JPH0463591A - Production of lipase - Google Patents

Abstract

PURPOSE:To obtain a lipase (a lipid-decomposition enzyme) on an industrial scale using a yeast other than Candida by culturing a specific yeast belonging to genus Hansenula in a nutrient medium. CONSTITUTION:A yeast belonging to genus Hansenula and capable of producing lipase (preferably Hansenula anomala) is cultured in a nutrient medium and the objective lipase is separated from the culture product.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for producing lipase using yeast.

[Prior Art] Lipase, which is a lipolytic enzyme, was discovered a long time ago, but its research has lagged behind compared to other enzymes. However, recently, its usefulness is beginning to be recognized again as its industrial use in various fields such as detergents, ester synthesis reagents, pharmaceuticals, and pitch decomposing agents for paper manufacturing is expanding.

Industrial production of lipase by microorganisms has been carried out in the genus Penicillium, Aspergillus (^s
pBgillus) genus, Rhitops (Rhitopns)
genus and other filamentous fungi, or Pseudomonas (Psen
Bacteria such as the genus Domonag are known. However, the production of lipase by yeast candida ((a
ndidg) and a few other strains are known.

[Problems to be Solved by the Invention] Production of lipase by filamentous fungi requires a long culture period due to the nature of the fungi, and has many disadvantages in terms of productivity.

Further, problems due to the elongation of hyphae are likely to occur even in liquid culture. On the other hand, production by bacteria requires a large amount of energy to separate the bacterial cells. Furthermore, the activity range of the produced lipase is mainly neutral to alkaline, and it is extremely unstable on the acidic side. Industrial production of lipase by yeast is thought to compensate for these drawbacks.

However, for yeast, the lipase production ability is
Candida cylindracea (Cxndid1c71
Only a few strains are known, such as C. indncej) and C.

Therefore, the present invention provides a method for producing lipase using a yeast strain other than Candida, that is, a method for producing lipase using a yeast strain that has not been known to have the ability to produce lipase.

[Means for Solving the Problems] The present inventors conducted extensive screening in the natural world for the purpose of obtaining lipase-producing strains of yeasts other than those of the genus Candida. As a result, they found that a strain belonging to the genus Hansenula isolated from wild animals produced highly active lipase.

The mycological properties of this strain are as follows.

Growth on 05% malt extract agar medium (25°C, 3 days)
; Cells are spherical and slightly elongated. The size is 1°9~4
．． IX is 2.1 to 6.1 μm. They are single cells, but sometimes they form small clusters that are grape-shaped. The growth is bu17rous and has a slightly yellowish brown color.

■Growth on Dalmau plate (25°C, 7 days): Growth under cover glass is vigorous and pseudohyphae are branched. It grows aerobically and is white to slightly yellowish white in color.
- Inside the shell is butyrous. The bacterial cells are smooth and shiny. Colonies are entire. Always produces a small amount of scent components.

■Spore formation: Performs heterothallic sexual reproduction, but forms diploid cells that become sporangia. Diploid cells directly become asci 1-4
Forms cap-shaped spores. Spores are observed on 5% malt extract agar and V8 agar.

■Fermentation of sugars; glucose + galactose ± sucrose + maltose, lactose, raffinose ± ■Assimilation of carbon compounds Nigalactose ± sucrose + maltose, cellobiose + trehalose + lactose + raffinose + soluble starch 10-xylose + L-arabinose ± D-ribose ± L-rhamnose erythrotol + ribitol ± D-mannitol + succinic acid decacitric acid deca-inositol L-sorbose melibiose melezitose + inulin D-arabinose α-methyl-D-glucoside + salicin + DL- Lactic acid 10 ■ Assimilation of nitrate; + ■ Growth in vitamin-free medium; + ■ Ferment and yeast vitrodiene basic medium + 1o ■ Growth at 37°C; ± The mycological properties listed above are The 7ea mountain; N, I ，
L Kreger-wan Riji Gronin
It was identified as Hansenula anomala (Mt. Hensen 1+1010) as described in Gen. Gen, 3rd edition (1984).

In addition, Hansenula anomalla, a strain that the present inventors successfully isolated, was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology, as part of the Microbiology Research Institute No. 11519 (FERM P-11519).
) has been deposited as.

Hansenula strains used in the present invention are grown under aerobic and facultative aerobic conditions in a medium consisting of components known in the art, assimilable carbon and nitrogen sources, together with other micronutrient sources. Can be cultured in a medium containing

Preferred carbon sources are carbohydrates, lipids and other esters. Further, the nitrogen source is preferably an inorganic substance such as nitrate, sulfate, or ammonium salt, or an organic substance such as yeast extract, corn steep liquor, soybean flour, or the like. The pH of the medium is 3~
9, preferably 5-8. Culture temperature is 10-40℃
, preferably 20 to 35°C. After culturing, the bacterial cells are removed by centrifugation or filter press to obtain a culture filtrate, which can be used as a crude lipase enzyme solution.

It can also be made into a concentrated crude enzyme solution by evaporation, reverse osmosis membrane, etc. These lipase crude enzyme solutions are
Purification can be carried out as appropriate using ammonium sulfate fractionation, dialysis, column chromatography, high performance liquid chromatography, or a water-soluble solvent such as acetone or ethanol.

[Example] The present invention will be explained in detail below with reference to Examples.

Note that the present invention is not limited to this.

(Example 1) 2% glucose, 2% soy bean mill, 2% olive oil
, (NH4)2SO40,1%, K2HPO40,5
%, MgSO4・7) 120.1%, CaCO30°5
After adjusting the pH of the liquid medium containing 5.0% to 5.0, 7ml was dispensed into an L-shaped test tube and sterilized in an autoclave. The above medium was inoculated with 0.7 ml of cell fluid of Anomala anomala, which had been previously cultured in the same medium at 30° C. for 24 hours with shaking in an L-shaped test tube. This was cultured with shaking at 30°C for 4 days,
Bacterial cells were separated by centrifugation and a filtrate was obtained. Using 1 ml of this filtrate as a crude enzyme solution, P.
Lipase activity was measured by the vA emulsion method. The lipase activity of the obtained crude enzyme solution was 10 U/ml.

(Example 2) Casein peptone 1.7%, soybean peptone 0.3%
, K 2 HPO 40.25%, glucose 0°25%
, NaCl0. The medium containing 5% was adjusted to pH 5.5,
The mixture was dispensed into 100 ml bottles and sterilized in an autoclave. In advance, in the same medium at 30℃, 2
882293-1 cultured with shaking in an L-shaped test tube for 4 hours.
10 ml of pre-cultured bacterial body fluid was inoculated into Colben. This was cultured with shaking at 30°C for 3 days, and the bacterial cells were separated by centrifugation to obtain a filtrate.

Lipase activity was measured in the same manner as in Example 1 using 1 ml of the filtrate as a crude enzyme solution. The lipase activity of the crude enzyme solution is 12 U/m
It was l.

[Effects of the Invention] The present invention provides yeasts of the genus Hansenula, particularly Hansenula spp.
This was the first to reveal that lipase could be produced by Anomara, and the fact that the possibility of industrial production of lipase using yeast other than Candida was demonstrated will greatly contribute to the production and utilization of lipase. It is.

Claims (2)

[Claims] (1) A method for producing lipase, which comprises culturing yeast that belongs to the genus Hansenula and has lipase-producing ability in a nutrient medium, and collecting lipase from the culture. (2) The yeast is Hansenula anomala (Hansenula anomala).
Claim 1 characterized in that:
Method for producing the lipase described.