KR102404872B1 - Degradation method for polycaprolactone polymer using Apiotrichum porosum - Google Patents

Abstract

The present invention discloses a method for decomposing polycaprolactone polymers using Apiotrichum porosum , particularly Apiotrichum porosum NIBR-WAE-1241 strain (accession number: KACC 83034BP).

Description

Degradation method for polycaprolactone polymer using Apiotrichum porosum}

The present invention relates to a method for decomposing polycaprolactone polymers using Apiotrichum porosum .

While plastics, which are widely used worldwide, have excellent convenience, they are a serious cause of environmental pollution due to waste generated after use.

There are several alternatives to replace these polymers, and one of them is bioplastics called eco-friendly plastics.

Bioplastic is a high molecular polymer manufactured using biomass, a renewable material, as a raw material. Generally, it is divided into biodegradable plastic and biomass-based plastics according to the content of biomass. Biodegradable plastics are biodegradable biomass plastics such as polylactic acid (PLA) and polyglycolic acid (PGA) and polycaprolactone ( It is divided into biodegradable petroleum-based plastics such as polycaprolactone and PCL).

PCL and PLA, known as representative biodegradable polymers, are known to take a long time to decompose in their natural state, although it varies depending on the content and processing conditions of the manufactured product. In particular, in the case of PLA, it is known that it takes a long time for decomposition in the soil to start.

While several microorganisms involved in decomposing PCL and PLA have been reported abroad, Pseudozyma jejuensis , a yeast belonging to the basidiomycete, has been reported to decompose PCL in Korea (Seo et al., 2007). ), and other fungi known to decompose PCL or PLA, there are no studies.

The present invention discloses the decomposition ability of polycaprolactone polymers of Apiotricum phorosom strains isolated and identified in domestic soil.

It is an object of the present invention to provide a method for decomposing polycaprolactone polymers using the Apiotricum phorosom strain.

Other objects or specific objects of the present invention will be set forth below.

As confirmed in the Examples and Experimental Examples below, the present invention isolates and identifies the Apiotricum phorosom NIBR-WAE-1241 strain, which is a yeast, in domestic soil, and the isolated and identified strain is poly It was completed by confirming that it has the decomposition ability of the caprolactone (Polycaprolactone, PCL) polymer.

In consideration of the above, the method for decomposing polycaprolactone polymer of the present invention may be understood to include the step of culturing by contacting the polycaprolactone polymer with Apiotricum phorosom.

In the method of the present invention, the polycaprolactone polymer is polycaprolactone obtained by using glycols as an initiator of a ring-opening polymerization reaction in addition to polycaprolactone obtained by ring-opening polymerization of caprolactone; It is meant to include polycaprolactone polyols such as polycaprolactone triol and polycaprolactone tetraol. Therefore, in addition to being applied to the decomposition of polycaprolactone, the method of the present invention can be usefully applied to the decomposition of polycaprolactone polyols such as polycaprolactone diol.

In the method of the present invention, in order to increase the decomposition capacity of polycaprolactone polymer by Apiotricum phorosum by promoting the culture and proliferation of Apiotricum phorosum, the culture solution obtained by culturing Apiotricum phorosum in a medium In the form of contact with the polycaprolactone polymer, or before and after the polycaprolactone polymer contact, a medium for promoting the culture and proliferation of Apiotricum phorosom may be added to the polycaprolactone polymer and mixed.

Such a medium generally contains a carbon source, a nitrogen source, a trace element, a growth promoter, and the like.

The carbon source is preferably a sugar such as a monosaccharide, disaccharide or polysaccharide. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, starch hydrolyzate, etc. can be used. Complex compounds such as molasses or by-products of sugar refining processes may also be used. In some cases, oils such as soybean oil and sunflower oil, organic acids such as acetic acid, glycerol, and the like may be preferable as the carbon source. These carbon sources may be included in the medium alone or as a mixture of two or more types, and whether included in the medium alone or in a mixture of two or more types, 2% (w/w) to 40% (w/w) It can be included in the medium to a range. When a natural product or processed product such as starch or starch hydrolyzate is used as a carbon source, these carbon sources can be sterilized and used to prevent unwanted fermentation by other microorganisms. Sterilization may be performed using methods known in the art, such as high-temperature/high-pressure sterilization, ultraviolet irradiation, and the like.

The nitrogen source may be an inorganic nitrogen compound, an organic nitrogen compound, or a complex compound including these compounds. Examples of the inorganic nitrogen compound include ammonia, ammonium salts (eg, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.) and nitrate. Examples of the organic nitrogen compound include urea and amino acids. As the complex compound, yeast extract, soytone, peptone, tryptone, malt extract, broth, soybean powder, soybean okara powder, cheonggukjang powder, soybean paste Natural nitrogen sources, such as powder, etc. are mentioned. One or more of these nitrogen sources may be mixed and used, and may be included in the medium in the range of 0.1% (w/w) to 8.0% (w/w). When using a natural nitrogen source, it is preferable to use it after sterilization for the same reason as described in relation to the case of using a natural carbon source.

Trace elements include calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper, iron, phosphorus, sulfur, etc. These trace elements may be added to the medium in the form of salt compounds, and the medium of these trace elements A chelating agent such as catechol, citric acid, etc. may be added together to increase solubility in the solution and to maintain a solution state. Examples of the salt compound form include sodium hydrogen phosphate, magnesium sulfate, iron chloride, calcium salt, manganese salt, cobalt salt, molybthenate, and chelate metal salt. One or more of these trace elements may be mixed and used, and may be included in the medium in the range of 0.001% (w/w) to 3.0% (w/w).

As the growth promoter, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate, pyridoxine, etc. may be used, and may be included in the medium in the range of 0.0001% (w/w) to 1.0% (w/w).

The medium can be prepared by mixing the aforementioned carbon source, nitrogen source, trace element, growth promoter, etc., but a medium of a known composition commonly used for yeast culture in the art, such as YPD medium (Yeast Extract-Peptone) -Dextrose Medium), SD medium (Sabouraud Dextrose Medium), SM medium (Sabouraud Maltose Medium), etc. may be used. There are many literatures in the art for optimization of medium composition, including Applied Microbiol. Physiology, A Practical Approach (Editors P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). It is known and reference can be made to these known documents.

The medium is preferably sterilized to prevent the proliferation of unnecessary microorganisms, and the method for sterilizing the medium is known in the art, and a suitable method may be selected from among the known sterilization methods. For example, high-pressure steam sterilization, a sterilization method using ultraviolet rays, etc. may be suitable.

The culture temperature is generally 15°C to 45°C, preferably 25°C to 40°C, more preferably 25°C to 30°C. The pH of the medium is 4 to 8.5, preferably 4.0 to 7.0. The pH of the medium can be adjusted by adding basic compounds such as sodium hydroxide, potassium hydroxide, aqueous ammonia, or the like, or acidic compounds such as phosphoric acid and sulfuric acid during the culture.

An antifoaming agent such as fatty acid polyglycol ester may be added to remove foam formed during culture.

Culture may be advantageous to create aerobic or anaerobic conditions depending on the propagation characteristics.

Cultivation may be performed batchwise, semi-batchwise or continuously. Suitable specific culturing methods are described, for example, in Bioprozeβtechnik 1. Einfuhrung in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to Bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991), Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Brunswick/Wiesbaden, 1994), the American Society for Bacteriology (Manual of Methods for General Bacteriology)" (Washington D.C., USA, 1981), etc. can be referred to.

In another aspect, the present invention relates to Apiotricum phorosom NIBR-WAE-1241 strain (Accession No.: KACC 83034BP).

Apiotricum phorosom NIBR-WAE-1241 strain according to the present invention is 28S rRNA gene can be understood as a strain having the sequence of SEQ ID NO: 3.

As described above, according to the present invention, it is possible to provide a method for decomposing polycaprolactone polymer using Apiotricum phorosom.

The method of the present invention can be usefully applied to the decomposition of polycaprolactone polymers that are difficult to decompose in nature.

1 is a photograph of 7 days of culture of Apiotricum phorosom NIBR-WAE-1241 strain in PDA medium.
Figure 2 is a phylogenetic tree of Apiotricum phorosom NIBR-WAE-1241 strain.
3 is a result showing the polycaprolactone polymer decomposition ability of the Apiotricum phorosom NIBR-WAE-1241 strain.
Figure 4 is the result of observation with an optical microscope around the transparent ring in the minimal medium containing polycaprolactone polymer of Apiotricum phorosom NIBR-WAE-1241 strain.

Hereinafter, the present invention will be described with reference to Examples. However, the scope of the present invention is not limited to these examples.

<Example 1> Isolation of Apiotricum phorosom NIBR-WAE-1241 strain

For the isolation of Apiotrichum porosum NIBR-WAE-1241 strain, 1 g of soil collected from the clove root area of Daecheong-do, Ongjin-gun, Incheon, Korea was added with 10 mL of sterilized water and stirred in a shaking incubator at 150 rpm for 30 minutes. shaken. Then, the shaken suspension was diluted 10-fold in sterile water to make a dilution, and 100 μL of the diluted solution was smeared on potato agar medium (Potato dextrose agar; PDA, Difco, USA), and dark culture was performed at 25°C. The mycelium observed after 1-2 days of culture was subcultured in a new PDA medium, named NIBR-WAE-1241, and used as a test strain.

The results of culturing the NIBR-WAE-1241 strain in PDA medium for 7 days are shown in FIG. 1 , and a wrinkled cream-colored colony was formed, and growing mycelium was observed at the edge of the colony (left front photograph, right rear photograph). .

<Example 2> Identification of Apiotricum phorosom NIBR-WAE-1241 strain

Apiotrichum porosum ( Apiotrichum porosum ) For molecular biological identification of the NIBR-WAE-1241 strain, 28S rRNA gene sequence was analyzed.

Genomic DNA of the test strain was extracted using HiGene ^TM Genomic DNA prep kit (BIOFACT, Daejeon, Korea) and forward primer (SEQ ID NO: 1, 5'-GCA TAT CAA TAA GCG GAG GAA AAG for amplification of the 28S rRNA gene) -3') and a reverse primer (SEQ ID NO: 2, 5'-GGT CCG TGT TTC AAG ACG G-3'), and the amplified PCR product was EXOSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA) was used for purification. For the purified PCR product, nucleotide sequence analysis was requested from Solgent (Daejeon, Korea), and the received data was then sequenced using SeqMan Lasergene software (DNAStar Inc., Madison, Wisconsin, USA) (28S rRNA). The gene sequence is SEQ ID NO: 3).

Based on the nucleotide sequence obtained from the test strain, the previously reported strain using the Blast program (Basic Local Alignment Search Tool) of the National Center for Biotechnology Information to analyze the phylogenetic relationship and sequence homology were compared. The phylogenetic relationship was created using MEGA7.0 software, and the created phylogenetic tree is shown in FIG. 2 .

The isolated NIBR-WAE-1241 strain shows 100% homology with Apiotrichum porosum CBS 2040 strain ( Apiotrichum porosum CBS 2040) and Apiotrichum porosum CBS 8397 strain ( Apiotrichum porosum CBS 8397) Tricum porosum ( Apiotrichum porosum ) was identified, deposited at the Korean Agricultural Culture Collection (KACC), and was given an accession number KACC 83034BP.

<Example 3> Degradation of polycaprolactone of Apiotricum phorosum NIBR-WAE-1241 strain

Polycaprolactone diol (PCL) (M.W. ~530, Sigma-Aldrich, USA) and polylactic acid film (PLA) (0.05mm Thickness, ME331050, GFM, Korea) were used in the experiment. .

The culture medium was prepared as follows using the method of Mao et al. (Mao et al., 2015). Minimal medium without carbon source (MgSO4·7H ₂ O 0.5 g/L, NH ₄ Cl 1.0 g/L, KH ₂ PO ₄ 5.54 g/L, Na ₂ HPO ₄ 4.78 g/L, CaCl ₂ 7H ₂ O 0.005 g /L, Agar powder 15 g/L) was prepared by mixing 10 g of PCL or 4 g of PLA film dissolved in dichloromethane with 1 mL of the emulsifier Plysurf in dichloromethane. The resolution was observed while culturing in an incubator at 25°C.

The observation results are shown in FIG. 3, and in the PCL-containing medium, PCL was uniformly dissolved and all was observed to be opaque. A clear zone was clearly observed. In addition, as a result of observation at 7-day intervals, it was observed that the size of the colony increased as the number of culture days elapsed, and thus the size of the transparent ring increased. However, no resolution was observed in PLA-containing media (results not shown). A portion indicated by a red arrow in FIG. 3 is a transparent ring in which PCL is decomposed.

In addition, the results of observation with an optical microscope around the transparent ring are shown in FIG. 4, the mycelium of the Apiotricum phorosom NIBR-WAE-1241 strain was observed, and the boundary with the PCL decomposed by the strain was clearly observed. became The part indicated by the red arrow in FIG. 4 is the mycelium.

<110> Republic of Korea (National Institute of Biological Resources) <120> Degradation method for polycaprolactone polymer using Apiotrichum porosum <130> PP20-000-NIBR-WAE-1241 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gcatatcaat aagcggagga aaag 24 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggtccgtgtt tcaagacgg 19 <210> 3 <211> 534 <212> DNA <213> Unknown <220> <223> Apiotrichum porosum NIBR-WAE-1241 <400> 3 aaaagaaact aacaaggatt cccctagtaa cggcgagtga accgggaaga gctcaaattt 60 gaaatctggc agtcttcgat tgtccgagtt gtaatctata gaagcgtttt ccgtgctgaa 120 ctgtgtataa gtcccttgga acagggtatc aaagagggtg acaatcccgt actttacaca 180 atcatcagtg ctctgtgata cgttctctac gagtcgagtt gtttgggaat gcagctcaaa 240 atgggtggta aattccatct aaagctaaat attggcgaga gaccgatagc gaacaagtac 300 cgtgagggaa agatgaaaag cactttggaa agagagttaa acagtacgtg aaattgttga 360 aagggaaacg aaattcatga atcatgttta ttggactcag ccgttcttcg gtgtacttcc 420 tttaaacggg tcaacatcag ttttgtccga tggataaagg tagtaggaag gtggctctct 480 cgggagtgtt atagcctatt gctgcataca ttggatgaga ctgaggactg cagc 534

Claims (5)

A method of decomposing polycaprolactone polymer comprising the step of culturing by contacting the polycaprolactone polymer with Apiotrichum porosum NIBR-WAE-1241 strain (accession number: KACC 83034BP).
The method of claim 1,
The method, characterized in that the Apiotricum phorosom NIBR-WAE-1241 strain has the sequence of SEQ ID NO: 3 in its 28S rRNA gene.
The method of claim 1,
The contact with the polycaprolactone polymer is made by contacting the polycaprolactone polymer in the form of a culture solution obtained by culturing Apiotricum forosum in a medium,
The method, characterized in that the medium comprises at least one of a carbon source, a nitrogen source, a trace element and a growth promoter.
The method of claim 1,
Before and after the contact with the polycaprolactone polymer, a medium for promoting the culture and proliferation of Apiotricum phorosum is added to and mixed with the polycaprolactone polymer,
The method, characterized in that the medium comprises at least one of a carbon source, a nitrogen source, a trace element and a growth promoter.
Apiotricum phorosom NIBR-WAE-1241 strain (accession number: KACC 83034BP).

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