KR100914475B1 - Novel Mortierella sp. DY-52 strain, chitin deacetylase produced therefrom, and uses thereof - Google Patents

Abstract

The present invention is a novel Mortierella sp. DY-52 (KACC 93037P) strain, chitin deacetylase (CDA) produced from the strain and its production method, Motiella sp. That produces chitin deacetylase, including the strain, its culture , Or a method for producing chitosan or chitosan oligosaccharides from a substrate such as chitin, chitin oligosaccharide, etc. using chitin deacetylase produced therefrom, and a motiella sp. That produces chitin deacetylase as an active ingredient thereof. Pesticides or nematicides containing chitin deacetylase produced therefrom.

Motiella, Chitin Deacetylase, Chitin, Chitin Oligosaccharide, Chitosan, Chitosan Oligosaccharide, Insecticide, Nematode

Description

New Motierella ｓｐ. Dv-52 strain, chitin deacetylase produced therefrom, and their use {Novel Mortierella sp. DY-52 strain, chitin deacetylase produced therefrom, and uses approximately}

1 is Motierella sp. Photos showing mycelial growth and spore morphology of DY-52;

2 is Motiella sp. 18S rRNA partial base sequence and homology of DY-52;

3 is Motiella sp. A graph showing the effect of carbon sources on CDA production of DY-52;

4 shows Motiella sp. A graph showing the effect of pH on CDA production of DY-52;

5 is Motiella sp. A graph showing the effect of shaking speed (rpm) on CDA production of DY-52;

6 shows Motiella sp. A graph showing growth of DY-52 and CDA production;

7A and 7B show Motiella sp. A graph showing the effect of pH and reaction temperature on the CDA activity of DY-52.

The present invention is a novel Mortierella sp. DY-52 (KACC 93037P) strain, chitin deacetylase (CDA) produced from the strain, and their use, and more specifically, Motiella sp. DY-52 strain, chitin deacetylase produced from the strain and a method for producing the same, motiella sp. Producing chitin deacetylase including the strain, culture medium thereof, or chitin deacetylase produced therefrom Method for producing chitosan or chitosan oligosaccharides and the like from a substrate such as chitin, chitin oligosaccharides, and the like, and Motiella sp. That produces chitin deacetylase, including the strain, as an active ingredient, its culture, or produced therefrom It relates to insecticides and nematicides containing chitin deacetylase.

Chitin, a raw material of chitosan, is a natural polymer present in shellfish such as shrimp and crabs. Producing a large amount of chitin to such an extent that the amount estimated to be about 100 billion tons of chitin are produced annually on Earth, and is not depleted resources (as described in gobyeongyeol, Kim Sang - Woo, Park, Young - Seo, 2002 Chitosan, Korea Institute of Science and Technology Information, 1- 5]). Currently, chitin and chitosan are used in various applications such as wastewater treatment, health supplements, livestock and fishery feeds, insecticides, fungicides, sewage treatment agents, cosmetic materials, various membranes, medical artificial skin, surgical sutures and chromatography resins. .

Chitosan is obtained by deproteination of crab and shrimp shells under alkaline conditions, demineralization under acidic conditions to prepare chitin, and then by deacetylation by chemical treatment with concentrated NaOH in a thermal bath (Chang, KL et. al., 1997 Heterogeneous N-deacetylation of chitin in alkaline solution, Carbohydr.Res . 303, 327-332]. However, currently used chemical methods have environmental problems such as generating a lot of energy and a large amount of waste alkaline solution, and it is difficult to produce chitosan having a specific molecular weight and deacetylation degree.

Chitin deacetylase (CDA: EC 3.5.1.41) is an enzyme that catalyzes the reaction of hydrolysis of N -acetamide bonds to convert chitin to chitosan, and has been described in many fungi (Heung-Sik Sohn et al. , 1999 Characterization of chitin deacetylase produced from Mucor rouxii, J. Korean Fish Soc . 32 (2), 121-126]; Aggeliki Martinou et al., 1993 Bioconversion of chitin to chitosan: Purification and characterization of chitin deacetylase from Mucor rouxii , Proc. Natl. Acad. Sci. USA 90, 2564-2568; NN Win et al., 2001 Shrimp chitin as substrate for fungal chitin deacetylase, Appl. Microbial Biotechnol . 57, 334-341; Iason Tsigos et al., 1995 Purification and characterization of chitin deacetylase from Colletotrichum lindemuthianum, The American Society for Biochemistry and Molecular Biology 270, 44, 26286-26291; and [A. Martinou et al., 1995 Chitin deacetylation by enzymatic means: monitoring of deacetylation processes, Carbohydrate Research 273 (2): 235-242])) And yeast (V. Bouriotis et al., 1996 Two sporulation-specific chitin deacetylase-encoding genes are required for the ascospore wall rigidity of Saccharomyces cerevisiae , J. Biol. Chem. 271, 31420-31425). Recently, bacteria that secrete chitin deacetylase have been isolated, and the enzyme has been purified and characterized (Jun Cai et al., 2006 Purification and characterization of chitin deacetylase from Scopulariopsis brevicaulis , Carbohydrate Polymers , In press , Corrected Proof, 1-7]).

CDA has emerged as an alternative to solve the shortcomings of the chitosan production process by chemical methods (Kuk, JH et al., 2005 Enzymatic characteristics and applications of microbial chitin deacetylase, Kor. J. Microbiol. Biotechnol. 33 ( 1), 9-15]). Currently, most of the studies have been done using CDA obtained from Mucor rouxii (Heung-Sik Sohn et al .; Aggeliki Martinou et al. And V. Bouriotis et al., Supra). have. In one example, WO 93/07262 (published on April 15, 1993) discloses the production and purification of immunoglobulins that react with chitin deacetylase isolated and purified from muco-luxi. However, research on better CDA resources than muco lux is needed. In addition, in order to industrially apply the enzymatic chitosan conversion method of chitin, it is necessary to develop a new strain that produces CDA with more selective and higher yield. In this case, it is more advantageous to develop fungi that have stable enzyme expression by growing for a long time than bacteria which have fast enzyme growth and fast killing time due to rapid growth.

On the other hand, Motiella sp. Is a fungus belonging to the fungus (Mucorales), previously known as arachidonic acid (achidonic acid) (Paek Jang et al., 2002 New Functional Biomaterial Technology Development Project; Commercial production of arachidonic acid using microorganisms Technology Development]; and [Lee, Seung-Kyo, 2002 Comparison of Growth Effect and Learning Ability by Ingesting Mortierella alpina Mycelia and Extracted Milk in Rats, Korean Journal of Food and Nutrition, Vol. 31, No. 6, 1084-1091]) γ-linolenic acid) (Yang Dong-hyun, 1989 production of γ-Linolenic Acid by Mortierella isabellina IFO 8183, Korean Journal of Food Science and Technology, 21, 6, 808-814), or α-glucosidase Or α-galactosidase. However, until now, no attempt has been made to use Motiella sp. In the production of chitin deacetylase.

The present inventors have carried out ongoing research to develop chitosan deacetylase and strains for producing chitosan by enzymatic methods. As a result, motiella sp. The DY-52 strain was isolated and identified for the first time, and the strain not only produces a highly active chitin deacetylase, but also secretes it into the medium, and the chitin deacetylase is used for the enzymatic production of chitosan, and microbial insecticides and pesticides. It confirmed that it can use efficiently as a nematode, and came to complete this invention.

Accordingly, a first object of the present invention is to make motiella sp. To provide a DY-52 strain.

A second object of the present invention is to provide a chitin deacetylase produced from the strain.

A third object of the present invention is a motiella sp. Having chitin deacetylase activity. To provide a culture of the DY-52 strain.

A fourth object of the present invention is to provide a method of producing chitin deacetylase by culturing the strain.

The fifth object of the present invention is to provide a substrate such as chitin, chitin oligosaccharides, and the like. The present invention is to provide a method for producing chitosan or chitosan oligosaccharide by treating with a strain, a culture solution thereof, or chitin deacetylase produced therefrom.

A sixth object of the present invention is Motiella sp. Which produces chitin deacetylase as an active ingredient. To provide a strain, a culture solution thereof, or an insecticide or nematicide containing chitin deacetylase produced therefrom.

First, the present invention is a Mortierella sp. Producing chitin deacetylase. DY-52 (KACC 93037P).

Second, the present invention is Motiella sp. Purified chitin deacetylase obtained by culturing DY-52 and having the following characteristics:

⒜ optimum temperature 50-60 ° C .;

⒝ optimum pH 4.5-5.0;

안정 stable for at least 1 hour at a temperature of 60 ° C. and pH 4.5;

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) measurement molecular weight 59 kDa and 54 kDa.

Third, the present invention has a motiella sp. It relates to a culture solution of DY-52.

Fourth, the present invention

1) Motiella sp. Shaking culture of DY-52 at 25-30 DEG C, pH 4.5-5.0, 100-150 rpm in a glucose-containing medium to obtain a culture solution;

2) separating and purifying chitin deacetylase from the culture solution;

It relates to a method for preparing chitin deacetylase, comprising the step. In the above method, it is more preferable to add cobalt ions (Co ²⁺ ) to the medium in step 1).

Fifth, the present invention provides a substrate selected from chitin, chitin oligosaccharide, Nod (Rhizobium nodulation) factor, xylan, or peptidoglycan, and more specifically, Motiella sp., Which produces chitin deacetylase. Erella sp. It relates to a method for producing chitosan or chitosan oligosaccharides and the like, comprising the step of treating with DY-52, its culture solution, or chitin deacetylase produced therefrom.

Sixthly, the present invention provides motiella sp., Particularly motiella sp., Which produces chitin deacetylase as an active ingredient. It relates to an insecticide or nematicide containing DY-52, its culture, or chitin deacetylase produced therefrom.

Hereinafter, the present invention will be described in detail.

In order to isolate a strain that produces highly active CDA capable of efficiently converting chitin to chitosan, the present inventors used dozens of soil samples collected from Damyang, Daegu, Gurye, Vietnam, Taiwan, etc. Species of CDA producing strains were isolated. Among them, Mortierella sp. DY-52 was isolated for the first time and classified by examining the morphological and genetic characteristics of this strain. Motiella sp. Of the present invention. DY-52 was deposited with the Korean Agricultural Culture Collection (KACC), Korea Agricultural Culture Research Institute, Rural Development Administration, 225, Seodun-dong, Suwon-si, Gyeonggi-do on May 8, 2006, and was assigned accession number KACC 93037P. .

Furthermore, the present inventors have identified the optimum culture conditions and chitin deacetylase production conditions of the strain, to provide a method for producing chitin deacetylase from the strain. In addition, by identifying the optimum temperature, optimal pH, activating factor and the like of chitin deacetylase produced from the strain, to provide a chitin deacetylase.

The strains according to the invention, their cultures and chitin deacetylases can be subjected to chitosan or chitosan oligosaccharide-producing substrates, from which chitosan or chitosan oligosaccharides can be efficiently produced. There is no particular limitation on the chitosan or chitosan oligosaccharide-producing substrate usable in the present invention, for example, any one of conventional substrates such as chitin, chitin oligosaccharide, Nod factor, xylan, peptidoglycan, or two or more thereof. Mixtures can be used. In addition, the strain and chitin deacetylase according to the present invention shows excellent insecticidal or nematicidal activity, it can also be utilized as a microbial insecticidal or nematicide.

Hereinafter, the present invention will be described in detail with reference to the following examples, but the scope of the present invention is not limited in any way by these examples.

Example 1 Isolation and Culture of CDA Producing Bacteria

To isolate CDA-producing microorganisms, approximately 100 soil samples were collected from Damyang, Daegu, Gurye, Vietnam and Taiwan, and the soil leachate was prepared using peptone-rose bengal agar medium (5.0 g peptone). , 10.0 g glucose, 1.0 g KH ₂ PO ₄ , 0.5 g MgSO ₄ · 7H ₂ O, 30.0 mg Rosebengal, 30.0 mg Streptomyces, 20.0 g agar and 1 L water) and incubated at 25 ° C. Strains were isolated first. The isolated fungi were inoculated in YPD medium (3.0 g yeast extract, 10.0 g peptone, 20.0 g glucose, 20.0 g agar and 1 L water), incubated at 28 ° C. and 150 rpm for 5 days, followed by centrifugation to incubate the culture supernatant and cells. The chitin deacetylase activity was investigated.

To measure chitin deacetylase activity, 0.1 ml of enzyme and 0.5 ml of water-soluble chitin (pH 4.5, DD 50%) in 25 mM sodium glutamate buffer as a substrate were mixed and incubated for 1 hour in a shaker at 50 ° C. Thereafter, the reaction was stopped by boiling in a water bath for 15 minutes and cooling with running water. The amount of acetic acid produced was measured using a Shimadzu GC 2014 (2 m × 2 mm glass column packed carbopack BDA, FID detector). One unit of enzyme was defined as the amount of enzyme that produced 1 μmol of acetic acid for one minute.

The culture supernatant and the cells obtained from the isolated strains were used to compare CDA activity by the above method, and finally, strain DY-52 having high enzymatic activity extracellularly was selected. The CDA activity and distribution of the DY-52 strain are shown in Table 1 in comparison to M. lux (ATCC 24905).

As shown in Table 1, the DY-52 strain showed much higher chitin deacetylase activity compared to M. luxi (ATCC 24905). Moreover, DY-52 strains secrete chitin deacetylase into the medium, unlike M. lux, which is advantageous in terms of enzyme purification and industrial application.

Example 2: Identification of Isolated Strains

Mycelial growth and spore morphology of the CDA producing strain DY-52 strain isolated from the soil were examined, and the results are shown in FIGS. 1 and 2.

In addition, in order to analyze the 18S rRNA sequence of the DY-52 strain, the DY-52 was isolated by shaking culture and the cells were recovered to separate genomic DNA. The 18s rRNA of the strain of the present invention uses 5'-CTTGGTCATTTAGAGGAAGT-3 '(SEQ ID NO: 1) as a forward primer and 5'-TCCTCCGCTTATTGATATGC-3' (SEQ ID NO: 2) as a reverse primer. Was cloned by polymerase chain reaction. 18S rRNA partial nucleotide sequence data (SEQ ID NO: 3) of the analyzed DY-52 strain was sequence homology using a Bethesda search in NCBI. 606 matched Motiella sp. 96% similarity with the strain (FIG. 2).

From these results, the DY-52 strain was found in Motiella sp. It was identified as DY-52, and it was deposited on May 8, 2006 by the Korea Agricultural and Biotechnology Research Institute, Rural Development Administration, Agricultural Biotechnology Research Center, 225, Seodun-dong, Suwon-si, Gyeonggi-do, and received accession number KACC 93037P.

Example 3: Motiella sp. Confirmation of CDA production condition of DY-52

1) Optimal culture conditions for CDA production

To investigate the CDA productivity according to the carbon source in the medium, the amount of CDA produced after shaking culture at 150 rpm at 28 ° C. with fructose, maltose, sucrose, chitin powder and swelling chitin was added instead of glucose (2.0%). Was measured. The results are shown in FIG. As shown in FIG. 3, the DY-52 strain showed the highest CDA production when glucose was added as a carbon source.

To determine the optimal pH of the medium, the amount of CDA produced after shaking culture at 28 ° C. was adjusted to pH 3.0, 4.0, 4.5, 5.0, 6.0, 7.0 and 8.0. The results are shown in FIG. As shown in Figure 4, it showed the highest CDA production in the medium pH 4.5 ~ 5.0.

In addition, in order to determine the optimum shaking rotation speed was adjusted to 50, 100, 150 and 300 rpm shaking shaking culture at 28 ℃ to measure the amount of CDA produced. The results are shown in FIG. As shown in Figure 5, it showed the highest CDA production at 100 ~ 150 rpm.

2) Motiella sp. CDA production of DY-52

Under optimum conditions, Motiella sp. The daily CDA activity of the DY-52 strain was measured. The results are shown in FIG. As shown in FIG. 6, the dry weight of the cells increased until 5-6 days after the start of the culture but decreased after 6 days, showing the highest CDA activity between 2-5 days.

Taken together, the results show that Motiella sp. Optimal culturing conditions for DY-52 strains were 100-150 rpm at 25-30 ° C., especially 28 ° C., pH 4.5-5.0, especially pH 4.5, in YPD medium containing 0.3% yeast extract, 1.0% peptone, 2.0% glucose, In particular, it can be seen that shaking culture at 150 rpm.

Example 4 Isolation and Characterization of Coenzymes

1) Isolation of Coenzyme

Motierella sp. DY-52 strains were inoculated in YPD (yeast extract / peptone / dextrose) medium and incubated at 28 ° C. and 150 rpm for 5 days. After 5 days, the culture was filtered with a vacuum filter to separate the culture supernatant and the cells. The culture supernatant from which the cells were removed was centrifuged again to remove the precipitate, and the remaining culture was used as coenzyme.

2) Optimum pH and Temperature

In order to determine the optimal pH and temperature of the coenzyme state CDA, enzyme activity was measured in the range of pH 3.5 to pH 7.5, 20 ℃ to 70 ℃. The results are shown in FIGS. 7A and 7B, respectively. As shown in Figures 7a and 7b, the optimum pH was 4.5-5.0, in particular pH 4.5, and the optimum temperature was 50-60 ° C, especially 60 ° C. From these results, Motiella sp. Since the optimal pH of CDA secreted by the DY-52 strain is different from the neutral and alkaline reported so far, it can be seen that the CDA secreted by the DY-52 strain is an enzyme different from the known CDA.

3) Influence of metal ions

The effect of metal ions on the enzyme activity was investigated and the results are shown in Table 3.

As can be seen from the results shown in Table 3, Ca ^{2 +} was increased, the activity was 26% when 10 mM added not affected at the time of 1 mM was added. Ni ²⁺ showed an increase of 11% at 1 mM addition but inhibited at 10 mM addition.

4) Activation of Enzyme by Cobalt Ion

The effect of CoCl ₂ on the activity of the enzyme was investigated and the results are shown in Table 4.

As can be seen from the results shown in Table 4, the addition of CoCl ₂ significantly increases the CDA activity, and the addition of 10 mM indicates that the activity is increased by 60%. From these results, Motiella sp. It can be said that DY-52 produces CoCl ₂ dependent CDA.

5) Molecular weight measurement

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was performed on the enzyme, and the molecular weight thereof was confirmed. The enzyme was found to have a molecular weight of 59 kDa and 54 KDa.

Example 5: Motiella sp. Application of DY-52

1) Motiella sp. Application of DY-52 CDA to Chitosan and Chitosan Oligosaccharide Production

In response to deacetylation of chitin and chitin oligosaccharides to bioconversion to chitosan and chitosan oligosaccharides, Motiella sp. DY-52 coenzyme was applied. At this time, N -acetyl-chitooligosaccharide and chitin having a polymerization degree of 2 to 7 were used as substrates. The results are shown in Table 5.

^* The literature [Jun Cai et al.]

^** supra, A. Martinou et al.]

As shown in Table 5, chitin oligosaccharides showed a deacetylation rate of 20.2 to 70.8% under the reaction conditions depending on the molecular weight. This is a higher deacetylation rate than the reported S. brevikaulis purified enzyme. Chitin, on the other hand, recorded a deacetylation rate of 9.1-18.3% under the same conditions. This is also higher than the reported deacetylation rate.

2) Motiella sp. Application of DY-52 as a pesticide and nematicide

For the purpose of applying the fungus to the control of pests and soil nematodes, first of all, it was infected with adult insects, eggs and pupa. Motierella sp. DY-52 grew slowly in eggs, chrysalis, and adult epidermis of Pabam moths. In particular, DY-52 inhibited egg hatching and pupa metamorphosis by 15-30%. This is Motiella sp. It is interpreted that CDA of DY-52 promotes softening of the cuticle layer of chitinous insects, thereby promoting the infiltration of mycelia and avoiding the host's resistance. In addition, Motiella sp. Even when inoculated with DY-52, the infection rate was 73.5% at 3 days, and mycelia were infiltrated into 33.9% of eggs to suppress hatching.

The results were obtained from Motiella sp. To demonstrate the applicability of DY-52 to microbial pesticides and nematicides.

As described above, the motiella sp. Of the present invention. DY-52 and the chitin deacetylase produced therefrom are capable of producing chitosan and chitosan oligosaccharides efficiently by enzymatic deacetylation of substrates such as chitin, chitin oligosaccharides, etc., as well as useful as microbial insecticides or nematicides. Can be utilized.

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Claims (9)

delete delete delete delete 1) Mortierella sp. DY-52 (KACC 93037P) was shaken in a glucose-containing medium in the presence of 0.01-10 mM cobalt ions (Co ²⁺ ) at 25-30 ° C., pH 4.5-5.0, 100-150 rpm to obtain a culture solution; 2) separating and purifying chitin deacetylase from the culture medium: Method comprising the step of, chitin deacetylase. 1) Mortierella sp. DY-52 (KACC 93037P) was shaken in a glucose-containing medium in the presence of 0.01-10 mM cobalt ions (Co ²⁺ ) at 25-30 ° C., pH 4.5-5.0, 100-150 rpm to obtain a culture solution; 2) Treating a substrate selected from chitin, chitin oligosaccharide, Nod (Rhizobium nodulation) factor, xylan or peptidoglycan with the culture obtained in step 1), or with chitin deacetylase separated and purified therefrom: Method of producing a chitosan or chitosan oligosaccharide, comprising the step. delete delete delete

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