KR102562447B1 - Xanthomonas sp. HY-71 strain having plastic degradation activity and uses thereof - Google Patents

Abstract

Xanthomonas sp. HY-71 strain of the present invention is excellent in decomposition and xanthan production of liquid polyester urethane, polyester urethane foam and polyether urethane foam, so it is a pretreatment process for plastic recycling or bioconversion It can be usefully used in related industries such as process.

Description

Xanthomonas sp. HY-71 strain having plastic degradation activity and its use {Xanthomonas sp. HY-71 strain having plastic degradation activity and uses thereof}

The present invention relates to Xanthomonas sp. HY-71 strain having plastic decomposition activity and its use.

Plastic is being used as a material for consumable goods or disposable items in many fields replacing existing natural materials due to its economic feasibility and ease of use. there is. Recently, a large amount of waste plastic is dumped in landfills and introduced into the ecosystem, causing serious environmental pollution problems.

Polyurethane is a general term for high molecular compounds having repetitive urethane bonds. It has excellent heat resistance, abrasion resistance and aging resistance, and is used as a material for synthetic fibers, rubber, paints, adhesives, etc., and water is added during polymerization. The foam produced by inducing foaming is used as a material for bed mattresses. In addition, polyurethane is composed of a soft segment and a hard segment, and by adjusting the ratio of them, various designs from elastic rubber to rigid engineering plastic are possible. The soft segment refers to a polyol having hydroxyl groups (-OH) at both ends, and is largely classified into polyether and polyester, and the hard segment has isocyanate at both ends. It refers to isocyanate having an isocyanate group (-N=C=O). Polyurethane is the sixth most used plastic in the world and is currently replacing existing synthetic materials such as polyvinyl chloride (PVC) and polystyrene. However, polyurethane waste is difficult to recycle or destroy due to heat curing properties that do not melt when heated and toxicity of some combustion products, and most of them are disposed of in landfills. Therefore, the importance of research on waste polyurethane treatment methods is growing worldwide.

One of the methods to solve this problem is biological degradation using microorganisms or enzymes. Recently, overseas researchers are conducting research on securing plastic decomposing microorganisms and using them to decompose plastics. Research infrastructure for commercialization and mass production is also lacking, so it is necessary to develop source technology for plastic decomposition derived from domestic materials and research and development for commercialization.

On the other hand, Korean Patent No. 2126889 discloses 'a novel microorganism having plastic decomposition activity and its use', and Korean Patent No. 2176506 discloses 'a recombinant PETase producing strain, a recombinant MHETase producing strain and for PET decomposition including the same' Composition' is disclosed, but there is no description of 'Xanthomonas sp. HY-71 strain having plastic decomposition activity and use thereof' of the present invention.

The present invention has been derived from the above needs, and the present inventors have found that Xanthomonas sp., which has excellent polyester urethane decomposition activity, among strains isolated from the intestines of adult Xylocopa appendiculata . The HY-71 strain was selected, and the selected strain showed resolution and xanthan of liquid polyester urethane, polyester urethane foam and polyether urethane foam The present invention was completed by confirming that the productivity was excellent.

In order to solve the above problems, the present invention provides a Xanthomonas genus ( Xanthomonas sp.) HY-71 strain having plastic decomposition activity and accession number KCTC14741BP.

In addition, the present invention provides a plastic decomposition composition containing the strain or its culture medium as an active ingredient.

In addition, the present invention provides a composition for producing xanthan containing the strain or its culture medium as an active ingredient.

In addition, the present invention provides a plastic decomposition method comprising the step of treating the strain or its culture medium to plastic.

In addition, the present invention provides a method for producing xanthan comprising the step of treating the strain or its culture medium to plastic.

Xanthomonas sp. HY-71 strain of the present invention is excellent in decomposition and xanthan production of liquid polyester urethane, polyester urethane foam and polyether urethane foam, so it is a pretreatment process for plastic recycling or bioconversion It can be usefully used in related industries such as process.

1 is a result of selecting strains having polyester urethane decomposition activity among strains isolated from the intestines of adult bees ( Xylocopa appendiculata ).
Figure 2 is a phylogenetic schematic diagram shown through comparison of 16s rRNA nucleotide sequences of selected strains.
Figure 3 is a result of confirming the liquid polyester urethane (liquid polyester urethane) decomposition ability of the Xanthomonas HY-71 strain of the present invention (A) and a result showing the liquid polyester urethane decomposition ability analyzed through absorbance measurement (B). . Control: strain-free group.
Figure 4 is a spectral result analyzed using a FT-IR (Fourier Transform Infrared Spectrometer) spectrometer after reacting the Xanthomonas HY-71 strain and liquid polyester urethane of the present invention. Control: strain-free group.
Figure 5 is a photograph (A) and weight loss of observing changes after treating the Xanthomonas HY-71 strain of the present invention with polyester urethane foam and polyether urethane foam is the result of analyzing (B). Control: strain-free group.
6 is a result of observing the degree of damage with a scanning electron microscope after treating the Xanthomonas HY-71 strain of the present invention with polyester urethane foam (A) and polyether urethane foam (B) am. Control: strain-free group.
7 is a result of measuring xanthan production after treating the Xanthomonas HY-71 strain of the present invention in a medium to which liquid polyester urethane, polyester urethane foam, and polyether urethane foam are added, respectively . Control: A control group in which medium was added only.

In order to achieve the object of the present invention, the present invention provides a Xanthomonas sp. HY-71 strain having plastic decomposition activity.

In the present invention, strains with polyester urethane decomposition activity were selected among strains isolated from the intestines of adult bees ( Xylocopa appendiculata ) and identified through the nucleotide sequence (SEQ ID NO: 1) of the 16S rRNA gene. Santo Monas genus ( Xanthomonas sp.) It was confirmed that the strain. The present inventors named the strain as Xanthomonas HY-71 strain, and deposited it with the Korea Research Institute of Bioscience and Biotechnology on October 22, 2021 (accession number: KCTC14741BP).

In the present invention, the plastic may preferably be polyurethane, more preferably liquid polyester urethane, polyester urethane foam or polyether urethane foam urethane foam), but is not limited thereto.

In addition, the Xanthomonas HY-71 strain of the present invention is characterized by producing xanthan, and when polyurethane is added during strain culture, the production of xanthan is increased.

The present invention also provides a plastic decomposition composition containing the Xanthomonas HY-71 strain or its culture solution having the accession number KCTC14741BP as an active ingredient.

In the plastic decomposition composition of the present invention, the plastic is as described above.

In the plastic decomposition composition according to an embodiment of the present invention, the Xanthomonas sp. HY-71 strain has excellent decomposition ability of liquid polyester urethane, polyester urethane foam and polyether urethane foam, so it is used as a plastic decomposition composition can be useful

In the composition of the present invention, the 'culture medium' may include a culture medium of Xanthomonas HY-71 strain, a concentrate of the culture medium, or a dried product of the culture medium, but is not limited thereto.

In addition, the culture solution may be used in the form of the culture itself containing the strain of the present invention, or may be subjected to centrifugation or filtration to remove the culture medium in the culture solution and recover only the concentrated cells, these steps can be performed as needed by those skilled in the art. Concentrated cells can be frozen or lyophilized according to conventional methods to preserve their activity.

The culture can be obtained by culturing in large quantities by a conventional microbial culture method, and a culture medium containing a carbon source, a nitrogen source, vitamins and minerals can be used, and the carbon source includes starch, sucrose, Citrate, maltose, glucose, mannitol, glycerol or xylose may be used, but is not limited thereto.

The method of culturing the strain of the present invention may be cultured according to a method commonly used in the art, and is not limited to a particular method.

When using the Xanthomonas HY-71 strain or its culture obtained in the step of culturing the strain of the present invention as an additive, the strain or the culture of the strain may be added as it is or used together with other additives, It can be used appropriately depending on the method. The mixing amount of the components can be suitably determined depending on the intended use thereof.

In addition, the composition of the present invention can be formulated in various forms. The formulation can be prepared, for example, by adding a solvent and/or a carrier.

In addition, the preparation method of the composition may use any method known in the art, and is not particularly limited to a specific method.

The present invention also provides a composition for producing xanthan containing, as an active ingredient, the Xanthomonas HY-71 strain or a culture solution thereof having the accession number KCTC14741BP.

In the composition for producing xanthan according to an embodiment of the present invention, the xanthan production of the Xanthomonas HY-71 strain increases with culturing time, and the xanthan production is further increased when polyurethane is added and cultured. Therefore, it can be usefully used as a composition for producing xanthan.

The present invention also provides a method of decomposing plastic comprising the step of treating the Xanthomonas HY-71 strain or its culture medium having the accession number KCTC14741BP in plastic.

In the plastic decomposition method according to one embodiment of the present invention, the plastic is as described above.

In addition, the treatment concentration and treatment time of the strain or its culture solution may vary depending on the type of the plastic.

The present invention also provides a method for producing xanthan, comprising the step of treating the Xanthomonas HY-71 strain or its culture medium having the accession number KCTC14741BP in plastic.

In the method for producing xanthan according to an embodiment of the present invention, the plastic is as described above.

The method for producing xanthan of the present invention may further include a step of recovering xanthan from the culture solution of the strain. In the step of recovering xanthan from the culture solution of the strain, conventional separation techniques such as distillation, electrodialysis, pervaporation, chromatography, solvent extraction, reaction extraction, etc. may be used, and usually high purity materials are separated. For this purpose, they can be used in combination.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited thereto.

Example 1. A bee ( Xylocopa appendiculata ) Isolation and selection of strains from adults

After immersion in 70% (v / v) ethanol for 1 minute to remove contaminants on the surface of the beetle, the adult larvae collected from Bomunsan, Daejeon were washed twice with distilled water. Thereafter, the intestine was taken out with tweezers, put in PBS (pH 7.4), and the extract obtained by crushing was diluted step by step to R2A solid medium (0.5% yeast extract, 0.5% yeast extract, 0.5% yeast extract) containing 1% (v / v) polyester urethane. % proteose peptone, 0.5% casamino acid, 0.5% dextrose, 0.5% soluble starch, 0.3% dipotassium phosphate heptahydrate, 0.05% magnesium sulfate, 0.3% sodium pyruvate, 15% agar) and incubated at 30°C for 7 days. Thereafter, by forming a transparent ring around the colony, a strain having the largest transparent ring among strains having polyester urethane decomposition activity was selected (FIG. 1).

Example 2. Identification of Selected Strains

In order to identify the strains selected in Example 1, 16s rRNA gene sequencing was performed. Specifically, in order to obtain the nucleotide sequence of the 16s rRNA gene of the strain, the genomic DNA of the strain was isolated using Qiagen's DNeasy Blood & Tissue kit and used as a template, and 27F (5'-AGAGTTTGATCCTGGCTCAG-3, SEQ ID NO: 2 ) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 3) PCR analysis was performed. After purification, the amplified PCR product was sequenced by requesting Macrogen Co., Ltd., and compared with the base sequence of the GenBank database using NCBI's BLAST (FIG. 2).

As a result, the selected strain was confirmed to be a Xanthomonas sacchari strain, named Xanthomonas genus HY-71, and deposited with the Korea Research Institute of Bioscience and Biotechnology on October 22, 2021 (accession number: KCTC14741BP).

Example 3. Evaluation of liquid polyester urethane degradability of Xanthomonas HY-71 strain

Xanthomonas HY-71 strain was inoculated into LB medium (1% bacto tryptone, 0.5% yeast extract, 0.5% NaCl), cultured at 30 ° C for 24 hours, filtered under reduced pressure with Whatman filter paper, and the culture medium was obtained. 10 ml of the obtained culture medium was concentrated to 200 μl using an acetone precipitation method, mixed with 5 ml of 10% (v/v) liquid polyester urethane, and allowed to stand for 1 hour.

(1) Absorbance measurement

For quantitative analysis of liquid polyester urethane decomposition, absorbance was measured at a wavelength of 600 nm using a spectrophotometer (DU 730 ^ⓡ Life Science UV/VIS spectrophotometer, USA) every 20 minutes.

As a result, it was confirmed that the liquid polyester urethane was not decomposed at all in the control group in which the strain was not added, while the experimental group in which the strain was added showed a decrease in absorbance due to decomposition of the liquid polyester urethane over time. In particular, when the strain is reacted for 1 hour, it is confirmed that liquid polyester urethane is degraded by 83.0 to 93.2%, effectively degrading liquid polyester urethane by the in vitro secretion enzyme of Xanthomonas HY-71 strain of the present invention It was found to be (Fig. 3).

(2) FT-IR analysis

The spectrum was measured at a resolution of 4 cm ^-1 and a wavelength of 400 to 4,000 cm ^-1 using an FT-IR spectrometer (Fourier Transform Infrared Spectrometer, Vertex 80V, USA).

As a result, it was confirmed that the liquid polyester urethane treated with the culture solution of the Xanthomonas HY-71 strain showed a change on the FT-IR spectrum. In particular, by confirming that the signal decreases at 1,729 cm ^-1 related to the elongation of the carbonyl group (C=O) of the polyester and urethane bond and at 1,261 cm ^-1 related to the elongation of the CNH bond of the urethane group, liquid polyester urethane It was found that it was decomposed (FIG. 4).

Example 4. Evaluation of polyurethane foam decomposition ability of Xanthomonas HY-71 strain

In order to evaluate the degradation ability of Xanthomonas HY-71 strain on polyurethane foam, two types of polyurethane foam, polyester urethane foam and polyether urethane foam, were used. was used. After measuring the weight of each piece of polyester urethane foam and polyether urethane foam, they were placed in LB medium (1% bacto tryptone, 0.5% yeast extract, 0.5% NaCl), and strain Xanthomonas HY-71 was 1×10 ⁶ Inoculated at CFU/mL. After culturing at 30℃ and 180 rpm for 14 days, the foam was taken out, bacteria remaining on the surface of the foam were removed with 1% (w/v) sodium hypochlorite, washed 5 times with distilled water, Weight was measured.

As a result, it was confirmed that the form treated with the Xanthoxanthomonas HY-71 strain changed in shape, size and weight compared to the form not treated with the strain (FIG. 5A). In addition, it was confirmed that the weight of the polyester urethane foam and polyether urethane foam of the strain-treated group decreased by 10.11 to 11.77% and 4.58 to 5.67%, respectively, compared to the strain-free group (FIG. 5B).

In addition, as a result of observing the degree of damage of polyester urethane foam and polyether urethane foam with a scanning electron microscope (SEM), the strain-treated foam in a panoramic view (70x magnification) had a destroyed network structure and loss of integrity. It was confirmed that this appeared, and in a closer view (1,500x magnification), the foam not treated with the strain showed a smooth surface, while the foam treated with the strain showed pores, erosion, breakage and bending (FIG. 6).

Example 5. Evaluation of xanthan production ability of Xanthomonas HY-71 strain

Xanthan is a water-soluble polysaccharide produced by microorganisms and is used in the food industry as a food additive for use as a gelling agent, stabilizer, viscosity increasing agent, and crystal inhibitor.

In order to analyze the xanthan production ability of Xanthomonas HY-71 strain using polyurethane, 10% (v / v) liquid polyester urethane, polyester urethane foam or polyether urethane foam was added to LBG medium (0.2% glucose , 1% bacto tryptone, 0.5% yeast extract, 0.5% NaCl) and inoculated with Xanthomonas HY-71 strain at 1×10 ⁶ CFU/ml. The culture solution was sampled at 6, 12, 18, 24, 36, 48 or 72 hours, respectively, and the dry weight of xanthan was measured. The dry weight of xanthan was measured using the supernatant obtained by centrifuging the culture medium sampled at each time point. Isopropane containing 0.1% calcium chloride was added to the supernatant at a ratio of 1:2 to precipitate, and the pellet was collected by centrifugation, dried at 50 ° C for 24 hours, and then dried at 50 ° C. ℓ) was measured.

As a result, it was confirmed that the xanthan production of the Xanthomonas genus HY-71 strain steadily increased during 72 hours of culturing the strain. In addition, the xanthan production of the strain added with liquid polyester urethane, polyester urethane foam, or polyether urethane foam was confirmed to increase more than the control group without adding liquid polyester urethane, polyester urethane foam, or polyether urethane foam (FIG. 7).

Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC14741BP

Entrusted date: 20211022

<110> Korea Research Institute of Bioscience and Biotechnology <120> Xanthomonas sp. HY-71 strain having plastic degradation activity and uses it <130> PN21262 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1365 <212> DNA <213> Xanthomonas sp. <400> 1 tctctgggtg gcgagtggcg gacgggtgag gaatacatcg gaatctacct tttcgtgggg 60 gataacgtag ggaaacttac gctaataccg cacacgacct tagggtgaaa gcggaggacc 120 ttcgggcttc gcgcggatag atgagccgat gtcggattag ctagttggcg gggtaaaggc 180 ccaccaaggc gacgatccgt agctggtctg agaggatgat cagccacact ggaactgaga 240 cacggtccag actcctacgg gaggcagcag tggggaatat tggacaatgg gcgcaagcct 300 gatccagcca tgccgcgtgg gtgaagaagg ccttcgggtt gtaaagccct tttgttggga 360 aagaaaagca gtcggttaat acccgattgt tctgacggta cccaaagaat aagcaccggc 420 taacttcgtg ccagcagccg cggtaatacg aagggtgcaa gcgttactcg gaattactgg 480 gcgtaaagcg tgcgtaggtg gttgtttaag tccgttgtga aagccctggg ctcaacctgg 540 gaattgcagt ggatactggg caactagagt gtggtagagg atggcggaat tcccggtgta 600 gcagtgaaat gcgtagagat cgggaggaac atctgtggcg aacggcggcc atctggacca 660 acactgacac tgaggcacga aagcgtgggg agcaaacagg attagatacc ctggtagtcc 720 acgccctaaa cgatgcgaac tggatgttgg gtgcaacttg gcacgcagta tcgaagctaa 780 cgcgttaagt tcgccgcctg gggagtacgg tcgcaagact gaaactcaaa ggaattgacg 840 ggggcccgca caagcggtgg agtatgtggt ttaattcgat gcaacgcgaa gaaccttacc 900 tggtcttgac atccacggaa ctttccagag atggattggt gccttcggga accgtgagac 960 aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1020 gcgcaaccct tgtccttagt tgccagcacg taatggtggg aactctaagg agaccgccgg 1080 tgacaaaccg gaggaaggtg gggatgacgt caagtcatca tggcccttac gaccagggct 1140 acacacgtac tacaatggta gggacagagg gctgcaagcc ggcgacggtg agccaatccc 1200 agaaacccta tctcagtccg gattggagtc tgcaactcga ctccatgaag tcggaatcgc 1260 tagtaatcgc agatcagcat tgctgcggtg aatacgttcc cgggccttgt acacaccgcc 1320 cgtcacacca tgggagtttg ttgcaccaga agcaggtagc ttaac 1365 <210> 2 <211> 20 <212> DNA <213> artificial sequence <220> <223> primer <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 19 <212> DNA <213> artificial sequence <220> <223> primer <400> 3 ggttaccttg ttacgactt 19

Claims (8)

Xanthomonas sp. HY-71 strain having a polyurethane decomposing activity and accession number KCTC14741BP. delete The HY-71 strain of Xanthomonas sp. according to claim 1, wherein the strain produces xanthan. A composition for degrading polyurethane containing the strain of claim 1 or claim 3 or its culture medium as an active ingredient. delete A composition for producing xanthans containing the strain of claim 1 or claim 3 or its culture medium as an active ingredient. A method for decomposing polyurethane comprising the step of treating the strain or culture thereof of claim 1 or 3 with polyurethane. A method for producing xanthans comprising the step of treating the strain of claim 1 or claim 3 or its culture medium with polyurethane.