JP3762990B2 - Biodegradable plastic decomposing agent and decomposition method - Google Patents

Description

【図面の簡単な説明】
【図１】リパーゼＣＳ２のアミノ酸配列（下段）及びそれをコードするＤＮＡの塩基配列（上段）を示す。
【図２】ポリ乳酸の分解実験の結果を示す。
【図３】ポリ乳酸分解反応後の反応溶液（３０℃、１０日）の状態を示す図面代用写真である。
【図４】ポリブチレンサクシネートの分解実験の結果を示す。
【図５】ポリブチレンサクシネート分解反応後の反応溶液（３０℃、２日）の状態を示す図面代用写真である。
【図６】ポリカプロラクトン分解反応後の反応溶液（３０℃、２日）の状態を示す図面代用写真である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to the degradation of plastics, particularly biodegradable plastics, and more particularly to a novel technique for degrading biodegradable plastics such as polylactic acid using a specific lipase.
[0002]
[Prior art]
The annual production of plastic in Japan is about 15 million tons (2000 statistics). Among them, the production of biodegradable plastics is low, about 2000 to 2500 tons. In this way, biodegradable plastics are still very useful in today's waste treatment and environmental pollution caused by waste, but their production remains low. However, the reason is mainly that the price is higher than that of conventional plastics, and that although it is biodegradable, a technique for efficiently decomposing has not been established.
[0003]
In recent years, polylactic acid (PLA) has been highlighted as a biodegradable plastic (biodegradable polymer). PLA is a polymer of lactic acid obtained by fermentation from corn starch and has excellent moldability, high transparency, and strength. Containers, packaging films, agricultural and horticultural films, civil engineering sheets, fibers It has a wide range of uses such as clothing, and is also used in the field of medicine as an implant material. It is also expected in terms of environment and resources as a plastic made from plants, and mass production has started in 2001. (Cargill Dow, USA).
[0004]
As mentioned above, polylactic acid has excellent properties as a plastic, and is expected to reduce its price and greatly expand its supply. On the other hand, although it is a biodegradable plastic, It is said to be an enzyme-type decomposing plastic, and there are virtually no known enzymes that effectively decompose it.
[0005]
Biodegradable plastics are thought to be decomposed by microorganism-derived secretory enzymes, but currently only degradation by proteases (proteolytic enzymes) has been confirmed for degradation of high-molecular polylactic acid. It is. So far, no high molecular polylactic acid degrading enzyme other than protease has been reported. Moreover, the degradation action by protease is not fully satisfactory. Furthermore, no examples of successful degradation of polylactic acid by lipase have been reported so far.
[0006]
[Problems to be solved by the invention]
As mentioned above, polylactic acid is an excellent biodegradable plastic derived from plants, and if its use is greatly expanded in the near future, no matter how biodegradable it can be, Therefore, it is necessary to construct an active and effective processing means. The same applies to other biodegradable plastics.
Therefore, the present invention is newly set for the purpose of proposing effective use of enzymes for biodegradable plastic degradation centering on polylactic acid.
[0007]
[Means for Solving the Problems]
As described above, polylactic acid-based plastics are said to be non-enzymatic decomposition type plastics while being classified as biodegradable plastics, and are difficult to decompose, and few enzymes are known to efficiently decompose them. In particular, there is no report of an enzyme belonging to lipase that has its degradability. In fact, as will be apparent from the following description, commercially available lipases (a commercially available lipase derived from Aspergillus niger, Candida rugosa, Rhizopus oryzae, Burkholderia sp., Penicillium roqueforti) are all decomposed. could not.
[0008]
In this way, plastics that are said to be biodegradable, such as polylactic acid-based plastics, are not well known for enzymes that effectively decompose them, and are usually not suitable for recycling because they decompose in the soil. Has been said to be.
[0009]
In the current state of this technology, that is, enzymes that effectively decompose biodegradable plastics are not well known, and no successful industrialization has been reported, and polylactic acid is degraded for conventional lipases. In spite of this, it has been confirmed that the polylactic acid is not degraded. Extensive screening was conducted.
[0010]
As a result, it was quite unexpected that an enzyme derived from a specific yeast and lipase CS2, which is a specific lipase, can degrade polylactic acid, and this enzyme has a higher ability to degrade polylactic acid in comparison with protease. It was confirmed newly to show. Conventionally, no high molecular polylactic acid-degrading enzyme has been reported other than protease, and this finding is completely new. As a result of further research, it was newly confirmed that lipase CS2 can also decompose other biodegradable plastics.
[0011]
The present invention was finally completed as a result of further research based on these useful new findings, and relates to a biodegradable plastic decomposing agent and a decomposing method.
Hereinafter, the present invention will be described in detail.
[0012]
Lipase CS2, which is an active ingredient in the present invention, is a novel enzyme developed by the present inventors, has the following physicochemical properties, and belongs to the genus Cryptococcus, such as Cryptococcus sp. It can be separated and collected from the culture of S-2 (Cryptococcus sp. S-2) (FERM P-15155) (Japanese Patent Laid-Open No. 2001-252072).
[0013]
The physicochemical properties of this enzyme are as follows.
[0014]
(1) Action
It has oil-degradability, acts on triglycerides, and hydrolyzes into glycerin and fatty acids.
[0015]
(2) Substrate specificity
Decomposes triptyline, tricaprylin, tripalmitin and triolein well. Triacetin, tricaprin, and trilaurin are moderately degraded. Degradability against trimyristin and tristearin is weak.
[0016]
(3) Position specificity
When acting on triolein, oleic acid and a small amount of 1,2 (2,3) -diolein are produced, and 1,3-diolein and monoolein are not detected.
[0017]
(4) Optimum pH and stable pH range
Optimum pH: 7.0
Stable pH range: 5-9
[0018]
(5) Reaction optimum temperature and temperature deactivation conditions
Optimal reaction temperature: 37 ° C
Conditions of deactivation due to temperature: Deactivation of activity due to temperature rise is moderate, and the activity is maintained even after heat treatment at 60 ° C. for 30 minutes.
[0019]
(6) Stability and activation against organic solvents
It is stable in organic solvents, and its activity is increased by dimethyl sulfoxide and diethyl ether.
[0020]
(7) Molecular weight
22 kDa (SDS-PAGE)
[0021]
The present enzyme having the above physicochemical properties can be isolated and obtained from a culture of a microorganism, for example, S-2 strain (FERM P-15155) (hereinafter sometimes referred to as S-2 strain) belonging to the genus Cryptococcus. it can. The strain created and isolated here is very characteristic in that it can produce and secrete a novel enzyme (lipase CS2), and this is referred to as Cryptococcus sp. It was named S-2 and deposited as FERM P-15155 at the Biotechnology Institute of Industrial Technology (currently the National Institute of Advanced Industrial Science and Technology (AIST)).
[0022]
This enzyme can be obtained by culturing the S-2 strain according to a conventional method, and separating and purifying from the culture. For example, pre-culture (5.4 to 5.8 × 10 6) at 25 ° C. in a YM medium (yeast extract 0.3%, malt extract 0.5%, peptone 0.5%, glucose 1%). ⁸ Cell / ml) is used as an inoculum for normal main culture.
[0023]
Yeast extract 0.5%, KH ₂ PO _Four 1% MgSO _Four ・ 7H ₂ The enzyme production medium was based on O and triolein 1%, and the nitrogen source, carbon source, and inducer were changed as required. A 100 ml enzyme production medium may be inoculated with 1% (v / v) of the pre-cultured bacteria, and the enzyme may be produced by shaking culture at 25 ° C. and 100 rpm. Triolein may not be added, but Cryptococcus sp. Lipase production by S-2 is about 3 times more lipase secretion than glucose medium by using oil such as olive oil and triolein as carbon source, and lipase production is superior by using oil as carbon source. Enhanced. As the oil and fat, it is preferable to use at least one selected from sardine oil, soybean oil, triglyceride and the like in addition to the above.
[0024]
The S-2 strain may be cultured in the same manner as the yeast culture. However, when oils and fats are used as a carbon source as described above, enzyme production increases. In addition, the use of yeast extract as a nitrogen source and lactose as a sugar further increases enzyme production.
[0025]
After culturing the S-2 strain, the enzyme is separated from the culture and purified. The method may be in accordance with a conventional method. For example, after concentrating the culture solution, a known purification method such as chromatography (cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, etc.) is repeated alone or repeatedly. May be appropriately implemented in combination.
[0026]
In the present invention, in addition to the above-described purified enzyme, a crude enzyme without being sufficiently purified can be used as an active ingredient. As a crude enzyme, in addition to various intermediate purified products obtained in the course of the purification process described above, cultures, culture supernatants, culture solutions, and processed products thereof (for example, concentrates, pastes, dried products, dried products) Powder etc.) can also be used as appropriate.
[0027]
As for lipase CS2, in addition to the fermentation method described above, that is, the production method by culturing yeast, the present inventors have further examined at the gene level, and as a result, the amino acid sequence of the enzyme and the encoding thereof. We have also succeeded in determining the base sequence of gene DNA. Therefore, an enzyme protein may be synthesized according to the amino acid sequence revealed this time, and a recombinant vector containing this gene has also been created, and this enzyme is industrially produced using recombinant DNA technology. It became possible. In the present invention, any enzyme produced by any method can be used as an active ingredient.
[0028]
Analysis of the lipase CS2 gene was performed as follows. The purified lipase CS2 was treated with a proteolytic enzyme (lysyl endopeptidase) and fragmented, and the amino acid sequence of several fragments was determined. A DNA primer (mixed primer) was designed based on the partial sequence and used to determine the internal sequence of the lipase CS2 gene. Next, Cryptococcus sp. MRNA was prepared from the cells of S-2, and cDNA was synthesized by reverse transcription.
[0029]
First, the internal sequence (about 180 bases) of the lipase CS2 gene was determined using the cDNA as a template and primers designed from the lipase CS2 fragmented peptide. A new primer was designed using the determined internal sequence, and the cDNA sequence of the lipase CS2 gene was determined by using the 3′- and 5′-RACE method. The entire base sequence of the lipase CS2 gene thus determined is shown in SEQ ID NO: 2 (FIG. 1), and the amino acid sequence of the protein deduced from this base sequence is shown in SEQ ID NO: 1 (FIG. 1).
[0030]
As described above, Cryptococcus sp. After obtaining cDNA of this enzyme from S-2, cloning was performed. The deduced amino acid sequence could be determined from the DNA sequence. The cDNA obtained above was inserted into the BamHI cloning site of Saccharomyces cerevisiae expression vector pG-1 (GPD promoter, PGK terminator, TRP1 marker) to prepare a recombinant vector. The obtained recombinant vector was named CS2 Lipase cDNA * pG-1 and deposited as FERM P-18939 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.
[0031]
When the recombinant plasmid CS2 Lipase cDNA * pG-1 (FERM P-18939) obtained above was transformed into the Saccharomyces cerevisiae YPH499 strain, YPD (yeast extract 1%, peptone 2 suspended in 1% concentration) was obtained. %, Glucose 2%) on the plate, the transformant formed a halo produced by butylene degradation. Thus, it was confirmed that the cDNA obtained had an oil-and-oil decomposition activity, and it was shown that it surely encodes the target lipase CS2.
[0032]
The present enzyme (lipase CS2: cutinase) can decompose various biodegradable plastics, and examples thereof include the following: polylactic acid plastic (PLA); succinic polyester plastic (polybutylene) Succinate (PBS), polyethylene succinate (PES) and others); polyesteramide plastic; polycaprolactone plastic (PCL); polyhydroxybutyrate plastic (PHB); Although this enzyme has been found to be very effective for polylactic acid degradation, this enzyme is a cutinase (ie, an excellent esterolytic enzyme) that degrades high molecular esters (cutins). The polyester-based plastic can also be effectively decomposed.
[0033]
In order to decompose the biodegradable plastic according to the present invention, the enzyme and the plastic may be brought into contact, the plastic is deposited outdoors, or buried in the ground, and the enzyme may be added thereto. When industrial treatment is performed, plastics that are shredded, coarsely crushed or pulverized as necessary are placed in a decomposition tank (reaction tank), an enzyme is added thereto, and the mixture is stirred as desired.
[0034]
The decomposition treatment can be carried out under either solid or liquid conditions. In the latter case, it is advantageous to add an emulsifier (for example, Prisurf (Daiichi Kogyo Seiyaku), etc.) and emulsify. As the enzyme, a purified enzyme as well as a crude enzyme can be used. Furthermore, yeast cell cultures, culture solutions, and processed products thereof can be used. Also, a synthetic enzyme, a recombinant enzyme, and the amino acid sequence of SEQ ID NO: 1 can be used. A protein having a sequence having high similarity and a protein having similarity to cutinase on the amino acid sequence may be used alone or in combination of two or more.
[0035]
In the liquid decomposition, the plastic is preferably emulsified with a surfactant, and the plastic concentration in the emulsion is appropriately determined depending on the type, the concentration of the enzyme used, and the like. A concentration of about 0001 to 1% and usually about 0.01 to 0.5% is usually used. However, if the amount of enzyme used is increased, a higher concentration can be obtained.
[0036]
The amount of enzyme added to such a plastic liquid varies depending on the type and concentration of the plastic to be treated. As a guideline, when decomposing the plastic liquid, the enzyme concentration in the liquid is 1 ng to 1 μg or more per ml. The enzyme addition amount may be set so as to be preferably 5 to 500 ng or more. If the cost is not taken into consideration, it is of course possible to use more than the above concentration range. In this way, the plastics can be efficiently decomposed, and, for example, when PLA is processed, components such as lactic acid are generated. By recovering these components again, resources can be recovered. Can be reused.
[0037]
Therefore, the preparation of the present decomposition agent may be carried out by using the present enzyme as an active ingredient in accordance with liquid preparations, wettable powders, emulsifiers, powders, tablets, granules and other commonly used pharmaceutical techniques. The amount may be appropriately set so that the enzyme concentration described above is achieved. Non-limiting examples include preparations with a content ratio of 1 to 1000 mg / ml, preferably 10 to 500 mg / ml. Examples of the enhancer include ethylene glycol, and as described above, the culture, the culture solution, and the processed product thereof can be used as they are as a preparation. In preparation of the preparation, an emulsifier for plastic emulsification may be separately prepared so as to be prepared at the time of use.
[0038]
As described above, the present invention has been described from the viewpoint of waste treatment using PLA as a representative example, but PLA is also used in the production of fibers, clothing, etc., and in that case, PLA is totally and / or locally. By disassembling, it is possible to improve the properties and improve the quality by softening the fibers and clothing or adding patterns. Therefore, the present invention is characteristic in that it has not only a waste decomposition process but also a new aspect of creating a new product.
[0039]
Examples of the present invention will be described below.
[0040]
[Example 1]
(1) Yeast extract 0.5%, KH ₂ PO _Four 1% MgSO _Four ・ 7H ₂ 1% (v / v) of yeast S-2 (FERM P-15155) pre-cultured in a liquid medium containing O, 1% triolein and 0.5% lactose, and shake culture at 25 ° C. and 100 rpm Went.
[0041]
(2) Next, the enzyme was purified as follows. A culture solution was obtained by removing yeast cells from the liquid-cultured culture as described above. The culture broth thus obtained was centrifuged at 8,000 rpm for 10 min, filtered through a 0.45 μm membrane filter, and concentrated by ultrafiltration. The lipase was purified by high performance liquid chromatography (gradient elution with pH 7.0 phosphate buffer and 0.5% NaCl added thereto) on a cation exchange resin TSK-gel SP-5PW column. The activity was 17.1 times and the yield was 11.4%. It became a single band on SDS-PAGE, and the molecular weight was found to be 22 kDalton by SDS-PAGE.
Its physicochemical properties are as described above.
[0042]
[Example 2]
(1) Lipase CS2 was produced by recombinant DNA technology. First, shaking culture was performed according to Example 1 (1), mRNA was extracted from the obtained cells, and a cDNA library was constructed based on this mRNA.
[0043]
(2) Example 1 (2) Accordingly, the purified lipase CS2 was treated with a proteolytic enzyme (lysyl endopeptidase) and fragmented, and the amino acid sequence of several fragments was determined. A DNA primer (mixed primer) was designed based on the partial sequence and used to determine the internal sequence of the lipase CS2 gene.
[0044]
(3) In order to clone the lipase CS2 gene, first, Cryptococcus sp. MRNA was prepared from the cells of S-2, and cDNA was synthesized by reverse transcription. First, the internal sequence (about 180 bases) of the lipase CS2 gene was determined using the above primers. A new primer was designed using the determined internal sequence, and the cDNA sequence of the lipase CS2 gene was determined by using the 3′- and 5′-RACE method. The entire base sequence of the lipase CS2 gene thus determined is shown in SEQ ID NO: 2 (FIG. 1), and the amino acid sequence of the protein deduced from this base sequence is shown in SEQ ID NO: 1 (FIG. 1).
[0045]
(4) The cDNA obtained above is inserted into the BamHI cloning site of the expression vector pG-1 (GPD promoter, PGK terminator, TRP1 marker) for Saccharomyces cerevisiae, and the recombinant plasmid CS2 Lipase cDNA * pG-1 (FERM P- 18939).
[0046]
(5) Saccharomyces cerevisiae YPH499 strain was transformed with the recombinant plasmid obtained above. The obtained transformed strain formed a halo produced by lysis of butylene on a YPD (yeast extract 1%, peptone 2%, glucose 2%) plate in which butylene was suspended at a concentration of 1%. Thus, it was confirmed that the cDNA obtained had an oil-and-oil decomposition activity, and it was shown that it surely encodes the target lipase CS2.
[0047]
(6) As described above, the entire base sequence (from position 1 (ATG) to position 720 (TAA)) of the lipase CS2 gene (cutinase) was determined (SEQ ID NO: 2 (FIG. 1)). It was revealed that the protein estimated from 239 is 239 residues.
[0048]
(7) In this way, the deduced amino acid sequence of lipase CS2 (SEQ ID NO: 1 FIG. 1) was determined from the DNA sequence. The molecular weight calculated from this amino acid sequence was consistent with the results of SDS-PAGE analysis of the purified enzyme. As a result of analyzing the homology between the amino acid sequence of this enzyme and a known protein sequence database using BLAST, similarity with cutinase which is a kind of lipase which is a lipolytic enzyme was confirmed, and the lipase CS2 according to the present invention Has the above-mentioned physicochemical properties and was confirmed to be a cutinase from the amino acid sequence. Further, the amino acid sequence and base sequence of the enzyme of the present invention determined this time have not been known so far, and it has been found that they are completely new.
[0049]
[Example 3]
(Establishment of enzymatic degradation measurement method)
As a method for measuring the activity of polylactic acid, a method using the decrease in absorbance (in this case, turbidity) using an emulsion was examined.
First, in order to verify the quantitative property of this method, the linearity of absorbance and PLA concentration was confirmed. That is, the linearity of absorbance and PLA concentration was confirmed using Shimazu UV-1600, and as a result, very good linearity and reproducibility were confirmed.
[0050]
Subsequently, in order to measure the enzyme reaction over time, an attempt was made to use a biophoto recorder (ADVANTEC TN-1506). In order to confirm the reliability of the measured value, the linearity was examined. As a result, although there was variation in each CH (total 6 CH), very good linearity could be confirmed.
[0051]
[Example 4]
PLA was emulsified with a surfactant (Plysurf (Daiichi Kogyo Seiyaku)), and then the solution state and the activity of each enzyme on the agarose plate were examined.
[0052]
(1) PLA degradation activity on an agarose plate containing PLA was examined as follows.
20 μl of each enzyme solution (100 mg / ml) was added to the plate and left at 30 ° C. for 12 hours. As a result, both the crude enzyme (concentrate of culture supernatant) and the purified enzyme degrade PLA, and the latter has a higher degree of degradation than the former, whereas the commercially available lipase is completely free of PLA. It was confirmed that no decomposition was shown.
[0053]
Commercial enzyme (derived from Aspergillus niger, Candida rugosa, Rhizopus oryzae, Burkholderia sp.): This enzyme (crude enzyme and purified enzyme derived from Cryptococcus sp. S-2).
[0054]
(2) The crude enzyme (50 μl: microliter) was added to the PLA emulsion and left at 37 ° C. for 21 hours. As a result, the PLA emulsion was clarified. In contrast, the control (50 μl: water) was not clarified and remained turbid, and the degradation activity by lipase CS2 on the PLA emulsion was also confirmed at the solution level.
[0055]
[Example 5]
(PLA degradation by lipase CS2: comparison of this enzyme with Proteinase K and other lipases)
[0056]
(1) A PLA decomposition experiment was performed under the following experimental conditions, and the results of FIG. 2 were obtained.
[0057]
(Experimental conditions)
0.04% PLA emulsion
20 mM TrisHCl (pH 8.0)
30 ° C
The degradation of polylactic acid was evaluated by the amount of decrease in absorbance at 660 nm.
* Proteinase K used Merck (No. 124568).
* Μg: microgram
[0058]
As is clear from the above results, it was quantitatively shown that this enzyme decomposes the polylactic acid emulsion.
Furthermore, it was also confirmed by the organic acid analysis that lactic acid monomers were produced as degradation products.
[0059]
Polylactic acid degradation experiments were carried out using Proteinase K (Tritrichium album) and other lipases. So far, degradation by proteinase K has been shown for the degradation of polylactic acid, but it has been found that this enzyme degrades polylactic acid with an efficiency of 500 times or more (per enzyme weight).
[0060]
Furthermore, under the same conditions, the same degradation experiment was performed for the following four lipases (0.4 mg / ml), but degradation of polylactic acid could not be confirmed.
(A lipase derived from Aspergillus niger, Candida rugosa, Burkholderia sp., Penicillium roqueforti (use commercially available product))
[0061]
(2) The decomposition reaction of PLA was further continued, and the state of the reaction solution after the PLA decomposition reaction after 10 days at 30 ° C. was visually observed. The state is shown in FIG. 3 (drawing substitute photograph). In the figure, the following items are shown from the left.
[0062]
Control (no enzyme)
Proteinase K (0.8 μg / ml)
Lipase CS2 (0.8μg / ml)
Proteinase K (400 μg / ml)
Lipase CS2 (0.08 μg / ml)
[0063]
As is clear from the above results, it can be seen that the polylactic acid emulsion treated with the present cutinase is decomposed and becomes transparent. When polylactic acid was treated at the same enzyme concentration (0.8 μg / ml), it was not completely degraded by Proteinase K, but completely degraded by this enzyme. Furthermore, even when this enzyme concentration is further set to 1/10 (0.08 μg / ml), it can be seen that the enzyme is completely decomposed.
[0064]
[Example 6]
(Degradation of other biodegradable plastics (PBS, PCL))
[0065]
(1) A decomposition experiment of PBS (polybutylene succinate) was conducted under the following experimental conditions, and the results of FIG. 4 were obtained.
[0066]
(Experimental conditions)
0.04% PBS
20 mM TrisHCl (pH 8.0)
20-80 ng / ml lipase CS2
30 ° C
The enzyme concentration was set to 1/10 or less of PLA.
(Enzyme concentration in the reaction solution is shown in the graph).
[0067]
As is clear from the above results, PBS was completely degraded by this enzyme within about 30 minutes under the conditions used. Although the enzyme concentration was set to 1/10 or less than in the case of polylactic acid, the decomposition reaction proceeded very rapidly.
Further, when the degradation experiment was performed with the enzyme concentration lowered to 1/5 (20 ng / ml), PBS was rapidly degraded within 1 hour.
[0068]
(2) The state of the reaction solution after the PBS decomposition reaction at 30 ° C. and 2 days later was visually observed. The state is shown in FIG. 5 (drawing substitute photograph). In the figure, the left represents control (no enzyme), and the right represents lipase CS2 (80 ng / ml).
[0069]
As is clear from the above results, it can be seen that the PBS emulsion treated with this enzyme is broken down and becomes transparent.
[0070]
(3) Similarly to PLA and PBS, polycaprolactone (PCL) was also subjected to a decomposition experiment with this enzyme, and the state of the reaction solution after the PCL decomposition reaction at 30 ° C. and 2 days later was visually observed. The state is shown in FIG. 6 (drawing substitute photograph). In the figure, the left represents control (no enzyme), and the right represents lipase CS2 (80 ng / ml).
[0071]
As is apparent from the above results, it can be seen that the PCL emulsion treated with the present enzyme is decomposed and transparent.
[0072]
【The invention's effect】
This enzyme showed enzymatic degradation over time of biodegradable plastics (PLA, PBS, PCL). The enzyme was found to be effectively degraded against any biodegradable plastic.
[0073]
In particular, this enzyme has been shown to be very effective in degrading high molecular polylactic acid. Even when compared with Proteinase K, which has been said to have polylactic acid resolving power, the decomposition efficiency was 500 times or more under the conditions used. Furthermore, it was found to be very effective against PBS degradation. Its degradability was much higher than that for polylactic acid. Similarly, this enzyme showed a very high decomposing ability for PCL. Although some lipases have the ability to degrade not only lipid degradation but also polyester, the enzyme has a very high ability to degrade high-molecular polyesters, and also acts on various substrates. It can be seen that the enzyme has a wide range of substrate specificities.
[0074]
As described above, the present invention can efficiently decompose biodegradable plastics by using this enzyme, and is excellent as a waste treatment technology. For example, PLA that is scheduled for mass production, Efficient treatment after its use is sufficiently possible, and at that time, lactic acid is produced, which can be reused.
[0075]
In addition, this enzyme acts on polylactic acid plastics and other plastics to be used for processing, or acts on polylactic acid plastics and other plastic fibers to improve the touch and dyeability. It improves the properties of plastics and enables new production of high-quality fibers and clothing.
[0076]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the amino acid sequence of lipase CS2 (bottom) and the base sequence of DNA encoding it (top).
FIG. 2 shows the results of a polylactic acid degradation experiment.
FIG. 3 is a drawing-substituting photograph showing the state of the reaction solution (30 ° C., 10 days) after the polylactic acid decomposition reaction.
FIG. 4 shows the results of a decomposition experiment of polybutylene succinate.
FIG. 5 is a drawing-substituting photograph showing the state of the reaction solution (30 ° C., 2 days) after the polybutylene succinate decomposition reaction.
FIG. 6 is a drawing-substituting photograph showing the state of the reaction solution (30 ° C., 2 days) after the polycaprolactone decomposition reaction.

Claims (9)

  A biodegradable plastic degrading agent characterized by comprising lipase CS2 as an active ingredient.   The degradation agent according to claim 1, wherein the lipase CS2 is derived from a cryptococcus yeast.   Cryptococcus yeast is Cryptococcus sp. It is S-2 (Cryptococcus sp. S-2) (FERM P-15155), The decomposition agent of Claim 2 characterized by the above-mentioned.   The degradation agent according to claim 1, wherein the lipase CS2 is an enzyme protein having an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.   The enzyme protein has high similarity to the amino acid sequence (SEQ ID NO: 1), or has similarity to cutinase on the amino acid sequence (SEQ ID NO: 1). Decomposing agent described in 1.   The biodegradable plastic is at least selected from polylactic acid plastic (PLA), succinic polyester plastic, polyester amide plastic, polycaprolactone plastic (PCL), polyhydroxybutyrate plastic (PHB), and polyester plastic. It is one, The decomposition agent of any one of Claims 1-5 characterized by the above-mentioned. A method for decomposing a biodegradable plastic, comprising treating the biodegradable plastic using the decomposing agent according to any one of claims 1 to 6 .   A method for degrading a biodegradable plastic, comprising culturing a lipase CS2-producing bacterium belonging to the genus Cryptococcus and bringing the obtained culture or a treated product into contact with the biodegradable plastic.   A recombinant vector comprising a DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 1 and having the base sequence represented by SEQ ID NO: 2 introduced into yeast, and the resulting transformation A method for degrading a biodegradable plastic, comprising bringing a protein having a lipase CS2 activity produced using the body or a content thereof into contact with the biodegradable plastic.

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