GB1592413A - Preparation of choline oxidase - Google Patents

Description

(54) PREPARATION OF CHOLINE OXIDASE (71) We, KYOWA HAKKO KOGYO CO., LTD., a Company organised and existing under the laws of Japan of 6-1 Ohtemachi Itchome, Chiyoda-Ku, Tokyo, Japan, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to a process for the preparation of choline oxidase by fermentation.

Heretofore, it has been known that choline oxidase is produced by a microorganism belonging to the genus Arthrobacter. Choline oxidase derived from the microorganism catalizes the conversion of choline chloride to betaine. By the conversion, hydrogen peroxide is also formed. A method for determination of choline by measuring amount of hydrogen peroxide formed by the conversion has been proposed [Rinsho Byori (Clinical Pathology) 24,461(1976)].

The present inventors have studied the preparation of choline oxidase by fermentation.

As the result, it has been found that a remarkable amount of choline oxidase is produced in the culture liquor, especially in the microbial cells, by culturing a microorganism capable of producing choline oxidase and belonging to the genus Brevibacterium or Corynebacterium in a nutrient medium.

The present inventors have further investigated properties of choline oxidase obtained by the present process.

As the result, it has been found that betaine aldehyde is produced by oxidation of choline in the presence of choline oxidase and that betaine aldehyde is further converted to betaine by the action of the enzyme.

Choline oxidase obtained by the present process may be used for determination of choline.

In accordance with the present invention, a process is provided capable of producing a remarkable amount of choline oxidase, this process comprising culturing a choline oxidaseproducing microorganism belonging to the genus Brevibacterium or Corynebacterium in a nutrient medium containing sources of assimilable carbon and nitrogen, and the inorganic salts necessary to support the growth of the microorganism, accumulating choline oxidase in the nutrient medium, and recovering the choline oxidase therefrom.

The invention is further described below with reference to the accompanying drawings, in which: Fig. 1 illustrates the result of identification by thin layer chromatography of the reaction solution obtained by oxidation of choline in the presence of choline oxidase prepared by the process of the present invention.

Fig. 2 illustrates the stable H range of choline oxidase prepared by the process of the present invention. (A), (B), (C) and (D) respectively exhibit test results of the enzymes obtained from microbial cells of Brevibacterium album KY 4319, Corynebacterium murisepticum KY 3505, Corynebacterium cholinoxydans KY 4707 and Corynebacterium choliniphilum KY 4706. (A), (B), (C) and (D) in the subsequent figures have the same significance as above.

Fig. 3 illustrates the optimum pH range for reaction of choline oxidase prepared by the process of the present invention.

Fig. 4 illustrates the optimum temperature range for reaction of choline oxidase prepared by the process of the present invention.

Fig. 5 illustrates the stable temperature range of choline oxidase prepared by the process of the present invention.

Fig. 6 illustrates the stabilizing activity of EDTA on choline oxidase prepared by the process of the present invention.

In accordance with the present invention, any microorganism capable of producing choline oxidase and belonging to the genus Brevibacterium or Corynebacterium is used. Particularly preferred microorganisms are found among those of the species Brevibacterium album, Brevibacterium cerinum, Corynebacterium murisepticum, Corynebacterium cholinoxydans (new species) and Corynebacterium choliniphilum (new species). The examples of favorable producers are Brevibacterium album KY 4319 (FERM-P No. 3777) (NRRL B-11,046 (ATCC 15,111), Brevibacterium cerinum KY 4320 (FERM-P No. 3778) (NRRL B-1 1,047 (ATCC 15,112), Corynebacterium murisepticum KY 3505 (FERM-P No. 3779) (NRRL B- 11,049) (ATCC 21,374), Corynebacterium cholinoxydans KY 4707 (FERM-P No. 4111 (NRRL B-11,158) and Corynebacterium choliniphilum KY 4706 (FERM-P No. 4110 (NRRL B-11,157). FERM-P means a deposit of a microorganism with the Fermentation Research Institute, Chiba-ken, Japan, NRRL means a deposit of a microorganism with the ARS Culture Collection Research Fermentation Laboratory, Peoria, Illinois, U.S.A. and ATCC is an abridgment of the American Type Culture Collection, Rockville, Maryland, U.S.A.

The bacteriological characteristics of Brevibacterium album KY 4319 and Brevibacterium cerinum KY 4320, and those of Corynebacterium murisepticum KY 3505 are described respectively in U.S. Patent No. 3,222,258 and U.S. Patent No. 3,630,842.

The above KY 4707 and KY 4706 strains have been isolated from soil and ascertained to belong to new species by the present inventors.

The bacteriological characteristics of the two microorganisms are detailedly described below.

Experiments for identification of the two strains were carried out mainly in accordance with Manual of Microbiological Methods (1957) edited by Society of American Bacteriologists' Committee on Bacteriological Technics and Biseibutsu no Bunrui to Dotei (Classification and Identification of Microorganisms) (1975) edited by T. Hasegawa and published by Tokyo Daigaku Shuppan-kai (Tokyo University Press).

KY 4707 Strain I. Morphology Cultured at 300C in nutrient agar medium: Generally short rods, 0.6 - 0.8 x 1.5 - 2.0,a; single or V-shaped in pairs, branching observed; in old culture becoming coccoid forms, that is pleomorphic; motile; having at least one sub-polar flagellum; Gram-positive; spore not formed; not acid-fast.

II. Cultural characteristics on various media 1. Nutrient agar plate (30"C) Circular; 4 - 5 mm in size after 2-3 days of culture; smooth; convex; entire in the edge; dull luster; opaquc; amorphous, butyrous; white at first and becoming yellow after 2-3 days of culture.

2. Nutrient agarslant (30"C) Abundant growth; filiform.

3. Nutrient broth (30 C) Abundant growth; flocculent sediment; pellicle not formed.

4. Nutrient stab (30 C) Abundant growth occurring only on the surface; 5. Nutrient gelatin stab (20 C) Growth on the surface and along stab; gelatin being liquefied.

6. Litmus milk Milk being alkalified and in 2 weeks becoming completely clear.

III. Physiological properties 1. Nitrate reduction: positive 2. Denitrification negative 3. Methyl red test : negative 4. Voges-Proskauci Reaction: negative 5. Indoleproduction: negative 6. H2Sproduction . positive 7. Hydrolysis of starch: negative 8. Utilization of citric acid: positive in Koser's medium and Christensen's medium 9. Utilization of inorganic nitrogen source: capable of utilizing ammonium salt and nitrate 10. Pigment formed : yellow; water insoluble 11. Urease : positive 12. Catalase : positive 13. Lecithinase : negative 14. Choline oxidase : positive 15. Optimum temperature range for growth: 15 - 37"C; scanty growth at 50C and 42"C.

16. pH range for growth: 5-9 17. Relation to oxygen: facultative anaerobes as determined from O-F (Oxidation-Fermentation) test using glucose as the carbon source 18. NH production from peptone: positive 19. Hydrolysis of esculin: negative 20. Decomposition of cellulose: negative 21. Salttolerancy: growthat 7 g/deof NaCe, none growth at 10 g/de of NaCt 22. Hemolytic activity: ss-hemolysis 23. Utilization of choline: positive 24. Utilization of creatine: positive 25. Utilization of betaine : positive 26. Utilization of uric acid: positive 27. Requirement for vitamins: none 28. Utilization of carbon source Utilized: D-glucose, D-fructose, D-mannose, D-galactose, D-xylose, L-arabinose, D-ribose, L-rhamnose, sucrose, maltose, trehalose, cellobiose, glycerol, D-mannitrol, D-sorbitol, inositol, acetic acid, DL-lactic acid, pyruvic acid, succinic acid, ethanol and inulin Not utilized: lactose, adonitol, cellulose and starch 29. Acid and gas production from carbohydrate 1 Acid is scarcely produced when peptone medium is used.

2 Acid is produced when a medium consisting of 2 g/e of tryptone, 5 g/e of Nacre and 0.3 g/ e of K2HPO4 is used.

(3) Acid is produced but no gas from D-glucose, D-mannose, D-fructose, D-galactose, maltose, sucrose, trehalose (weak), D-mannitol and glycerol.

(4) No acid nor gas is produced from L-arabinose, D-xylose, lactose, D-sorbitol, inositol and starch.

IV. Cytochemical analysis 1. Guanine: Cytosine G-C ratio of DNA: 67.8% 2. Diamino acid composing cell wall: lysine 3. Sugar composing cell wall: rhamnose, galactose and mannose The bacteriological characteristics of KY 4706 strain are almost the same as those of KY 4707 strain except the following properties.

1. non-motile: having no flagellum 2. Hydrolysis of starch and acid production from starch: positive 3. Lactose is utilized as a sole carbon source.

The above two strains KY 4707 and KY4706 are compared with known microorganisms in bacteriological characteristics referring to Bergey's Manual of Determinative Bacteriology 8th edition (1974) and 7th editio (1957) [hereinafter merely referred to as Bergey's Manual 8th edition (or 7th edition)], Yamada, Komagata et al., J. Gen. Appl. Microbiol. 16, 103 (1970) and 16, 215 (1970), Cummins, J. Gen. Microbiol. 28, 35 (1962) and U.S. Patent No.

3,222,258(1963), The two strains are Gram-positive, forming no spore, not acid-fast, facultatively anaerobic, pleomorphic rods. Therefore the two strains should be classified as Coryneform Group of Bacteria described in Part 17 of Bergey's Manual 8th edition. In this group, 4 genera, that is, Cellulomonas, Kurthia, Arthrobacter and Corynebacterium are contained. The two strains are distinguished from those belonging to the genus Cellulomonas, Kurthia orArthrobacter in the following properties.

The strains belonging to the genus Cellulomonas are capable of decomposing cellulose and require biotin and thiamine for growth. G-C ratio of DNA in those strains is 71.7 - 72.7% [Yamada, Komagata et al.: J. Gen. Appl. Microbiol. 16, 215(1970)]. On the other hand, KY 4706 and KY 4707 strains are incapable of decomposing cellulose and having no requirement for biotin and thiamine for growth. G-C ratio of DNA in KY 4707 and KY 4706 strains is 68 + 0.5 %. Therefore, KY 4707 and KY 4706 strains do not belong to the genus Cellulomonas.

The strains belonging to the genus Kurthia are regularly shaped, not branched and form chains in the logarithmic growth phase. Further, these strains are strict aerobes and do not reduce nitrates. On the other hand, KY 4707 and KY 4706 strains are irregularly shaped, partly branched (branching is observed in some cells) and do not form chains in the logarithmic growth phase. Further, these strains are facultative anaerobes and reduce nitrates. Therefore, KY 4707 and KY 4706 strains do not belong to the genus Kurthia.

The strains belonging to the genus Arthrobacter are generally Gram-negative in the logarithmic growth phase. Further, these strains are strict aerobes and have only galactose as cell wall-composing sugar.

Many strains belonging to this genus are incapable of growing at 37"C. On the other hand, KY 4707 and KY 4706 strains are Gram-positive during the whole culturing. Further, these strains are facultative anaerobes, grow abundantly at 370C and have galactose, rhamnose and mannose as cell wall-composing sugar. Therefore, KY 4707 and KY 4706 strains do not belong to the genusArthrobacter.

The bacteriological characteristics of KY 4707 and KY 4706 strains are not inconsistent with those of the strains belonging to the genus Corynebacterium described in page 599-617 of Bergey's Manual 8th edition. Therefore, KY 4707 and KY 4706 strains should be classified as the genus Corynebacterium.

It is mentioned on page 599 of Bergey's Manual 8th edition that there are the genera Brevibacterium and Microbacterium as those which are close to the genera Corynebacterium andArthrobacter but of which classification is uncertain. An opinion of Fiedler et. al. that the strains belonging to the genus Brevibacterium should be classified as members of the genus Corynebacterium is described on pages 625 and 626 of Bergey's Manual 8th edition. Further, an opinion of Jensen that Microbacterium lacticum and Microbacterium liquefaciens should be classified as the genus Corynebacterium is described on page 628 of Bergey's Manual 8th edition.

Under the above circumstances, the present inventors compared KY 4706 and KY 4707 strains with those belonging to various species of the genus Corynebacterium, Brevibacterium or Microbacterium referring to various literature such as Bergey's Manual 8th and 7th editions, etc. Further, experiments for the comparison were carried out as occasion demands.

Among the strains belonging to known species of the genera Corynebacterium, Brevibacterium and Microbacterium, those which are capable of growing abundantly in a medium containing choline as a sole carbon source like KY 4707 and KY 4706 strains belong to the species Corynebacterium murisepticum, Brevibacterium album, Brevibacterium cerinum or Brevibacterium maris. Further, the strains which are incapable of growing or capable of only scantily growing in a medium containing choline as a sole carbon source, contain lysine as diamino acid composing cell walls and fairly resemble KY 4707 and KY 4706 strains in other bacteriological characterics belong to the species Corynebacterium poinsettiae, Brevibacterium fulvum, Brevibacterium heIvohm, Brevibacterium imperiale or Brevibacterium sulfureum.

Comparison of KY 4707 and KY 4706 strains with the strains of the above-mentioned various species in bacteriological characteristics is shown in Table 1.

Table 1

Bacteriological Oc o ri-.

-I::, "a 0 istics ll - istics Cb II i;m 0 9 "o . :o:: OoO ; :- E 0o 8 %. & $ "9 e r" O,(p n 0 0 OO - 09' vi m CD C. choliniphilum + Lys + yellow - + + + + KY 4707 Key 4706 KY 4706 Strains belonging *1 to C. murisepticu + Strains belonging *1 *2 *2 * *2 *2 to B. album + ish brown *2 pale Strains belonging *1 yellow- *2 *2 *2 *2 to B. cerinum + ish - - + + brown Strains belonging *1 *3 *4 *4 *4 *4 to B. maris + DL- orange - - + DAP yellow *3 Strains belonging *1 *5 *1 4*1 4*1 *4*1 *1 *1 *5 to C. poinsettiae - + orange + - + - + - Lys Strains belonging *1 *3 deep *4 *4 *4 *2 *4 to B. fulvum - Lys yellow - + + Strains belonging *1 *3 *1 *1 *4 *1 *1 *1 to B. helvolum - Lys gray - + - + Strains belonging *1 *3 *1 *1 *1 *1 *1 *1 to B. imperiale - Lys orange + + - Strains belonging *1 *3 *4 *4 *4 ~it *4 to B. sulfureum - Lys yellow + + Note C : Corynebacterium B : Brevibacterium, Lys: Lysine DL-DAP : DL-diaminopimelic acid *1 : Experimental data *2 : U.S. Patent No. 3,222,258 *3 : J. Gen. Appl. Microbiol. 16, 103 (1970) *4 : Bergey's Manual 7th edition *5 : J. Gen. Microbiol. 28, 35 (1962) As shown in Table 1, KY 4707 and KY 4706 strains do not coincide with any strains belonging to the analogous species in bacteriological characteristics. From the foregoing, the present inventors have decided that both KY 4707 and KY 4706 strains should be classified as new species, and named the species to which KY 4707 strain belongs Corynebacterium cholinoxydans and the species to which KY 4706 strain belongs Corynebacterium choliniphilum.

In the present invention, either a synthetic medium or a natural medium may be used so long as it properly contains a carbon source, a nitrogen source, inorganic materials, and small amount of other nutrients which may be required by the specific strains used.

As the carbon source, choline or a salt thereof may be most preferably used. However, carbohydrates such as glucose, fructose, sucrose and molasses, hydrocarbons such as n-paraffins and kerosene, organic acids such as acetic acid, lactic acid, pyruvic acid and fumaric acid, and amino acids such as glutamic acid and aspartic acid etc., may be used depending upon the utilization by the microorganisms to be employed.

As the nitrogen source, ammonia, inorganic and organic ammonium salts such as ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium carbonate and ammonium acetate, urea, monosodium glutamate, glycine, monosodium aspartate, choline, and other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, fish meal, soybean meal and other nitrogenous organic materials may be used depending upon the utilization by the microorganisms to be employed.

As the inorganic materials, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, potassium chloride and calcium carbonate may be used.

If a microorganism to be employed in the present invention requires specific nutrients for growth such as biotin and thiamine, appropriate amounts of such nutrients must, of course, be supplemented to the medium. Sometimes, the nitrogenous organic materials to be employed as the nitrogen source may also serve as the source of the required nutrients. When such nitrogenous organic materials are employed, it is not necessary that the required nutrients be separately supplemented to the medium.

In the present invention, productivity of choline oxidase is increased by supplementing choline or a salt thereof to the medium as the inducer of the formation of choline oxidase. In the event, choline or a salt thereof may be used both as a carbon source and as the inducer. As the salt of choline, choline chloride, choline bromide, choline bicarbonate, choline citrate and choline ascorbate may be used. When choline or a salt thereof is used as the inducerofcholine oxidase, it is preferred that the concentration in the medium is 7-800 mmol/l. In adding choline or a salt thereof as the inducer, it may be present in the initial medium or added during the culturing. When choline or a salt thereof is added during the culturing, it is preferable that the feeding is completed by the end of the logarithmic growth phase of the microorganism.

The following experimental example is illustrative of the determination of a preferred range of the amount of the inducer to be added to the medium.

The activity of choline oxidase in the experimental examples and the examples is determined by oxidizing choline with choline oxidase to produce hydrogen peroxide, decomposing hydrogen peroxide with peroxidase in the presence of phenol and 4-aminoantipyrine to produce the pigment, and measuring absorbancy of the pigment solution. The detail of the determination is described below.

0.05 mol/C Tris buffer solution (pH 8.0) containing 0.01 mol/ e of 4-aminoantipyrine, 0.01 mol/ e of phenol and 5000 U/e of peroxidase is prepared and used as color-forming liquid. To 3 me of the color-forming liquid are added 0.1 me of enzyme solution to determine the activity and 0.1 me of 1/30 mol/e choline chloride solution. Reaction is carried out at 370C for 20 minutes and then absorbancy of the solution is measured at 500 nm. The amount of the reacted choline chloride is calculated based on the measured value. The enzyme activity is expressed by "U". A "U" is defined as the amount of enzyme which will decomposes 1 ,umole of choline in 1 minute under the above-described conditions.

Experimental Example In this experimental example, Brevibacterium album KY 4319, Corynebacterium murisepticum KY 3505, Corynebacterium cholinoxydans KY 4707 and Corynebacterium choliniphilum KY 4706 are used. 10 me portions of a nutrient medium having the following composition are poured into 70 matest tubes and sterilized at 120"C for 15 minutes. choline chloride various concentration shown in Table 1 corn steep liquor 0.5 g/de yeast extract 0.5 g/de K2HPO4 0.1 g/de MgSO4.7H20 0.05 g/de monosodium glutamate 0.5 g/de (pH 7.2) One loopful of the above strains are separately inoculated into the medium in the test tubes and are cultured with shaking at 300C for 24 hours.

After the completion of culturing, each of the culture liquors is subjected to centrifugation.

The resultant microbial cells are suspended in 10 me of 0.05 mol/ e Tris buffer having a pH of 8.0 and disrupted by ultrasonic generator. The resultant mixture is subjected to centrifugation and choline oxidase activity of the supernatant is measured. The results are shown in Table 2.

Table 2

Activity of choline oxidase (U/de) Con cen- I ' B. album I C. I C. I C. of choli KY 4319 murisepticum cholino- choliniof 4319 KY3505 xydans xydans philum chloride KY 4707 KY 4706 0 2.8 2.1 2.7 2.3 (7.2 mmol/d) 0.1 13.0 6.4 23.0 25.0 (3.6 mmol/t) 0.5 35.4 9.3 60.0 54.0 (72 mmol/e) 1.0 32.7 17.1 57.0 48.0 (144 mmol/e) 2.0 17.5 - (360 mmol/e) 5.0 23.4 12.8 47.0 39.0 (720 mmol/e) 10.0 15.1 6.7 27.0 21.0 Culturing of the present choline oxidase-producing microorganisms is carried out under aerobic conditions, for example, with shaking or with aeration and stirring. The preferred temperature for culturing is 25-35"C. The pH of the medium is preferably maintained in the range of 7.0 - 8.5 during culturing. Usuaully, after 1 - 3 days of culturing, a considerable amount of choline oxidase is accumulated in the culture liquor, mainly in the microbial cells.

After the completion of culturing, the microbial cells are isolated from the culture liquor, for example by centrifugation and disrupted by suitable means, for example using ultrasonic generator. The resultant mixture may be subjected to centrifugation to obtain the supernatant. To recover choline oxidase from the supernatant, various methods usually used in purification of enzymes such as salting out, precipitation by organic solvent, dialysis, column chromatography by Sephadex (Sephadex is Trade Mark for molecular sieve, derivatives of polysaccharide dextran, produced by Pharmacia Fine Chemicals Inc., U.S.A.), column chromatography by ion exchange Sephadex (Sephadex having groups for ion exchange, produced by Pharmacia Fine Chemicals Inc., U.S.A.) and column chromatography by ion exchange cellulose, may be used.

Enzymatic properties of the present choline oxidase derived from Brevibacterium album KY 4319, Corynebacterium murisepticum KY 3505, Corynebacterium cholinoxydans KY 4707 and Corynebacterium choliniphilum KY 4706 are illustrated in the following Table 3.

Table 3

ase Choline * * Derived Derived Derived Derived from from from from from B. C. C. C. album muri- cholino- cholini Enzymatic Key 4319 septicum xydans philum properties Key 3505 KY 4707 KY4706 Catalyzes oxidation of choline to betaine through betaine aldehyde.

The reaction process is exhibited as follows.

Action o2 choline ) betaine aldehyde + H202 O2 betaine aldehyde betaine + H202 H20 Stable pH range around (pH) 7.0- 8.3 8.0 7.0- 8.0 7.0- 8.0 Optimum pH range for reaction (pH) 7.5 - 9.0 7.5-9.5 7.5-9.5 7.5-9.5 Optimum temperature range for reaction 20 - 35 - 20-35 20 - 35 ( C) choline chloride 100 100 100 100 acetyicholine Sub- chloride 0 0 0 0 strate sarcosine 0 0 0 0 speci ficity . betaine 0 0 0 0 (rela tive glycine 1 0 0 0 0 acti vity 2-dimethyl 570) aminoethanol 7.3 8.5 8.7 7.9 2-methyl aminoethanol 1.0 0 - 0 0 ethanolamine hydrochloride 0 0 0 0 Stable temperature range 20-45 45 Stabilizer EDTA - - Inhibitor sodium - - azide Molecular weight about - - 97,000 Isoelectric point (pH) 4.05 - - * As choline oxidases derived from KY 4319, KY 3505, KY 4707 and KY 4706 strains, those obtained respectively in Examples 1, 3,4 and 5 are used.

Enzymatic properties in Table 3 are measured respectively by the following methods.

(1) Action In the following Experiment (1) and (2), choline oxidase, derived from Brevibacterium album KY 4319 is used.

The same results are also obtained using choline oxidases derived from the other strains.

Experiment (1) Identification of the products by the enzyme reaction by thin layer chromatography Choline oxidase is dissolved in 0.05 mol/# Tris buffer having a pH of 8.0 to make the concentration of 17.0 U/m#, 0.1 m# of the solution, 3.0m# fo 0.2 mol/# borate buffer having a pH of 9.0,5 m# of 1 mol/# aqueous solution of choline chloride and 5 mg of peroxidasc are mixed and allowed to react with shaking at 30 Covernight.

The following sample solutions including the above reaction solution are respectively spotted in an amount of 5 -- Part 2 Formation of hydrogen peroxide - 0.05 mol/e Tris buffer (pH 8.0) containing 0.001 mol/e of 4-aminoantipyrine, 0.001 mol/e of phenol and 50 mg/e of peroxidase is used as color-forming liquid. To 1.9 me of the color-forming liquid are added 0.1 me of aqueous solution of substrate (containing choline chloride or betaine aldehyde in an amount shown in Table 5), 0.2 me of 0.05 mol/e Tris buffer (pH 8.0) containing 3.5 U/me of choline oxidase and 0.8 me of water, and reaction is carried out at 370C for 20 minutes. Absorbancy of the reaction mixture is measured at 500 nm. The amount of the produced hydrogen peroxide is calculated based on the measured value. The ratio of the amount of the produced hydrogen peroxide (,umol) to the amount of the charged substrate ( mol) is shown in Table 5.

Table 5 Amount of Amount of Substrate substrate A'] hydrogen [B'] [B']/[A'] (,umol) peroxide (itmol) 0.045 0.081 1.80 Choline 0.090 0.150 1.67 chloride 0.180 0.310 1.72 0.045 0.035 0.78 Betaine 0.090 0.084 0.93 aldehyde 0.180 0.168 0.93 From Table 4, it is found that the amount of oxygen consumed by 1 mol of choline chloride is 1.87 - 1.91 mol and from Table 5, it is found that the amount of hydrogen peroxide produced from 1 mol of choline chloride is 1.67 - 1.80 mol. Therefore, it should be concluded that 2 mol of hydrogen peroxide is produced from 1 mol of choline chloride consuming 2 mol of oxygen. Similarly, it should be concluded that 1 mol of hydrogen peroxide is produced from 1 mol of betaine aldehyde consuming 1 mol of oxygen.

From the results of Experiments (1) and (2), it is understood that the present choline oxidase has the action described in Table 3.

(3) Stable pH range Choline oxidase is dissolved in 0.05 mol/e Tris buffer having a pH of 8.0 to make a specific concentration (1.5, 0.9, 1.4 and 1.4 U/me respectively in case of choline oxidase derived from KY 4319, KY 3505, KY 4707 and KY 4706 strains). 0.5 me portions of the solution are mixed with 1 me of each of 0.05 mol/e Tris buffers having various pH values to make solutions having different pH values between 5 and 10. The thus prepared solutions are kept at 45"C for 30 minutes and then subjected to determination of choline oxidase. Relative enzyme activities are calculated defining the highest enzyme activity among the activities obtained with the solutions having different pHs. as 100. The results are shown in Fig. 2.

In Fig. 2, (A), (B), (C) and (D) respectively exhibit test results of the enzymes obtained from cells of Brevibacterium album KY 4319, Corynebacterium murisepticum KY 3505, Corynebacterium cholinoxydans KY 4707 and Corynebacterium choliniphilum KY 4706.

This is the same in the subsequent figures.

(3) Optimum pH range for reaction About 4 me-reactor for measurement of oxygen absorption (produced by Kyusui-Kagaku Kenkusho Co., Ltd., Japan) is kept at specific temperature (30, 30, 37 and 37"C respectively in case of choline oxidase derived from KY 4319, KY 3505, KY 4707 and KY 4706 strains) and oxygen electrode is inserted therein.

Choline oxidase is dissolved in 0.05 mol/ Tris buffer having a pH of 8.0 to make a specific concentration (2.3, 1.2, 1.6 and 1.6 U/me respectively in case of choline oxidase derived from KY 4319, KY 3505, KY 4707 and KY 4706 strains). 0.1 me of the resultant enzyme solution, 0.1 me of 0.1 mol/e choline chloride aqueous solution and 3.6 me of 0.05 mol/e Tris buffer having different pH values between 6 and 11 are poured into the reactor and stirred.

Concentration of oxygen dissolved in the solution is recorded continuously by recorder (Desk Top Recorder, produced by Okura Denki Co., Ltd., Japan). Decrease in oxygen concentration between 30 seconds and 3 minutes after start of the reaction is calculated.

Relative enzyme activites are calculated defining the maximum decrease among the decreases obtained with the solutions having different pHs as the enzyme activity of 100. The results are shown in Fig. 3.

(4) Optimum temperature range for reaction Choline oxidase is dissolved in 0.05 mol/e Tris buffer having a pH of 8.0 to make a specific concentration (1.2, 0.5 and 0.5 U/mt respectively in case of choline oxidase derived from KY 4319, KY 4707 and KY 4706 strains). The resultant solutions are respectively subjected to the same determination of choline oxidase as described above except that the reaction is carried out at different temperatures of 20 - 60"C. Relative enzyme activities are calculated defining the highest enzyme activity among the activities obtained at the different temperatures as 100. The results are shown in Fig. 4.

(5) Substrate specificity Choline oxidase is dissolved in 0.05 mol/ e Tris buffer having a pH of 8.0 to make a specific concentration (1.5, 0.4, 0.9 and 0.9 U/me respectively in case of choline oxidase derived from KY 4319, KY 3505, KY 4707 and KY 4706 strains). The resultant solutions are respectively subjected to the same determination of choline oxidase as described above except that various substances other than choline chloride shown in Table 3 are used as a substrate. Relative enzyme activities on the various substances are calculated defining the enzyme activity on choline chloride as 100.

(6) Stable temperature range Choline oxidase obtained from Brevibacterium album KY 4319 is dissolved in 0.05 mol/e Tris buffer having a pH of 8.0 to make a concentration of 2.2 U/me. Portions of the resultant solution are respectively kept at 30, 40, 45, 50, 55, 60 and 65"C for 30 minutes and then cooled. The cooled solutions are subjected to the determination of choline oxidase. Relative enzyme activities at the various temperatures are calculated defining the enzyme activity at 30"C as 100. The results are shown in Fig. 5. From Fig. it is found that about 84% of loss in activity is caused by the treatment at 650C for 30 minutes.

(7) Stabilizers Choline oxidase, from Brevibacterium album KY 4319 and EDTA are dissolved in 0.05 mol/e Tris buffer having a pH of 8.0 to make respective concentrations of 2.2 U/mt and 10-3mol/e. Separately, choline oxidase is dissolved in 0.05 mol/e Tris buffer having a pH of 8.0 to make a concentration of 2.2 U/ mt (control) . The resultant solutions as they are, or the solutions obtained by keeping the above solutions at 45"C for 15, 30, 40 or 60 minutes and then cooling are subjected to the determination of choline oxidase. Relative enzyme activities are calculated defining the enzyme activity in case of no heat treatment as 100. The results are shown in Fig. 6. In Fig. 6, o o exhibits the case of EDTA addition and o o exhibits the case of no EDTA addition. It is found from Fig. 6 that EDTA has a choline oxidase stabilizing activity.

(8) Inhibitors Abot 4 m#-reactor for measurement of oxygen absorption is kept at 37 C and oxygen electrodc is inserted therein. Choline oxidase is dissolved in 0.05 mol/# Tris buffer having a pH of 8.0 to make a concentration of 0.8 U/me. 0.1 my of the resultant enzyme solution, 50 e of 1 mol/e choline chloride aqueous solution, 0.1 me of 1.7 x 10-3mol/e sodium azide aqueous solution and 3.5 me of 0.05 mol/ e Tris buffer having a pH of 8.0 are poured into the reactor. Concentration of oxygen dissolved in the solution is recorded continuously by recorder while stirring the solution. Decrease in oxygen concentration for 5 minutes after the start of the reaction is calculated.

The same procedure as above is repeated except that sodium azide is not added (control).

As the result, it is found that the enzyme activity is inhibited by 44.4% by 1.7 x 10-3 mol/ sodium azide as compared with the control.

(5) Molecular weight The molecular weight of choline oxidase is determined according to the gel-filtration method [Biochemical Journal 96 595 (1965)] using Sephadex G 200 (Trade Mark for molecular sieve, produced by Pharmacia Fine Chemicals Inc., U.S.A.). As the result, the enzyme is determined to have a molecular weight of about 97,000.

(10) Isoelectric point Isoelectric point of choline oxidase is determined according to electrofocusing method under the following conditions.

Carrier ampholite having pHs of 3.5-5.0 Column volume : 110 me Time charged with electricity : 24 hours fraction : 2g As the result, the enzyme is determined to have an isoelectric point of pH 4.05.

Now the present invention is further illustrated by the following examples, but the scope of the present invention is not limited thereto.

Example 1 In this example, Brevibacterium album KY 4319 is used as a seed strain. 10 me of a seed medium having the following composition is poured into a 70 me-test tube and sterilized at 120for 15 minutes.

Composition of the seed medium: Choline chloride 2 g/de corn steep liquor 0.5 g/de, yeast extract 0.5 g/de, monosodium glutamate 0.5 g/de, K2HPO4 0.1 g/de, MgS04.7H20 0.05 g/de (pH 7.2) One loopful of the strain is inoculated into the seed medium and cultured with shaking at 30"C for 48 hours. The whole resultant culture liquor is transferred to 300 me of the same seed medium as above in 2 e-Erlenmeyer flask and cultured with shaking at 300C for 48 hours.

The whole resultant culture liquor is again transferred to 3.0 e of a medium having the same composition as that of the seed medium in a 5 e-jar fermenter. Culturing is carried out at 30"C for 24 hours with aeration of 1 e/e of medium/min and stirring at 500 r.p.m. After the completion of culturing, 0.62 U/mt of choline oxidase is accumulated in the culture liquor.

The culture liquor is subjected to centrifugation to obtain microbial cells. The obtained cells are suspended in 1 e of 0.05 mol/e Tris buffer having a pH of 8.0. The suspension is treated with Dyno Laboratory mill (KDL type) (produced by Willy A, Bachofen Inc., Switzerland) to obtain a mixture containing disrupted cells. Then, the mixture is subjected to centrifugation to obtain a supernatant (The aforesaid amount of the enzyme accumulated in the culture liquor is calculated based on choline oxidase activity of the supernatant). To the supernatant is added ammonium sulfate to 30%saturation to form precipitates. The resultant mixture is subjected to centrifugation, and to the obtained supernatant is added ammonium sulfate to 60% saturation. The resultant precipitates are separated by centrifugation and dissolved in 0.05 mol/ Tris buffer having a pH of 8.0. The solution is dialysed against the same Tris buffer as above at 5"C overnight using a "Cellophane" (Registered Trade Mark) tube. After the dialysis, the solution is charged onto a column of 1 e of DEAE cellulose equilibrated with 0.05 mol/e Tris buffer (pH 8.0) containing 0.05 mol/e sodium chloride.

After washing the resin with 1 e of 0.05 mol/e Tris buffer (pH 8.0) containing 0.05 mol/e sodium chloride, gradient elution is carried out with 0.05 mol/e Tris buffer containing sodium chloride varying the concentration of sodium chloride from 0.05 to 0.45 mol/ e.

The eluate is taken in fractions and the fractions containing choline oxidase are combined.

To the combined solution is added ammonium sulfate to 60% saturation. The resultant precipitates are separated by centrifugation and dissolved in 0.05 mol/e Tris buffer having a pHof8.0.

The solution is charged onto a column of 500 me of Sephadex G-150 (Trade Mark for molecular sieve; produced by Pharmacia Fine Chemicals Inc., U.S.A.) equilibrated with 0.05 mol/e Tris buffer (pH 8.0).

Elution is carried out using the same buffer as above and the eluate is taken in fractions.

The fractions containing choline oxidase are combined. To the combined solution is added ammonium sulfate to 60% saturation. The resultant precipitates are separated by centrifugation and dissolved in 0.05 mol/e Tris buffer having a pH of 8.0. The solution is dialyzed against the same buffer as above at 50C overnight using Cellophane tube. After the dialysis, the solution is charged onto a column of 500 me of DEAE Sephadex A-50 (Trade Mark for weakly basic anion exchange resin; produced by Pharmacia Fine Chemicals Inc., U.S.A.) equilibrated with 0.05 mol/e Tris buffer (pH 8.0) containing 0.1 mol/e sodium chloride.

After washing the resin with 500 me of 0.05 mol/e Tris buffer p H 8.0) containing 0.1 mol/e sodium chloride, gradient elution is carried out with 0.05 mol/e Tris buffer containing sodium chloride varying the concentration of sodium chloride from 0.1 to 0.5 mol/ e.

The eluate is taken in fractions. The fractions containing choline oxidase are combined and dialyzed against 0.05 mol/ e Tris buffer having a pH of 8.0 at 5"C overnight using Cellophane tube. After the dialysis, the solution is freeze-dried, whereby choline oxidase is obtained in about 10% activity yield. The product exhibits a specific activity of 2.2 U/mg of protein.

Example 2 In this example, Brevibacterium cerinum KY 4320 is used as a seed strain. 300 me of a medium having the following composition is poured into a 2 e Erlenmeyer flask and sterilized at 120 Cfor 15 minutes.

Composition of the medium: Choline chloride 2 g/db, corn steep liquor 1 g/de, monosodium glutamate 0.2 g/de, K2HPO4 0.1 g/de, MgSO4.7H2O 0.05 g/de (pH 7.2). One loopful of the strain is inoculated in the medium and cultured with shaking at 300C for 48 hours. After the completion of culturing, 0.36 U/mt of choline oxidase is accumulated in the culture liquor.

Example 3 In this example, the procedures described in Example 1 are repeated except that Corynebacterium murisepticum KY 3505 is used.

After the completion of main culturing 0.56 U/me of choline oxidase is accumulated in the culture liquor.

After the purification, choline oxidase preparate is obtained in about 10% activity yield.

The product exhibits a specific activity of 3.5 U/mg of protein.

Example 4 In this example, the procedures described in Example 1 are repeated except that Corynebacterium cholinoxydans KY 4707 is used and that main culturing is carried out for 18 hours(cf.: 24 hours in Example 1).

After the completion of main culturing, 1.21 U/mE of choline oxidase is accumulated in the culture liquor.

After the purification, choline oxidase preparate is obtained in about 13% activity yield.

The product exhibits a specific activity of 4.5 U/mg of protein.

Example5 In this example, Corynebacterium choliniphilum KY 4706 is used as a seed strain. 300 me of a seed medium having the same composition as described in Example 1 is poured into a 2 e Erlenmeyer flask and sterilized at 1200C for 15 minutes. One loopful of the strain is inoculated in the medium and cultured with shaking at 300C for 48 hours.

The whole seed culture is transferred to 3.0 e of the main medium having the same composition as that of the seed medium in 5 t-jar fermenter and cultured at 300C for 22 hours with aeration of 1 e/e of medium/min and stirring at 500 r.p.m. After the completion of culturing, 1.10 U/me of choline oxidase is accumulated in the culture liquor.

The culture liquor is subjected to the same purification process as described in Example 1.

As the result, choline oxidase preparate is obtained in about 12% activity yield. The product exhibits a specific activity of 4.2 U/mg of protein.

Choline oxidase obtained by the present process is used for the determination of choline.

That is, choline may be determined by measuring the amount of hydrogen peroxide formed by the oxidation of choline in the presence of choline oxidase.

Claims (17)

WHAT WE CLAIM IS:

1. A process for preparing choline oxidase which comprises culturing a choline oxidaseproducing microorganism belonging to the genus Brevibacterium or Corynebacterium in a nutrient medium containing sources of assimilable carbon and nitrogen, and the inorganic salts necessary to support the growth of the microorganism, accumulating choline oxidase in the nutrient medium and recovering the choline oxidase therefrom.

2. A process according to claim 1, wherein said nutrient medium contains choline or a salt thereof as inducer of the formation of choline oxidase.

3. A process according to claim 2, wherein said nutrient medium contains 7 to 800 mmol/ e of choline chloride or a salt thereof as said inducer.

4. A process according to claim 1, 2 or 3, wherein the microorganism is cultured at a temperature of from 250 to 350C and at a pH of 7.0 to 8.5.

5. A process according to any one of the preceding claims, wherein the microbial cells are separated from the culture medium at the end of the fermentation process and ruptured prior to the recovery of the choline oxidase therefrom.

6. A process according to any one of the preceding claims, wherein said microorganism belongs to the genus Brevibacterium.

7. A process according to claim 6, wherein the microorganism is Brevibacterium album or Brevibacterium cerinum.

8. A process according to claim 7, wherein said microorganism is Brevibacterium album KY 4319 (FERM-P No. 3,777) (NRRL B-11,046) (ATCC 15,111) or Brevibacterium cerinum KY 4320 (FERM-P No.3,778) (NRRL B-1 1,047) (ATCC 15,112).

9. A process according to any one of claims 1-5, wherein the microorganism is of the genus Corynebacterium.

10. A process according to claim 9, wherein the microorganism is Corynebacterium murisepticum.

11. A process according to claim 10, wherein the microorganism is Corynebacterium murisepticum KY 3505 (FERM-P No.3,779) (NRRL B-il ,049)ATCC 21,374).

12. A process according to claim 9, wherein the microorganism is Corynebacterium cholinoxy ans KY 4707 (FERM-P No. 4,111) (NRRL B-11,158) or Corynebacterium choliniphilum KY 4706 (FERM-P No. 4,110) (NRRL B- 11,157).

13. A process according to claim 1, substantially as hereinbefore described in Example 1 or2.

14. A process according to claim 1, substantially as hereinbefore described in Example 3.

15. A process according to claim 1, substantially as hereinbefore described in Example 4 or5.

16. Choline oxidase, when prepared by a process claimed in any one of the preceding claims.

17. A choline oxidase according to claim 16 which is (a) capable of catalyzing the oxidation of choline to betaine aldehyde and that of betaine aldehyde to betaine, (b) having a stable pH range of 7.0 to 8.3, (c) having an optimum pH range for reaction of 7.5 to 9.0, (d) having an optimum temperature range for reaction of 20 to 350C, (e) having a stable temperature range of 20 to 450C, (f) having enzymic activity on choline chloride and on 2-dimethyl-aminoethanol but only about 1 % on 2-methylaminoethanol and none on acetylcholine chloride, sarcosine, betaine, glycine or ethanolamine hydrochloride, (g) being stabilized by EDTA, (h) being inhibited by sodium azide, (i) having a molecular weight of about 97,000 and (j) having an isoelectric point of pH 4.05.