WO2003038081A1

WO2003038081A1 - Method for production of chitinase using metarhizium genus microorganism

Info

Publication number: WO2003038081A1
Application number: PCT/KR2002/002041
Authority: WO
Inventors: Ho Yong Park; Kwang Hee Son; Dong Ha Shin
Original assignee: Insect Biotech Co.,Ltd.
Priority date: 2001-11-02
Filing date: 2002-11-02
Publication date: 2003-05-08
Also published as: KR20030035709A

Abstract

The present invention relates to a method for production of chitinase using a Metarhizium genus microorganism and a culture medium to produce said microorganism. The medium of the present invention can be used for the mass-production of a Metarhizium genus microorganism or chitinase produced by the microorganism to control the soil pests.

Description

METHOD FOR PRODUCTION OF CHITINASE USING METARHIZIUM GENUS MICROORGANISM

FIELD OF THE INVENTION

The present invention relates to a method for controlling soil pests, more particularly to a method for controlling soil pests using a Metarhizium genus microorganism producing chitinase.

BACKGROUND ART OF THE INVENTION

Insects have excellent ability to adapt to environment and have the widest species on earth (Wilson, 0. and F.N. Peter., Biodiversi ty. , 1989, Washington, D.C. Natl. Acad. Press) . Some of those insects harm various major crops. Chemical insecticides have been used to control such pests. But such chemical insecticides have killed not only pests but also useful insects and parasites living in pests because of their wide spectrum. In addition, target pests cannot be controlled anymore by having resistance against chemical insecticides owing to the repeated exposure on them. Further, chemical insecticides are harmful for human body as well.

Various plants, trees and grasses are growing widely in golf courses, so that the appearance of pests is unique and the number of pests adapting to such ecosystem is increasing year after year. As a kind of soil pests, Mimela splendems (gold bug) is a euryphagous pest harming plants (42 families, 186 species), especially plants of Rossaceae, Salicaceae, Fagaceae, Betulaceae and Aceraceae . Some adults of gold bugs harm leaves of crops and plants and larvae do roots of various crops and grasses, making them major target pest of golf clubs. Indirectly, adults of the gold bugs steal onto greens and away with discharges during the night, causing interruption of a course of a golf ball (Lee et al, Korean Journal of Applied Entomology, 1997, 36, 2, 156-165; Lee, PhD Thesis, 2000) . Particularly, the larvae of soil pests including gold bugs harming major crops and grasses are troublesome since they damage the roots of plants and grasses. The larvae of gold bugs developing and living in golf courses directly harm grass roots to death and indirectly provide themselves as feed for birds, causing digging up the grass. Therefore, they have a bad effect on the preservation of grass quality.

To control such soil insects like gold bugs, fenitrothion emulsion, chlorpyrifos-methyl emulsion and ethoprophos granules have been used. But those chemical pesticides have effects on only just-hatched larvae (the first larva stage) . Thus, catching the right time is essential for controlling those insects. By the way, those chemical pesticides weaken the grass and cause overdose (Korea Patent Application #1999- 15472) .

Great efforts have been made to control the soil pests like gold bugs, Encarcia formosa , Eretmocerus eremicus , Plutella xylostella , Spodoptera litura and Nilaparvata lugens . But using the conventional chemical pesticides causes not only high expense but also such problems that the destruction of ecosystem, the under water contamination, the residual toxicity in agricultural products and the appearance of insects having resistance. Thus, it is urgently required to develop an environment-friendly controlling method for controlling soil pests in order to minimize such problems .

As one way of environment-friendly controlling methods for pests, insect pathogenic microorganisms are now being used, which is characterized by working selectively for target pests only and no harming human, animals and plants.

Protoderm taking most part of the cuticle of insect is composed of chitin, a kind of carbohydrates, and proteins in the main, and includes other miner factors such as inorganic ions, lipids, pigments and enzymes. The property of protoder varies with insect species and types of cuticles. That is because the characteristics are much influenced by the chitin or protein content and consolidation process. Precisely, proteins control the mechanical properties of the cuticle and chitin forms the backbone of the cuticle, thus the characteristics of the cuticle depend on the length or the structure of chitin molecules.

Exoskeleton of every arthropod including insects and cell wall of fungi are rich in chitin. Chitin is a long chained carbohydrate in which N-acetylglucosamine units are bound by β -1,4 binding. 30-50% of protoderm of insects is chitin. Chitin is also a component of the cuticle of body wall, organ system, foregut and hindgut of an insect and particularly, body wall cuticle is composed of α -chitin in which most chitin molecules run opposite direction. Hydrogen bonds among molecules of chitin fiber produce a thin chitin plate. Every OH groups and amide groups of molecules build up hydrogen bonds. Generally, 6-7 chitin molecules make one chitin plate and 3 chitin plates form a chitin microfilament (2.5-3.0 mm in diameter). Hydrogen bonds are formed between chitin molecules from adjacent chitin plate, resulting in the stable structure. Chitinase is an enzyme decomposing chitin and is divided into three classes, which are basic plant chitinase, exo-cleaving β -N-acetylglucosaminidase and chitinase of bacteria, yeast and cucumber (Henrissat, B., Drot . seq. data Anal . , 1990, 3:523-526).

It is required for an insect pathogenic microorganism having insecticidal activity to decompose protoderm of insects containing chitin to the extent of about 30-50% in order to have excellent insecticidal effect. That is, using insect pathogenic microorganisms producing chitinase is an effective way to control pests.

The present inventors found out that a Metarhizium genus microorganism produced chitinase, and accomplished this invention by preparing medium for mass-production of the above microorganism and chitinase that the microorganism produced, and by using the same for controlling soil pests.

SUMMARY OF THE INVENTION

The present invention relates to a method for production of chitinase using a Metarhizium genus microorganism, a culture medium to produce said microorganism, a method for mass-production of the microorganism and chitinase using the medium and a method for controlling soil pests using the same. The medium of the present invention can be used for the mass-production of a Metarhizium genus microorganism or chitinase produced by the microorganism to control the soil pests.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing Metarhizium anisopliae HY-2 observed by an electron microscope,

FIG. 2 is a photograph showing that Metarhizium anisopliae HY-2 is decomposing chitin on chitin- contained medium,

FIG. 3 is a graph showing the quantity of Metarhizium anisopliae HY-2 cultured in a liquid medium of the present invention, I : collected 4 days after beginning culture, : collected 8 days after beginning culture,

1 : Saccharose 3%, NaN0₃ 0.3%,

2 : Saccharose 3%, NaN0₃ 0.3%, Yeast extract 0.5%,

3 : Colloidal chitin 2%, NaN0₃ 0.3%, Yeast extract 0.5%,

4 : Wheat bran 2%, NaN0₃ 0.3%,

5 : Rice bran 2%, NaN0₃ 0.3%,

6 : Starch 3%, NaN0₃ 0.3%,

7 : Glucose 3%, NaN03 0.3%,

8 : Saccharose 3%, Soybean meal 0.74%,

9 : Saccharose 3%, Pharmamedia 0.52%, 10 : Saccharose 3%, P. brevi tarsis powders 2%,

FIG. 4 is a graph showing the level of chitinase produced by Metarhizium anisopliae HY-2 cultured on the liquid medium of the present invention,

I : collected 4 days after beginning culture, HI : collected 8 days after beginning culture,

1 : Saccharose 3%, NaN0₃ 0.3%,

2 : Saccharose 3%, NaN0₃ 0.3%, Yeast extract 0.5%, 3 : Colloidal chitin 2%, NaN0₃ 0.3%,

Yeast extract 0.5%,

4 : Wheat bran 2%, NaN0₃ 0.3%,

5 : Rice bran 2%, NaN0₃ 0.3%,

6 : Starch 3%, NaN0₃ 0.3%, 7 : Glucose 3%, NaN03 0.3%,

8 : Saccharose 3%, Soybean meal 0.74%,

9 : Saccharose 3%, Pharmamedia 0.52%,

10 : Saccharose 3%, P. brevi tarsis powders 2%,

FIG. 5 is a graph showing the quantity of

Metarhizium anisopliae HY-2 cultured on the solid medium of the present invention and the level of chitinase produced by the bacteria as well. • : Propagules, A : Spore, I : Chitin, ♦: Chitinase DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for production of chitinase using a Metarhizium genus microorganism. The present invention also provides a medium for mass-production of a Metarhizium genus microorganism and chitinase produced thereby.

The present invention further provides a method for controlling soil pests using a Metarhizium genus microorganism and chitinase produced thereby.

Further features of the present invention will appear hereinafter.

The present invention provides a method for production of chitinase using a Metarhizium genus microorganism.

As an insect pathogenic microorganism having effective insecticidal activity to soil pests, a Metarhizium genus microorganism of the present invention can be separated from insects killed by the infection with insect pathogenic microorganisms or from soil samples where soil pests are inhibiting. The present inventors named the separated insect pathogenic microorganism "Metarhizium anisopliae HY-2" and deposited at Gene Bank of Korea Research Institute of Biosciene and Biotehnology on March 10, 1995 (Accession No : KCTC 0156BP ) .

When being cultured on chitin-contained medium, Metarhizium anisopliae HY-2, a kind of insect pathogenic microorganisms, produces chitinase and grows by using degradation product of chitin as a carbon source. Therefore, it is possible to produce chitinase by using the Metarhizium genus microorganism that is growing using chitin as a carbon source (see FIG. 2) .

The present invention also provides a medium for mass-production of a Metarhizium genus microorganism and chitinase produced thereby.

The medium of the present invention for mass- production of a Metarhizium genus microorganism and chitinase produced thereby is prepared by mixing carbon source, nitrogen source and inorganic elements with water.

As a carbon source, one or more components selected from a group consisting of glucose, cornstarch, saccharose, molasses, starch, colloidal chitin and rice bran can be used in this invention and it is preferable to use glucose, cornstarch, saccharose, molasses, wheat bran or rice bran. It is more preferable to use wheat bran or rice bran. As a nitrogen source, one or more components selected from a group consisting of yeast extract, soybean flour, corn-immersion, malt extract, P. brevi tarsis powders and peptone can be used in this invention and it is preferable to use yeast extract, soybean flour, corn-immersion, malt extract or peptone. As an inorganic element, one or more components selected from a group consisting of potassium chloride (KC1) , dipotassium phosphate (K₂HP0₄) , magnesium sulfonate (MgS0₄) , ferroic sulfate (FeS0₄) and sodium nitrate (NaN0₃) are preferably used and sodium nitrate is more preferred. The preferable ratio of carbon source to nitrogen source to inorganic elements is 2- 30% weight% : 1-10 weight% : 0.01-2 weight% .

The medium of the present invention for mass- production of a Metarhizium genus microorganism and chitinase produced thereby is prepared by mixing wheat bran and rice bran with water. At this time, nitrogen source and inorganic elements might not be included. The preferable ratio of wheat bran to rice bran to water for the medium of the present invention is 1-10 : 1-10 : 1-10.

The mass-production of a Metarhizium genus microorganism and chitinase produced thereby is possible by culturing the Metarhizium genus microorganism in the medium prepared above.

As being cultured in the medium of the present invention, Metarhizium anisopliae HY-2 and chitinase produced thereby were continuously produced even after

25^th day from the beginning of the culture. Thus, it was confirmed that economical mass-production of the bacteria and chitinase was possible by culturing the bacteria in the medium of the present invention (see FIG. 5) .

The present invention further provides a method for controlling soil pests using the above Metarhizium genus microorganism and chitinase produced thereby

The insecticidal activity can be inferred by the capacity to decompose chitin, a major component that is forming cuticle of insects. Metarhizium genus microorganism of the present invention produces chitinase (see FIG. 2) -decomposing chitin that takes large part of protoderm of insects, that is, the microorganism has insecticidal activity to soil pests. Therefore, the Metarhizium genus microorganism and chitinase produced thereby can be effectively used for controlling soil pests.

EXAMPLES

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Separation of microorganisms from soil

In order to separate microorganisms that are useful for controlling soil pests, the present inventors collected dead insects infected with insect pathogenic microorganisms and at the same time, picked insects from soil samples where soil pests are inhibiting as well. Particularly, the present inventors crushed soil pests dead by infection with insect pathogenic microorganisms and diluted with sterile water. Took 0.1 m& of suspension and smeared on microorganism test medium (Dermatophyte test medium) as presented in Table 1. Suspended 0.1 g of soil samples with 5 mi of sterile water and smeared on microorganism test medium as well. Cultured thereof at 30 ^°C for 5 days and made a selection, which was inoculated on new DTM medium and further cultured under the same condition above.

As a result, the present inventors separated a microorganism showed excellent controlling effect on soil pests, and identified thereof. The microorganism was identified as a kind of Metarhizium anisopliae (FIG. 1) , named as "Metarhizium anisopliae HY-2" and deposited at Gene Bank of Korea Research Institute of Biosciene and Biotehnology on March 10, 1995 (Accession No: KCTC 0156BP) . A photograph of Metarhizium anisopliae HY-2 observed with an electron microscope is presented in FIG. 1.

Example 2: Preservation and cultivation of Metarhizium anisopliae HY-2

For the pre-culture of Metarhizium anisopliae HY- 2 of the present invention separated in the above Example 1 for further mass-culture and preservation, CDAY medium prepared by adding yeast extract to Czapek medium (Czapek-Dox broth, DIFCO) was used (Table 2) .

CDAY plate medium containing 2% agar was used for the preservation of microorganisms, which was cultured at 26°C for about 15 days until spores were formed. Cut the medium on which hyphae of microorganisms were blooming by sterilized stick-like test spoon with making blocks. Set the test spoon straight, so that three dimension on medium were touched by that. Untouched one side was taken and preserved in a container containing distilled water including 15% glycerol at -70^°C.

CDAY medium was autoclaved at 121 ^°C for 20 minutes, after which bacteria were inoculated thereto and shaking-cultured at 26^°C with 180 rpm for 4-5 days.

Example 3: Analysis of chitinase production of Metarhizium anisopliae HY-2

In order to confirm whether the Metarhizium anisopliae HY-2 separated in the above Example 1 can produce chitinase, the present inventors prepared colloidal chitin medium that had chitin only as a carbon source. In order to prepare colloidal chitin, dissolved 20 g of chitin collected from shrimp shells with 200 ml of hydrochloric acid at 40 ^°C for about 30 minutes. After complete dissolution, added 2 I of sterilized water at 5^°C and precipitated thereof. Filtered floating materials of colloidal chitin solution using a filter paper and then repeatedly washed thereof until pH was adjusted to 5.0, from which colloidal chitin was prepared. Next, added 2% agar to 10% colloidal chitin solution and sterilized thereof at 1210 for 20 minutes, resulting in the preparation of colloidal chitin medium.

The present inventors inoculated Metarhizium anisopliae HY-2 into the colloidal medium prepared above. Colloidal chitin medium contains chitin only as a carbon source. Therefore, in case a microorganism produces chitinase, it's growth by using degradation product of chitin as a carbon source can be detected. Thus, a microorganism that survives in the medium is proved to produce chitinase.

The present inventors concluded that the Metarhizium anisopliae HY-2 separated in the above

Example 1 was the microorganism producing chitinase after confirming that a transparent ring was formed when the Metarhizium anisopliae HY-2 was inoculated onto agar medium containing colloidal chitin (FIG. 2) .

Example 4 : Preparation of medium for production of Metarhizium anisopliae HY-2 and chitinase

The present inventors prepared a medium for economical mass-production of the Metarhizium anisopliae HY-2 separated in the above Example 1 and chitinase produced thereby.

In order to mass-produce Metarhizium anisopliae HY-2, prepared a medium for liquid culture, which contributed to easy mass-production, using Czapek medium, yeast extract, chitin, wheat bran, rice bran, starch, glucose, cottonseed and P. brevi tarsis powder as seen in Table 3. Particularly, prepared the medium by adding the components presented in Table 3 to the basic medium that was prepared by eliminating bacto saccharose, NaN0₃ and yeast extract from Czapek medium of the above Example 2.

Example 5: Analysis of the proliferation of Metarhizium anisopliae HY-2

The present inventors investigated the extent of proliferation of Metarhizium anisopliae HY-2 using the medium prepared in the above Example 4. Particularly, inoculated the microorganism into 100 ml of the medium, followed by culturing thereof in 1 Erlenmeyer flask at 26°C with 180 rpm. At that time, the concentration of inoculum was 3%. Collected the microorganism twice on the 4^th day and the 8^th day of culture, and measured dry weight thereof. For the measurement of dry weight, centrifuged 10 ml of culture solution at 5,000 rpm for

15 minutes. Then, removed supernatants and collected precipitates. Added 5 ml of distilled water to the precipitates, followed by centrifuging thereof at 5,000 rpm for 15 minutes. Removed supernatants and then added 2 ml of distilled water to the collected precipitates, which was then put in a dish whose weight was already measured, and dried thereof at 50 ^°C for 2-4 days. Finally, measured the weight.

As a result, the production of Metarhizium anisopliae HY-2 on the 4^th day of culture was higher than that on the 8^th day in every test groups except for the culture on Czapek basic medium and wheat bran- added medium, implying that the productivity was improved by shortening the culture period due to the liquid culture. P. brevi tarsis powder containing medium showed the highest productivity and yeast extract containing medium, cottonseed containing medium, rice bran containing medium, soybean meal containing medium and glucose containing medium followed in order. Each medium containing chitin, starch or wheat bran showed low productivity in liquid phase. When 2% P. brevi tarsis powder was added to a medium, the bacteria productivity was especially high on it (more than 0.5 g of bacteria/10 ml of medium) owing to its similar composition of insects in natural status (FIG. 3) .

Example 6: Analysis of chitinase productivity

The power to decompose chitin, a major component that is forming cuticle of insects, implies the insecticidal activity. Based on the thought, the present inventors analyzed the productivity of chitinase using the medium prepared in the above Example 4 for mass-production of chitinase.

To measure the level of chitinase, Yanai's method was used (Yanai, K. , et al., J. Bacteriol . , 1992, 174; 7398-7460) . Particularly, the inventors just left reaction mixture containing 250 μl of 0.5% colloidal chitin, 0.2 M sodium acetate buffer (pH 4) and 500 μl of coenzyme solution at 37 ^°C for 2 hours. After centrifuging the mixture, obtained 500 μl of supernatants. Added 100 μl of 0.8 M boric acid and then added 100 μ of 1 M NaOH solution to adjust pH to 10.2. Warmed the test tube in a double boiler for 3 minutes and then added 3 ml of p-dimethylaminobenzaldehyde (DMAB) solution (prepared by melting 1 g of DMAB in 100 ml of glacial acetic acid containing 1% hydrochloric acid) thereto, which was left for 20 minutes until coloring reaction was done. Finally, measured OD at 585 nm. One unit of chitinase was determined to be the amount of enzyme producing 1 μ mole of N-acetyl- glucosamine per minute.

As a result, the enzyme productivity was high when chitin was used for medium. That is, chitinase production was induced by chitin that was used as a matrix. As yeast extract, rice bran and P. brevitarsis powder were used for medium, the chitinase productivity was also good (FIG. 4), suggesting that the kind of a carbon source and a nitrogen source could be an important factor for the production of chitinase.

The present inventors compared the results of bacteria productivity test and chitinase productivity test. It was confirmed from the results that chitin was recommended to be added into medium in order to induce the production of chitinase only, while yeast extract and rice bran were preferable components for economical and industrial medium for the production of chitinase and bacteria to increase insecticidal activity to soil pests.

Example 7 : Preparation of medium composition for the production of chitinase and for the increase of bacteria, and analysis of the productivity thereof

The present inventors prepared solid medium composition in order to mass-produce bacteria and chitinase at the same time. Particularly, mixed 4 kg of wheat bran, 2 kg of rice bran and 3 kg of water for 6 minutes for the preparation of industrial solid medium composition and then weighted out the mixture by 750 g. Put that in a vinyl pack for autoclave and sealed thereof. Autoclaved thereof at 121 ^°C for 30 minutes. Inoculated 0.01-1% of pre-cultured liquid medium onto the completely cooled industrial medium and cultured thereof at 26^°C with 50% humidity and light condition. Inoculated with 10 ml of pre-cultured culture solution (3.38 x 10⁴ cfu/g) and further cultured in a culture room in which temperature was regulated to 28 ^°C. Observed the production of chitinase and measured the number of live bacteria, the demand of propagule and the level of chitin, a major component of fungi cells.

In order to measure the number of live bacteria, diluted general medium and antibiotics-containing medium from 10^~3 to 10^~7 respectively and then inoculated with 100 μl of pre-cultured solution. Cultured thereof at 26^°C. Counted the number of live bacteria from the next day. In order to prepare antibiotics-containing medium, put 1 . 5%/l oxygall, a growth inhibitor, in Czapck-Dox agar medium (DIFCO) and added antibiotics thereto as presented ratio in Table 4.

Confirmed the extent of general contamination in a general medium and measured the number of Metarhizium anisopliae HY-2 and contaminating bacteria in a microorganism test medium containing antibiotics. It was confirmed that Metarhizium anisopliae HY-2 grew slower than contaminating bacteria and could be detected at least 2-3 days later.

Measured the number of propagules by counting the number of spores observed with a 400X optical microscope using hemocytometer (100 μ depth) . Quantified fungal cells indirectly by measuring the amount of chitin. Precisely, dried chitin and crushed thereof with a mortar, after which filtered thereof using a 40 mesh sieve. Decomposed the obtained sample with 10 N HCl at room temperature for 3 hours. Reacted thereof at 80°C for 24 hours again and then decomposed with N-acetylglucosamine. Broiled the decomposed materials obtained above for 20 minutes in the presence of acetyl acetone reagent. After cooling down, induced coloring reaction with ethanol and Erhlich reagent at 65^°C for 10 minutes, so that measured OD at 530 nm.

As a result, Metarhizium anisopliae HY-2 was confirmed to keep growing until the 25^th day from the beginning of culture and chitinase was confirmed to be produced continuously even after the 25^th day of culture, lx 10⁸ cfu/g of the Metarhizium anisopliae HY-

2 and 370 mU/g of chitinase were produced in a medium composed of wheat bran and rice bran. Therefore, it was conclusively confirmed that microorganisms could economically be mass-produced by culturing with a medium composition of the present invention (FIG. 5) .

INDUSTRIAL APPLICABILITY

As described hereinbefore, the medium of the present invention can be effectively used for the mass- production of a Metarhizium genus microorganism and chitinase produced thereby to control the soil pests.

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Claims

What is claimed is

1. A method for production of chitinase using a Metarhizium genus microorganism.

2. The method for production of chitinase as set forth in claim 1, wherein the Metarhizium genus microorganism is Metarhizium anisopliae HY-2

(Accession No; KCTC 0156BP) .

3. A medium for production of a Metarhizium genus microorganism and chitinase produced thereby prepared by mixing carbon source, nitrogen source and inorganic elements with water.

4. The medium as set forth in claim 3, wherein the medium contains one or more carbon sources selected from a group consisting of glucose, cornstarch, saccharose, molasses, wheat bran, starch, colloidal chitin and rice bran.

5. The medium as set forth in claim 3, wherein the medium contains one or more nitrogen sources selected from a group consisting of yeast extract, soybean flour, corn-immersion, malt extract, P. brevi tarsis powders and peptone.

6. The medium as set forth in claim 3, wherein the medium contains one or more inorganic elements selected from a group consisting of potassium chloride (KC1) , magnesium sulfate ( gS0₄) , ferroic sulfate (FeS0) , dipotassium phosphate (K₂HP0₄) and sodium nitrate (NaN0₃) .

7. The medium as set forth in claim 3, wherein the medium comprises 2-30 weight% of carbon sources,

1-10 weight% of nitrogen sources and 0.01-2 weight% of inorganic elements.

8. The medium as set forth in claim 3, wherein the medium is prepared by mixing wheat bran and rice bran with water.

9. The medium as set forth in claim 8, wherein the ratio of wheat bran to rice bran to water is 1- 10 : 1-10 : 1-10.

10. A method for mass-production of a Metarhizium genus microorganism of claim 1 and chitinase produced thereby using the medium composition of claim 3.

11. A method for controlling soil pests using a Metarhizium genus microorganism or chitinase produced thereby.