KR890004029B1 - New microorgamism for degradation of toxic compound - Google Patents

Abstract

A method for breeding microorganisms containing multi-plasmid to degrade contaminants is presented. Thus, microorganism which can be used for naphthalene as a sole source of carbon is isolated from waste water and identified as Pseudomonas putida N3 (KCTC 8287 P). Pseudomonas putida CST3 (KCTC 8288P) containing TOL plasmid and CAM plasmid simultaneously is conjugated with KCTC 8287P strain to give a new strain Pseudomonas putida Conj 26 (KCTC 8289 P), which can degrade toluene, naphthalene, and camphor.

Description

Environmentally-purified microbial strains by genetic engineering (conjugation) and breeding methods thereof.

1 is a growth curve under optimal growth conditions using naphthalene of isolated strain Pseudomonas N3 as the only carbon source.

Figure 2 Photograph on agarose gel of naphthalene degrading plasmid isolated from strain Pseudomonas putida N3 strain isolated.

FIG. 3 A photograph on agarose gel of a toluene and camphor plasmid of strain Pseudomonas putida CST 3 strain bred.

Figure 4 Photograph of growth confirmation in camphor and metatoluic acid medium of strain Pseudomonas putida CST 3 strain.

Fig. 5 Photograph of growth confirmation in naphthalene, camphor and metatoluic acid medium of strain Pseudomonas putida Conh 26.

Fig. 6 Plasmid photographs on aganose gels of strain Pseudomonas putida Conj 26 strain.

7 is a diagram showing the change in the chemical oxygen demand of each strain in the metatoluic acid medium.

8 shows the change in chemical oxygen demand of each strain in camphor medium.

9 is a diagram showing the change in the chemical oxygen demand of each strain in naphthalene medium.

10. Changes in Chemical Oxygen Demands of Isolated and Sarcoma Strains in the Complex Medium of Naphthalene, Camphor, and Toluene.

The present invention relates to a method for rearing a microorganism strain having enhanced environmental purification ability and contaminated with a known microorganism strain Pseudomonas putida PpGl (KCTC 1646) and mt-2 (Pseudomonas putida mt-2, KCTC1643). A method for breeding multi-plasmid microbial strains capable of degrading a large number of contaminants with a single microbial strain by transferring the decomposition-related plasmids of the naphthalene decomposing strains isolated from soil to one strain by conjugation. will be.

Environmental pollution is a phenomenon in which wastes generated by human activities or phenomena resulting from the activities cause changes in the environment and ecosystem, which impedes the survival, growth and reproduction of plants and animals.

Today, with the development of science, it is possible to artificially produce all consumer goods, accelerate the consumption of large amounts of natural resources for human survival and economic activities, and as a result, environmental pollution becomes more serious by releasing large amounts of waste. Was done.

The types and emissions of pollutants vary and increase with the development of science and technology, the change and the capacity of consumer materials. In other words, the increasing demand for synthetic fibers, synthetic resin-based daily necessities, the use of synthetic detergents, the consumption of liquid fuels, the development of pesticides, the development of radioactive materials, and the use of many other organic and inorganic materials have not been seen in the past. As new materials enter the environment, they become new sources of pollution.

The damage caused by environmental pollution is widespread, including human body, livestock, wild flora and fauna, fish and shellfish, buildings and other property, soil, etc., and acute damage, chronic damage, and also indirect damage caused by ecological change.

Many types of artificial compounds are present in broth, seawater, cropland and other soils. Many of them can be transformed into harmful or harmful substances in nature.

Organic compounds that enter land or sewage are mostly mineralized by microorganisms in the soil or sewage. Microorganisms increase the number of cells and the amount of cells by biological synthesis using energy generated at the same time by magnetizing them into their constituents by using other sources obtained by decomposing and mineralizing organic compounds or artificial compounds. In the end, mineralization is closely related to growth, and as a result toxic is removed.

On the other hand, many chemicals, microorganisms can not use these materials as energy or nutrients, but they can also be converted into non-toxic substances that can eventually be degraded.

Among the microorganisms having such environmental purification ability, Pseudomonas strains are famous as microbial strains that are widely distributed in soil or sewage and can use various hydrocarbons. Pseudomonas genus strains are environmentally diverse, including dominant chromosomal genes and very large molecular weight plasmids. Plasmids are genetic material other than chromosomes in microbial strains, and are materials capable of self-replication independently from chromosomes.

In general, the plasmid of the microorganism is not necessary, but in terms of the natural environment, the plasmid plays an indispensable role. Resolving plasmids allow host cells to adapt to the ecological status of nature. However, plasmids that give host cells resistance to antibiotics or the ability to produce toxins are found in enterobacteriaceae living in relatively stable environmental conditions.

On the other hand, microorganisms that live on the surface of the complex that contain many complex compounds, such as wood, fiber and pectin, which make up the plant, do not need all of the decomposition systems necessary for their decomposition even if they use these compounds. Do not. This is because of the plasmid's interdependence, Chakrabarty explains that this information is distributed from an economic point of view to avoid undue burden on an organism in a group (Annual Review of Genetice, vol. 10, p. 7). , 1976). Therefore, the genetic information that is urgently required by each microorganism in the whole microbial population can be exchanged and complemented, and this is explained as the reason that various species of Pseudomonas show the ability to adapt to the surrounding environment using various substances. Pemberton, Journal of Bact eriology, Vol. 119, p. 748, 1974.

Many types of degradation plasmids have been identified so far, and their characteristics are well studied for plasmids with the ability to degrade salicylic acid, naphthalene, toluene, camphor and octane. Degradation of these substances in Pseudomonas strains resulted in rapid progress as the plasmids were directly isolated, revealing almost completely the structural properties of the plasmids. However, biochemical studies have shown that many enzymes are involved in the degradation process and are listed in several genes (Clarke and Richmond, "Genetics and biochemistry of Pseudomonas", 1975).

Therefore, using genetic engineering techniques, which have begun to develop rapidly in recent years, in order to make recombinant plasmids having multiple genes in all metabolic processes, analysis of the gene composition involved in metabolic processes is preceded. There are many problems that must be addressed.

On the other hand, the United States proposed by Chakrabarty and Friello in 1975, based on the transfer of digested plasmids by the transfer plasmid in the genus Pseudomonas in 1974, by Chakrabarty (Journal of Bacterio10gy, Vol. 118, p. 815). Patent No. 3,923,603 uses a transfer plasmid (here, "factork") to shift hydrocarbon degradation pathways labeled on chromosomal genes of the Pseudomonas genus, resulting in plasmid aggregates in other Pseudomonas strains. Conjugation was used.

Since then, many researchers have been working on the production of plasmids and the transfer of plasmids by conjugation.

In the present invention, a known microbial strain Pseudomonas putida mt-2 (KCTC 1643) containing a TOL plasmid that degrades toluene in order to grow a strain enhanced in environmental purification capacity and a CAM plasmid that degrades camphor Of microbial strain Pseudomonas putida PpGl (KCTC 1646) was used. In addition, Pseudomonas putida N3 (KCTC 8288p) was isolated by selecting microbial strains using naphthalene as the sole carbon source from car washes and gas stations near metropolitan areas contaminated with waste oil to give the breeder the ability to break down naphthalene. It was confirmed that the strain contained an NAH plasmid that degrades naphthalene.

The strains Pseudomonas putida CST3 (TCTC 8288p), which has toluene and camphor plasmid at the same time, were bred using the three microbial strains above. PpGl having CAM plasmid with mt-2 strain having TOL plasmid as donor The breeding strain was selected by conjugation on plate medium using the strain as a receptor, and the streptomycin resistance of PpGl strain and metatoluic acid resolution of TOL plasmid to be transferred were used as indicators for conjugation. The strained strain CST3 contained both a TOL plasmid and a CAM plasmid.

On the other hand, strain Pseudomonas putida Conj26 (KCTC 8288p), which simultaneously contains naphthalene, toluene, and plasmid capable of decomposing camphor, was also bred. Sarcoma strains were selected by inducing conjugation on plate medium using Pseudomonas putida N3 as a receptor. As an indicator of conjugation, various antibiotic resistances of N3 strain and camphor and metatoluic acid resolution of CAM and TOL plasmid to be transferred were used. The resulting sarcoma strain Conj26 contained CAM, NAH and TOL plasmid.

The present invention, unlike the method of transferring to another Pseudomonas strain using a hydrocarbon degradation pathway labeled in a chromosome gene used in US Pat. No. 3,923,603, issued by Chakrabarty and Friello in 1975, exists in the presence of plasmid aggregates. Eggplant is a method of developing a sarcoma strain in which a plasmid is transferred using a direct conjugation method so that it exists in the same strain.

Example 1

Soil and wastewater from gas stations and car washes in Seoul and large cities were collected to isolate the growing strain using naphthalene as the sole carbon source.

The selection process of the strain was performed by adding 0.1% of yeast extract to the liquid minimal medium as shown in Table 1 and growing the cells while supplying naphthalene in a vapor state, and lowering the concentration of yeast extract to 0.05%, 0.03%, and 0.01%. Afterwards, naphthalene was first selected for growth in liquid minimal media, the only carbon source.

TABLE 1

The first selected mycelium is grown in batch culture in a continuous culture apparatus until it reaches a certain mass, and then slowly supplied with the culture solution at a dilution rate of 5.2-5.8 / min to dilute the strain whose growth rate is lower than the dilution rate, and is excluded from the incubator. Only the species with generation time of 7.2-8.0 hours were secondary selected.

These secondary selection strains were transferred three times in a solid minimal medium containing naphthalene as the only carbon source, and then grown, and finally a pure colony showing uniform growth was selected as a naphthalene decomposing strain.

The final selected strains were gram-negative rod-shaped aerobic bacteria with strong motility and showed optimum growth in oxidase positive neutral maize (PH). Pseudomonas) was identified as Pseudomonas putida N3 (KCTC 8287P) because it has physiological and biochemical characteristics as shown in Table 2.

Pseudomonas putida N3 (TCTC 8287P) strains showed optimal growth at a temperature of 30 ° C and pH 7.0.If growing with naphthalene as the only carbon source, it grew for three days after four days of incubation. Later, growth was complete (Figure 1), and it also contained a plasmid (NAH Plasmid) that degrades naphthalene (Figure 2).

TABLE 2

Example 2

If a strain having several kinds of plasmids in one strain is developed at the same time, it is possible to use several kinds of substrates with only one strain, and thus it may be very preferable in terms of environmental purification. Therefore, Pseudomonas putida mt-2 (TCTC 1643) strain, a known strain known to have a TOL plasmid that degrades toluene, is used to obtain a strain capable of using both camphor and toluene. Conjugation was attempted using a strain of Pseudomonas putida PpGl (KCTC 1646), a known strain having a plasmid that degrades camphor as a donor.

As an indicator for conjugation, the antibiotic streptomycin preparation of Pseudomonas putida KCTC 1646 strain was induced as a natural mutation, and then the transfer of the known plasmid 117 kilobase (kb) -sized TOL plasmid from Pseudomonas putida KCTC 1643 strain was observed. Induced on solid medium containing streptomycin at 100 ug / ml and having m-toluicacid as the only carbon source.

The strain Pseudomonas putida CST3 (KCTC 8288P) thus obtained contained both a plasmid that degrades camphor and a plasmid that degrades toluene (Figure 3), and the only carbon source was camphor and metatoluic acid. The branches showed the same growth in the minimal medium (Fig. 4).

Example 3

Pseudomonas putida CST3 (Pseudomonas putida CST3, KCTC 8288P) containing both the previously bred TOL plasmid and CAM plasmid as donors, and Pseudomonas putida N3, KCTC which decomposes naphthalene isolated from nature Pseudomonas putida Conj26 (Pseudomonas putida Conj26, KCTC 8289P) capable of simultaneously decomposing naphthalene, camphor and toluene was bred by the method of conjugation using 8287P) as a receptor.

As an indicator for conjugation, various antibiotic resistances of Pseudomonas putida N3 (KCTC 8288P) strains were used. A strain containing three types of plasmids that decompose naphthalene, camphor, and toluene together with 200 ug / ml of antibiotic streptomycin, showing a uniform growth simultaneously in medium containing camphor and metatoluic acid as the only carbon source Was selected.

Pseudomonas putida Conj26 (KCTC 8288P) of the bred splicing strain showed uniform growth in a medium containing naphthalene, camphor, and toluene as the only carbon source (figure 5). Three plasmids, CAM and TOL, were contained (Fig. 6).

Multi-plasmid containing benzene ring-based naphthalene, toluene, and plasmid that can decompose camphor with complex ring structure together as a major component of environmental pollutants through the above process The strain Pseudomonas putida Conj26 (KCTC 8288P) was bred.

Example 4

TOL plasmids contained in known strains Pseudomonas putida mt-2 (KCTC 8288P) and sarcoma strains Pseudomonas putida CST3, Conj26 (Pseudomonas putida CST3, KCTC 8288P and Conj26, KTCT 8289P) Degradation activity was compared based on chemical oxygen demand. The change in chemical oxygen demand was observed in a minimal medium (see Table 1) to which metatoluic acid was added as a substrate (see Table 1) with shaking at 30 ° C (100,100 rpm).

Pseudomonas putida mt-2 strains were completely grown within 24 hours, and metatoluic acid was digested and used for their growth. Therefore, a decrease in metatoluic acid in the medium resulted in a decrease in chemical oxygen demand. The final chemical oxygen demand was 210 mg / 1. The breeding strain CTS3, which contains two types of plasmids that degrade camphor and toluene, was the same as the strain mt-2. The breeding strain Conj26, which contains three kinds of plasmids up to naphthalene plasmid, was found to be mt-2 and CST3. Growth was slower for about 12 hours compared to the strain. Therefore, the decrease in chemical oxygen demand was small at the beginning of culture, but the final chemical oxygen demand after 3 days was the lowest as 180 mg / l.

Example 5

The activity of the CAM plasmid that degrades camphor was determined by the known strain Pseudomonas putida PpGl (KCTC 1646) and by strains Pseudomonas putida CST3, conj26 (Pseudomonas putida CST3, KCTC 8288P and Conj26, KCTC8289P). Camphor was added to compare the decrease in chemical oxygen demand (see Figure 8). Growth conditions were shaken at a temperature of 30 degrees as in Example 4 at a split rotation speed of 100 (100 rpm).

Growth of Pseudomonas putida PpGlrbs strains was completed within 23 hours, and the reduction of chemical oxygen demand was continued for two days after incubation, maintaining the final chemical oxygen demand of 200 mg / 1. The sarcoma strain CST3 containing two types of plasmids decomposing camphor and toluene showed slightly higher growth rate than the PgGl strain, and the decrease in chemical oxygen demand was similar to that of the PpGl strain. The value of oxygen demand was as low as 170 mg / 1.

In the case of strain Conj26 containing three kinds of plasmids, unlike the decomposition of metatoluic acid in Example 4, the growth was immediately performed without undergoing a long incubation period to decompose camphor. The growth and chemical oxygen demand decreased in the same manner as in the PpGl and CST1 strains, and the final chemical oxygen demand after 3 days was 160 mg / 1.

Example 6

Activity of NAH plasmid that degrades naphthalene was obtained by adding Pseudomonas putida N3 (KCTC 8287P) isolated in Example 1 and Pseudomonas putida Conj26 (KCTC 8289P) bred in Example 3 with 2.5 mM naphthalene as a substrate. Growth in the medium was compared with the decrease in chemical oxygen demand (see Figure 9). Culture conditions were the same as in Examples 4 and 5.

The growth of Pseudomonas putida N3 strain in taphthalene was significantly lower than that of naphthalene in water solubility in water and camphor and metatoluic acid as substrates. The initial value of chemical oxygen demand of 350 mg / l remained about 15%. In the case of breeding strain Conj26, the growth rate and final cell weight were about 90% compared to the N3 strain, but the rate of chemical oxygen demand was slow at the beginning but accelerated later in 48 hours, and the final chemical oxygen demand value was 250mg. It was fairly low at / 1.

Example 7

In Example 1 and Examples 2 and 3, the degradation activity against the composite substrate of the isolates and breeding strains was compared with the trend of decreasing the chemical oxygen demand (see FIG. 10). The composite substrate was naphthalene, toluene, camphor were used at a concentration of 2.5mM, respectively, and the culture conditions were the same as in Example 4,5,6.

Pseudomonas putida N3 (KCTC 8287P), a naphthalene degrading strain isolated in Example 1, grew only slightly because naphthalene was not used and camphor and toluene were not used as substrates, thus reducing the amount of chemical oxygen. In other words, the N3 strain contains only naphthalene-degradable NAH plasmid, so only naphthalene is slightly decomposed among the three composite substrates, and the chemical oxygen demand is reduced by about 30%.

The CST3 (KCTC 8288P) strain, the camphor and toluene degrading strains bred in Example 2, has TOL and CAM plasmids that can use both toluene and camphor, so that toluene and camphor except naphthalene are decomposed to decompose camphor. Indicated. The degree of growth was about 90% of the strain containing only one plasmid, and thus the chemical oxygen demand was reduced by about half.

The growth of three complex substrates of strain Conj26, which had three kinds of plasmids capable of degrading all of naphthalene, toluene and camphor bred in Example 3, was similar to that of N3, a naphthalene-decomposing strain. This is because strain and Conj26 strain are the same homework cells. On the other hand, since the three substrates can be used by digestion by three plasmids, the final cell weight after three days of culture was slightly increased compared to the N3 strain, and the chemical oxygen demand was reduced by 60%.

Claims (5)

Pseudomonas putida N3 (KCTC 8287P) with a plasmid that degrades naphthalene. Pseudomonas putida CST3 (KCTC 8288P) with respective plasmids that degrade toluene and camphor. Pseudomonas putida Conj26 (KCTC 8289P) with respective plasmids to degrade naphthalene, toluene and camphor. It is known that Pseudomonas putida mt-2 (KCTC 1646) with a plasmid having toluene resolution as a donor and Pseudomonas putida PpGl (KCTC 1646) with a plasmid having a long brain resolution as a acceptor are conjugatioin-conjugated with toluene. A method for preparing a novel Pseudomonas putida CST3 (KCTC 8288P) with camphor resolution. A novel toluene, camphor, and naphthalene resolution characterized by conjugation with a novel Pseudomonas putida N3 (KCTC 8288P) with acceptor as Pseudomonas putida CST3 (KCTC 8288P) and naphthalene resolution isolated from soil. Process for preparing Pseudomonas putida Conj26 (KCTC 8289P).

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