KR20050088696A - Enzyme decomposing ahl, and method for controlling pathogenic disease using thereof - Google Patents

Abstract

The present invention uses a novel microorganism isolated from soil to decompose acylation homoserine lactone (AHLs), a density recognition signal substance, an enzyme having an activity to decompose AHLs derived from the microorganism, It is related to the control method.

The ecological approach of gram-negative microorganism control using actinomycetes culture medium according to the present invention will be able to effectively control pathogenic microorganisms while solving existing problems such as antibiotic resistance and pesticide residues.

Description

Acylated homoserine lactone degrading enzyme and disease control method using the same {Enzyme Decomposing AHL, and Method for Controlling Pathogenic Disease Using Thereof}

The present invention is a novel actinomycetes isolated from soil that decomposes acylated homoserine lactone (AHL), an enzyme having an activity of degrading AHLs derived from the actinomycetes and the isolated microorganisms. From a method for biologically controlling pathogenic microorganisms using enzymes.

Many Gram-negative bacteria are known to monitor population density by the Quorum-sensing mechanism and to regulate the expression of specific genes according to bacterial cell density. This signaling mechanism is determined by AHLs, which are small and diffuse signal molecules, which have a common structure of homoserine lactone rings, but differ in the length of acyl side chains and their substitution.

Quorum-sensing systems play an important role in regulating the expression of the pathogenic genes of pathogenic bacteria, including Vibrio, Erwinia carotovora and Pseudomonas aeruginosa . AHLs-mediated signaling mechanisms are widespread in many pathogenic bacteria and their mechanisms are very conserved, making them attractive targets for new anti-infective therapies.

Recently, in order to discover enzymes that inactivate AHL, AHL degradation strains were first separated from soil, and AHL degrading enzyme genes were identified from some strains. AHL can be decomposed into a substance that has no function as a signal by acylhomoserine lactonase (AHLase), which breaks down the ester bonds of AHL's lactone ring, and AHL acylase, which breaks down the amide bonds between the lactone ring and the acyl side chain of AHL. Various Bacillus cereu s bacteria, Arthrobacter sp. Caused by E. carotovora by Gram-positive bacteria, including IBN110 and Rhodococcus Plant-pathogenicity was attenuated, and transgenic plants expressing AHLase AiiA from Bacillus cereu s showed resistance to the plant pathogen E. carotovora . In addition, Gram-negative Variovorax parodoxus , which metabolizes AHL as the only carbon source, was found to be able to break down AHL with an enzyme that is believed to be an aminoacylase.

Quorum-sensing is known as a signaling mechanism that modulates a variety of biological functions mediated by AHLs in Gram-negative bacteria. Recently, there have been reported signal degradation bacteria and degradation enzymes that can control this. However, there is no known AHL degrading enzyme to date. The present invention relates to novel actinomycetes that degrade AHL, and AHL degrading enzymes secreted therefrom with high stability.

To date, antibiotics and chemical pesticides have been commonly used to control bacterial diseases in farms and crops. However, the use of such antibiotics and chemical pesticides have problems such as drug residues in the soil, killing of useful microorganisms, and emergence of antibiotic resistant strains. As a way to overcome these problems, a new method is emerging that targets the density recognition signal transmission system of pathogenic microorganisms.

Just as insects secrete a chemical called pheromone to attract spouses or signal aggression, microbes also secrete signs of disease-related pathogens in Pseudomonas aeroginosa . A representative signal material in gram-negative bacteria is AHL, which is mainly involved in the expression of genes that cause pathogenic flora and fauna pathogenic bacteria, thereby causing disease.

Types of AHL include OHHL (N-β-oxo-hexanoyl-L-homoserine lactone), OOHL (N-β-oxo-octanoyl-L-homoserine lactone), and ODHL (N-β-oxo-decanoyl-L-homoserine lactone), HHL (N-hexanoyl-L-homoserine lactone), and BHL ( N- butanoyl-homoserine lactone).

In the case of Pseudomonas aeroginosa , which causes respiratory infections in patients with fibrocystic cysts, the concomitant signaling mechanism by the acylated homoserine lactones has been elucidated. This is in progress.

As a prior patent on the control of microorganisms using perturbation of acylated homoserine lactone signaling system, a method for controlling pathogenic microorganisms by using acylated homoserine lactone as a medium for signaling between microorganisms (Korean Patent Application No. 10 -1997-706614) and a method for controlling bacterial pathogenic genes using proteins that inactivate AHL (PCT / SG99 / 00128). However, the Korean Patent Application No. 10-1997-706614 patent does not control pathogenic microorganisms through inhibition of acylated homoserine lactone itself, but uses furanone and derivatives thereof which are structurally similar to acylated homoserine lactone. The present invention relates to a method for controlling pathogenic microorganisms by competitively interfering with the binding of acylated homoserine lactones with regulatory proteins. The PCT / SG99 / 00128 patent also relates to a method for controlling pathogenic microorganisms using expression of signal genes. There has been no study on concomitant control using acylated homoserine lactone degrading enzymes themselves.

An object of the present invention is to provide a microorganism containing an enzyme that decomposes acylated homoserine lactone, which is a density recognition signal substance between microorganisms.

It is another object of the present invention to provide an enzyme having an activity of degrading AHLs derived from the microorganism.

In another aspect, the present invention is to provide a method for blocking the signal transmission between the microorganisms by decomposing the parallel signal substance using a stable enzyme from the isolated microorganisms.

In order to achieve the above object, the present invention provides Streptomyces sp. Strain M664 (KCTC 10605BP) having the ability to degrade acylated homoserine lactone, which is a microbial density recognition signal.

The strain was selected and separated by the following procedure.

First, about 600 actinomycetes samples were obtained from soils of various regions. Samples were incubated in G.S.G medium and G.S.M medium (see Example below), and then the culture was loaded onto a CV026 bioassay plate to find strains that reduce violacein pigment. Among the 12 samples in which the violacein pigment was found to be reduced, the samples showing the violacein pigment reducing activity in the aqueous layer after butanol and ethyl acetate solvent extraction were selected and the activity disappeared after 10 minutes of heat treatment at 100 ° C. This fact can be inferred that violacein pigment reduction is due to enzymatic activity. One actinomycete that finally produced a signal lyase was selected and named this strain M664.

The isolated strain M664 was identified as belonging to the genus Streptomyces by analysis of the 16S rDNA sequence.

As described in the following examples, the signal degrading enzyme from the isolated strain according to the present invention was a secreted protein secreted in the medium, and had a high resolution for acylated homoserine lactones having a long acyl side chain, and an acyl side chain. Regardless of the length and the substitution, the various acylated homoserine lactones such as OHL, DHL, and OdDHL can be decomposed.

The present invention provides a protein (enzyme) that catalyzes the degradation of AHL. AHL-degrading enzymes were relatively stable even at high temperatures of 50 ° C. and maintained for 5 days in seawater. Therefore, it can be seen that the culture of the strain having high stability can be used for the purpose of preventing, suppressing or treating diseases caused by pathogenic microorganisms.

To determine the pathogenic microorganism control ability of the culture of the strain Vibrio sp. Application to PT2 resulted in Vibrio sp. It was confirmed that the protease activity of PT2 was decreased. Indeed, the cultures of the strains were from Vibrio sp. It was confirmed that pathogenic microorganisms can be controlled by reducing the incidence caused by PT2.

In addition, Pseudomonas aeroginosa , which causes respiratory infections in patients with fibrocystic cysts, has been identified as OdDHL and BHL, and the production of BHL is known to be controlled by OdDHL. Therefore, the use of a degrading enzyme having excellent resolution to AHLs having long acyl side chains strongly suggests that it is possible to suppress and control animal diseases caused by Pseudomonas et al.

Therefore, the present invention, by spraying or inoculating the above-mentioned strain M664 or the AHL-degrading enzyme to the surface of the plant or animal, etc. to induce or enhance the resistance to pathogenic microorganisms to prevent the development of bacterial diseases of the plant or animal Provide a method of inhibiting or treating.

In another aspect, the present invention provides the above-mentioned strain M664 or the above-mentioned AHL-degrading enzyme as a main component of a plant or animal bacterial disease suppression-treatment, animal feed, fertilizer, pesticide containing the same.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1 Separation of Acylated Homoserine Lactone Degrading Microorganisms

(1) Separating Signal Substance Strains

In order to search for actinomycetes that degrade acylated homoserine lactone, CV026 bioassay plate was prepared by overlaying on HB to the final concentration of 50 nM Chromobacterium vioraceum CV026 strain cultured overnight at 30 ℃. About 600 actinomycetes isolated from the soil were collected in GSG medium (1% soluble starch, 2% Glucose, 2.5% soybean meal, 0.1% beef extract, 0.4% yeast extract, 0.2% NaCl, 0.025% K2HPO4, 0.2% CaCO3 / pH 7.2) and GSM medium (1% soluble starch, 2% glucose, 0.5% Molassese, 0.5% yeast extract, 0.5% peptone, 0.2% CaCO3 / pH 7.2) medium at 30 ℃, 7 days. Actinomycetes cultures were loaded on a paper disk, or directly on the CV026 bioassay plate, and the strains that reduced violacein pigments were examined.

As a result, a decrease in violacein pigment was confirmed from 12 cultures of about 600 actinomycetes samples. Among them, one actinomycetes cultured in G.S.M medium showed violacein pigment reduction activity in the aqueous layer after butanol and ethyl acetate solvent extraction, and the activity disappeared when the culture was heat-treated at 100 ° C for 10 minutes. This fact can be inferred that violacein pigment reduction is caused by enzymatic activity and not by low molecular weight substances. Thus, the actinomycetes, which produce a signal lyase, were selected and named M664. 1 shows that as the amount of M664 actinomycetes increased, the production of violacein pigment was decreased, that is, the inhibitory effect on HHL signaling was increased (FIG. 1). A is 10 μl in the figure; B is 20 μl; C is 30 μl; D is 40 μl; E is 50 μl; F is 60 μl; G, 70 μl; H means 80 μl of culture.

In addition, M664 strain was cultured in YEME medium for 5 days to investigate the AHL degradation activity. 50 μl of the M664 supernatant and 20 μM of DHL were mixed in the same volume. The reaction was stopped at 30 ° C. for 1 hour with slow stirring, and then the reaction solution was heated at 95 ° C. for 5 minutes. Reaction Solution 10 Μl was loaded into the holes of A. tumefaciens NT1 (PDCI51E33) -overlaid plate (bioassay solid medium) and cultured to determine the diameter (x) of the blue circle formed. The specific method of bioassay is described in (2) below.

The residual concentrations of AHLs were calculated using the following correlations obtained by analyzing the relationship between the diameter (x) of the generated violet circle (D) according to the DHL concentration in the bioassay solid medium. The correlation between AHLs and each type is as follows.

OHHL concentration (pmole) = 0.0335e ^1.3679x (reliability R ² = 0.9952)

OHL concentration (pmole) = 0.0137e ^1.7206x (reliability R ² = 0.99973)

DHL, pmole = 2.1114e ^1.3595x (Reliability R ² = 0.99873)

OdDHL concentration (pmole) = 0.0195e ^1.5185x (reliability R ² = 0.9937)

As the M664 strain grew, the activity of signal degrading enzymes from the culture medium increased, showed the maximum activity in the initial exponential growth phase, and maintained the degradation activity for 5 days of culture (FIG. 2).

So far, signal lyase has been reported to exist in the cytoplasm or cell membrane. The signal lyase from M664 is an extracellular secreted enzyme unlike conventional signal lyase.

(2) bioassay methodology

A. tumefaciens NT1 (pDCI41E33) incubated for 24 hours using AB minimal medium at 30 ° C. was overlaid on a bioassay solid medium made of AB minimal medium. After making a hole in a bioassay solid medium, 20 μl of the culture solution sampled for each time period was placed in the hole. After incubating the solid medium for bioasaay for 24 hours at 30 ° C., the diameter of the blue biolasin zone (violacein zone) formed around the hole was obtained to measure the amount of AHL remaining.

Example 2 Identification of Acylated Homoserine Lactone Degrading Microorganisms

16S rDNA sequencing and other analyzes were performed to identify the AHL degradation strain M664 isolated and selected in Example 1 above.

(1) 16S rDNA Sequencing

As a result of analyzing the 16S rDNA sequence of the strain isolated by the conventional method, the 16S rDNA sequence of the M664 strain showed 99% identity with Actinomycetes Streptomyces griseus .

(2) other analysis

Gram staining revealed that the isolated strain was Gram positive. In the actinomycetes containing Bennet medium, isolated strains formed hard colonies, which are common forms of actinomycetes, and aerial mycelium and spores during 2-3 days growth.

As a result of the above analysis, it was confirmed that the selected and isolated strain belongs to Streptomyces sp. Therefore, AHL-degrading M664 strains were isolated from Streptomyces sp. It was named M664 and was deposited on February 20, 2004 with the accession number KCTC 10605BP to the Biotechnology Research Institute Gene Bank (KCTC).

Example 3: Various AHL-degrading activity bioassay of M664 strain

Streptomyces sp. To determine the substrate range for AHL of the M664 strain, the culture was used to assay the resolution for various AHLs.

Among the substrates belonging to AHL, N-octanoyl-L-homoserine lactone (OHL), N-decanoyl-L-homoserine lactone (DHL) and OdDHL (N-3-oxododecanoyl-lactone) were purchased from Quorum Sciences, N-hexanoyl-L-homoserine lactone (HHL) and N-3-oxohexanoyl-L-homoserine lactone (OHHL) are known methods (Zhang, L., PJ Murphy, A. Kerr, and ME Tate. 1993. Agrobacterium conjugation and gene regulation by N- acyl-L-homoserine lactones.Nature (London) 362: 446-448).

The M664 strain was reacted with 50 μl of the supernatant obtained after 3 days in YEME medium and 20 μM of each substrate OHHL, OHL, DHL, OdDHL at 30 ° C. for 1 hour and heated at 95 ° C. for 5 minutes to stop the reaction. The culture medium was stopped in the reaction was loaded in the hole of A. tumefaciens NT1 (pDCI41E33) -overlaid plate and further cultured, and the size of the color zone was measured. The amount of residual AHLs is shown in FIG. 3 calculated from the relationship between the size of the color zone and the amount of AHLs (see Example 2 above).

As can be seen in Figure 3 strain M664 according to the present invention had the highest degradation activity against DHL. The enzyme unit for DHL was 640 U, and showed 150 U, 120 U, and 67 U of enzyme activity in the order of OdDHL, OHL, OHHL (FIG. 3). In this case, U was defined as the amount of AHLs decomposed for 1 hour by the culture supernatant Iμl (pmole). M664 seems to have a relatively low resolution for a short signal of the acyl side chain.

Since P. aeruginosa and Bulholderia glumae, which are known as major Dong-pathogenic strains, produce OdDHL and OHL, respectively, as signaling materials, AHL-degrading enzymes from the strain M664 according to the present invention may be useful for the treatment of these bacterial diseases.

Example 4: Temperature and pH Effects on AHL Degrading Enzyme Activity

Streptomyces sp. In order to investigate the temperature stability of AHL degrading enzyme from M664, the coenzyme concentrated with 60% ammonium sulfate was left for 30 minutes at each temperature, and the residual activity was examined using a DHL substrate (FIG. 4).

As a result, the enzyme activity of AHL degrading enzyme was maintained up to 50 ° C, and the activity was rapidly decreased at 60 ° C. Therefore, AHL degrading enzyme of M664 was found to be a stable enzyme up to 50 ℃.

The optimum reaction temperature was determined as the enzyme activity after 1 hour of reaction at each temperature. The AHL-degrading enzyme from M664 strain showed the maximum activity at the culture temperature of 30 ° C, and the degradation activity was 70% of the optimum activity even at 50 ° C. The degree was maintained.

The pH stability was measured by enzyme activity after leaving M664 coenzyme at 4 ° C. for 1 day at each pH, and the optimum pH by enzyme activity after 1 hour reaction at each pH. From M664 AHL-degrading enzyme was most stable at pH 6.0 and activity decreased rapidly at other pH (FIG. 5).

Meanwhile, AHL-degrading enzyme showed optimal activity at pH 7.0, and showed almost similar activity over pH 4.5-8.0 (FIG. 5).

From the experimental results of Examples 1, 2 and 3 above, it can be confirmed that the actinomycetes have the ability to decompose various ranges of AHLs by enzyme reaction. In addition, it can be seen that the signal decomposing enzyme has a high commercial stability because of its high stability against heat.

Therefore, by spraying or inoculating by injecting the recombinant microorganisms secreting the AHL-degrading enzyme and AHL-degrading enzyme according to the present invention onto the surface of a plant or animal, or injecting the AHL-degrading enzyme according to the present invention into an animal body, In the case of taking, etc., it can be inferred that the degrading enzyme can induce resistance to diseases or enhance resistance by decomposing signal material of pathogenic microorganisms. In addition, the strain according to the present invention, AHL-degrading enzymes and AHL-degrading enzymes, including the recombinant microorganisms that secrete the bacterial disease occurrence-treatment of plants or animals, the animal feed containing the same, fertilizers, pesticides, etc. It will be possible.

In addition to the use of the recombinant microorganism which secrete the AHL- lyase and AHL- lyase according to the invention, by a germ-based industrial (Pseudomonas aeroginosa) in Pseudomonas this to the respiratory tract of an animal, including humans induce fibrous cyst increase It would also be possible to manufacture pharmaceuticals that inhibit-treat disease development.

Example 5: HPLC Analysis of Reactive Vapor

The previously reported AHL degrading enzymes can be divided into AHL-acylase and AHL-lactonase types. AHL-acylase decomposes the acyl chain of AHL to homoserine lactone (HSL), and AHL-lactonase decomposes the homoserine lactone ring of AHL to produce acyl-homoserine.

The reaction product was analyzed by HPLC to determine what kind of enzyme the AHL degrading enzyme of M664 was. 100 μl of coenzyme dialyzed on 100 mM Tris (pH 7.0) was concentrated with 60% ammonium sulfate and reacted with 5 mM DHL for 1 hour. By standard OPA analysis, the standard HSL was observed as a minor peak at 12.5 minutes of retention time and a main peak at 20 minutes (FIG. 6A). Since HSL is unstable and hydrolyzed under alkaline conditions, the minor peak of 12.5 minutes is estimated to be a homoserine (HS) in which the lactone ring of HSL is degraded. Practically OPA analysis for HS was able to observe the peak of 12.5 components (Fig. 6B). The reaction product of M664 coenzyme with DHL showed a main peak at 20 minutes of retention time and a minor peak at 12.5 minutes. Therefore, it can be estimated that the AHL degrading enzyme produced by the M664 strain is acylase (FIG. 6C).

Example 6: Streptomyces  sp. Fish disease inhibition experiment using M664 culture

(1) Stability of AHL Degrading Enzyme in Seawater

The culture temperature at which Vibrio or Edward's disease occurs is more than 20 ° C. In order to investigate the stability of AHL degrading enzyme in seawater, the enzyme degrading activity was measured every day while leaving the enzyme in seawater for 5 days at room temperature.

As shown in FIG. 7, the AHL degrading enzyme maintained almost the initial activity of 45U even after 3 days of incubation, and maintained more than 70% of activity at 33.8 U even after 5 days. Therefore, MHL's AHL degrading enzyme is very stable in seawater and is expected to be of great value as a fish disease control agent.

(2) Fish disease inhibitory effect by AHL degrading enzyme

Vibrio sp., A bacterium isolated from diseased fish. PT2 was used.

Vibrio sp. Protease activity, a presumed concomitant factor of PT2, was found in Streptomyces sp. Of AHLase and B. thuringiensis of M664 It was reduced by the expression of AiiA AHL lactonase (not shown).

Substantially Vibrio sp. Whether the involvement of PT2 is controlled by quorum sensing, or if the involvement is controlled by quorum sensing. Concomitant experiments were performed on the olive flounder ( Paralichthys olivaceus ) to determine if it was controlled by AHL degrading enzymes. Vibrio sp. PT2 was directly inoculated into seawater to vibrio sp. The disease caused by PT2. On the flounder as a control, Vibrio sp. Vibrio sp. Only PT2 and Vibrio sp. Long-term analysis of pathogen survival, flounder mortality and dead flounder was performed when the flounder was treated with PT2 and M664. In both cases, fish were treated with Vibrio sp. The number of PT2 remained at 103, similar to the initial dose after 5 days (Table 1).

In seawater, Vibrio sp. The viability of PT2 remained constant. However, the mortality rate of the flounder occurred only 2 days after treatment, and all 6 animals died after 4 days. In contrast, Vibrio sp. For PT2 / M664 Vibrio sp. There was no mortality on the flounder for the same 6 days as in the control group not treated with PT2 (Table 2).

8 is a figure comparing the corpse of the olive flounder within 3 hours of our death. Vibrio sp. The internal organs of the olive flounder killed by PT2 treatment showed a typical vibriopathy with organs filled with water and a shape that is difficult to distinguish between liver types (Fig. 8B). While the control group (FIG. 8A) and Vibrio sp. The corpse of the flounder randomly killed in the PT2 / M664 treatment experimental group (Fig. 8C) was confirmed that the organ and liver are very clean (Fig. 8). From these results, Streptomyces sp. It can be concluded that the culture medium of M664 is effective in controlling fish disease of Vibrio , and its mechanism may be due to the decrease in the APL degrading enzyme of M664, which regulates the cofactor protease activity regulated by the signal substance AHL of quorum sensing.

In conclusion, M664 signaling inhibitors are expected to be used in the development of fish disease control agents.

Pathogenic microorganisms can be biologically controlled using a recombinant microorganism that produces a degrading enzyme and a degrading enzyme that degrades acylated homoserine lactone, which is a density recognition signal of a pathogen according to the present invention.

The new ecological approach to controlling pathogenic microorganisms is useful as an effective and environmentally friendly pathogenic microorganism control method while solving the problems of pesticide residue and antibiotic resistance, which are problems of conventional biochemical pathogenic microorganism control.

1 is a graph showing the inhibition of violacein production of C. violaceum CV026 according to the culture medium of the isolated M664 strain.

Figure 2 is a graph showing the AHL degradation activity according to the growth and growth of the isolated M664 strain.

Figure 3 is a photograph showing the degradation activity of various AHLs by the culture medium of M664 strain.

4 is a graph showing the effect on temperature of AHL degrading enzyme from M664 strain.

5 is a graph showing the effect on pH of AHL degrading enzyme from M664 strain.

6 is Streptomyces sp. Photo showing the mechanism of AHL degrading enzyme reaction from M664.

7 is Streptomyces sp. Graph showing stability of AHL degrading enzyme against seawater from M664.

8 is Streptomyces sp. Photograph showing pathogenic attenuation caused by Vibrio by M664 culture.

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Claims (9)

Streptomyces strain M664 (KCTC 10605BP), which has the ability to degrade acylated homoserine lactones, a microbial density signaling signal. AHL-lysed protein produced by the strain according to claim 1. A method of inhibiting-treating bacterial disease occurrence by treating a plant or animal with the strain or protein according to claim 1. An agent for inhibiting bacterial disease development in a plant or animal by using the strain or protein according to claim 1 or 2. The method of claim 4, wherein Inhibition-treatment of bacterial disease caused by Pseudomonas aeroginosa , which causes fibrocystic cystosis in the respiratory tract of animals including humans. Animal feed containing a bacterial disease inhibiting-treatment according to claim 5. Fertilizer containing the bacterial disease development inhibition-therapeutic agent of Claim 5. A pesticide containing a bacteriostatic suppression-treatment agent according to claim 5. A method for inhibiting-treatment of bacterial disease by treating a plant according to claim 2 with a plant or animal.