JP2523700B2 - Method for producing ester hydrolase - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing an ester hydrolase, and more specifically, culturing a plant pathogenic filamentous fungus in a medium containing higher fatty acids or their esters, The present invention relates to a method for efficiently producing a hydrolase.

[Prior Art and Its Problems] Generally, filamentous fungi that infect plants are known to produce an ester hydrolase that hydrolyzes an ester compound present on the plant surface. It has a function of degrading the above long-chain fatty acid ester and the long-chain fatty acid ester polymer.

That is, the filamentous fungus dissolves the surface of the plant and therefore is used as an agrochemical potency enhancer to enhance the agrochemical efficacy by simultaneously spraying the agrochemical at the time of agrochemical application (JP-A-61-178907) and decomposition of solid fatty acid ester, Utilization for organic synthesis reaction is considered. However, it is known that these filamentous fungi do not produce esterases under normal culture conditions, and it has been considered to add a polyester layer on the plant surface as an enzyme inducer to the medium for obtaining the enzyme. However, there are problems that it is difficult to obtain a large amount of the polyester layer and the production efficiency of the enzyme is low.

[Means for solving problems]

As a result of intensive studies to solve these problems, the present inventors have found that these filamentous fungi can efficiently produce an ester-degrading enzyme in a short time by culturing in a medium containing a higher fatty acid or its ester. Then, the present invention has been completed.

That is, the present invention relates to Colletotrichum
Genus, and culturing plant pathogenic filamentous fungi selected from the group consisting of the genus and Greosporium (Gloeosporium) in a medium containing higher fatty acids or their esters, and collecting the ester hydrolase fraction from the culture, The present invention also provides a method for producing an ester hydrolase fraction that hydrolyzes an ester compound present on the surface of a plant.

The phytopathogenic filamentous fungus according to the present invention is a filamentous fungus that invades while degrading the polyester layer on the plant surface, and is selected from the group consisting of the genus Colletotrichum and the genus Gloeosporium.

The higher fatty acid used in the present invention is preferably a saturated or unsaturated fatty acid having 12 to 26 carbon atoms, and more preferably
16 to 24 unsaturated fatty acids, higher fatty acid esters as esters of the above fatty acids and various alcohols, such as lauryl alcohol, nonyl phenyl alcohol,
Examples thereof include esters with alcohols such as pentaerythritol, ethylene glycol, glycerin, sorbitol, mannitol, glucose and mannose, and higher fatty acid esters of glycerin are preferable.

In the present invention, the medium for culturing the filamentous fungus can optionally contain a component other than the above, for example, an inorganic salt, a carbon source, a nitrogen source and the like. As the inorganic salt, a combination of sodium nitrate, magnesium sulfate, potassium phosphate, sodium phosphate, potassium chloride, ammonium sulfate, ferrous sulfate, calcium pantothenate, calcium chloride, boric acid, zinc sulfate, copper sulfate, molybdic acid, etc. ,
As the carbon source and the nitrogen source, potato extract, yeast extract, gazein peptone, soybean peptone, tributypeptone, casamino acid, meat extract, malt extract, potato extract, ammonium salt, starch, dextrin, glucose, fructose, mannose. , Sucrose, lactose, glycerin and the like.

In the present invention, the concentration of higher fatty acid or its ester in the medium varies depending on the type of bacterium and the type of ester, but is preferably 0.1 to 50 g /, and the concentration of the inorganic salt is
The concentration of the carbon source and the nitrogen source is preferably 50 g / g to 0 to 50 g /. The culture temperature may be in the range of 20 ° C to 34 ° C, preferably 24 ° C to 28 ° C. The pH in the medium is preferably in the range of 3.5 to 8.5.

In the culture of the present invention, the main culture may be carried out directly, but it is preferable to carry out the preculture to sufficiently proliferate the bacterial cells, and then to transfer them to the main culture medium. When performing preculture, higher fatty acids or their esters may be added to the medium from the beginning, but in the preculture, the cells are cultured in a carbon source other than higher fatty acids or their esters, and the cells are cultured according to a conventional method. After separation from the above, the cells may be washed with an aqueous medium, such as a phosphate buffer, and then the cells may be transplanted to the main culture medium. The ester hydrolase produced in the medium may be isolated and purified using a conventional method such as an ion exchange resin column if necessary, but the medium is concentrated by performing ultrafiltration or the like. You can use it.

Next, a method for measuring the activity of the ester hydrolase (esterase) obtained in the present invention is shown.

(1) Esterase activity Using para-nitrophenyl butyrate as a substrate, the amount of para-nitrophenol produced after the enzymatic reaction is quantified by measuring the absorbance at 405 nm.

<Method> Paranitrophenyl butyrate (manufactured by Hanai Chemical) 0.42 mM
(In the final reactor), Emulgen 810 (made by Kao) 2m
g, pH 6.8, using 2.8 ml of 100 mM phosphate buffer and 0.2 ml of enzyme solution or culture solution, and using a reaction solution having a final volume of 3 ml, the change in absorbance at 405 nm is recorded at 28 ° C.
The absorbance of para-nitrophenol at 405 nm is measured in advance, and the amount of para-nitrophenol produced by hydrolysis is produced from the amount of change in absorbance during the enzymatic reaction.

The activity was indicated by the amount of enzyme that produced 1 U = 1 µmol of para-nitrophenol in 1 minute.

(2) Plant surface polyester-degrading activity 0.2 mg of tritium-labeled cucumber epidermis was treated with pH.
After adding 50 μ of 7.8, 100 mM phosphate buffer, add 50 μ of enzyme solution or concentrated culture solution and perform the reaction at 30 ° C.
After completion of the reaction, an appropriate amount of hydrochloric acid is added to acidify the reaction solution, and then ether extraction is performed, and the amount of tritium as an enzymatic decomposition product transferred to the ether layer is measured by a liquid scintillation counter.

For activity, Was calculated and compared as relative activity.

〔Example〕

Next, the present invention will be described in more detail with reference to Examples.
The invention is not limited to these examples.

Example 1 2 g of a yeast extract (manufactured by Difco) was added to a hot water extract of 200 g of potato, and the mixture was adjusted to 1 with distilled water.
Sterilize for 15 minutes at ℃, 1 × 10 ⁴ spores of Colletotrichum lagenarium
The cells were inoculated at a concentration and shake-cultured at 27 ° C for 2 days to give a preculture. The culture was filtered, MgSO ₄ · 7H ₂ O only cells
0.5g, K ₂ HPO ₄ 1.0g, KCl 0.5g, NaNO ₃ 2.0g, FeSO ₄ / 7H ₂ O
After transplanting into a sterilized medium containing 0.01 g, olive oil 1.5 g, and water 1, it was cultured at 27 ° C. for 10 hours.

The esterase activity was measured by the method described above using 200 μl of the culture solution. The plant surface polyester degrading activity was measured by the method described in the preceding paragraph using the supernatant of the culture solution treated with 80% acetone, dialyzing the resulting precipitate and dissolving it in 20 ml of a phosphate buffer as an enzyme solution.

 Table 1 shows the results.

Example 2 Colletotrichum laginalium
enarium) spores were pre-cultured for 2 days in the same manner as in Example 1, and then 1.5 g of triolein was added to the culture solution.
It was cultured at 27 ° C for 10 hours.

The esterase activity and plant surface polyester degrading activity of the culture solution were measured in the same manner as in Example 1.

 Table 1 shows the results.

Example 3 Colletotrichum Greospolioides
chum groeosporioides) spores were precultured for 2 days in the same manner as in Example 1, and then 1.0 g of eicosapentaenoic acid was added.
Was added to the medium and the cells were further cultured for 10 hours at 27 ° C.

The esterase activity and plant surface polyester splitting activity of the culture broth were measured in the same manner as in Example 1.

 Table 1 shows the results.

Comparative Example 1 Colletotrichum laginalium
Enarium) spores were pre-cultured for 2 days in the same manner as in Example 1, and then the cells were separated from the pre-cultured solution, and only the cells were transferred to a medium containing a potato extract and a yeast extract. After transplantation, the cells were cultured at 27 ° C for 10 hours.

The esterase activity and plant surface polyester degrading activity of the culture solution were measured in the same manner as in Example 1.

 Table 1 shows the results.

Comparative Example 2 Colletotricum Greos Polioides
Spores of chum groeosporioides) were pre-cultured for 2 days in the same manner as in Example 1, then 1.0 g of plant epidermal polyester membrane was added, and the mixture was cultured at 27 ° C. for 10 hours.

The esterase activity and plant surface polyester degrading activity of the culture solution were measured in the same manner as in Example 1.

 Table 1 shows the results.

Claims (1)

(57) [Claims] 1. A phytopathogenic filamentous fungus selected from the group consisting of the genus Colletotrichum and the genus Gloeosporium is cultivated in a medium containing higher fatty acids or their esters, and the ester hydrolase is extracted from the culture. A method for producing an ester hydrolase fraction for hydrolyzing an ester compound existing on the surface of a plant, which comprises collecting the fraction.