JPS6010714B2 - Method for producing choline oxidase by fermentation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing choline oxidase by fermentation.

More specifically, the present invention provides a new bacterial species, Corynebacterium corinoxidans.
holinoxyda ship) or the new bacterial species Corynebacterium corinophilum (Corynebacterium corinophilum).
choliniphilum) in a nutrient medium.

Choline oxidase is used to quantify choline.

Conventionally, choline oxidase originating from microorganisms has been reported to be caused by bacteria of the genus Arthrobacter [Clinical Pathology 24 (Okehatsu) 461 (1976) (Clinical Pathology Society Abstracts); Lipid Biochemistry Research Group Lecture Proceedings Collection (19
June 16th and 17th, 1977 (Kyoto)].

The present applicant previously filed an application for a method for producing choline oxidase using choline oxidase-producing bacteria belonging to the genus Brevibacterium or Corynebacterium (Samurai Application No. 51-139120 (Special Publication No. 43075)) , 51
1155655 (Special Publication No. 57-13046y)).

In order to obtain even better choline oxidase-producing bacteria, the present inventors newly isolated microorganisms with excellent choline assimilation ability from soil, inoculated these strains into a nutrient medium, and cultured them. As a result, the inventors found that the following two strains belonging to the genus Corynebacterium have excellent ability to produce choline oxidase, and completed the present invention.
Next, the mycological properties of the strains referred to as Corynebacterium corinoxidans KY4707 and Corynebacterium corinophilum KY4706 used in the present invention will be explained in detail.

Identification experiments for these strains are mainly conducted in the Manual of Microbiological Methods (American Society for Bacteriology, 1999).
57) and “Classification and Identification of Microorganisms” (Takeji Hasegawa paper)
The study was carried out in accordance with the University of Tokyo Press, 1975).

Corynebacterium corinoxidans KY4707 (Corynebacterium Corinoxidans No. 4111) (NRRL
B-11, 1 spot) 1 Cell morphology Broth agar, 30℃ culture: Usually 0.6-0.8 x 1.5-
2.0 French short camphor.

Branched cells, either singly or in pairs in a V-shape, were also observed, and in older cultures they became spherical. That is, they were pleomorphic. They were motile. The flagellum was one non-polar flagella. Gram positive. No spores. Non-acid-fast. B Cultural properties 1 Cultured on broth agar plate, 30oo round.

The size is 4-5 sides in 2-3 days. The surface is smooth.

Convex circular shape. Entire periphery. Has a dark luster. Opacity. The content is homogeneous buttery. It is white at first and turns yellow after 2-3 days. 2 Meat juice agar slant culture “30oo Good growth.

Stringy. 3 Meat juice liquid culture, 30qo Good growth.

Hair mist-like next thin. No film formation. 4 Meat juice puncture culture, 3 pi○ Good growth on the surface.

Internal growth is sparse and filamentous. 5 Meat juice gelatin puncture culture, 2
0qo Growth on the surface and along the puncture line.

Liquefy gelatin.

6 litmus milk The milk is alkalized and becomes completely clear in two weeks.

m Physiological properties 1 Nitrate reducing ability: Positive 2 Denitrification reaction: Negative 3 M old test: Negative 4 VP test: Negative 5 Indole production: Negative 6 Hydrogen sulfide production: Enteric 7 Starch hydrolysis: Negative 8 Citric Utilization of acids: positive and negative in Kosar medium and Christensen's soil 9 Utilization of inorganic nitrogen sources: Both ammonium salts and nitrates can be used.

10 Pigment formation: yellow, non-water-shedding.

11 Urease: Positive 12 Catalase: intestinal 13 Lecithinase: Negative 14 Choline oxidase: intestinal 15 Minimum growth temperature range: 15-370.

Growth was weak at 5 and 42°C.

16 Growth pH range: 5-9 17 Attitude towards oxygen: Glucose used as carbon source ○
- Appropriate anaerobic based on results such as F test.

18 Production of ammonia from beptone: positive 19 Hydrolysis of esculin: negative 20 Decomposition of cellulose: negative 21 Salt tolerance: Grows at 7 t', non-grows at 10 t'.

22 Hemolysis: 3-Hemolysis 23 Choline utilization: Enteric 24 Creatine utilization: Positive 25 Petine utilization: Enteric 26 Uric acid utilization: Enteric 27 Vitamin requirement: None 28 Carbon source utilization Three D-glucose , D-fructose, D-mannose to D-galactose, D-xylose, L-arabinose, D-ribose, L-rhamnose, sucrose, maltose, trehalose, cellobiose, glycerin, ○-mannite, 0-solpit, ○-inosyt L-inosit, acetic acid, DL-lactic acid, pyruvic acid, succinic acid, ethanol, and inulin are used.

Does not use lactose, adonite, cellulose or starch.

29 Production of acids and gases from carbohydrates■ When using Bepton tower, almost no acid production is observed.

■ Tryptone 2x, NaC15x, K2HP
Acid production was detected at the foundation tower on 040.3 evening/evening.

■ D-glucose, D-mannose, D-fructose, D-galactose, maltose, sucrose,
Treha.

- Produces acid from gas (weak), D-mannite, and glycerin, but does not produce gas.

■ L-arabinose, D-xylose, lactose,
Does not produce acids or gases from D-sorbit, inosites, or starch.

W Cytochemical analysis I DNA G-C ratio: 67.8% 2 Cell wall diamino acids: Lysine 3 Cell wall bran: Rhamnose, mannose Corynebacterium corinophilum KY4706 (Corynebacterium coryniphyllum KY4706) )(NRRL
8-111157) is almost identical to Corynebacterium corinoxidans KY4707 except for the following points.

1 Non-motile and does not have flagella.

2 Hydrolysis of starch and production of acid from starch: Positive 3 Utilizes lactose as the sole carbon source.

Based on the above-mentioned mycological properties, Purge's Manual of Determinative Bacteriology 8th Edition (1974) and 7th Edition (1957) [hereinafter referred to simply as Purge-8th Edition (or 7th edition)], Yamada,
Komagata: J. Gen. Appl. Microbiol. 1
6, 103 (1970) and 16 “215 (19
70), Cmmmlns, J. bag n. Microbio
l. 28, 35 (1962), and USP 32222
Differences with known bacterial groups were examined with reference to $ (1963). The two bacterial strains entrusted above are Gram-positive, non-sporulating, non-acid-fast, facultatively anaerobic, and pleomorphic birch bacteria.
It is considered that this should be included in ria in fBac.

This group includes four genera: Cellulomonas, Kurtia, Arthrobacter, and Corynebacterium. These two bacterial strains are distinguished from cocoon strains belonging to the genera Cellulomonas, Kurtia, and Arthrobacter in the following points.

Box strains belonging to the genus Cellulomonas have the ability to decompose cellulose, require biotin and thiamine for growth, and have DN
A's G-C ratio is 71.7-72.7% [Yamada/Komagata: J
．． snake n. Appl. Microbiol. 16, 215
(1970)].

In contrast, these two strains do not have the ability to decompose cellulose,
Biotin and thiamine are not required for growth, and DNA
The total G-C ratio is ±0.5%. Therefore, these two strains do not belong to the genus Cellulomonas. Strains belonging to the genus Curtia have a regular shape during the logarithmic growth phase, and are unbranched and chain-like. It is also obligately aerobic and does not reduce nitrate.

On the other hand, the present two strains have an irregular shape during the logarithmic growth phase, some are branched, and do not form a chain. It is also facultatively anaerobic and reduces nitrates. Therefore, book 2
The strain does not belong to the Curthia genus. Strains belonging to the genus Arthrobacter are generally Gram-negative in the logarithmic growth phase.

It is also obligately aerobic, and many strains belonging to this genus cannot grow in 370.

Furthermore, it has only galactose as a cell wall constituent sugar.
On the other hand, the present two strains are gram enteric throughout the culture. It is also suitable anaerobic and grows well at 370°C.
Furthermore, it has glasstose, rhamnose, and mannose as cell wall sugars. Therefore, these two strains do not belong to the genus Arthrobacter. On the other hand, these two strains are a purge of strains belonging to the genus Corynebacterium - 8th edition (
599-61). therefore,
It is considered appropriate that these two strains belong to the genus Corynebacterium. On the other hand, Purge - 8th edition, page 599 states that Plebibacterium and Microbacterium are genera that are close to Corynebacterium and Arthrobacter but whose classification is uncertain.

Purge - 8th edition, pages 625-626
It is stated that the strain belonging to the genus Previbacterium by R et al. should be assigned to the genus Corynebacterium.

In addition, on page 628 of Purge - 8th edition, Jesen's assertion that Microbacterium techteicum and Microbacterium lychefaciens should belong to the genus Corynebacterium is stated. So book 2
Strains belonging to various species of the genus Corynebacterium as described in Strains and Purges - 8th Edition, and cocoons belonging to various species of the genera Previbacterium and Microbacterium as described in Purges - 7th Edition. A comparison was made with reference to the descriptions in these documents.

Further, as necessary, experiments were conducted to compare the two bacterial strains with the strains belonging to the various species mentioned above. Among the strains belonging to the known species of the genera Corynebacterium, Brevibacterium, and Microbacterium, the strains that can grow vigorously in a medium containing only choline as a carbon source, like these two strains, are Corynebacterium muricepteicum, It belonged to Brevibacterium album, Brevibacterium serinum and Brevibacterium maris.

Also, although it cannot grow or can only grow weakly in a medium containing only choline as a carbon source, it contains lysine as a diamino acid in its cell wall like these two strains, and has other properties that are relatively similar to these two strains. belonged to Corynebacterium poinsettiae, Brevibacterium fulhum, Brevibacterium herleborum, Brevibacterium imbelli**are and Brevibacterium sulfureum. Table 1 shows a comparison of the mycological properties of these two strains and strains belonging to these species.

Table 1 Note ○:○orynebaCterlum'B:Bre
uibacterlum, Lys: Lysine, DL-
DAP: DL-diaminopimelic acid *1: Experimental value *2:
USP 3222258 *3: J. Gen. Appl. Microbiol. 1
6, 103 (1970) *4: Purge - 7th edition *5: J. Gen. Microbiol. 28, 35 (
1962) As can be seen from Table 1, KY4707 and K
The Y4706 strain does not match any of the strains belonging to related species in terms of mycological properties.

Therefore, both strains are considered to belong to new bacterial species, and KY
The bacterial species to which 4707 belongs was named Corynebacterium corinoxidans, and the bacterial species to which KY4706 belonged was named Corynebacterium corinophilum. Next, culture conditions for enzyme production using both bacteria will be explained.

The culture medium used in the present invention may be either synthetic or natural, as long as it contains adequate amounts of carbon sources, nitrogen sources, inorganic substances, and other necessary nutrients. Carbon sources for the culture medium include carbohydrates such as glucose, sugar, low sugar, and honey, lactic acid,
Organic acids such as acetic acid and succinic acid; choline, etc. are used.

Ammonium chloride, ammonium sulfate,
Ammonium phosphate, urea, monosodium glutamate,
Inorganic organic nitrogen compounds such as glycine, sodium aspartate, choline, etc. can be used.

Furthermore, nitrogen-containing natural products such as beptone, meat extract, yeast extract, corn steep liquor, etc. can also be used as nitrogen sources. As the inorganic substance, potassium phosphate, dipotassium phosphate, magnesium sulfate, etc. can be used.

Next, in the present invention, by allowing choline to exist in the medium as an inducer of choline oxidase,
It can increase the amount of choline oxidase produced. As choline as a discussion guide substance, choline acid salts such as choline chloride and choline sulfate, free choline, and the like are used. As for the timing of adding choline to the medium, it may be present in the medium from the beginning, or it may be added to the beach during culture. When added during culture, it is preferably added before the end of the logarithmic growth phase of the bacteria used. Next, an example of an experiment conducted to determine a suitable concentration range of choline as the inducer in a medium will be shown. The activity of choline oxidase in the experimental examples and the examples shown later is determined by a method in which hydrogen peroxide produced when choline is oxidized by choline oxidase is decomposed by peroxidase, leading to a phenol-4-aminoantipyrine coloring system. Measure.

The details of this measurement method are as follows. That is, a 0.08 hol solution containing 0.01 mol of 4-aminoantipyrine and phenol each (pH
7.0) peroxidase per 100 ships of the solution
The liquid containing 00U is used as a coloring liquid. of the coloring liquid 3
0.1', 1/3 of the enzyme solution whose activity is to be measured
Add 0.1M choline chloride solution and reduce the
After a minute reaction, the absorbance of 50 skin kites is measured. The unit of activity, U, is the enzyme titer that decomposes 1 Amol of substrate per minute. Example 1 Corynebacterium corinoxidans KY as inoculum
4707 and Corynebacterium coriniphyllum K
Use Y4706.

Choline Chloride 0, 0.1, 0.5, 1.0, 5.0 or 10.0 t/d, Corn Steep Liquor 0.5
Yu/d', yeast extract 0.5 Yu/d', KH2P040
．． 1 evening/d', MgS04/7th 200.05 evening/d'
and 0.5 ta'd' of sodium glutamate (pH 7.2
) was dispensed into 70 large test tubes and sterilized at 120°C.

One platinum loop of the above bacterial strain was inoculated into each tower, and cultured at 30°C for two hours.

The cultured solution was centrifuged to obtain bacterial cells, which were adjusted to pH 8.0.
Suspend in 0.0 ml of Tris buffer-10' and sonicate.

The lysate is subjected to centrifugal separation to obtain a supernatant. Use the supernatant as an enzyme solution and measure choline oxygose. The results are shown in Table 2.

Table 2 From the above experimental examples, etc., the concentration of choline oxidase inducer in the medium is 0.1 to 10.
．． 0 evening/d' is appropriate.

The choline oxidase production activities of Corynebacterium corinoxidans KY4707 and Corynebacterium corinophyllum KY4706 shown in Table 2 are based on conventional strains (Hakama Gansho No. 51-113912).
Special Publication No. 54-143075) "Special Publication No. 51-15565
5 (see Tsubaki Koukai No. 57-3046)), making it possible to produce choline oxidase industrially more advantageously. The culture of choline oxidase-producing bacteria in the present invention is usually carried out by shaking moat culture or aerated fly culture.

The culture temperature is preferably 25 to 35 qo. The pH of the medium is preferably in the range of 7.0 to 8.5. Culture is 1
Do this for ~3 days. By culturing as described above, choline oxidase is produced and accumulated in the culture, mainly in the bacterial cells.

After completion of the culture, bacterial cells are obtained from the culture solution by centrifugation or the like, and then the cells are crushed by a suitable means such as using a bacterial cell crusher, and a supernatant liquid is obtained from the crushed liquid by centrifugation or the like.

Choline from the supernatant.

To collect oxidase, use any method commonly used for enzyme purification, such as salt folding, organic solvent precipitation, dialysis, ion-exchange cellulose column chromatography, ion-exchange sepadex column chromatography, sepadex column chromatography, and freeze-drying. can be used. Next, the properties of choline oxidase obtained for each strain according to the present invention will be shown. m Action Oxidizes choline to generate betaine via betaine aldehyde.

This reaction route is shown by the following formula. Choline 02 Petainaldehyde + Ratio 02 Petainaldehyde Hyebetaine + Q.

2 [2) Stable pH range Day 20 Corynebacterium corinoxidans KY4707 obtained in Examples 1 and 2 and Corynebacterium corinoxidans KY4707 obtained in Examples 1 and 2
1. each of the enzyme preparations of Coriniphyllum KY4706.
Dissolve in 0.0 l/miffer of pH 8.0 to a concentration of 4 U/'.

The resulting solution 0.5 mol' and 0.05 mol' of various pH
Mix with Cutris Buffer to pH 6.0, 7.0,
Obtain a solution of 8.0, 9.0 or 10.0. These solutions were then kept at 45:00 for 30 minutes. Using the treated solution as an enzyme solution, absorbance is measured according to the method for quantifying choline oxidase. The results are shown in Figure 1. From A and B in FIG. 1, it can be seen that there is almost no difference in the stability of choline oxidase between the two strains, and that the stability axis is 7.0 to 8.0, respectively. Mackerel kept at the thinnest range of 370,
Insert an oxygen electrode into a reaction vessel for oxygen absorption measurement (manufactured by Shusui Ibaku Kenkyusho Co., Ltd.) for approximately 4 minutes.

On the other hand, the enzyme preparations of both strains obtained in Examples 1 and 2 were diluted with 0.05 mol of pH 80 to a concentration of 1.6 U/'.
/dissolve in its Tris buffer. The resulting enzyme solution was 0.1 [, 0.1 mol/so choline chloride aqueous solution 0.
1 secretion, and 6.5, 7.0, 8.0, 8.5, 9.
0.05 mol' of 25, 10.0 or 10.5' of Tris buffer is placed in the reaction vessel. While stirring the solution with a stirrer, measure the decrease in dissolved oxygen [Product name: Desktop recorder (skTo
pRecorder), manufactured by Okura Electric Co., Ltd.]. The slope of the decrease in dissolved oxygen from sand 3 to 5 minutes after the reaction is determined. Figure 2 shows the relative activity when the maximum decreasing slope at each pH is defined as 100 activity.From A and B in Figure 2, choline oxidase of both strains has almost no (reaction) activity in the optimal range. There is no difference and both are 7.
．． It turns out that it is 5 to 9.5. (4) Range of suitable temperature for action The enzyme preparations of both strains obtained in Examples 1 and 2 were heated at 0.05 m
ol' is dissolved in Tris buffer to prepare an enzyme solution.

Using the enzyme solution, the reaction temperature (OC) was set to 20"35".
Choline oxidase activity is measured in the same manner as the choline oxidase assay method, except that the choline oxidase activity is 40 or 45 or 50.

The results are shown in Figure 3. From A and B in FIG. 3, it can be seen that there is almost no difference in the optimal temperature range for choline oxidase of both strains, and both are 20 to 35°C. {5}
Substrate specificity The enzyme preparations of both strains obtained in Examples 1 and 2 were
An enzyme solution is prepared by dissolving 0.08 hol' of Tris buffer at pH 8.0 so that U/I.

Using this enzyme solution, the measurement is carried out in the same manner as the method for quantifying choline oxidase, except that each substrate shown in Table 2 is used instead of choline chloride. The quantitative value for choline chloride is 1
Table 3 shows the relative activity of each substrate when it is set as 00. Table 3 Next, the present invention will be specifically explained with reference to Examples.

Example 1 Corynebacterium corinoxidans KY as inoculum
Use 4707.

Choline Chloride 2/d', Corn Steep Liquor 0.
5 t/d', yeast extract 0.5 t/d', sodium glutamate 0.5 t/d', dipotassium phosphate 0.1 t', magnesium sulfate heptahydrate 0.05 t/d' (PH7
．． 2) Seed culture tower consisting of 1 to 70 large test tubes
0 [Dispense and sterilize at 120:00 for 15 minutes.

One platinum loopful of the above-mentioned strain was inoculated into the medium and cultured in a shaking moat at 30°C for 4 hours. Then, all of the obtained culture solution was inoculated into the same seed culture medium 300' as above, which was dispensed into two Erlenmeyer flasks, and cultured in a shaking moat for 3 and 4 periods. After the completion of the culture, all of the culture solution was inoculated into 3.0 ml of the same medium in a 5.0 ml jar fermenter. p.
Production culture is carried out with aeration of 1 night/night (medium)/min, and the culture is completed in 1 hour. The amount of choline oxidase produced was 1.21 U per kite of culture solution. In order to collect choline oxidase from the culture solution, the solution was centrifuged to obtain bacterial cells, which were collected in 0.08 hol' at pH 80.
After suspending it in a Dino Laboratory Mill,
) KDL type (Wmy A, Bachofan Inc, S
(manufactured by wipMand) to obtain a disrupted bacterial cell solution. Centrifuge the disrupted bacterial cells to obtain a supernatant (the amount of choline oxidase produced in the cultured solution was determined by measuring the activity of choline oxidase using this supernatant as an enzyme solution) ). Ammonium sulfate is added to this supernatant to make it 30% saturated with ammonium sulfate, and the precipitate is removed by centrifugation to obtain a supernatant.

Ammonium sulfate was added to this supernatant to make it 60% saturated with ammonium sulfate, and the precipitate was collected by centrifugal separation. This precipitate was dissolved in 0.05 mol of Plaque 8.0 in Tris buffer, and dialyzed against the same buffer overnight at 5°C using a cellophane tube as a dialysis membrane. The solution in the dialysis tube was equilibrated with Tris buffer (pH 8.0) containing 0.0 ml of sodium chloride and DEA.
Charge the cellulose column with Tris buffer (pH 8.0) containing 0.0 ol/so sodium chloride.
After washing with 1 sleeve, sodium chloride 0.05-0.48ho
Minato extraction is carried out at a concentration gradient of 1/l.

The eluted fraction of choline oxidase is collected, saturated with ammonium sulfate to 60%, and the precipitate is collected by centrifugation.
This precipitate was mixed with 0.05 mol of Tris at pH 8.0.
Pharmacia Fi
neChemicalslm. , U. S. A. (manufactured by ) and elute with the same buffer. The eluted fraction of choline oxidase is collected, saturated with ammonium sulfate to 60%, and the precipitate is collected by centrifugation. This precipitate is dissolved in 0.05 mol/cutris buffer of pH 8.0, and dialyzed against the same buffer overnight at 5° C. using a cellophane tube as a dialysis membrane. After dialysis, 0.
DEA equilibrated with 0.08 hol/Sotris buffer (pH 8.0) containing 1 mol/sodium chloride
E-Sephadex A-50 (trade name of weakly basic anion exchange resin; Phannacia Fine Chemical)
to alsl. , U. S. A. manufactured by )5
Charge the 00' column. Wash the resin with 500 ml of 0.08 hol/so Tris buffer (pH 8.0) containing 0.1 mol/so sodium chloride.

Next, elution is performed with a concentration gradient of 0.1 to 0.5 hol/sodium chloride. The choline oxidase elution fraction was collected, dialyzed using a cellophane tube as a dialysis membrane with 0.05 mol/cutris buffer at pH 8.0, and then lyophilized. Choline oxidase was collected with an activity yield of approximately 13%.Activity is 4.5 U'mg protein.Example Corynebacterium corinophilum K as the second seed strain
Use Y4706.

The seed culture tower shown in Example 1 was dispensed into two Erlenmeyer flasks and sterilized at 12,000 °C for 15 minutes.

One platinum loopful of the above-mentioned strain was inoculated into the medium, and cultured for 4 hours at 30 pm. After the cultivation was completed, all of the culture solution was inoculated into a medium having the same composition as the 3.0 seed culture medium in a 5-spore jar fermenter, and 3030, 50. p. m. ,
Main culture is carried out with aeration of 1 hour/night (medium)/min, and the culture is completed after 2 hours of aeration.

The amount of choline oxidase produced is 1.1 per secretion of culture solution.
It is 0U. Choline oxidase was purified from the culture solution in the same manner as in Example 1, and choline oxidase was collected with an activity yield of about 12%.

Activity 4.2U/female protein.

[Brief explanation of drawings]

FIG. 1 shows the stable pH range of the choline oxidase of the present invention. A is Corynebacterium corinoxidans KY470
Figure 2 shows the optimum pH range of the choline oxidase of the present invention. Explanation of A and B is the same as in FIG.
FIG. 3 shows the optimal temperature range for the action of the choline oxidase of the present invention. The explanation of A and B is the same as in FIG. English drawing technique z drawing g3 drawing

Claims (1)

[Scope of Claims] 1. A method for producing choline oxidase, which comprises culturing a microorganism belonging to Corynebacterium corinoxidans or Corynebacterium corinophilum in a nutrient medium. 2. The production method according to claim 1, wherein the nutrient medium contains choline or an acid salt of choline.