KR20030035709A - Method for production of chitinase using Metarhizium genus microorganism - Google Patents

Abstract

PURPOSE: A method for producing chitinase using a microorganism Metarhizium sp. is provided, thereby the chitinase can be mass-produced, so that the chitinase can be useful for prevention of the growth of harmful insects in soil. CONSTITUTION: The method for producing chitinase comprises culturing a microorganism Metarhizium sp. in a medium, wherein the microorganism Metarhizium sp. is Metarhizium anisopliae HY-2(KCTC 0156BP); the medium contains carbon sources, nitrogen sources and inorganic substances; the carbon source is at least one material selected from the group consisting of glucose, corn starch, saccharose, molasses, wheat bran, starch, colloidal chitin and rice bran; the nitrogen source is at least one material selected from the group consisting of yeast extract, soybean flour, corn steep liquor, malt extract, P. brevitarsis powder and peptone; and the inorganic substance is at least one material selected from the group consisting of KCl, MgSO4, FeSO4, K2HPO4 and NaNO3.

Description

Method for production of chitinase using Metarhizium genus microorganism}

The present invention is a method for producing chitinase using a microorganism of Metarhizium , a carbon source, a nitrogen source and a mineral for producing the chitin microorganism or chitinase produced by the same A medium, a method for mass production of microorganisms in the medium and chitinases produced by using the medium, and a method for controlling soil pests using the microorganisms in the medium and chitinases produced by the medium .

Insects are adaptable to the environment and are currently the largest species of life on earth (Wilson, O. and FN Peter., Biodiversity ., 1989, Washington, DC Natl. Acad. Press). Some of these insects are causing a great deal of loss in a variety of commercially important agricultural crops, and chemical pesticides have long been used to control these pests, but chemical pesticides are beneficial parasites in beneficial insects and pests due to their broad spectrum. Undesired microorganisms such as In addition, when the pesticide is often used, the target pest repeatedly exposed to the pesticide may have resistance to the chemical pesticide, and the chemical pesticide component has a disadvantage in that it is harmful to the human body.

On the other hand, the golf course is composed of a large area of grass, various wood and herbaceous plants not only differ in the appearance of pests, but also the trend of increasing the number of pests that adapt to this particular ecosystem each year. Among them, the Scarab is a photogenic pest that affects 42 and 186 species of plants, including the species Rossaceae, Salicaceae, Fagaceae, Betaceae, and Aceraceae. It adds to the leaves of trees and crops, and the larvae add to the roots of grass and are recognized as the main pests of golf courses. In addition, adult insects sneak into the green at night and escape, causing indirect problems that impede the flow of golf balls and hinder the flow of golf balls (Lee et al., 1997. Host plants and host preferences of mule beetles. (2): 156-165; Lee Dong-Woon 2000. A Study on the Correlation between Adoretus tenuimaculatus and Host Plants in Golf Course; Doctoral dissertation). Soil pests, including scarabs, which cause grass and other commercially important agricultural crops, are becoming problematic. The larvae of the scarab pests that live in the grass of the golf course not only kill the grass by directly applying the grass roots, but also indirectly destroy the grass by the algae that feed on the larvae. It's bad.

Organophosphorus insecticides that are currently reported for grasses to control pests of scarabs include mep emulsion, grome emulsion, and etope preparation, but the pesticides must be treated in egg incubators (1st stage). If the control period is missed, the control is not good, and the soil is weakened by pesticide treatment by soil irrigation in order to facilitate the penetration of pesticides in the turf soil, and there is a problem such as an increase in pesticide use due to frequent use of the pesticide. (Korean patent application 1999-15472).

In order to control soil pests in Korea, we are working hard to control not only scarab larvae that affects golf course grass and ginseng fields, but also greenhouse dust, cabbage moth, castrated moth, and rice hopper. Due to the problem of environmental pollution caused by the problem is difficult. Accordingly, the necessity of environmentally friendly pest control to minimize the damage caused by soil and water pollution by chemical pesticides used in the past, toxic and environmental hormone damage to humans, the appearance of resistant pests, reduction of useful natural enemies, etc. are increasing. .

As an environmentally friendly pest control, there is a method using insect pathogenic microorganisms. These insect pathogenic microorganisms have insecticidal properties specific to insects and can effectively control target pests without harm to humans, livestock or plants. There is a characteristic.

The epidermal layer, which accounts for most of the insect epidermal layer, is composed mainly of carbohydrate chitin and protein, but inorganic ions, lipids, pigments and enzymes are also found. The properties of the original epidermal layer are very different depending on the species of the insect species or the epidermal layer, because the properties of chitin and protein vary greatly depending on the amount of chitin and protein. Proteins influence the mechanical properties of the epidermal layer and chitin forms the backbone of the epidermal layer, so the properties of the epidermal layer vary depending on the length and tissue of the chitin molecules.

Chitin is abundant in the exoskeleton and fungal cell walls of all arthropods, including insects, and is a long-chain carbohydrate in which N-acetylglucosamine units are composed of β-1,4 bonds. Account for 30-50% of the total. In the case of insects, chitin is a constituent of the epidermal layer in the body wall, organ system, full length and rear intestine, and the body wall epidermal layer of the insect is composed of alpha-chitin in which chitin molecules run in opposite directions. In the chitin fibers, hydrogen bonds are formed in the same molecule as well as between molecules to form a thin chitin plate. All hydroxyl groups in the molecule as well as amide groups form hydrogen bonds. Normally 6-7 chitin molecules make one chitin plate, and three chitin plates form one chitin microfiber (2.5-3.0 mm in diameter) and maintain a stable structure by hydrogen bonding between chitin molecules in neighboring chitin plates. do.

An enzyme that degrades chitin is chitinase, which is generally a basic plant chitinase or exo-cleaving β-N-acetylglucosaminidase. And bacteria, yeast and cucumber chitinases (Henrissat, B., Drot. Seq. Data Anal ., 1990, 3: 523-526).

If insecticide microorganisms with insecticidal properties can decompose the outer epidermal layer of insects containing about 30-50% chitin, it can show an effective insecticidal effect. In other words, using insect pathogenic microorganisms that produce chitinase can effectively control pests.

Accordingly, the present inventors found that the microorganisms of Metarhizium produce chitinase, and produced a medium capable of mass-producing the microbial cells and chitinases produced by the microorganisms of Metarhizium and its microorganisms. The present invention has been completed by producing a large amount of chitinase to be produced and used to control soil pests.

An object of the present invention is a method for producing chitinase using a microorganism of Metarhizium , a medium for mass production of the microorganism and chitinase produced by the microorganism, And it provides a method for mass production of chitinases he produces and a method for controlling soil pests using the microorganisms in the metastasis and chitinases he produces.

Figure 1 is a photograph of the electron microscope ( Metharhizium anisopliae ) HY-2 strains observed,

Figure 2 is a photograph showing that the HY-2 strain in the medium anisophyll decomposes chitin in the chitin-containing medium,

Figure 3 is a graph showing the cell mass of the HY-2 strain in the medium anisophyl cultured in the liquid medium of the present invention,

■: 4 days after culture, 배양: 8 days after culture,

1: Saccharose 3%, NaNO ₃ 0.3%,

2: saccharose 3%, NaNO ₃ 0.3%, yeast extract 0.5%,

3: colloidal chitin 2%, NaNO ₃ 0.3%, yeast extract 0.5%,

4: Bran 2%, NaNO ₃ 0.3%, 5: Rice bran 2%, NaNO ₃ 0.3%,

6: starch 3%, NaNO ₃ 0.3%, 7: glucose 3%, NaNO ₃ 0.3%,

8: saccharose 3%, soybean meal 0.74%,

9: 3% saccharose, 0.52% of Pharmamedia,

10: saccharose 3%, white spotted flower powder 2%,

Figure 4 is a graph showing the degree of production of chitinase in the HY-2 strain cultured in the medium anisophyl cultured in the liquid medium of the present invention,

■: 4 days after culture, 배양: 8 days after culture,

1: Saccharose 3%, NaNO ₃ 0.3%,

2: saccharose 3%, NaNO ₃ 0.3%, yeast extract 0.5%,

3: colloidal chitin 2%, NaNO ₃ 0.3%, yeast extract 0.5%,

4: Bran 2%, NaNO ₃ 0.3%, 5: Rice bran 2%, NaNO ₃ 0.3%,

6: starch 3%, NaNO ₃ 0.3%, 7: glucose 3%, NaNO ₃ 0.3%,

8: saccharose 3%, soybean meal 0.74%,

9: 3% saccharose, 0.52% of Pharmamedia,

10: saccharose 3%, white spotted flower powder 2%,

5 is a graph showing the cell mass of the HY-2 strain and chitinase produced by the strain in the medium anisophyl cultured in the solid medium of the present invention.

●: propagules, ▲: spores,

■: chitin, ◆: chitinase

In order to achieve the above object, the present invention provides a method for producing chitinase by using a microorganism of the genus Metarhizium .

In another aspect, the present invention provides a medium for producing a large amount of the microorganisms in the medium and chitinase produced therein.

In addition, the present invention provides a method for controlling soil pests by using the microorganisms in the medium and chitinase produced therein.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for producing chitinase by using a microorganism of the genus Metarhizium .

The microorganism of the genus of the present invention is an effective insectogenic pathogenic microorganism having insecticidal properties against soil pests, and can be separated from soil samples in which insects or soil pests infected by insect pathogenic microorganisms inhabit. The inventors named the insect pathogenic microorganism isolated by the above method " Metarhizium anisopliae HY-2" and deposited it on March 10, 1995 to the Korea Biotechnology Research Institute Gene Bank ( Accession No .: KCTC 0156BP).

HY-2, which is isolated from the above-described insect pathogenic microorganisms, meta-analysium, produces chitinase, which degrades chitin when cultured in a medium containing chitin, and grows using chitin degradation products as a carbon source. As described above, chitinase is produced using a microorganism in the metabium that can grow using chitin as a carbon source (see FIG. 2 ).

In another aspect, the present invention provides a medium for producing a large amount of the microorganisms in the medium and chitinase produced therein.

The medium for producing a large amount of microorganisms in the metabium of the present invention and chitinase produced by the medium is prepared by mixing a carbon source, a nitrogen source, and a mineral with water.

In the present invention, one or more selected from the group consisting of glucose, corn starch, saccharose, molasses, bran, starch, colloidal chitin and rice bran can be used as a carbon source, and among them, glucose, corn starch, saccharose and molasses. It is preferable to use bran or rice bran, and more preferably to use bran or rice bran. In addition, as a nitrogen source, one or two or more selected from the group consisting of yeast extract, soy flour, corn immersion, malt extract, white spotted flower powder and peptone can be used, and among them, yeast extract, soy flour, corn immersion, malt extract Or it is preferable to use peptone, the mineral group is composed of potassium chloride (KCl), dicalcium phosphate (K2HPO4), magnesium sulfate (MgSO ₄ ), ferrous sulfate (FeSO ₄ ) and sodium nitrate (NaNO ₃ ) It is preferable to select one or more than from and use. The medium of the present invention has a concentration of 2-30% by weight of the carbon source; 1-10% by weight of the nitrogen source; It is preferable to include the concentration of the mineral in 0.01-2% by weight.

In addition, the medium for producing a large amount of microorganisms in the metabium of the present invention and chitinase produced by the medium is prepared by mixing bran and rice bran in water, and when using the bran and rice bran as described above, the nitrogen source and minerals are It does not need to be included. The medium of the present invention is preferably prepared by mixing the ratio of bran: rice bran: water in a ratio of 1-10: 1-10: 1-10.

When culturing the microorganisms in the medium using the medium prepared above, the mass of the microorganisms in the medium and the chitinase produced by the microorganisms in the medium.

In the medium of the present invention, strains and chitinases produced by the medium were continuously produced in the medium anisophyll until 25 days later, and the cultures of the strains using the medium of the present invention can be economically produced in large quantities. (See FIG. 5 ).

In addition, the present invention provides a method for controlling soil pests by using the microorganisms in the medium and chitinase produced therein.

The ability to decompose chitin, which is the main component that forms the outer wall of insects, can be used as an indirect indicator of insecticidal activity. The microorganism in the metabium of the present invention produces chitinase (see FIG. 2 ), and the chitinase Decomposes chitin, which occupies most of the outer epidermal layer of insects, and shows insecticidality against soil pests. Therefore, the microorganism of the genus of the present invention and chitinase produced by the present invention can be usefully used for soil pest control.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Isolation of Microorganisms from Soil

In order to isolate effective insect pathogenic microorganisms having insecticidal properties against soil pests, the present inventors combined a method of collecting insects killed by insect pathogenic microorganisms in the open air and a method of separating from a soil pest inhabited soil sample. Soil pests infected with insect pathogenic microorganisms were diluted in sterile water, 0.1 ml of the suspension was plated on microbial assay medium (DTM medium; Dermatophyte assay medium) as shown in Table 1 . In order to separate the microbial strain from the soil sample, 0.1 g of the collected soil sample was suspended in 5 ml sterile water and plated on a microbial assay medium. The microbial assay medium was incubated at 30 ° C. for 5 days, then separated and inoculated in a new microbial assay medium and cultured under the same conditions.

ingredient Volume (%) Glucose 2 Soytone One Phenolic Red 0.02 Cycloheximide 0.05 Chloheximide 0.01 Gentamycine 0.01 Agar 1.5 Acetone (w / w) 2 Distilled water 93.41

As a result, an effective insect pathogenic microorganism having insecticidal properties against soil pests was selected and identified as Metarhizium anisopliae . The present inventors named the insect pathogenic microorganism isolated above as "HY-2 in mesodium anisophyl", and deposited it on March 10, 1995 to the Gene Bank in Korea Research Institute of Bioscience and Biotechnology (Accession Number; KCTC 0156BP). ). The photo of observation of the HY-2 strain on the metaphysis anisophyl under an electron microscope is shown in FIG. 1 .

Example 2 Storage and Cultivation of HY-2 Strains in Metasidium Anisome

The present inventors stored the strains of the metastasis isolated in Example 1, yeast in a zapak (Czapek-Dox broth; DIFCO Co., Ltd.) as shown in Table 2 for pre-culture for culturing the strain in large quantities CDAY medium containing the extract was used.

ingredient Volume (%) Jacek-Dox broth Bacto saccharose 3 Sodium nitrate (NaNO ₃ ) 0.3 Dipotassium phosphate (K ₂ HPO ₄ ) 0.1 Manganese sulfate (MgSO ₄ ) 0.05 Potassium chloride (KCl) 0.05 Ferrous sulfate, FeSO ₄ 0.001 Yeast extract 0.5

Storage of the strains of the metabium was used CDAY plate medium containing 2% agar (agar). After 15 days of incubation at 26 ° C until the spores are formed, the medium containing the mycelia of microorganism is bloomed using a sterile stick reagent spoon to cut a block, and then the reagent spoon is perpendicular to each other. The three sides were taken and one side was taken slightly, and then placed in a container containing 10% glycerol and stored at a low temperature of -70 ° C. Microbial culture was autoclaved for 20 minutes at 121 ℃ in CDAY medium, and then inoculated with bacteria and shake culture at 180 rpm for 4 days at 26 ℃.

Example 3 Analysis of Chitinase Production of HY-2 Strain in Metaphysis Anisome

The present inventors made and identified a colloidal chitin medium containing chitin as the carbon source in order to determine whether HY-2 produces chitinase in the metaphysis anisofil isolated in Example 1 above. First, to prepare colloidal chitin, 20 g of chitin isolated from shrimp shells were dissolved for about 30 minutes by adding 200 ml of hydrochloric acid at 40 ° C. After the chitin was completely dissolved, 2 L of sterile water at 5 ° C. was added to precipitate, and the suspension of colloidal chitin was filtered through a filter paper, and the washing was repeated until pH was 5.0 to prepare colloidal chitin. Next, a colloidal chitin medium was prepared by mixing 2% agar with 10% colloidal chitin solution and sterilizing at 121 ° C. for 20 minutes.

The present inventors plated HY-2 strain in the metaphysis anisophyl to the colloidal chitin medium prepared above. Since colloidal chitin medium is the only medium containing chitin as a carbon source, microorganisms that produce chitin-degrading enzymes produce chitinase to decompose chitin and use chitin-decomposition products as carbon sources to confirm that microorganisms grow. By doing so, it can be seen that the microorganisms capable of surviving in the medium produce chitinase.

The present inventors confirmed that when the HY-2 strain was smeared on the metabolic anisophil in the colloidal chitin medium, a transparent ring was formed on the agar medium containing the colloidal chitin, and thus, the mesorium anisofil separated in Example 1 The HY-2 strain was found to be a microorganism producing chitinase ( FIG. 2 ).

Example 4 Preparation of Medium for Production of HY-2 Cells and Chitinase in Metadium Anisome

The present inventors prepared a medium capable of economically mass-producing chitinases produced in HY-2 cells in the meth-2 anisofil isolated in Example 1 and HY-2 in the meth-2 anisofil.

In order to mass-produce HY-2 cells in the metaphysis anisophyl, a medium for easy cultivation of liquid culture was prepared as Czapeck, yeast extract, chitin, wheat bran, rice bran as shown in Table 3 below. It was prepared using bran), starch, glucose (Glucose), cottonseed foil and white spotted flower powder (P. brevitarsis powder). Specifically, the medium was prepared by adding the constituents of Table 3 to the basic medium except for the bacto saccharose, sodium nitrate, and yeast extract in the zappa medium of Example 2.

Badge composition Configuration One Saccharose 3%, NaNO ₃ 0.3% 2 Saccharose 3%, NaNO ₃ 0.3%, Yeast Extract 0.5% 3 Colloidal Chitin 2%, NaNO ₃ 0.3%, Yeast Extract 0.5% 4 Bran 2%, NaNO ₃ 0.3% 5 Rice bran 2%, NaNO ₃ 0.3% 6 Starch 3%, NaNO ₃ 0.3% 7 Glucose 3%, NaNO ₃ 0.3% 8 Saccharose 3%, Soybean Meal 0.74% 9 Saccharose 3%, Pharmamedia 0.52% 10 3% saccharose, 2% white spotted flower powder Default medium K ₂ HPO ₄ 0.1%, MgSO ₄ 0.05%, KCl 0.05%, FeSO ₄ 0.001%

Example 5 HY-2 Cell Growth Proliferation in Metadium Anisome

The present inventors used the medium prepared in Example 4 to determine the degree of proliferation of HY-2 in mesorium anisophyl. Specifically, the strain was inoculated into 100 ml of each medium composition and then cultured in a 1 L Erlenmeyer flask. The spawn concentration was 3%, and cultured at 180 rpm and 26 ° C. The dry weight was quantified by recovering on the 4th and 8th day of culture. The dry weight was measured by centrifugation of 10 ml of the culture at 5,000 rpm for 15 minutes, after which the supernatant was removed and a precipitate was obtained. 5 ml of distilled water was added to the obtained precipitate, followed by centrifugation at 5,000 rpm for 15 minutes, the supernatant was discarded, and 2 ml of distilled water was added to the collected precipitate. After drying for 4 days the weight was measured.

As a result, the productivity of HY-2 cells was higher than the 8th day of cultivation in the medium anisophyl on the 4th day of culture except the Zappa base medium and the medium to which bran was added, thereby improving productivity by reducing the culture time by liquid culture. This was done ( FIG. 3 ). Cell production was highest in the medium containing P. brevitarsis powder, followed by yeast extract, cottonseed meal, rice bran, soybean meal, and glucose. Chitin, starch and bran had low cell productivity in the liquid phase. In the case of 2% white spotted flower powder, the yield of cells was particularly high due to the composition similar to that of natural insects. At this time, the amount of cells over 0.5 g per 10 ml medium was observed.

Example 6 Productivity Analysis of Chitinase

The ability to decompose chitin, which is the main component that forms the outer wall of insects, can be used as an indirect indicator of insecticidal activity. The present inventors have used chitin decomposition using a medium composition for mass production of chitinase prepared in Example 4. The productivity of the enzyme was analyzed.

The determination of chitinase was performed according to Yanai's method (Yanai, K., et al., J. Bacteriol ., 1992, 1747398-7460). Specifically, the reaction mixture containing 250 μl of 0.5% colloidal chitin, 250 μl of 0.2 M sodium acetate buffer (pH 4) and 500 μl of coenzyme solution was left at 37 ° C. for 2 hours. The mixture was centrifuged to obtain 500 μl of supernatant, then 100 μl of 0.8 M boric acid was added and 100 μl of 1 M NaOH solution was added to adjust the pH to 10.2. After the reaction tube was stirred for 3 minutes, 3 ml of DMAB (p-dimethylaminobenzaldehyde) solution (prepared by dissolving 1 g of DMAB in 100 ml of glacial acetic acid containing 1% hydrochloric acid) was added and allowed to stand for 20 minutes, followed by color development at 585 nm. Absorbance was measured. One unit of chitinase was defined as the amount of enzyme that produces N-acetyl-glucosamine corresponding to 1 μmole per minute.

As a result, the use of chitin as a medium showed a high enzyme production capacity. The results show that enzyme production was induced by chitin used as a substrate. And yeast extract, rice bran, white spotted radish ( P. brevitarsis ) powder showed good productivity ( Fig. 4 ), and also the carbon source and nitrogen source was found to play an important factor in the production of chitin degrading enzyme Could.

From the above results, in comparison with the cell productivity test and chitin degrading enzyme productivity test, it is preferable to add chitin to the media components under the condition of producing only chitin degrading enzyme, but in order to improve the bactericidal power for controlling pests, Considering the production amount of the enzyme and the media components that are easy to use industrially and economically, it can be seen that yeast extract and rice bran are good media.

<Example 7> Preparation and productivity analysis of the medium composition for cell growth and chitinase production

In the present invention, in order to mass-produce cell mass and chitinase simultaneously, a solid medium composition was prepared. Specifically, the industrial solid medium composition was mixed with 4 kg of wheat bran, 2 kg of rice bran and 3 kg of water for 6 minutes, and then sealed in a high-pressure sterilized plastic pack at 750 g. Thereafter, the mixture was autoclaved at 121 ° C. for 30 minutes and inoculated with 0.01-1% of the precultured liquid medium to a sufficiently cooled industrial medium. Cultivation of the strain was incubated with a constant temperature and humidity control at 26 ℃, 50% humidity and light conditions. 10 ml (3.38 X 10 ⁴ cfu / g) of pre-cultured cultures were inoculated and cultured in a constant temperature room at 28 ° C. Measurement of viable cell count, demand of propagules, and indirect by the amount of chitin, the main component of fungal cells Quantitative and production of chitinases were observed.

In order to measure the number of viable cells, antibiotic medium and general medium containing antibiotics were diluted from 10 ^-3 to 10 ^-7 times, respectively, and then inoculated with 100 µl and incubated at 26 ° C. Antibiotic medium was prepared by adding 1.5% / l growth inhibitor Oxygall (Oxygall) to Zappa agar medium (Czapck-Dox agar, DIFCO Co., Ltd.) and adding antibiotics in the ratios shown in Table 4 .

ingredient Amount (mg / ml) Ampicillin 50 Nakidix acid 10 Rose Bengal 50

In general medium, general contamination level was confirmed, and in microbiological assay medium containing antibiotics, the number of HY-2 strains was measured in the actual contaminant and metabium anisofil. Metaphysis anisole HY-2 strain was found to grow slower than contaminants including bacteria and after 2-3 days.

The number of propagules was calculated using a hemocytometer (hemocytometer, 100 μm depth) observed in a 400 magnification optical microscope. Indirect quantification of fungal cells by chitin amount was used as follows. The chitin was dried, ground in a mortar and then sieved through a 40 mesh sieve, and the sample was decomposed with 10 N HCl for 3 hours at room temperature, and then reacted at 80 ° C. for 24 hours to react with N-acetylglucosamine (N -acetylglucosamine). After the acetyl acetone reagent was added to the digested product and boiled for 20 minutes, the resultant was cooled and developed with ethanol and an Erhlich reagent for 10 minutes at 65 ° C., and the absorbance was measured at 530 nm.

As a result, it was confirmed that the HY-2 strain grows in the metaphysis anisophyl until after 25 days, and chitinase was continuously produced after 25 days. In the medium of wheat bran and rice bran, which is easy to use for industrial use, cells were produced at 1 × ^{10 8} cfu / g and chitinase was 370 mU / g. It can be seen that it can produce ( FIG. 5 ).

As described above, the microorganism of the genus of the present invention, when culturing the microorganisms of the medium using the medium of the present invention economically mass production of the microbial cells of the medium and chitinase produced by the microorganisms It can be easily used for soil pest control.

Claims (11)

Method of producing chitinase by using Metatarzium sp. The method of claim 1, wherein the genus microorganism is Metarhizium anisopliae HY-2 (Accession Number; KCTC 0156BP). A medium for producing a microorganism of claim 1, which is produced by mixing a carbon source, a nitrogen source, and a mineral with water, or a chitinase produced by the same. 4. The medium according to claim 3, wherein the carbon source is one or more selected from the group consisting of glucose, corn starch, saccharose, molasses, bran, starch, colloidal chitin and rice bran. 4. The medium according to claim 3, wherein the nitrogen source is one or more selected from the group consisting of yeast extract, soy flour, corn immersion, malt extract, white spotted powder powder and peptone. The method of claim 3, wherein the mineral is selected from the group consisting of potassium chloride (KCl), magnesium sulfate (MgSO ₄ ), ferrous sulfate (FeSO ₄ ), dicalcium phosphate (K2HPO4) and sodium nitrate (NaNO ₃ ). A medium characterized by one or more than one. 4. The method of claim 3, wherein the carbon source, the nitrogen source and the inorganic material have a concentration of 2-30 wt%; 1-10% by weight of the nitrogen source; A medium comprising the inorganic concentration of 0.01-2% by weight. A medium for producing a microorganism of the metabium of claim 1, which is prepared by mixing bran and rice bran in water or a chitinase produced by the same. The medium according to claim 8, wherein the medium is prepared by mixing a ratio of bran: rice bran: water in a ratio of 1-10: 1-10: 1-10. A method of producing a large amount of microorganisms in the metabium of claim 1 or chitinases produced by the medium composition of claim 3. A method of controlling soil pests using microorganisms in metarium or chitinase produced by them.