KR102454448B1 - Novel microorganism having polycaprolactone decomposition activity and use thereof - Google Patents

Abstract

The present invention provides a microorganism having polycaprolactone decomposition activity, which is a polymer plastic, and provides a method for selecting a strain having excellent polycaprolactone decomposition activity, and Pseudomonas putida sp. ) to provide an optimal culture condition method to improve the polycaprolactone decomposition activity of the.

Description

Novel microorganism having polycaprolactone decomposition activity and use thereof

The present invention relates to a novel microorganism having polycaprolactone degrading activity and uses thereof.

Polycaprolactone (PCL), which can be produced relatively inexpensively among aliphatic polyesters synthesized from petrochemical raw materials, has a low melting point, but is stable even at a high temperature of 200°C or higher, so it has excellent processability and compatibility with other degradable plastics. It has a good advantage and also occupies a position as a standard material for biodegradable plastics because of its excellent biodegradability. Because polycaprolactone has good compatibility with other polymers, it is a new material that has biodegradability even in mixtures with polyester, polyamide, polyurethane, etc. or copolymers with those monomers. It is processed into fishing line, etc.

As it has been revealed that not only the physical properties of high molecular weight polycaprolactone but it is completely decomposed by microorganisms, research is being conducted to add the function of degradability to solve the problem of convenience and environmental pollution of plastics. Although not much is known about polycaprolactone degrading enzymes, polycaprolactone-degrading microorganisms are widely distributed in various environments such as aerobic and anaerobic conditions, soil and aquatic conditions, and polycaprolactone-decomposing microorganisms include fungi Fusarium sp., Aspergillus fumigutus and other bacteria include Bacillus pumilis , Paenibacillus sp. Pseudomonas sp. etc have been reported. The decomposition of polycaprolactone proceeds in two stages. In the first stage, molecular weight reduction occurs due to main chain cleavage due to random hydrolysis, and in the second stage, weight decrease occurs due to the decomposition of low molecular weight fragments and small fragments. .

The degradability of this microorganism by polycaprolactone degrading enzyme is affected by the decomposition activity and culture conditions of the microorganism, and in order to improve the polycaprolactone decomposition efficacy, it is necessary to study the optimal culture conditions and culture technology of the decomposing microorganism. For practical use of polycaprolactone degrading enzyme, it is necessary to study optimal culture conditions and culture technology for degrading microorganisms for mass production of degrading enzyme and to develop enzyme mass production technology.

Numerous references are referenced throughout this specification and citations thereof are indicated. The disclosures of the cited documents are hereby incorporated by reference in their entirety to more clearly set forth the content of the present invention and the level of the art to which the present invention pertains.

Republic of Korea Patent Publication No. 10-2126889

The present invention has been devised to solve the above problems, and provides an optimal culture condition method to select and provide a strain with excellent decomposition activity of poly (ε-caprolactone) and improve the decomposition activity of the present microorganism is to do

More specifically, the present invention provides a microorganism having excellent polycaprolactone decomposition ability even in a wet environment through esterase expression, and provides culture conditions for improving the decomposition activity of the microorganism, more specifically Pseudomonas putida ( Pseduomonas putida sp.) by confirming the esterase activity of the polycaprolactone degrading enzyme and comparing and confirming the activity according to culture conditions such as temperature and pH to improve its activity, especially polycaprolactone decomposition activity in a wet environment It's about how to improve.

One aspect of the present invention is to provide a method for decomposing polycaprolactone using a Pseudomonas sp. strain expressing an esterase.

Both esterases and lipases are enzymes that hydrolyze ester bonds, and lipases exhibit high activity on the aggregated state of substrates, whereas esterases generally exhibit the highest activity on the soluble state of substrates.

Previously known polycaprolactone decomposing strains mainly degrade polycaprolactone through lipase activity, so they are suitable for decomposition of agglomerated substrates such as compost, but are not suitable for decomposing polycaprolactone in a solubilized state in a wet environment. Thus, the strain of the present invention is suitable for decomposing polycaprolactone contained in a wet environment through esterase expression.

In a preferred embodiment, the Pseudomonas sp. strain expressing the esterase is Pseudomonas putida , and more preferably, Pseudomonas putida KRICT-1 newly isolated by the present inventors.

The present inventors extracted and amplified the DNA of the newly isolated microorganism, confirmed the gene sequencing of 16S rRNA, and performed phylogenetic analysis. As a result, it was identified as a novel microorganism closest to Pseudomonas putida sp. The present inventors deposited this as Pseudomonas putida KRICT-1 (Pseudomonas putida KRICT-1) at the Korea Institute of Biotechnology and Biotechnology Biological Resources Center (KCTC) on October 23, 2020, and was given an accession number KCTC18857P.

According to the present invention, the Pseudomonas putida KRICT-1 strain is a strain isolated from a wet environment, such as soil, sludge, food wastewater, domestic sewage, industrial wastewater, livestock wastewater and manure, etc., through esterase expression It was confirmed as a strain having a very high polycaprolactone decomposition activity.

The Pseudomonas putida KRICT-1 strain of the present invention can produce polycaprolactone in at least one moist environment selected from soil, sludge, food wastewater, domestic sewage, industrial wastewater, livestock wastewater and manure at room temperature within 3 days and within 4 days. , within 5 days, within 6 days, within 7 days, within 10 days, within 15 days, within 20 days, within 25 days, or within 30 days.

Therefore, the novel strain can be usefully used for biological purification of a wet environment contaminated with polycaprolactone.

Another aspect of the present invention is to provide a composition for decomposing polycaprolactone comprising a Pseudomonas sp. strain expressing an esterase as an active ingredient.

The composition may further contain nitrogen and oxygen-causing substances such as phosphorus and hydrogen peroxide, a surfactant to promote surface contact between the contaminants and microorganisms, and an enzyme that appropriately activates various stages of biochemical degradation of the contaminants. . In addition, the composition may include additional additives such as nutrients necessary for the growth of microorganisms, carbon sources, and electron acceptors such as oxygen for effective decomposition of contaminants. In addition, the composition of the present invention is not limited to the components listed above, and may include various components known in the art as components that promote microbial activity and decomposition of contaminants.

Another aspect of the present invention comprises the steps of: (a) diluting a sample collected in a wet environment; (b) smearing and culturing the diluted sample on a plate medium containing emulsified polycaprolactone (emulsified-PCL) and distilled water; (c) obtaining a single colony strain forming an active zone (clear zone) on the plate medium; and (d) monitoring the polycaprolactone decomposition ability of the strain on a polycaprolactone-containing liquid medium. A strain having an activity of decomposing polycaprolactone through esterase expression It provides a way to sympathize with

The plate medium further includes LB medium (Luria-Bertani broth) and agar (Agar), and is saturated with emulsified polycaprolactone. can be made selectable.

In addition, the method for identifying a microorganism having a polycaprolactone degrading ability provided in the present invention comprises: acquiring a sample indicating a region on a plate medium containing polycaprolactone in which at least one colony of the microorganism is disposed; Identifying at least one colony of the microorganism by indicating an active zone (clear zone) in the region on the plate medium containing polycaprolactone, and monitoring the ability of the microorganism to decompose polycaprolactone on the liquid medium containing the polycaprolactone film may include.

The plate medium may consist of LB medium (Luria-Bertani broth), agar, polycaprolactone and distilled water, and the emulsion polycaprolactone is saturated therein, so the activity formed around the microorganisms when decomposing polycaprolactone. By identifying the strain, it is possible to select a strain having a degradability.

The plate medium enables high-speed separation and culture of microorganisms having polycaprolactone decomposition ability. Based on 100 mL of distilled water, 2 g of LB medium (Luria-Bertani broth) and 1.5 g of agar powder are used as a nutrient medium. adding and dissolving, mixing 1 g of polycaprolactone powder in 1 ml, autoclaving the two solutions at 121° C. for 15 minutes, adding 1 g of polycaprolactone powder based on 100 ml of the medium of the sterilization step It can be prepared by a method comprising the step of turbiding with an ultrasonic sprayer, and coagulating the turbid medium in an amount of 20 ml in a Petri dish.

In one embodiment, E. coli without polycaprolactone degrading ability in the region on the plate as a negative control, and Alcaligenes faecalis sp. as a strain having polycaprolactone degrading ability as a positive control. control), by checking the active zone generated in the polycaprolactone plate medium according to the polycaprolactone decomposition ability, it is also possible to separate microorganisms having the polycaprolactone decomposition ability.

In the present invention, the microbial sampling means soil, sludge, food wastewater, domestic sewage, industrial wastewater, livestock wastewater prepared to screen microorganisms having an industrially useful enzyme, polycaprolactone depolymerase activity. and extracting microorganisms from samples derived from manure, etc.

In one embodiment of the present invention, in the step of culturing the strain having polycaprolactone decomposition ability, it contains LB medium at a concentration of 1 to 2 volume %, weighs and contains polycaprolactone film sterilized with ethanol and UV. can The culturing step may be culturing for 48 to 120 hours at 50 ml to 2.0L of LB medium, 20 °C to 37 °C.

In a preferred embodiment, the culture may be performed by a batch culture method, but is not limited thereto.

Through the present invention, Pseudomonas putida , an excellent microorganism having polycaprolactone decomposition activity, is isolated, and polycaprolactone degrading enzyme activity is improved by optimizing the culture conditions of the microorganism. can be used.

1 shows a schematic diagram of a method for selecting strains having polycaprolactone-decomposing activity on a plate medium made by turbiding polycaprolactone in an agar medium through the formation of an active zone.
2 is a result showing the phylogenetic tree of Pseudomonas putida KRICT-1 having a polycaprolactone decomposition ability identified in the present invention.
3 is a graph showing the polycaprolactone film resolution and growth in aerobic culture conditions of Pseudomonas putida KRICT-1 strain.
4 is a result of confirming the polycaprolactone film degraded by Pseudomonas putida KRICT-1 strain with a scanning electron microscope (SEM).
5 is a result of confirming the polycaprolactone film degraded by Pseudomonas putida KRICT-1 strain with Fourier Transform Infrared spectroscopy (FT-IR).
6 is a diagram showing the results of SDS-PAGE electrophoresis by purifying polycaprolactone degrading enzyme of Pseudomonas putida KRICT-1 strain.
7 is a result of confirming the active zone of polycaprolactone-degrading enzyme of Pseudomonas putida KRICT-1 strain in turbid polycaprolactone-agar plate medium.
8 is a graph confirming the activity and stability of polycaprolactone degrading enzyme activity according to temperature and pH of Pseudomonas putida KRICT-1 strain.

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

Example

Example 1. Isolation, selection and identification of polycaprolactone-degrading microorganisms

(One) Isolation of polycaprolactone-degrading microorganisms

To identify polycaprolactone-degrading microorganisms, samples collected from soil, sludge, and food wastewater were diluted with physiological saline at various magnifications (10 ^-1 , 10 ^-2 , 10 ^-3 and 10 ^-4 ) to dilute mixed strains A sample was obtained. Then, based on 100 mL of distilled water, adding and dissolving 2 g of LB medium (Luria-Bertani broth) and 1.5 g of agar powder as a nutrient medium, and mixing 1 g of polycaprolactone powder in 1 ml Then, the two solutions were autoclaved at 121° C. for 15 minutes in an autoclave, 1 g of polycaprolactone powder was added based on 100 ml of the medium in the sterilization step, and the turbidity was turbid using an ultrasonic sprayer, and the turbid medium was prepared in a Petri dish (Petri dish). ) in an amount of 20 ml and solidified to produce a polycaprolactone-agar plate medium, streaking the diluted sample on the plate medium, and culturing at various temperatures (25° C., 30° C., 37° C.) for 2 days. Single colony strains forming (clear zone) were obtained. 1A is E. coli without polycaprolactone decomposition ability as a negative control, and FIG. 1B is a polycaprolactone decomposable strain with Alcaligenes faecalis sp. strain as a positive control. control) and by checking the active zone generated in the polycaprolactone plate medium according to the polycaprolactone decomposition ability, the standard of the method for isolating microorganisms having the polycaprolactone decomposition ability was established.

(2) Selection and identification of polycaprolactone-degrading microorganisms

The polycaprolactone-agar plate medium is sterilized with a liquid medium containing 20 g of LB medium (Luria-Bertani broth) based on 1 L of distilled water for colonies showing the active zone, and then 5 ml of a conical tube with a volume of 50 ml. LB medium and polycaprolactone film (about 300g) were put in, cultured at 30° C. for a week, and the polycaprolactone film weight loss was compared to compare the polycaprolactone decomposition ability, and excellent decomposing strains were selected.

Among them, the DNA of the microorganism with the highest resolution was extracted and amplified, and the 16s rRNA sequence was analyzed to determine the species of the microorganism. 2 shows a phylogenetic tree similar to the nucleotide sequence of the present microorganism, the strain KRICT-1 strain identified in this patent showed the closest match to Pseudomonas putida sp.

Example 2. Confirmation of polycaprolactone resolution of Pseudomonas putida

After sterilizing the liquid medium containing 20 g of LB medium (Luria-Bertani broth) based on 1 L of the identified Pseudomonas putida KRICT-1 (Accession No. KCTC18857P) strain, 50 ml of the identified Pseudomonas putida KRICT-1 (Accession No. The strain was inoculated with a lactone film so that the initial OD (600 nm) was 0.2, and cultured at 30° C. for about 5 days using a shaking incubator. In addition, only LB medium and polycaprolactone film were put in an Erlenmeyer flask and cultured at 30° C. using a shaker incubator, and this was used as a control. Cell growth was measured by sampling the culture medium as shown in FIG. 3, and the weight loss of the polycaprolactone film was measured. The weight reduction of the polycaprolactone film was insufficient until about 3 days, and then rapidly decomposed after the 3rd day, and it was confirmed that all the decomposition of the polycaprolactone film on the 5th day occurred. In addition, the polycaprolactone decomposition of Pseudomonas putida by recovering the initial polycaprolactone film and the polycaprolactone film on the 4th day and checking the surface of the polycaprolactone film by taking a scanning electron microscope (SEM) as shown in FIG. 4 . It was checked whether 5 is a result of confirming whether the sample is decomposed by using a Fourier-Transform Infrared spectrometer (FTIR) It was confirmed that the N-H protein/peptide bond was increased.

Example 3. Purification of polycaprolactone degrading enzyme of Pseudomonas putida strain

In order to purify the polycaprolactone depolymerase of Pseudomonas putida KRICT-1 (Accession No. KCTC18857P) strain having the polycaprolactone decomposition ability, this microorganism was incubated with a shaking incubator in LB medium at a stirring speed of 200 rpm, 2 at 30 ° C. Incubated for one day. 100 mL of this culture solution was centrifuged at 13000 rpm, 4° C. for 10 minutes to take only the supernatant of the culture solution, and 50% of the supernatant volume ratio ((NH ₄ ) ₂ SO ₄ ) was added at 4° C. and stirred well for 4 hours. After centrifuging the enzyme solution to collect only the precipitate, the precipitate was dissolved in 20 mM potassium phosphate buffer pH 7.5 and dialyzed overnight in the same buffer solution. This enzyme solution was centrifuged again to remove insoluble substances, and then purified with a 0.22 μm filter and used as an enzyme solution.

In order to measure the purity and molecular weight of the enzyme purified in the present invention, electrophoresis was performed on 10% SDS-polyacrylamide gel, and the molecular weight was calculated by comparing the movement speed with the protein marker. SDS-PAGE was prepared according to Laemmli's method, and Bio-rad's protein marker (10-250 kDa) was used. As shown in FIG. 6 , a single band was exhibited, and as a result of measuring the molecular weight compared to molecular weight markers composed of standard proteins, the molecular weight of the polycaprolactone degrading enzyme produced by Pseudomonas putida was confirmed to be about 62.5 kDa. The activity of polycaprolactone degrading enzyme can be confirmed by confirming that the enzyme purified solution shows an activity zone as shown in B of FIG. It was confirmed that the polycaprolactone degrading enzyme has an esterase activity.

Example 4. Optimization of polycaprolactone degrading enzyme activity of Pseudomonas putida strain

In order to determine the optimum reaction temperature for the purified polycaprolactone degrading enzyme having the present purified esterase activity, the reaction was performed at 5°C intervals in the range of 15 to 65°C for 10 minutes, and the relative activity of the enzyme was expressed. For thermal stability, the enzyme solution was heat-treated at intervals of 5° C. in the range of 15 to 65° C., and the enzyme solution was heat-treated in units of 1 hour, and the residual activity of the enzyme was measured. The optimal pH measurement of this enzyme is 0.1 M acetate buffer (pH 3.5-5.5), 0.1 M phosphate buffer (pH 5.5-8.0), 0.1 M Tris-HCl buffer (pH 8.0-9.0), 0.1 M glycine-NaOH buffer (pH 9.0-12.0) was used. The stability of the pH was measured by adding a buffer of each pH to the enzyme solution and leaving it at room temperature for 1 hour, and then the activity of the remaining enzyme was measured. . To check the activity, 100 μl of a sample in which p -nitrophenyl butyrate ( p -NPB) was dissolved in isopropanol to 10 mM as a substrate solution and 100 μl of the enzyme solution were mixed with 800 μl of 50 mM tris-HCl buffer (pH 8.0) to 37 After reacting at ℃ for 5 minutes, it was confirmed by measuring the absorbance at 405 nm. The results of the activity change with respect to the temperature and pH of the enzyme purified in the present invention are shown in FIG. 8 . The activity against temperature was maintained at more than 80% of the maximum activity up to 50 °C, and the stability to heat decreased sharply at a temperature of 40 °C or higher. The activity to pH was the highest at pH 8.0, and it was confirmed that the relative activity was maintained at 70% of the maximum activity at pH 5.0 ~ pH 9.0.

Example 5. Comparison of polycaprolactone degradation with other Pseudomonas sp. strains

Pseudomonas putida NRRL B-14875 (Agriculture Research Service Culture Collection (USDA)) as another Pseudomonas genus strain in order to compare the Pseudomonas putida KRICT-1 strain of the present invention and polycaprolactone decomposition ability and Pseudomonas pseudoalkali, a gram-negative soil bacterium Genius (Pseudomonas pseudoalcaligenes) was used.

After sterilizing the liquid medium containing 20g of LB medium (Luria-Bertani broth) based on 1L of each of these strains, put 50ml into an Erlenmeyer flask with a volume of 300ml, put the weighed polycaprolactone film, and set the strain to the initial OD (600nm) It was inoculated so that it became 0.2, and incubated for about 5 days at 30°C using a shaking incubator. In addition, only LB medium and polycaprolactone film were put in an Erlenmeyer flask and cultured at 30° C. using a shaker incubator, and this was used as a control. Cell growth was measured by sampling the culture medium, and the weight loss of the polycaprolactone film was measured.

As a result of the experiment, in the case of Pseudomonas putida KRICT-1 strain, the decomposition occurred rapidly after 3 days at 30°C, and the weight reduction of the polycaprolactone film reached 100% within 5 days, indicating that it actively decomposes polycaprolactone at room temperature. Confirmed. On the other hand, in Pseudomonas putida NRRL B-14875, the weight reduction of the polycaprolactone film at 30° C. was hardly observed after 5 days, and slight deformation of the film began to be observed after 7 days. In Pseudomonas 　 Pseudo Alkali Genies, the weight reduction of the polycaprolactone film at 30° C. was hardly observed until 5 days had elapsed, and it was confirmed that the weight reduction of the polycaprolactone film started after 7 days.

Korea Institute of Bioscience and Biotechnology KCTC18857P 20201023

Claims (11)

As a method of decomposing polycaprolactone using a Pseudomonas sp. strain expressing an esterase,
The method, characterized in that the strain Pseudomonas putida KRICT-1 (Accession No. KCTC18857P). delete The method according to claim 1, wherein the strain decomposes polycaprolactone contained in a wet environment through esterase expression. The method according to claim 3, wherein the wet environment is at least one selected from soil, sludge, food wastewater, domestic sewage, industrial wastewater, livestock wastewater, and manure. A composition for decomposing polycaprolactone comprising a Pseudomonas sp. strain expressing an esterase as an active ingredient,
The strain is a composition, characterized in that Pseudomonas putida KRICT-1 (Accession No. KCTC18857P). delete Pseudomonas putida KRICT-1 (Accession No. KCTC18857P) strain. The strain according to claim 7, wherein the strain has an activity of decomposing polycaprolactone through expression of esterase. The strain according to claim 7, wherein the Pseudomonas putida KRICT-1 strain is a strain isolated from at least one wet environment selected from soil, sludge, food wastewater, domestic sewage, industrial wastewater, livestock wastewater and manure. delete delete

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