JP4997504B2 - Polylactic acid degradation method, polylactic acid degradation composition and microorganism used therefor - Google Patents

Description

  The present invention relates to a method for decomposing polylactic acid, a polylactic acid decomposing composition, and a microorganism used therefor.

In recent years, as the amount of plastic used increases, biodegradable plastics that can be decomposed in a natural environment have been developed in consideration of the environment. For example, polylactic acid is a polymer that can be hydrolyzed in an aqueous environment, and an enzyme may be used for the degradation. Several microorganisms that directly degrade polylactic acid have also been identified.
Known microorganisms capable of degrading polylactic acid include, for example, Amycolatopsis actinomycetes, Saccharomyces actinomycetes, and Streptomyces actinomycetes isolated from soil at 30 ° C. (for example, patent documents) 1-4). Further, Penibacillus bacteria having the resolution of plastics having an ester bond in the molecular structure have also been isolated from soil at 30 ° C. (Patent Document 5). These bacteria decompose polylactic acid by treatment at 30 ° C. for about 4 hours. Patent Documents 6 to 7 describe Bacillus subtilis, Bacillus circulans, Bacillus sterothermophilus, Actinomadura actinomycetes, and Staphylococcus bacteria isolated from soil at 50 ° C. Yes. These bacteria decompose polylactic acid by treatment at 50 ° C. for about 2 weeks.
JP 9-37776 A JP 2000-60540 A JP 2001-128693 A JP-A-10-108669 JP 2004-166542 A Japanese Patent Laid-Open No. 11-4680 JP 11-46755 A

However, although the plastic decomposition rate generally increases depending on the temperature, the decomposition activity by the microorganisms decreases if the microorganisms grow at a temperature suitable for growth. On the other hand, the resolution of microbial polylactic acid varies depending on the microbial species.
Accordingly, an object of the present invention is to provide a method for decomposing polylactic acid, a polylactic acid decomposing composition, and a microorganism that can be used therefor, which can efficiently decompose polylactic acid.

Decomposition method of the polylactic acid of the present invention, actinomycete Akuchinomadeyu la or with polylactic acid resolution using the crushed material, is intended to include the decomposition of polylactic acid.
Further, polylactic acid degradation composition of the present invention, actinomycete Akuchinomadeyu la or with polylactic acid resolution is intended to include the crushed material.
Microorganisms available present invention in the cracking process and the degradation composition of the polylactic acid, actinomycetes activator having a polylactic acid resolution Roh Madeyura-Atsurahabasea T16- 4 (Actinomadura atraherbacea T16- 4, accession number FERM P-21063) is .

  ADVANTAGE OF THE INVENTION According to this invention, the polylactic acid degradation method which can decompose | disassemble polylactic acid efficiently, a polylactic acid degradation composition, and the microorganisms which can be utilized for this can be provided.

The method for decomposing polylactic acid of the present invention includes decomposing polylactic acid using actinomydula aturahabasea T16 new strain having polylactic acid degrading ability or a crushed material thereof.
Moreover, the polylactic acid decomposition | disassembly composition of this invention contains actinomycetes actinomadura aturahabasea T16 new strain which has polylactic acid resolution | decomposability, or its crushed material.

The polylactic acid in the present invention may be a polymer mainly composed of lactic acid, and is composed of homopolymers such as poly L-lactic acid and poly D-lactic acid, poly L / D-lactic acid, and other components. Can be mentioned. In the case of a copolymer, the lactic acid component preferably has a weight ratio of 10% or more. Examples of other components constituting the copolymer include ε-caprolactone, glycolide, and depsipeptide. These other components may be one type or two or more types.
Although there is no restriction | limiting in particular in the molecular weight of the polylactic acid which can be decomposed | disassembled by this invention, For example, it can be preferably 50,000-300,000 from a viewpoint of a number average molecular weight 10,000-100,000 and a decomposition rate.

Actinomycetes Actinomadura Atsurahabase A in the present invention is a new species with a polylactic acid resolution.
This Actinomadura Atsurahabasea T16, in particular, Actinomadura Atsurahabasea T16-4 share (Higashi 1-1-1 center Tsukuba, Ibaraki Prefecture sixth, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, consignment number FERM P- 21063, deposited on receipt of Oct. 19, 2006).

As described later, Actinomadura T16-4 is a bacterium isolated from forest soil as a microorganism capable of degrading polylactic acid under a temperature condition of 50 ° C., and shows Gram-positive, TSB (Trypticase Soy Broth, Becton Dickinson) The agar cultured at 50 ° C. is a filamentous fungus having a width of about 1 μm, and forms a spore chain in which 10 to 15 spheroidal spherical spores are linked (FIG. 2). It also forms serrated circular colonies, has wrinkles on its surface, no gloss, and has an excellent ability to decompose polylactic acid.
As described later, this microorganism can be distinguished from other closely related bacterial species from the viewpoints of morphological properties, culture properties, physiological properties, homology of 16S rDNA sequences, and DNA-DNA hybridization. It has the common properties of degrading polylactic acid and degrading cellulose and starch, assimilating mannose and growing ability at 60 ° C.

The microorganisms used in the present invention can be cultured, maintained and propagated under the conditions usually used. Such culture conditions are pH 4 to 10, preferably from the viewpoint of growth rate, pH 6.0 to 8.0, 40 to 60 ° C., and preferably from 45 to 55 ° C. from the viewpoint of growth rate. be able to.
The basic culture medium (hereinafter simply referred to as “medium”) capable of maintaining the microorganism according to the present invention includes, for example, ammonium sulfate, ammonium phosphate, ammonium carbonate and the like as a nitrogen source, and other inorganic salts such as diphosphate. Each of them contains potassium, monosodium phosphate, magnesium chloride, calcium chloride, ferrous sulfate, sodium molybdate, manganese chloride, etc., and any solid or liquid medium that is usually used can be preferably used. . In addition to the above components, the medium may contain various vitamins, minerals, and other nutritional components for promoting the growth of microorganisms.

  This microorganism is used for the degradation of polylactic acid in various forms. As such a form, it can select suitably from each forms, such as a microbial cell, a crushed cell of a microbial cell, and a culture supernatant of a microorganism. In the case of microbial cells, any of live microbial cells obtained by collecting bacteria such as centrifugation from the culture, dry powder obtained by freeze-drying the microbial cells, or a culture solution containing microorganisms may be used. The crushed material may be any material that has been crushed by physical means or chemical means. Examples of physical means include physical means such as an ultrasonic grinder and a cell crusher using glass beads. Examples of chemical means include bacterial enzymes such as lysozyme. When crushing bacterial cells using physical means or chemical means, it is preferable to carry out under mild conditions that do not impair the degradation activity of the crushed material. The culture supernatant is not particularly limited as long as the microorganism is cultured in the liquid medium described above. From the strength of the decomposing activity in the culture supernatant, the culture is preferably performed after the second day of culture, more preferably after the third day of culture. The supernatant can be used.

The decomposition process of polylactic acid is performed by bringing the microorganism or its crushed material prepared in an appropriate form into contact with the polylactic acid to be processed under aerobic conditions. The pH during the decomposition treatment is preferably pH 4 to 10, and preferably pH 6.0 to 8.0 from the viewpoint of the decomposition rate. The temperature during the decomposition treatment is preferably 40 to 60 ° C., and preferably 45 to 55 ° C. from the viewpoint of the treatment rate of polylactic acid. The treatment time can be 2 days to 10 days, and preferably 3 days to 7 days from the viewpoint of treatment efficiency.
Moreover, the form of polylactic acid at the time of decomposing | disassembling polylactic acid can mention a film (sheet), a molded object, a crushed material, a powder, a suspension, etc., and any of these may be sufficient. Further, the polylactic acid during the decomposition treatment may be the above-described polylactic acid alone or a mixture with other plastics.

The amount of polylactic acid in the decomposition treatment varies depending on the form of the target polylactic acid and the form of the microorganism used for the treatment, but can be 0.1 to 5.0 grams per 100 ml of the bacterial culture solution. From the viewpoint of the decomposition rate, it can be 0.5 to 1.0 gram.
The treatment may be carried out by adding a basic culture medium, polylactic acid to be treated, a microorganism having a resolution or a pulverized product thereof, a tablet, a culture solution to the culture tank, and the microorganism is added to activated sludge and compost. It may be done by incorporating.

The polylactic acid-decomposing composition according to the present invention only needs to contain the microorganism, a crushed product thereof, or a mixture thereof, and is mixed in a dry form composed only of the microorganism or the crushed product, or an appropriate liquid carrier. It may be in liquid form.
The liquid carrier used for constituting the liquid form is not particularly limited as long as it is usually used in this application, and examples thereof include water and a buffer solution. The pH of the liquid carrier may be 6.0 to 9.0, and preferably 7.0 to 8.0 from the viewpoint of decomposition activity.
In addition, as conditions applicable to the degradation of polylactic acid, known conditions can be applied as they are as long as the activity of the microorganism according to the present invention is not impaired.

Examples of the present invention will be described below, but the present invention is not limited thereto.
[Example 1]
1. Isolation of microorganisms that degrade polylactic acid (PLA) (1) Prepare 400 ml of polylactic acid (PLA) -containing basic medium with the composition shown in Table 1, divide it into equal parts, and add 6 g of agar and heat Sterilize (120 ° C., 20 minutes). After heat sterilization, PLA (Lacia H400, Sumitomo Chemical) dissolved in 16 ml of dichloromethane was added to the medium to which agar was not added, and sufficiently emulsified (10 minutes) using an ultrasonic crusher. The emulsified PLA was added to a medium to which agar was added and stirred sufficiently, and then dispensed into a petri dish to prepare a PLA emulsified agar medium.

(2) A small amount of soil sample is collected from the forest soil using a micro spatula and suspended in 3 ml of sterile water dispensed into a test tube. Next, the test tube is treated with an ultrasonic cleaner for 30 seconds, and further suspended (3 minutes) using a stirrer. 0.1 ml of this suspension was smeared on the PLA emulsified agar medium and cultured at 50 ° C. As a result, 11 strains forming a clear zone by PLA degradation on the agar medium were obtained. One of them was named T16-4 stock.

2. Various properties of strains according to this example (1) Morphological, cultural and physiological properties of T16-4 Table 2 shows the morphological, cultural and physiological properties of the new strain T16-4 according to the present invention. Shown in As the growth medium, yeast / malt agar medium (ISP medium No. 2), oatmeal agar medium (ISP medium No. 3), starch / inorganic salt agar medium (ISP medium No. 4), glycerin / asparagine agar medium (ISP) Medium No. 5) was used.
In addition, in order to clarify the properties of the new strain T16-4, it was compared with the related bacteria Actinomadura viridilutea (A. vilidilutea NBRC 14480) and Actinomadura rubrobrunea (A. rubrobrunea NBRC 15275).

(2) Cell lipid composition of T16-4 Next, the fatty acid composition analysis of T16-4 was performed by Miller's method using MIDI system (category and identification of actinomycetes, edited by Japanese Society for Actinomycetes, p72-76, Japan According to the Academic Affairs Office). Briefly, cell fatty acids are converted to fatty acid methyl esters using alkali methanol and hydrochloric acid methanol and analyzed by gas chromatography. The results are shown in Table 3.
In addition, Kroppenstedt, RM et al., (1990) System.Appl.Microbiol., Vol.13, pp148-160 was referred for the cell fat composition of Actinomadura villageidte in the table. The cell fat composition of Actinomadura rubrobrune is Agre and Guzeva. (1975) Mikrobiologiya. Vol.44, pp518-522.

(3) Classification and identification by 16S rDNA Next, classification and identification based on the 16S rDNA sequence of T16-4 was performed according to a conventional method as described below. The following 16S rDNA was used: Actinomadura rubrobrunea [GenBank Accession No. AF134069], Actinomadura viridilutea [GenBank Accession No. D86943], Actinomadura・ Actinomadura oligospora [GenBank Accession No. AF163118], Actinomadura hibisca [GenBank Accession No. AJ293705], Actinomadura atramentaria [GenBank Accession No. AJ420138] -Actinomadura namibiensis (GenBank Accession No. AJ420134), Actinomadura kijaniata [GenBank Accession No. X97890].

  Target bacteria including T16-4 were streaked on a TSB (Becton Dickinson) plate medium and cultured at 50 ° C. for 24 hours. Colonies were picked up with a toothpick and suspended in 20 μl of sterile distilled water. At the tip of the toothpick, the mixture was crushed and 180 μl of InstaGene Matrix (BIO-RAD) was added. The mixture was gently stirred by hand and heated at 100 ° C. for 15 minutes. After heating, the mixture was stirred for 10 seconds or more and centrifuged at 12,000 rpm (13,000 × g) for 2 to 3 minutes. 20 μl of the supernatant was used as a 50 μl scale PCR template.

  Amplification of the 16Sr DNA gene of the T16-4 strain was performed using 9F (SEQ ID NO: 1) and 1541R (SEQ ID NO: 2) described in Table 4 described later as primers. As the thermal cycler, TaKaRa PCR Thermal Cycler Dice Standard (Takara Bio Inc.) was used. As the polymerase, Hot Star Taq DNA polymerase (Qiagen Co., Ltd.) was used. The reaction solution was prepared according to the manual. PCR reaction was performed at 96 ° C for 5 minutes for 1 cycle, 97 ° C for 45 seconds, 50 ° C for 30 seconds, 74 ° C for 1 minute, 5 cycles, 96 ° C for 45 seconds, 50 ° C for 30 seconds, 74 ° C for 1 minute. The PCR product was purified using PCR Purification Kit -Spin Type- (manufactured by BIONEX).

DNA sequencing was performed by the dye terminator method. ABI PRISM 3100 DNA Sequencer (Applied Biosystems) was used for the determination of the base sequence. Cycle sequencing, purification of sequencing products, and sequencing conditions were carried out by the following method according to the manual method.
1) Preparation of sequence sample For the preparation of the sequence sample, a dedicated kit BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) was used. For the sequence, 9F, 1541R, 785F (SEQ ID NO: 3) described in Table 4 were used. ), 802R (SEQ ID NO: 4) was used. Cycle sequence conditions and sample preparation methods were partially modified from the manual method, and a reaction solution was prepared according to the manual, and a PCR reaction was performed. PCR conditions were 96 ° C for 1 minute for 1 cycle, 96 ° C for 10 seconds, 50 ° C for 5 seconds, and 60 ° C for 4 minutes for 25 cycles.

2) Purification of sequencing sample To the sample obtained above, 10 μl of Clean SEQ (Agencourt) was added while mixing. 62 μl of 85% ethanol was added and after pipetting, placed on a magnetic plate and allowed to stand for 3 minutes. The solution was discarded after confirming that it was clear. 100 μl of 85% ethanol was added and allowed to stand for 30 seconds. The solution was discarded and the same operation was repeated. After incubating at 37 ° C. for 10 minutes, 40 μl of 0.1 mM EDTA (pH 8.0) was added and allowed to stand for 5 minutes.

The 16S rDNA partial nucleotide sequence was determined as follows. The base sequence obtained from each primer was edited by Genetix-ATGC ver.10.1 (manufactured by Software development), and the 16S rDNA partial base sequence was determined (SEQ ID NO: 5: 1450 bp, see FIG. 1). BLAST search was performed on the determined nucleotide sequence from the National Institute of Genetics, Japan DNA Data Bank (DDBJ: http://www.ddbj.nig.ac.jp/), the National Institute of Genetics, National Institute of Genetics .
In addition, in this classification and identification method using 16S rDNA, it can be estimated that the species is the same with a homology of 99.8% or more.
The results are shown in Table 5.
As shown in Table 5, none of the strains showed 99% or more homology with T16-4, and 98.0% and 97% for Actinomadura ruburobrunea and Actinomadura viridutte, which had the highest homology, respectively. 9%.

(4) DNA-DNA homology test Next, the classification and identification based on the DNA-DNA homology of T16-4 was performed using the fluorescence method of Ezaki et al. (Category and identification of actinomycetes, edited by the Japanese Society for Actinomycetes, p134-137, This was done in accordance with the Japan Society for Administrative Affairs. Briefly, after extracting the total DNA, the total DNA is immobilized on a microplate according to a conventional method, and after adding photobiotin (Vector Laboratories, Inc., # SP-1000), the DNA is irradiated with a mercury lamp. Label. After hybridizing the labeled DNA with the other DNA, a predetermined amount of a streptavidin-enzyme (β-galactosidase) solution is added, and then a fluorescent substrate (4-MUF-β-D-galactopranoside) is reacted. Then, the fluorescence intensity is measured with a microplate reader, and the homology is calculated based on the fluorescence intensity.
DNA was prepared using Instagene Matrix (Bio-Rad). As related bacteria, Actinomadura rubrobrunea NBRC 15275 and Actinomadura Viridilutea NBRC 14480, which showed homology of about 98% or more by the 16S rDNA classification and identification method described above, were used. did.
In this DNA-DNA homology test, if the DNA homology is 70% or more, it is the same bacterial species and can be estimated as a subspecies with a similarity of 60 to 70%.
The results are shown in Table 6.
As shown in Table 6, the homology was 50% or less for each of the two strains that were considered to be the most homologous in the classification and identification method using 16S rDNA and considered to be related species.

From these things, it became clear that the T16-4 strain has the following characteristic properties.
(1) Aerobic, Gram-positive, non-acid / anti-alcohol, non-motile actinomycetes.
(2) Forms cream-yellow base mycelium in ISP-2, 4 and 5 and does not produce diffusible pigments in ISP-5.
(3) Air hyphae are rare, but when present, they are green-gray for ISP-4 and white for ISP-2 and 3.
(4) Scanning photomicrograph image of T16-4 (FIG. 2) shows spherical spores with short helical strands after growth on vitamin humate (HV) agar medium.
(5) Grows in the presence of 6% NaCl at 30 to 60 ° C.
(6) Cellulose, starch, oatmeal and polylactic acid can be decomposed, and nitrate is reduced to nitrous acid.
(7) The key amino acid of peptidoglycan is meso-DAP.
(8) Cellular hydrolysates include galactose, glucose, madulose, mannose and ribose, and phospholipids include diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol and phosphatidylinositol mannoside.
(9) The main fatty acid methyl esters are isoC16: 0 and isoC17: 0.
(10) The main menaquinones are MK-9 (H ₄ ), MK-9 (H ₆ ) and MK-9 (H ₈ ).
(11) G + C content is 72 mol%.

  As a result, T16-4 is a bacterium that can be distinguished from other closely related bacterial species from the viewpoint of 16S rDNA sequence homology and DNA-DNA hybridization, and degrades polylactic acid and cellulose and Nov. (Actinomadura atraherbacea T16-4 sp. Nov.) Was determined as a New Species based on starch resolution, mannose assimilation ability and viability at 60 ° C. ).

3. PLA degradation by T16-4 strain-1
The PLA resolution of the T16-4 strain obtained above was examined.
The T16-4 strain was transplanted to the 0.1% PLA emulsified agar medium used in 1 above and cultured at 50 ° C. The results are shown in Table 7 and FIG. In the medium after 5 days at 50 ° C., a clear zone with a diameter of about 3 cm was formed by the T16-4 strain (see FIG. 3).
In the culture at 50 ° C. for 3 days, the T16-4 strain formed a clear zone having a diameter of about 2 cm on the PLA emulsified agar medium.

  Conventional PLA-degrading active microorganisms (Streptarothecus hindustanus, Saccharomonospora azrea, Kibdelos porangiium arism, Saccharopolis pora erythrae, Saccharopolispora hordeii, Streptarotecus hindus stanus , Rentzea Albidocapyrata, Actinoquineospora liparia, Actinopolispora halophylla, Actinopolispora Mortivaris, Actinomadura Viridis, Streptomyces bioaceus niger, Streptomyces cyanes, Amicoraptops Mediterranei Since it takes 2 weeks to form a clear zone of about 3 cm, it can be seen that the PLA-degrading activity of the T16-4 strain is superior to that of conventional strains.

[Example 2]
From a TSB (Becton Dickinson) agar slant medium, one platinum loop of T16-4 strain cells was inoculated into 5 ml of TSB medium (test tube, φ18 mm), and cultured with shaking at 50 ° C. (180 rpm, 20 hours). . 0.1 ml of this culture solution is 5 ml (test tube, φ18 mm) of M229 medium (0.5% yeast extract, 0.1% calcium chloride, pH 7.3) containing 0.1 g of PLA film, and the final concentration of PLA is 0. 0.5%) and inoculated at 50 ° C. with shaking (180 rpm).
As a result of the culture, PLA in the medium was completely degraded after 72 hours by T16-4.

[Example 3]
The PLA resolution of T16-4 was evaluated in the same manner as in Example 2 except that the PLA final concentration 0.1% PLA liquid medium was used. As a result, PLA was completely decomposed in about 1-2 days.
In the conventional PLA-degrading bacteria, PLA in a 0.1% PLA liquid medium cannot be completely degraded even if it takes 3 weeks or more, so that the PLA-degrading activity of the T16-4 strain is superior to the conventional strains. I understand.

  Thus, the PLA-degrading bacterium T16-4 of the present invention has a high PLA resolution and was clearly a useful microorganism in the field where PLA degradation is required.

It is a 16S rDNA partial base sequence of the T16-4 strain of the present invention. It is an electron micrograph (9000 times) of T16-4 stock of the present invention. It is a photograph which shows PLA degradation activity by T16-4 stock | strain of this invention.

Claims (5)

Actinomycetes activator having a polylactic acid resolution Roh Madeyura-Atsurahabase A or using the crushed material, method for decomposing polylactic includes decomposing polylactic acid. The actinomycete Actinomadura Atsurahabase authors, actinomycetes Actinomadura Atsurahabasea T16-4 (Actinomadura atraherbacea T16-4, accession number FERM P-21063) is an exploded method of claim 1, wherein the polylactic acid. Actinomycetes activator having a polylactic acid resolution Roh Madeyura-Atsurahabase A or polylactic acid degradation composition containing the crushed product. The actinomycete Actinomadura Atsurahabase authors, actinomycetes Actinomadura Atsurahabasea T16-4 (Actinomadura atraherbacea T16-4, accession number FERM P-21063) in a third aspect polylactic acid degradation composition. Actinomadura atraherbacea T16-4 ( Acinomadura atraherbacea T16-4 , accession number FERM P-21063) having polylactic acid resolution.