JPH0632610B2 - Lipase manufacturing method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing lipase by yeast.

[Prior Art] Regarding the lipase, which is a lipolytic enzyme, although the discovery was old, there was a delay in research compared to other enzymes. However, recently, industrial use is expanding in various fields such as detergents, ester synthesis reagents, pharmaceuticals, and pitch decomposers for papermaking, and their usefulness is beginning to be recognized again.

Penicillium is used for industrial lipase production by microorganisms.
(Penicillium) genus, Aspergillus (Aspergillus) genus, Rhizopus (Rhizopus) genus and other filamentous fungi, Pseudomonas (Pseudomonas) genus and other bacteria are known. However, yeast production of lipase
(Candida) and other few strains are known.

[Problems to be Solved by the Invention] The production of lipase by a filamentous fungus has a long culture period due to the nature of the fungus, and there are many disadvantages in terms of productivity. Also, in liquid culture and the like, troubles due to elongation of hyphae are likely to occur. On the other hand, bacterial production requires a large amount of energy to separate bacterial cells. 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-producing ability is
Candida cylindrace
a) or Candida lipholy
Only a few strains such as tica) are known.

Therefore, the present invention provides a method for producing a lipase by a yeast strain other than Candida, that is, a method for producing a lipase by a yeast strain that is not known to have lipase-producing ability in the past.

[Means for Solving the Problem] The present inventors conducted a wide-range screening in the natural world for the purpose of obtaining a lipase-producing strain by a yeast other than the genus Candida. As a result, it was found that a strain belonging to the genus Hansenula isolated from wild bird feces produces highly active lipase.

The mycological properties of this strain are as follows.

Growth on 5% malt extract agar medium (25 ° C., 30 days); Cells are spherical and slightly elongated. The size is 1.9 ~
It is 4.1 × 2.1 to 6.1 μm. It becomes a single cell, but sometimes it forms a small lump and becomes a grape shape. It grows butyrous and has a slight tan color.

Growth on Dalmau plate (25 ° C, 7 days); Growth under cover glass is vigorous, and pseudohyphae diverge. It grows aerobically and is white to slightly yellowish-brown in color, generally butylow. The cells are smooth and have bright light. The colony is whole-edged. Always produce a little scented ingredients.

Sporulation: Heterozygous sexual reproduction occurs but forms a diploid that becomes a sporangia. Diploid cells directly become asci and 1-4
Form hat-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; galactose ± sucrose + maltose + cellobiose + trehalose + lactose + raffinose + soluble starch + D-arabinose + ± D-ribose ± L-rhamnose-erythritol + ribitol ± D-mannitol + succinic acid + citric acid + inositol-L-sorbose-melibiose-merezitose + inulin-D-arabinose-α-methyl-D-glucoside + salicin + DL-lactic acid + assimilation of nitrate; + growth on vitamin-free medium; + yeast nitrogen basic medium + 10% Nacl + 5%
Growth on glucose; ± growth at 37 ° C; ± The mycological properties are: The yeasts; NJWKreger-van Ri
ji Groningen, 3rd edition (1984) described various properties of Hansenula anomala (Hansenula anomala).

In addition, Hansenula anomala, which is a strain that the present inventors have succeeded in isolating, was reported to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Microbiology Research Institute No. 11519 (FERM P-11519).
Has been deposited as.

The Hansenula strain used in the present invention comprises a medium consisting of components known in the art under aerobic and facultative aerobic conditions, an assimilable carbon source and a nitrogen source, and other micronutrient sources. It can be cultured in the medium containing. The preferred carbon sources are carbohydrates, lipids and other esters. The nitrogen source is preferably an inorganic substance such as nitrate, sulfate or ammonium salt, or yeast extract, corn steep liquor, soybean powder or other organic substance. The pH of the medium is 3-9, preferably 5-8. The culture temperature is 10 to 40 ° C, preferably 20 to 35
℃. 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. A concentrated crude enzyme solution can also be prepared by evaporation, reverse osmosis membrane, or the like. These crude lipase enzyme solutions can be purified by ammonium sulfate fractionation, dialysis, column chromatography, high performance liquid chromatography, or the like, or can be appropriately purified using a water-soluble solvent such as acetone or ethanol.

[Examples] The present invention will be described in detail with reference to Examples. The present invention is not limited to this.

(Example 1) Glucose 2%, soybean mill 2%, olive oil 2
_{%, (NH 4) 2 SO} 4 0.1%, K 2 HPO 4 0.5%, MgSO 4 · 7H 2
A liquid medium containing 0.1% and CaCO ₃ 0.5% was adjusted to pH 50, poured into an L-shaped test tube for 7 ml and sterilized by an autoclave. Preliminarily in the same medium at 30 ℃ for 24 hours,
The above medium was inoculated with 0.7 ml of Hansenula anomala cell fluid cultured by shaking in an L-shaped test tube. This was cultivated at 30 ° C. for 4 days with shaking, and the bacterial cells were separated by centrifugation to obtain a filtrate. Using 1 ml of this filtrate as a crude enzyme solution, the lipase activity was measured by the PVA emulsion method using olive oil as a substrate. The crude enzyme solution obtained had a lipase activity of 1 ml.
It was 10 U per unit.

Example 2 Casein Peptone 1.7%, Soybean Peptone 0.3
%, K ₂ HPO ₄ 0.25%, glucose 0.25%, Nacl
The medium containing 0.5% was adjusted to pH 5.5, 100 ml was poured into 500 ml Sakaguchi Kolben, and sterilized by an autoclave. 10 ml of pre-cultured bacterial cell fluid of AN2293-J that had been shake-cultured in the same medium at 30 ° C for 24 hours in an L-shaped test tube.
Was inoculated into Kolben. This was cultivated at 30 ° C. for 3 days with shaking, 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 crude enzyme solution has a lipase activity of 12 U / ml
Met.

EFFECTS OF THE INVENTION The present invention is the first clarification that a lipase can be produced by a yeast of the genus Hansenula, particularly by Hansenula anomala. The fact that the sex has been proved greatly contributes to the production and use of lipase.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kunio Hata 5-21-1 Oji, Kita-ku, Tokyo Inside the Central Research Laboratory, Jujo Paper Manufacturing Co., Ltd.

Claims (2)

[Claims] 1. A method for producing a lipase, which comprises culturing a yeast belonging to the genus Hansenula and having a lipase-producing ability in a nutrient medium, and collecting the lipase from the culture. 2. The yeast is Hansenula anomala.
The method for producing lipase according to claim 1, wherein the lipase is nomala).