KR102404871B1 - Degradation method for polylactic acid polymer using Talaromyces pinophilus - Google Patents

Abstract

The present invention discloses a method of decomposing polylactic acid polymers using Talaromyces pinophilus , particularly Talaromyces pinophilus KNUF-20-PDG06 strain (accession number: KACC 83035BP).

Description

Degradation method for polylactic acid polymer using Talaromyces pinophilus

The present invention relates to a method for decomposing polylactic acid polymer using Talaromyces pinophilus .

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 capacity of polylactic acid polymers of Talaromyces pinophilus strains isolated and identified in domestic soil.

It is an object of the present invention to provide a method for decomposing polylactic acid polymer using a Talaromyces pinophilus 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 Talaromyces pinophilus KNUF-20-PDG06 strain, which is a fungus, from domestic soil, and the isolated and identified strain It was completed by confirming that it has the decomposition ability of polylactic acid (PLA) polymers.

Considering the above, the decomposition method of the polylactic acid polymer of the present invention can be understood to include the step of culturing by contacting the polylactic acid polymer with Thalalomyces pinophilus.

In the method of the present invention, in order to increase the decomposition capacity of polylactic acid polymers by Talaromyces pinophilus by promoting the culture and proliferation of T. In the form of the obtained culture solution, contact with the polylactic acid polymer, or before and after contact with the polylactic acid polymer, a medium for promoting the culture and proliferation of Talaromyces pinophilus may be added to the polylactic acid 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 above-mentioned carbon source, nitrogen source, trace element, growth promoter, etc., but a medium of a known composition commonly used for culturing fungi in the art, such as BHI liquid medium (Brain- heart Infusion Broth Medium), Czapek liquid medium (Czapek Broth Medium), PDB medium (Potato Dextrose Broth 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 Thalalomyces pinophilus It relates to the KNUF-20-PDG06 strain (Accession No.: KACC 83035BP).

Talaromyces pinophilus according to the present invention The KNUF-20-PDG06 strain is The RNA polymerase II large subunit 2 (RPB2) gene, the beta-tubulin (TUB) gene, and the calmodulin (CAL) gene may be understood as strains having the sequences of SEQ ID NOs: 7, 8, and 9, respectively.

As described above, according to the present invention, it is possible to provide a method for decomposing polylactic acid polymer using Talaromyces pinophilus.

The method of the present invention can be usefully applied to the decomposition of polylactic acid polymers that are difficult to decompose in a natural state.

1 is a talaromyces pinophilus This is a picture of the KNUF-20-PDG06 strain cultured in PDA medium for 7 days.
2 is a talaromyces pinophilus The phylogenetic tree of the KNUF-20-PDG06 strain.
3 is a talaromyces pinophilus This is a result showing the polylactic acid polymer decomposition ability of the KNUF-20-PDG06 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> Thalalomyces pinophilus Isolation of KNUF-20-PDG06 strain

Talaromyces pinophilus ( Talaromyces pinophilus ) To isolate the KNUF-20-PDG06 strain, 1 g of soil collected from the rhizome of a plant inhabiting Sangyeok-dong, Buk-gu, Daegu-si, Korea was added with 10 mL of sterile water and shaken at 150 rpm for 30 minutes in a shaker incubator. 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 KNUF-20-PDG06, and used as a test strain.

The results of culturing the KNUF-20-PDG06 strain in PDA medium for 7 days are shown in FIG. 1 , the center of the colony is raised, the mycelium is light green to green, the edges are white, and the back side (left photo) is dark orange to brown color.

<Example 2> Thalalomyces pinophilus Identification of KNUF-20-PDG06 strain

Talaromyces pinophilus ( Talaromyces pinophilus ) For molecular biological identification of the KNUF-20-PDG06 strain, RNA polymerase II large subunit 2 (RPB2) gene, beta-tubulin (TUB) gene, and CAL (calmodulin) gene sequences were analyzed.

Genomic DNA of the test strain was extracted using HiGene ^TM Genomic DNA prep kit (BIOFACT, Daejeon, Korea), and in RPB2 gene amplification, forward/reverse primers (RPB2-5f; 5'-GAY GAY MGW of SEQ ID NOs: 1 and 2) GAT CAY TTY GG-3'/RPB2-7cR: 5'-CCC ATR GCT TGY TTR CCC AT-3'), for TUB gene amplification, forward/reverse primers of SEQ ID NOs: 3 and 4 (Bt2a; 5'-GGT AAC CAA) ATC GGT GCT GCT TTC-3'/Bt2b;5'-ACC CTC AGT GTA GTG ACC CTT GGC-3'), forward/reverse primers of SEQ ID NOs: 5 and 6 (Cl1;5'-GAR TWC AAG) for CAL gene amplification PCR was performed using GAG GCC TTC TC-3'/Cl2a;5'-TTT TGC ATC ATG AGT TGG AC-3') and the amplified PCR product was EXOSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA) was purified using 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) (RPB2 gene). is SEQ ID NO: 7, the TUB gene is shown in SEQ ID NO: 8, and the CAL gene is shown in SEQ ID NO: 9).

Based on the TUB, CAL, and RPB2 gene sequences obtained from the test strain, the Blast program (Basic Local Alignment Search Tool) of the National Center for Biotechnology Information was used to analyze the phylogenetic relationship. was used to compare sequence homology with previously reported strains. The phylogenetic relationship was created using MEGA7.0 software, and the created phylogenetic tree is shown in FIG. 2 .

The isolated strain was confirmed to show more than 99% homology with the Talaromyces pinophilus NRRL 62136 strain, and formed the same group as the Talaromyces pinophilus on the phylogenetic tree to form the same group as the Talaromyces pinophilus NRRL 62136 strain. It was identified as phyllus, and it was deposited with the Korean Agricultural Culture Collection (KACC) and assigned an accession number KACC 83035BP.

<Example 3> Degradation of polylactic acid of Talaromyces pinophilus KNUF-20-PDG06 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 PLA-containing medium, the PLA film was uniformly dissolved, and the medium was observed to be opaque, and some undissolved PLA film particles were observed on the medium. When the Talaromyces pinophilus KNUF-20-PDG06 strain was dented and cultured, a clear zone was observed around the colony on the 8th day of culture, and on the 18th day, the clear ring was enlarged around the colony, and cultured After 45 days, it was confirmed that all the remaining white film particles were decomposed. However, no degradation was observed in PCL-containing media (results not shown). The portion indicated by the red arrow in FIG. 3 is a transparent ring in which PLA is decomposed.

<110> Republic of Korea (National Institute of Biological Resources) <120> Degradation method for polylactic acid polymer using Talaromyces pinophilus <130> PP20-000-KNUF-20-PDG06 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gaygaymgwg atcayttygg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cccatrgctt gyttrcccat 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ggtaaccaaa tcggtgctgc tttc 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 accctcagtg tagtgaccct tggc 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gartwcaagg aggccttctc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ttttgcatca tgagttggac 20 <210> 7 <211> 891 <212> DNA <213> Unknown <220> <223> Talaromyces pinophilus KNUF-20-PDG06 <400> 7 gccagccact ctcacaggtg gtttgaaata tgctctcgct accggaaact ggggtgagca 60 gaagaaggca atgagctcga aagcaggtgt ttctcaggtg ctcagtcgat acacctttgc 120 ctctactttg tctcatttga gacgtaccaa tacacctatt ggtcgtgatg gaaaaattgc 180 caagcctcgt cagctacata acactcactg gggtctggtt tgtcctgccg agactcctga 240 aggtcaagct tgtggtttgg tcaagaactt ggctttgatg tgttcaatca ctgtcggttc 300 tcctagcgag cctattgttg atttcatgat tcagcgaaac atggaagtac ttgaggaatt 360 cgaaccgcta gttacacctc atgccaccaa ggtctttgtc aatggtgttt gggttggcgt 420 gcatcgtgat ccagcgcatt tggtcagcac tgtccagtca cttcgtcgac gaaacatgat 480 ttccccacgaa gtcagtttag ttcgtgatat tcgtgaccga gagtttaaga tcttcacaga 540 tgctggtcgt gtttgtcgac cacttttcgt cattgacaac gatccacgaa gtgaaaactg 600 cggctctttg gtgctcaaca aagaccatat tcgcagactt gaagcagacc gtgagcttcc 660 accagacctc gatcctgaag agcgaaggga acaatactac ggttgggagg gccttgttaa 720 gtcgggagtc attgagtatg ttgatgctga agaggaggaa accattatga ttgcgatgtc 780 tccggaagat ctcgaaattt cgaaacaact acaagccggt tatgctctgc ctgaagacaa 840 cagtgatccg aataagcgtg tccggtctgt tctgagtcaa agggcgcata t 891 <210> 8 <211> 611 <212> DNA <213> Unknown <220> <223> Talaromyces pinophilus KNUF-20-PDG06 <400> 8 tgtaagtttg gaaatctggt tgtcgcaatg ttgtggtgga tggttagctg actagccgtt 60 ttgatgagta ggacaaggat ggagatggtg agtccgccac gaacacgacg atatttgtct 120 cgaacaaagg tttttttcac gagcatatat tgataaaatc taataggcca aattacaact 180 aaggaactgg gcaccgttat gcgttccctc ggccagaacc cctccgaatc cgaactgcag 240 gacatgatca acgaagtcga cgctgacaac aacggcacaa tcgatttccc tggtatgata 300 taattgttca cgggtttata cgatggcagt actaactgcc gcagaattct tgacaatgat 360 ggcccgcaaa atgaaggata ccgactccga ggaagagatc cgtgaagctt tcaaggtgtt 420 tgaccgtgac aacaatggtt ttatctctgc tgctgaactg cgccacgtca tgacctcgat 480 tggcgagaag ttgaccgacg atgaggttga tgagatgatt cgcgaggctg accaggacgg 540 tgatggaagg attgactgta agcctcgctg tgttttcatg aaatcagagc cattctaaca 600 agtattttct a 611 <210> 9 <211> 425 <212> DNA <213> Unknown <220> <223> Talaromyces pinophilus KNUF-20-PDG06 <400> 9 tctggtgagt tgggctctcg acctggaatt tctatcaatt gttgcgacag cacgttgact 60 tttccaggca aatcatctct gctgagcatg gcctcgatgg ctctggtgtg taagtattac 120 acgatccgaa tgcagctaca atccgacaag atctgataat caacagctac aatggctcct 180 ccgacctcca gttggagcgt atgaacgttt acttcaacga ggtgcgtcga accaatccat 240 tgtataacgg aacaaagctc atactggtgt aggcctccgg caacaaatac gttccccgtg 300 ccgtcctcgt cgacttggag cccggtacca tggacgccgt ccgcgctggt ccctttggtc 360 agctcttccg tcccgacaac tttgttttcg gtcagtccgg tgctggtaac aactgggcca 420 agggt 425

Claims (5)

Decomposition method of polylactic acid polymer comprising the step of culturing by contacting the polylactic acid polymer with Talaromyces pinophilus KNUF-20-PDG06 strain (accession number: KACC 83035BP).
The method of claim 1,
The Talaromyces pinophilus KNUF-20-PDG06 strain is A method, characterized in that the RNA polymerase II large subunit 2 (RPB2) gene, the beta-tubulin (TUB) gene, and the CAL (calmodulin) gene have the sequences of SEQ ID NOs: 7, 8, and 9, respectively.
The method of claim 1,
The contact with the polylactic acid polymer is made by contacting the polylactic acid polymer in the form of a culture solution obtained by culturing Thalalomyces pinophilus 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 contact with the polylactic acid polymer, a medium for promoting the culture and proliferation of Thalalomyces pinophilus is added to the polylactic acid polymer and mixed,
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.
Thalalomyces pinophilus KNUF-20-PDG06 strain (accession number: KACC 83035BP)

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