GB2042548A - Novel actinomycetes and process for producing vitamin B12 from microbial cells thereof - Google Patents

Abstract

Vitamin B12 is obtained from novel actinomycetes of the genus Pseudonocardia which have the ability to assimilate methanol. Cells of these actinomycetes and vitamin B12 are obtained by cultivating them in a culture medium containing methanol as a major carbon source, a source of cobalt also being required to obtain vitamin B12. Novel strains which are disclosed are P. methanolignica (ATCC 31596) and P. yukotes (ATCC 31597).

Description

SPECIFICATION Novel actinomycetes and process for producing microbial cells thereof This invention relates to novel actinomycetes of the genus Pseudonocardia which have the ability to assimilate or utilize methanol. This invention also relates to a process for producing the cells of these actinomycetes by cultivating them in a culture medium containing methanol as a major carbon source.

Vitamin B12 is an essential factor in the metabolization of nucleic acids, proteins, lipids and carbohydrates in the living body, and is also in widespread use as medicines, e.g. a therapeutic agent for malignant anemia in humans. Its utilization would further increase if it can be produced at a lower cost.

Because vitamin B12 has a complex structure, its complete synthesis is difficult, and commercially it is now produced mainly by a fermentation method and a biochemical method. These two methods will continue to be predominant in the future.

Conventionally known vitamin B12-producing microorganisms are those of the genera Propionibacterium, Bacillus, Corynebacterium, Arthrobacter, Rhodopseudomonas, Pseudomonas, Protaminobacter, Streptomyces, Rhodospirillum, Actinomyces, and Nocardia. All of these microorganisms produce vitamin B12 by utilizing carbohydrates as carbon sources.

There are also known vitamin B12-producing microorganisms which utilize hydrocarbons as carbon sources, such as microorganisms of the genera Corynebacterium, Arthrobacter, Pseudomonas and Nocardia.

In recent years, production of microbial cells or microbial metabolites using methanol obtainable at low cost from natural gases and other materials has attracted attention. Fukui et al. reported the production of vitamin B12 by microorganisms of the genera Protaminobacter and Pseudomonas (Japanese Laid-Open Patent Publication No. 133596/74). Likewise, Kamikubo et al. reported the production of vitamin B12 by microorganisms of the genus Klebsiella (Japanese Laid-Open Patent Publication No.

132186/75).

However, no actinomyces having the ability to assimilate methanol has been known in the genus Pseudonocardia. Moreover, none of the known actinomycetes having the ability to produce vitamin B12 have the ability to assimilate methanol.

It is a primary object of this invention to provide novel actinomycetes which can assimilate as a major carbon source methanol which is commercially available easily and in stable amounts, and to produce microbial cells utilizable as protein sources in high yields by cultivating these novel actinomycetes.

A second object of this invention is to produce microbial cells utilizable as vitamin B12-containing protein sources or microbial cells utilizable for extraction of vitamin B12 by growing the aforesaid actinomycetes on methanol as a major carbon source and causing vitamin B12 to be accumulated in the microbial cells.

The present inventors widely searched in nature for actinomycetes which would utilize methanol as a major carbon source, and consequently, discovered two novel actinomycetes of the genus Pseudonocardia which preferentially assimilate methanol and have the ability to produce vitamin B12 in a high output and which gives a good yield of microbial cells based on methanol. The two novel actinomycetes have been named Pseudonocardia methanolignica and Pseudonocardia yukotes.

Microbial cells are produced in high yields when actinomycetes of the genus Pseudonocardia having the ability to assimilate methanol, typified by these two strains, are cultivated in a culture medium containing methanol as a major carbon source.

Accordingly, the present invention makes it possible to obtain microbial proteins at low cost using as a material methanol which is commercially available easily at low cost. In addition, microbial cells having vitamin B12 accumulated in large amounts can be obtained by using the aforesaid two actinomyces strains having the ability to assimilate methanol and produce vitamin B12. These microbial cells are highly valuable as microbial proteins having a unique nutrient value and as materials for extracting vitamin B12.

The microbiological properties of the novel strains isolated by the present inventors are described in detail below.

Pseudonocardia methanolignica A. Morphological characteristics Good growth on glucose-asparagine agar. Colonies yellow to orange with a thick cover of snow-white aerial mycelia. Hyphae often zig-zag shaped and bear straight chains of blastspores. Under certain conditions fragmentation spores produced in the aerial hyphae in a basipetal direction starting at the terminal segment or, more rarely, intercalary in the hyphae. Spores are smooth-walled and vary greatly in size, usually 0.4 - 0.6 ,um wide by 1.0 - 1.6 ,um long.

Spores are cylindrical and short-oval to long-oval in scanning electron microphotographs.

B. Cultural characteristics Cultivated at 3PC for 2 weeks.

(1) Sucrose-nitrate agar Growth moderate; thin cover of scanty white aerial mycelium; no soluble pigment.

(2) Glucose-asparagine agar Growth good; substrate mycelium yellow to orange; snow-white aerial mycelium abundant; yellow to orange soluble pigment.

(3) Glycerol-asparagine agar Growth good; substrate mycelium yellow to orange; thick cover of white aerial mycelium abundant; yellow to orange soluble pigment.

(4) Starch agar Growth moderate; thin cover of scanty white aerial mycelium; no soluble pigment.

(5) Tyrosine agar Growth moderate to good; substrate mycelium ivory; white aerial mycelium; no soluble pigment.

(6) Nutrientagar Growth good; substrate mycelium yellow; scanty white aerial mycelium; no soluble pigment.

(7) Yeast-malt extract agar Growth good; substrate mycelium yellow to orange; thick cover of white aerial mycelium abundant; no soluble pigment.

(8) Oatmeal agar Growth moderate to good; substrate mycelium white to ivory; thin cover of white aerial mycelium; no soluble pigment.

(9) Glucose-pepton gelatin (28'C) Growth good; thick cover of substrate mycelium; white aerial mycelium abundant; brown soluble pigment.

(10) Pridham-Gottlieb agar Growth moderate; thin cover of scanty white aerial mycelium; no soluble pigment.

(11) Peptone-yeast extract iron agar Growth good; no aerial mycelium; substrate mycelium yellow to orange; no soluble pigment.

C. Physiological properties (1) Growth temperature range: 25 to 45 C (2) Optimal growth temperature: 35 to 430C.

Even at 43DC, the growth is as good as or better than that at 3PC.

(3) Liquefaction of gelatin: positive (4) Hydrolysis of starch: negative (5) Coagulation of skim milk: partly coagulated (6) Peptonization of skim milk: negative (7) Formation of melanine-like pigment Tyrosine agar medium: negative Peptone-yeast extract iron agar medium: negative (8) Reduction of nitrate: positive (9) Assimilation of carbon sources (on the Pridham-Gottlieb agar) (a) Positive: L-arabinose, D-xylose, D-glucose, D-fructose, inositol, L-rhamnose, D-mannitol, methanol, ethanol.

(b) Negative: Sucrose, raffinose.

(10) Chemical composition of the cellular wall 'determined by the method of M. P. Lechevalier et al, International Journal of Systematic Bacteriology, Vol. 20, 435-443 (1970)1 (a) Type of the cellular wall: type IV (b) Its principal ingredients: meso-DAP, arabinose, galactose.

D. Source of isolation: Soil Pseudonocardia yukotes A. Morphological characteristics Good growth on glucose-asparagine agar and yeast-malt extract gar. Colonies yellow to orange with a thick cover of snow-white aerial mycelia.

Hyphae often zig-zag shaped and bear straight chains of blastspores. Under certain conditions fragmentation spores produced in the aerial hyphae in a basipetal direction starting at the terminal seg ment or, more rarely, intercalary in the hyphae.

Spores are smooth walled and vary greatly in size, usually 0.4 - 0.6 ,am wide by 1.0-1.6 ,am long. Spores are cylindrical and short-oval to long-oval in scan ning electron microphotographs.

B. Cultural characteristics: Cultivated at 3PC for 2 weeks.

(1) Sucrose-nitrate agar Growth moderate; thin cover of scanty white aerial mycelium; no soluble pigment.

(2) Glucose-asparagine agar Growth good; substrate mycelium white to ivory; snow-white aerial mycelium abundant; no soluble pigment.

(3) Glycerol-asparagine agar Growth good; substrate mycelium brown; thick cover of white aerial mycelium abundant; yellow to orange soluble pigment.

(4) Starch agar Growth moderate; thin cover of scanty white aerial mycelium; no soluble pigment.

(5) Tyrosine agar Growth moderate to good; substrate mycelium ivory; snow-white aerial mycelium; no soluble pigment.

(6) Nutrient agar Growth good; substrate mycelium ivory; scanty white aerial mycelium; no soluble pigment.

(7) Yeast-malt extract agar Growth good; substrate mycelium yellow to orange; thick cover of snow-white aerial mycelium abundant; no soluble pigment.

(8) Oatmeal agar Growth moderate; substrate mycelium white to ivory; thin cover of white aerial mycelium; no soluble pigment.

(9) Glucose-peptone gelatin (28"C) Growth moderate; no soluble pigment.

(10) Pridham-Gottlieb agar Growth moderate; thin cover of scanty white to gray aerial mycelium; no soluble pigment.

(11) Peptone-yeast extract iron agar Growth good; no aerial mycelium; substrate mycelium ivory to yellow; no soluble pigment.

C. Physiological properties: (1) Growth temperature range: 24 to 49oC (2) Optimal growth temperature: 37 to 4PC (3) Liquefaction of gelatin: negative (4) Hydrolysis of starch: negative (5) Coagulation of skim milk: negative (6) Peptonization of skim milk: negative (7) Formation of a melanine-like pigment Tyrosine agar medium: negative Peptone-yeast extract-iron agar medium: negative (8) Reduction of nitrate: positive (9) Assimilation of carbon sources (on the Pridham-Gottlieb agar medium): (a) Positive: L-arabinose, D-xylose, D-glucose, D-fructose, L-rhamnose, D-mannitol, methanol, ethanol, n-paraffin.

(b) Negative: Sucrose, inositol, raffinose.

(10) Chemical composition of the cellular wall 'determined by the method of M. P. Lechvalier et al, International Journal of Systematic Bacteriology, Vol. 20, 435-443 (1970)1: (a) Type of the cellular wall: type IV (b) Principal ingredients: meso-DAP, arabinose, galactose.

D. Source of isolation: Soil The properties of the two novel strains are summarized as follows: The growth on various media exhibits a thick cover with an ivory to pale yellow color.

The aerial mycelia are white or snow-white, and grow straight or zig-zag with formation of branches.

Basipetal growth of the aerial mycelia causes the spores to be fragmented, and the surfaces of the spores are smooth.

No melanine-like pigment is formed on the peptone-yeast extract-iron agar medium.

The principal ingredients of the cellular wall are meso-DAP, arabinose and galactose.

These two strains are very similar to microorganisms of Streptomyces in view of the results of observation on various media. But they differ from the genus Streptomyces because the principal ingredients of the cellular wall are meso-DAP (diaminopimelic acid), arabinose and galactose. The strains are also different from the genus Nocardia because long spore chains form as a result of the basipetal growth. This suggests the propriety of assigning the strains to the genuspseudonocardia.

The above microbiological properties have been studied with reference to Bergey's Manual of Determinative Bacteriology, 8th edition. This has led to the confirmation that the two strains of this invention are novel strains.

Pseudonocardia thermophlla rArchiv. fur Mikrobiologie, Vol.26,373-414 (1957)1,Pseudonocardia Spinosa 'International Journal of Systematic Bacteriology, Vol.21,29-34(1971)1, and Pseudonocardia fastidiosa sp. nov. Rutien (U.S. Patent No. 4,031,2061 are known to be species of the genus Pseudonocardia. Among them, Pseudonocardia thermophila is most analogous to the two strains of this invention in respect of the growth temperature range and the optimal growth temperature. Thus, the two strains of this invention and Pseudonocardia thermophila IFO 12133 were cultivated under the same conditions, and comparatively studied. The results are shown in Table 1.

As is seen from Table 1,Pseudonocardia methanolignica and Pseudonocardia yukotes differ from the known Pseudonocardia thermophila in regard to their growth on a sucrose-nitrate agar medium and their growth on a glycerol-asparagine agarmedium, the liquefaction of gelatin, the peptonization of skim milk and the assimilation of carbon sources.

Table 1

Pseudonocardia Pseudonocardia Pseudonocardia methanolignica yukotes thermophila Growth moderate Growth moderate Growth good Sucrose-nitrate with scanty with scanty with abundant agar white aerial white aerial snow-white mycelia. mycelia around powdery aerial the colonies. mycelia.

Growth good Growth good Growth moderate Glycerol- with a thick with a thick with white asparagine agar cover. White cover. Snow- aerial mycelia.

aerial mycelia white powdery around the aerial mycelia colonies. with crevices.

Growth tem perature range 28-4C 24-49"C 28- 60"C Optimal growth temperature 35-43"C 37-470C 42 - 50 > C Liquefaction of gelatin Positive Negative Negative Peptonization of skim milk Positive Negative Negative Inositol, L-rhamnose, Sucrose L-rhamnose, methanol, (positive).

Assimilation of methanol, ethanol and Inositol, carbon sources ethanol and n-paraffin L-rhamnose, n-paraffin (positive). methanol, (positive). Inositol and ethanol and Sucrose sucrose n-paraffin (negative). (negative). (negative).

The Pseudonocardia methanolignica and Pseudonocardia yukotes of this invention have been 45 deposited in Fermentation Research Institute, Agency of Industrial Science and Technology, Japan under accession numbers FERM-P No. 4747 and FERM-P No.4746, respectively, and also in American Type Culture Collection under accession numbers ATCC No.31596 and ATCC No. 31597.

The aforesaid two strains are preferred in the production of actinomyces cells in accordance with this invention. These are not limitative examples, how ever, and any strain belonging to the genus Pseudonocardia which assimilates methanol can be used in this invention.

Any culture media which contain carbon sources, nitrogen sources, inorganic salts and other nutrient sources required by the microorganisms, as exemplified hereinbelow, can be used in this invention.

In addition to methanol, ethanol, acetic acid, n-paraffin, etc. can also be used as carbon sources.

Pseudonocardia methanolignica or Pseudonocardia yukotes produces an especially large amount of vitamin B12 when it is cultivated on a medium having methanol as a major carbon source.

The suitable concentration of methanol in the culture medium is not more than 2% by weight because higher concentrations of methanol inhibit the growth of the microorganism and the formation of vitamin B12. Good results are obtained when the early stage of cultivation is carried out at a low methanol concentration and methanol is added as desired during the laterstage of cultivation.

Useful nitrogen sources include ammoniac nitrogen, nitric acid nitrogen, and urea. Other nitrogen-containing organic materials, such as peptone, malt extract, meat extract, yeast extract and casein hydrolyzate, can also be used.

Dipotassium phosphate, monosodium phosphate and magnesium sulfate are examples of the inorganic salts.

The presence of a metal ion greatly affects the production of vitamin B12. In particular, the presence of Co++ is essential. Various cobalt salts such as cobalt chloride or cobalt sulfate are added to the culture medium as sources of supplying the cobalt ion.

Other nutriments for growth such as L-asparagine, or yeast extract as a trace nutriment may also be added to the culture medium.

One specific example of the culture medium that can be used in this invention is prepared by dissolving the ingredients indicated in Table 2 below in 1 liter of tap water, adjusting the pH of the solution to 7.0, autoclaving it, and adding methanol to a predetermined concentration, for example 1% by weight.

Table 2 Ammonium chloride 4 9 Dipotassium phosphate 8 9 Monosodium phosphate dihydrate 4 9 Magnesium sulfate heptahydrate 0.5 g Ferric chloride hexahydrate 1 mg Copper sulfate pentahydrate 0.1 mg Zinc sulfate heptahydrate 0.1 mg Manganese sulfate heptahydrate 0.1 mg Cobalt chloride hexahydrate 3 mg Yeast extract 0.03g L-asparagine 1 g Of course, if desired, other suitable nutriments, a trace element, and a precursor (e.g., 5, 6-dimethyl benzimidazole betaine, etc.) can be added.

The cultivation can be performed by a method of stationary cultivation, shaking cultivation, aerationagitation cultivation, etc. Usually, the cultivation is carried out at a temperature of 25 to 480C, preferably 37 to 45"C and a pH of 5.5 to 9.6, preferably 6.5 to 7.0.

In 2 to 5 days, usually in 3 to 4 days, the amounts of the microbial cells and vitamin B,2 reach maximum levels.

In the resulting fermentation product, vitamin B,2 is contained mainly in the microbial cells. The amount of vitamin B12 in the fermentation product is measured by a conventional method which involves adjusting the pH of the fermentation broth to 5 to 6 with sulfuric acid, adding potassium cyanide so that its final concentration reaches 0.002 to 0.004%, extracting it in an autoclave at 121"C for 10 minutes, and determining the amount of vitamin B12 using Lactobacillus leichimannii ATCC 7830 rKudo: Theory and Basic Procedures of the Method of Microbiological Determination of Vitamins and Amino Acids (a Japanese-language Publication),19691.

For example, when the two strains of this inven- tion are cultivated on a culture medium of the composition cited above as one specific example (with a methanol concentration of 1% by weight), large amounts of vitamin B12 are accumulated in the microbial cells, as shown in Table 3. For comparison, the results obtained by using a cobalt chloride-free medium are also shown in Table 3.

Table3

Amount of Amount of Amount of cobalt the micro- vitamin Brain chloride bial cells the fermented added formed product Actinomyces strains (mg/l) (girl) (g/l) Pseudonocardia 3.8 ~ methanolignica 4.3 230 Pseudonocardia 2.5 6 yukotes 3 2.8 190 The following Examples specifically illustrate the process of this invention.

Example 1 The ingredients shown in Table 2 were dissolved in 1 liter of tap water. 100 ml of the solution was put into a 500 ml. shaking flask and heat-sterilized, and then methanol was added aseptically to a concentration of 1% by weight. Pseudonocardia methanolignica was inoculated in the resulting culture medium, and cultivated with shaking at 40"C for 5 days.

The amount of vitamin B12 formed in the cultivated product was determined by the microbiological method using Lactobacillus leichimanniiATCC 7830, and was found to be 240,ug/l of culture broth. The yield of the microbial cells was 43% based on methanol.

Example 2 Pseudonocardia yukotes was inoculated in the same culture medium as used in Example 1, and cultivated with shaking at 37 C for 5 days.

The amount of vitamin B12 in the cultivated product, determined by the microbiological method using Lactobacillus leichimannii ATCC 7830, was 195 ,ug/l of culture broth. The yield of the microbial cells was 29% based on the methanol.

Claims (10)

1. An actinomycetes of the genusPseudonocardia having the ability to assimilate methanol.

2. An actinomycetes according to claim 1 having the ability to produce vitamin B12.

3. An actinomycetes according to claim 1 or 2 which is Pseudonocardia methanolignica (ATCC No.

31596).

4. An actinomycetes according to claim 1 or 2 which is Pseudonocardia yukotes (ATCC No.31597).

5. A culture of an actinomycetes as claimed in any one of the preceding claims in a culture medium containing a source of assimilable carbon, a source of assimilable nitrogen and inorganic salts, and substantially free from other micro-organisms.

6. A process for producing actinomycetes cells, which comprises cultivating an actinomycetes as claimed in any one of claims 1 to 4 in a culture medium containing methanol as a major carbon source, and recovering the resulting microbial cells.

7. A process according to claim 6 wherein a cobalt source and other components essential to the production of vitamin B12 are added to the culture medium so as to allow vitamin B12 to be accumulated in the microbial cells.

8. A process according to claim 6 or 7 wherein the cultivation is carried out at a temperature of 37 to 45fC.

9. A process according to claim 6,7 or 8 wherein the concentration of methanol in the culture medium is not more than 2% by weight.

10. A process according to claim 6 substantially as described by reference to Example 1 or Example 2.