc., and freeze drying. can be used alone or in combination.

Next, examples of the present invention will be shown. Example 1 Pseudomonas florescens KYY4 as the inoculum
032 (Collaboration Research Institute No. 3656) (NRRLB-11
001) is used.

Bepton 1/d', meat extract 0.5/d', salt 0
．． Dispense 10 times the primary seed medium consisting of 3 days/d' (pH 7.2) into large test tubes and sterilize at 12,000 for 15 minutes (hereinafter, all culture media will be sterilized under these conditions before use). )
.

One platinum loopful of the above strain was inoculated into the medium, and cultured with shaking at 28 oo for 2 hours. The primary seed culture thus obtained was transferred at a rate of 10% to a secondary seed medium 300' with the same composition as above in two Erlenmeyer flasks, and incubated at 28o0 for 24 hours.
Incubate with shaking for an hour.

Next, the second seed culture 1 obtained in this way was placed in a fermenter of 15 in a Jarfamentor.
', meat extract 0.5 t/d', salt 0.3 t/d', choline chloride 1.5 t/d', Kararin 102 (trade name of an antifoaming agent whose main component is a polyalkylene glycol derivative)
(pH 7.2)] and the number of flies was 30. p. m. Cultivate for 24 hours at 2800 mA with an aeration rate of 15 min. About 16 minutes of the culture solution thus obtained was centrifuged to collect the bacterial cells. After washing the bacterial cells with 0.1 M Tris buffer, the cells were washed with 0.1 M Tris buffer containing 10 M mercaptoethanol and 10 mM magnesium chloride. Fill up the solution 1 and crush the bacterial cells using a Manton-Garlin bacterial cell crusher.

The cocoon crushed liquid was centrifuged to obtain a supernatant liquid, and this supernatant liquid (containing 2.1 mU' of choline dehydrogenase) had a saturation level of 0.4.
A corresponding amount of ammonium sulfate (approximately 300 g) is added and the resulting precipitate is collected by centrifugation. This precipitate was dissolved in 50' of the above-mentioned buffer containing mercaptoethanol and magnesium chloride, and this was added to the buffer containing mercaptoethanol and magnesium chloride using a cellophane tube as a dialysis membrane.
The solution is used as an external solution and dialyzed at 4°C overnight.

Freeze-dry the dialyzed fluid and use it as a crude enzyme preparation. In this way, a crude enzyme preparation of 3.6 days was obtained.

After dissolving this crude enzyme preparation in water, choline dehydrogenase activity was measured according to the enzyme measurement method described above. As a result, choline dehydrogenase activity equivalent to 3 U was present in 1 ta of this crude enzyme preparation. . Example 2 As a result of measuring the enzyme activity in the culture supernatant obtained by removing bacterial cells and other solids from the culture supernatant obtained in Example 1, it was found that 3.74 U per top of the culture supernatant. The presence of choline dehydrogenase was observed.

Example 3 10% of the primary seed culture obtained by the method of Example 1
In a Sakaguchi flask, 50' of fermentation medium [bepton 1/d', meat extract 0.5/d', salt 0.3/d', choline chloride 1.5/d', biotin 100y
/ evening (pH 7.2)] and cultured with shaking at 2800 °C for 24 hours.

After completion of the culture, bacterial cells were obtained from the culture solution and used in Example 1.
The supernatant was obtained and the enzyme activity was measured. As a result, the presence of 5.43 U of choline dehydrogenase per 1 volume of culture supernatant was confirmed.

Example 4 As a fermentation medium, choline chloride 1.5 t/d', dipotassium phosphate 0.1 t/d', magnesium sulfate 0.05 t/d'
', Salt 0.1 Y/d', Biotin 50 Y/L, Corn Steep Liquor 1 T'd', Sodium Glutamate 0
．． 5/d', glycerin 0.5/d' (PH7.2
) in the same manner as in Example 3, except that a beach consisting of
It was cultured in a shaking moat for two years.

Bacterial cells were obtained from the obtained culture solution, an enzyme solution was prepared in the same manner as in Example 1, and the choline dehydrogenase activity was measured. The presence of enzyme was observed.

Example 5 The various strains shown in Table 1 were cultured in the same manner as in Example 1, the cells were obtained, an enzyme solution was prepared, and the choline dehydrogenase activity was measured. As a result, the results are shown in Table 1. The presence of choline dehydrogenase activity was observed.

Table 1 Bacterial strain name (Culture solution of skin U/1) Pseudomonas aeroginosa A-16 (NRRLB-8199
) (Choken Bacteria Collection No. 3654) 0.80 Pseudomonas Iodeinum (NRRL-B-141)
1.10 Pseudomonas
Mela nogenum (ATCC#17806)
2.00 Pseudomonas obalis (IF○112051) 0.81 Pseudomonas seuilkiriensis (NRRL-B-6)
1.20 Pseudomonas cinquisanta (ATCC #9890) 7.83 Agrobacterium tumhuaciens (ATCC #4720)
0.70 Agrobacterium radiobacter (ATCC #4718)
0.20 Arthrobacter
Citreus (ATCC #11624)
0.80 earth.

Bacter paraphineus (ATCC #21191)
0.80 Bacillus megaterium (ATCC #19380) 0.45 Bacillus.
Satillus (ATCC #21554) 0.10 Brepibacterium devaricatum (ATCC #14020
) 0.20 Brevibacterium liquefaciens (ATCC #14929)
1.00 Brevibacterium lyticum (ATCC #15921)
6.50 Erwinia force 0 Topora (KY3242) 1.02 Erwinia alloyi (ATCC #15390) o. lo Microbacterium flavum (ATCC #10340)
0.34 Microbacterium sp. (ATCC #21376)
0. Group Paracorbacterium coliform (A
TCC#21186)
0.71 Pseudobacterium sp. (ATCC#
21284) 0.25
Salmonella chihoza (ATCC #9992) 0.
45 Sarcina lutea (ATCC#9341)
5.07 Zarcina flava (ATCC #21550)
0.80 Asbergillus niger (KYI14)
0.10 Asbergillus flavus (ATCC
#136 Group) 0.06 Mukor Jiyabanikas (ATC
C#15242) 0.17 Mucor Alternance (
ATCC #20132) 0.15 Example 6 A culture solution was obtained in the same manner as in Example 3, except that the culture time was changed to 9 hemorrhoids.

The bacterial cells and the contents of the culture solution are separated by centrifugation, and the bacterial cells are treated in the same manner as in Example 3 to obtain an enzyme solution.

As a result of measuring the choline dehydrogenase activity in the enzyme solution obtained from the bacterial cells and the culture supernatant, it was found that the enzyme activity from the disrupted bacterial cell solution and the culture solution supernatant was 2 per culture solution, respectively.
．． 00 U and 1.40 skin U.

[Brief explanation of drawings]

Figures 1 and 2 show the state action curve and temperature action curve of this enzyme, respectively. Ta {Rokuta 2 illustration

Claims (1)

[Claims] 1 Belongs to the genus Agrobacterium, Arthrobacter, Bacillus, Brevibacterium, Erwinia, Microbacterium, Paracolobactrum, Salmonella, Sarcina, Aspergillus, or Mucor, and has choline dehydrogenase A method for producing choline dehydrogenase, which comprises culturing a strain capable of producing choline in a medium containing choline, and collecting choline dehydrogenase oxygen from the culture.