JPS5822195B2 - Novel yeast fungus - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel yeast belonging to the genus Hansenula that can utilize methanol and grow at relatively high temperatures. It is an object of the present invention to provide a thermophilic bacterium that can effectively utilize methanol produced as a raw material for fermentation, and to advantageously industrially produce bacterial cells, bacterial cell proteins, etc. using the bacterium.

Conventionally, as raw materials for producing yeast cells, molasses, sulfite pulp waste liquid, n-paraffin, etc. have been mainly used as carbon sources.

However, these carbon sources have problems in terms of supply amount and price for use as industrial raw materials.

On the other hand, methanol has recently attracted attention as a new fermentation raw material.

Methanol has come to be produced in large quantities due to the development of the organic synthetic chemical industry, so it can be obtained at low cost, and since methanol is water-soluble, it is suitable as a growth medium for yeast. It is also considered preferable as a raw material for fermentation.

By the way, when yeast is used to industrially produce various metabolic products, a large amount of fermentation heat is generated due to culturing a large amount of yeast at high speed. The cost of cooling to maintain the temperature cannot be underestimated.

Therefore, if we can discover bacteria that can grow at higher temperatures,
Furthermore, it can be advantageous in industrial production.

Therefore, the present inventors conducted a wide search in nature for microorganisms that can strongly assimilate methanol as a main carbon source and grow at high temperatures, and as a result, they found a specific yeast that can assimilate methanol and developed the present invention. It has been completed.

That is, the present invention relates to the thermophilic bacterium Hansenula xyloensis (Hansenula xyloensis), which belongs to the genus Hansenula and has the ability to assimilate methanol.
The present invention relates to a novel yeast strain having characteristics identified as F. nulaxyloensis) No. 5679 or identified therefrom.

Next, the mycological properties of the microorganisms according to the present invention isolated and collected by the present inventors will be described in detail.

(1) Growth status in each medium (a) MY liquid medium = cultured at 28°C for 4 to 7 days.

Cells are (l, 5-20 x 2.5-4.0) microns, oval or oval.

Can be used singly or in pairs.

Forms a slight film. It does not form meristems and grows by multipolar budding method.

(b) MY agar medium = cultured at 28°C for 4 days.

The colony is pale yellowish white and opaque, conical in shape, with a smooth surface and a golden edge.

(c) Slide culture on corn extract agar medium:
Culture at 28°C.

Hyphae and pseudohyphae are not formed.

(c) Methanol-containing medium = culture at 37°C.

The colony is pale yellow and opaque.

The ridge is conical, with a smooth surface and a golden edge.

(2) Formation of ascospores: 1 to 4 cap-shaped spores are formed in Gorodkova medium or sodium acetate medium.

(3) Formation of extruded spores No formation of extruded spores was observed in the two-MY agar surface culture.

(4) Physiological Properties (a) Optimum Growth Conditions It grows well at 34-42°C, and grows and proliferates fairly well at 30-44°C.

Good growth is observed at pH 4.0 to 55.

(b) Range of growth No growth is observed below 5°C and above 45°C.

It grows fairly well up to pH 6.0, but does not grow above pH 7.0.

(c) Nitrate assimilability: Yes (d) Fat degradability: None e) Degradability of urea: Yes (f) Liquefiability of gelatin: None g) Salt tolerance: Grows at 10% or less.

(h) Carotenoid production: None i) Insect acidity: None j) Formation of starch-like substances: None k) Vitamin requirements: Requires hyothine.

(1) Methanol assimilation ability: Grows well using methanol as a carbon source.

- Formation of esters: None n) Degradability of arbutin: Yes (o) Lidomus milk reaction: None 5) Fermentability of sugars D-glucose + D-galactose - Maltose + Sucrose - Lactose - Cellobiose - Melibiose-trehalose + Fructose - Inulin - Raffinose - α-Methyl-D-glucoside - Soluble starch - (6) Carbon source assimilation D-arabinose + L-arabinose + D-lipose + D-xylose + D-glucose + D-galactose + L-rhamnose + D-fructose + L-sorbose + maltose + sucrose + lactose - melibiose-cellobiose + trehalose + raffinose - fructose + α-methyl-D-glycoside + aesculin + dextrin - soluble starch - inulin ethanol + Methanol + Atonite + Erythritol Inosit - D- Mannit + D - Sorbitol Zurcit + D- Gluconic Acid Decoglycerin + Salicin + DL - Lactic Acid - Citric Acid Decasuccinic Acid Isolation Source: Soil Collection Site: Tochimoto Prefecture As mentioned above, the yeast according to the present invention belongs to the spore-bearing yeast class, and is described in the book The Yeasts A Taxonomic Study by Lodder.
According to micStudy1970) J, Hansenula was identified based on spore shape, nitrate assimilation ability, film formation, etc.
It is considered to belong to the genus.

Comparing the yeast according to the present invention with the known bacterial species described by Roder, it is found that it ferments glucose and maltose, its spores are cap-shaped, and it requires vitamins;
Hansenula, which can assimilate erythritol,
Hansenula subpelli
It is mentioned as a similar bacterium to S. culosa).

However, L-sorbose, raffinose, inulin,
- It differs from Hansenula subpeliculosa in many respects, including the ability to assimilate carbon sources such as rhamnose, DL-lactic acid, soluble starch, galactose, melezitose, and D-arabinose, and the ability to produce esters.

Therefore, it does not match the known yeast described by Roder.

Furthermore, among the yeasts that can assimilate methanol, Hansenula belongs to the genus Hansenula, and is thought to have the most similar properties.
Hansenulathilandi
a) MT-3150 (see Japanese Patent Application Laid-Open No. 53-41478) differs in the size of vegetative cells in YM liquid medium, and in YM agar medium, Hansenula thailandea MT-3150 has a radial colony surface. In contrast to this yeast, which has a trapezoidal shape with striations, this yeast has a smooth surface, a conical shape, and a completely rounded periphery. Differences in sodium chloride concentration in osmotic pressure tests, assimilation of carbon sources such as galactose, L-sorbose, inulin, D-arabinose, L-arabinose, L-rhamnose, surgit, α-methyl-D-glycoside, and gluconic acid They differ in many ways, including points.

Therefore, since this bacterium does not match any known yeast, it was determined to be a new fungal species belonging to the genus Hansenula, and was named Hansenula xyloensis.

This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbial Deposit No. 5679.

Next, a method for separating and collecting yeast from soil according to the present invention will be described.

The new bacterial species of the present invention can be obtained by conventional means of separating yeast from soil samples, and for example, enrichment culture method can be used as the separation means.

As the separation medium, a normal yeast growth medium containing only methanol as a carbon source and appropriately supplemented with a nitrogen source, inorganic salts, and various growth factors may be used.

The concentration of methanol as a carbon source is preferably 2% by volume in consideration of transpiration during culture.

Examples of the nitrogen source include ammonium sulfate, ammonium chloride, ammonia, urea, sodium nitrate, yeast extract, peptone, and the like, which are commonly used in fermentation.

In addition, calcium salts, magnesium salts, phosphates,
Add various inorganic salts such as potassium salts and iron salts, as well as essential ingredients for growth such as vitamins and amino acids.

The culture is carried out aerobically at a temperature of 40° C. and a pH of 4.0.

Table 1 shows an example of the composition of the separation medium used in the present invention.

After enrichment culture using a separation medium, the accumulated yeast is made into a single bacteria on a MY plate.

Thereafter, this fungal species can be pure cultured by sub-planting it onto a methanol-containing medium.

The new bacterial species Hansenula xyloensis obtained in this way can grow at high temperatures using methanol as the main carbon source, which is inexpensive and can be supplied in large quantities. In some cases, bacterial cells can be obtained at low cost and in a short time.

The medium for culturing the collected new bacterial strain may be either a synthetic medium or a natural medium, as long as it contains appropriate amounts of carbon sources, nitrogen sources, inorganic salts, vitamins, and other growth-promoting substances.

As the carbon source for the medium, in addition to the above-mentioned methanol, any utilizable carbon source such as glucose, xylose, sucrose, molasses, and ethanol can be used.

In a culture method using methanol as a carbon source, this yeast can grow up to a methanol concentration of 4% by weight in the medium, but in order to reduce the evaporation loss of methanol due to aeration during culture, Keep the initial concentration as low as possible and add as methanol is consumed.
It is preferable to maintain a low concentration of methanol in the culture solution.

The culture temperature is preferably 34 to 42°C in consideration of the growth rate of the bacteria, the methanol cell yield, the protein content in the cells, and the like.

Growth is possible at a pH of 3 to 6.5, but in order to carry out continuous industrial cultivation over a long period of time without contamination with bacteria other than the target yeast, it is especially recommended to culture at a pH of 4 to 5. preferable.

Furthermore, in order to collect yeast cells from the culture solution, the cells are separated by a conventional method such as filtration and centrifugation, and then washed if necessary.

The method for pure isolation of the microorganism of the present invention from soil and the reproducibility of the microorganism will be described in detail below with reference to Examples.

Example 1 The separation medium shown in Table 1 was dispensed into an L-shaped tube in an amount of 10 TIl, and sterilized by autoclave at 121° CI for 5 minutes.

At this time, methanol is prepared separately and added at 2% by volume before the start of culture.

Approximately 0.5 g of a soil sample was added to this, and cultured with shaking at 40°C for 7 days.

After 7 days, 0.5 rul of the culture solution was collected and cultured again with shaking for 7 days using the same medium and conditions as the -th time.

After two enrichment cultures, the accumulated yeasts were spread on MY agar medium and cultured at 40°C for 3 days.

All colonies on the medium after culture were of the same type.

Five of these colonies were added to the medium composition shown in Table 1 on agar 1.
Each was inoculated onto a slant medium supplemented with 5% and cultured at 40°C for 3 days.

The growth status and physiological properties of the bacteria on each of the five slanted media were examined, and it was confirmed that these bacteria were the same.

The properties and physiological properties of each of the above-mentioned media are as described above.

Next, a dispersion medium consisting of 10% skim milk, 5% sucrose, and 1% sodium glutamate was added to the present yeast strain cultured on MY slant medium for 3 to 7 days to prepare a uniform cell suspension.

The cell concentration at this time was 10 g cells/ml.

This suspension was dispensed into ampoules in an amount of 0.2 ml and freeze-dried.

The drying method is slow freezing to -30 to -40℃ and then drying 10 tons.
It was dried for 18 to 20 hours at room temperature under orr.

Then, it was vacuum sealed with a gas burner and stored at 5°C.

The freeze-dried bacteria thus obtained were reconstituted after 3 months.

At this time, sterile distilled water was used as the condensate, and MY culture medium was used as the culture medium.

The morphological and physiological properties of the restored bacteria were the same as before freezing.

Next, an example in which yeast cells were produced using the yeast of the present invention will be given as a reference example.

Reference example 1 (NH4)2S043g, KH2PO42g, Mg50
4-7H200,5g, FeSO4・7H201～,C
acI12・2H22H2O5, BiotinlOμg
1 Tap water 11. 50 TLl of medium consisting of pH 5.0
After dispensing into a 001111 volume Sakalo flask, it was heated to 121°C.
Sterilize for 0 minutes, cool, add 2% by volume of methanol aseptically, inoculate the methanol-assimilating yeast Hansenula xyloensis (Feikokenbacterium GS5679) into the round culture medium,
Shaking culture was performed at 0°C for 45 hours.

After culturing, the culture solution was centrifuged, washed with water, and then heated at 100°.
It was dried at C for 24 hours.

4 g of yeast cells were obtained from No. 11 culture solution.

Reference Example 2 Culture medium 51 having the same composition as that shown in Reference Example 1 was placed in a 101-volume jar fermentor, and after sterilization, 1% by volume of methanol was added thereto.

To this was added 50 ml of a culture solution of Hansenula xyloensis (Feikoken Bokuyori No. 5679), which had been cultured in the same manner as in Example 1 above, as a seed yeast, and the mixture was heated at 40°C with 51/2 aeration.
The cells were cultured at a stirring speed of 1 min.

0.0 when the methanol concentration reaches 0.05% by weight.
The operation of adding methanol equivalent to 5% by weight was repeated, and the culture was continued for 72 hours while keeping the methanol concentration at a low level.

The pH was automatically adjusted to around 4.5 with 7% aqueous ammonia.

After 72 hours of culture, the amount of methanol added reached 9% by volume.

After culturing, the bacteria were collected by centrifugation and freeze-dried to obtain 23 g of dry yeast cells per 11 of the culture solution.

The yield based on the amount of methanol added was 32%, and the maximum specific growth rate was 0.17 hr'.

Claims (1)

[Scope of Claims] Han3enula xyloensis, a thermophilic fungus belonging to the genus Hansenula and capable of assimilating methanol, No. 5679, or a novel yeast having characteristics identified therewith.