JP4915593B2 - Biodegradable plastic-degrading bacteria and method for producing the degrading enzyme - Google Patents

Description

  The present invention relates to a microorganism that degrades a biodegradable plastic and a method for producing the novel biodegradable plastic degrading enzyme.

Plastics are used in large quantities in pipehouse coverings and mulch as an indispensable material at agricultural sites, and the amount of used plastic for agricultural and forestry is currently estimated to be about 150,000 tons. This used plastic not only takes effort to collect, but it also has an adverse effect on the environment such as global warming due to the generation of CO ₂ and dioxins due to combustion. Therefore, from the viewpoint of using renewable resources, research on biodegradable plastics has progressed in recent years and has begun to be put into practical use.
At present, domestic production of biodegradable plastics, including those produced for purposes other than agricultural materials, is estimated to have exceeded 100,000 tons. In addition, biodegradable plastic-degrading enzymes have been isolated from microorganisms collected from soil, sludge, air, etc. (Patent Documents 1, 2, and 3). However, it is difficult to balance the strength and biodegradability required for agricultural materials, and there is a problem that the biodegradability cannot be sufficiently exhibited if the materials are given strength. In addition, it is difficult to obtain many enzymes that can be used for biodegradable plastic degradation.

JP 2004-261102 A JP 2004-75905 A JP-A-2005-304388

  Accordingly, an object of the present invention is to provide a microorganism that can be biodegradable plastics quickly and inexpensively, and a method for efficiently producing a novel degrading enzyme from the microorganism.

In order to solve the above problems, the present applicant provides a biodegradable plastic-degrading microorganism and a method for producing the novel degrading enzyme. Plant leaves and fruits are covered with wax, which is a solid fat ester, and resists changes in weather conditions such as rainfall and invasion of pathogenic bacteria. There is a high possibility that the microorganisms isolated from the leaf surface and the fruit skin will contain a strain having a leaf surface and fruit skin wax assimilating ability. On the other hand, the composition of biodegradable agricultural materials is polyhydroxybutyric acid, polycaprolactone, polyesters such as polybutylene succinate and polylactic acid, and the structure is similar to wax.
As a result of repeated research based on the assumption that the microorganisms that have the ability to decompose wax and can inhabit leaf surfaces and fruit epidermis have a high density of microorganisms capable of degrading biodegradable plastics. The present invention has been completed.

That is, this invention provides the manufacturing method of a biodegradable plastic degrading enzyme including the following processes.
1) preparing a screening sample collected from yeast, filamentous fungi or bacteria that can inhabit the leaf surface of the plant;
2) screening the biodegradable plastic from the yeast, filamentous fungus or bacteria,
3) A step of culturing the screened yeast, filamentous fungus or bacterium in a medium containing biodegradable plastic to secrete biodegradable plastic degrading enzyme into the culture solution,
4) A step of purifying the biodegradable plastic degrading enzyme from the culture solution.

  The present invention also provides a biodegradable plastic degrading enzyme obtained by the previous method. The present invention also includes the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, has a molecular weight of about 22-28 kDa by SDS electrophoresis, and is polybutylene succinate (PBS) and polybutylene succin Provided is a protein having nate adipate (PBSA) degradation activity.

  Furthermore, the present invention provides a method for degrading a biodegradable plastic, comprising contacting the biodegradable plastic degrading enzyme or protein with a biodegradable plastic.

  Furthermore, the present invention provides a biodegradable plastic degradation formulation comprising yeast, filamentous fungi or bacteria screened in step 2 of the method of the present invention.

  Furthermore, the present invention provides a polypeptide encoded by the nucleotide sequence at positions 501-1172 of SEQ ID NO: 13.

  Furthermore, the present invention comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 80%, more preferably at least 90%, particularly preferably at least 95% homology with said sequence, and PBS and PBSA degradation Proteins having activity are provided.

  Furthermore, the present invention relates to a PBS and PBSA degradation comprising the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 80%, more preferably at least 90%, particularly preferably at least 95% homology with said sequence. Proteins having activity are provided.

  Furthermore, the present invention provides a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 14.

  Furthermore, the present invention provides a polynucleotide according to claim 18, which is represented by the nucleotide sequence at positions 501 to 1172 of SEQ ID NO: 13.

  Furthermore, the present invention includes a step of transforming a polynucleotide encoding the protein of the present invention or a vector incorporating the polynucleotide of the present invention into an appropriate host cell, and the conditions under which the polynucleotide is expressed. A method for producing a biodegradable plastic degrading enzyme comprising the step of culturing host cells.

  According to the method of the present invention, biodegradable plastic-degrading enzymes can be efficiently obtained by collecting and screening microorganisms from the surface of plant leaves.

In the first step of the method for producing a biodegradable plastic-degrading enzyme of the present invention, a screening sample collected from yeast, filamentous fungi or bacteria that can live on the surface of a plant leaf is prepared. The surface of plant leaves is generally covered with wax, so there are no particular restrictions on the type of plant. For example, rice, wheat (wheat, barley, etc.), pasture (Italian ryegrass, etc.), sorghum, corn, leek, abragaya Monocotyledonous plants such as cabbage, tomato, bell pepper, tea, camellia, beet, soybean, cauliflower and the like. A screening sample may be prepared by collecting microorganisms such as yeast from leaves of a plurality of types of plants. Further, “being able to live on the leaf surface” means being able to live on the wax component on the leaf surface and the hydrophobic surface of the cuticle layer. While not intending to be bound by theory, it is presumed that this microorganism is typically based on the ability to utilize wax.
Those skilled in the art can collect yeast and the like that can inhabit the surface of the leaves of the plant by any method. For example, plant leaves are collected, placed in a suitable container with 10 mM sodium phosphate buffer (pH 7.0) and beads, and shaken at 1500 rpm while cooling, 10 mM sodium phosphate buffer (pH 7.0) And a method in which the mixture is placed in a flask and shaken at 180 rpm for about 1 hour, placed in a mortar with 10 mM sodium phosphate buffer (pH 7.0), and completely ground with a pestle. As described above, it is preferable to collect yeast or the like by washing the leaf surface with an appropriate buffer solution, water, an isotonic solution or the like.
In addition, microorganisms that inhabit other than the surface of the leaf may be collected by these methods, but this is not a problem as long as the conditions include the yeast that inhabit the surface of the leaf. Microorganisms may be mixed in the screening sample.

Any microorganism can be used as long as it can inhabit the leaves of plants, and it is typically yeast, filamentous fungus, or bacteria.
The yeast may be any kind that is generally known as a kind that can inhabit the leaf surface, or any yeast that is actually collected from the leaf surface. Here, the types of yeast that can inhabit the leaf surface include, for example, Pseudozyma genus (P.antarctica, P.ruglosa, P.parantarctica, P.aphidis, etc.), Cryptococcus genus (Cryptococcus laurenti, Cryptococcus flavus, etc.), and others, Rhodotorula glutinis, Rhodotorula mucilaginosa, Sakaguchia dacryoidea, Sporidiobolus pararpseus and (Allen et al, Can. J. Microbiol. (2004)) Ustilago maydis are known (Kurtzman and Fell, The Yeasts a taxonomic study (1998). Among the yeasts, yeasts belonging to the genus Pseudozyma or Cryptococcus are preferred, such as Pseudozyma antarctica JCM3941, Pseudozyma antarctica JCM10317, Pseudozyma antarctica JCM3941, Pseudozyma ruglosa JCM10323 and Pseudozyma parantarctica Jtic117

  The filamentous fungus may be any one that is generally known as a type that can inhabit the leaf surface, or that is actually collected from the leaf surface. For example, Acremonium genus, Alternaria genus, Arthrinium genus, Aspergillus genus, Aureobasidium genus, Cladosporium genus, Epicocccum genus, Exophiala genus, Fusarium genus, Leptosphaeria genus, Paecilomyces genus, Penicillium genus, Phoma genus, Trichoderma genus, Pseudotaeniolina genus, U Examples include Phaeosphaeriopsis genus and Galactomyces genus fungi, and among these, Cladosporium genus, Penicillium genus, Leptosphaeria genus and Alternaria genus fungi are preferred. For example, Alternaria alternata, Cladosporium cladosporioides, Cladosporium oxysporum, and Penicillium pinophilum filamentous fungi are particularly preferred.

  The bacteria may be any bacteria that is generally known as a kind that can inhabit the leaf surface, or that is actually collected from the leaf surface. For example, bacteria belonging to the genus Bacillus can be mentioned, and among these, Bacillus pumilus is preferable.

  Insects prey on yeasts and other microorganisms and plants (sites to which plant-inhabited microorganisms such as sap are attached) and are therefore the most important mediator of yeasts and other microorganisms in nature. Is known (Phaff et al. Yeast Life 1982). Therefore, yeast, filamentous fungi and bacteria that can inhabit the foliage can also be collected from the body surface and digestive tract of insects that prey on plants. For example, from Drosophila isolated from the citrus fruit, the genus Galactomyces geotrichum (known to occur on the surface of lemon fruit) and the genus Acremonium (isolated well from the leaves of wheat leaves, etc.) Filamentous fungi such as Phaeosphaeriopsis spp. (Known as the pathogen of Yucca, an ornamental plant of the Agave family) can be isolated from the digestive tract of stag beetles that prey on wood.

In the second step of the method for producing a biodegradable plastic degrading enzyme of the present invention, those that degrade the biodegradable plastic are screened from the yeast, filamentous fungus or bacteria collected in the first step.
In the present invention, the term “biodegradable plastic” means “when used it has a function similar to that of a conventional petroleum-derived plastic, and after use, it eventually becomes water or carbon dioxide by microorganisms in the soil or water in the natural world. Has the meaning commonly understood in the art, “plastic that is decomposed into Specific examples include polyhydroxybutyric acid, polycaprolactone, polyesters such as polybutylene succinate and polylactic acid.
The type of biodegradable plastic used for the screening of the method of the present invention can be arbitrarily selected by a person who performs the method. For example, many of the multi-films are made of succinic polyester [mixture of polybutylene succinate (PBS) and polybutylene succinate adipate (PBSA)], and it is preferable to use PBS and PBSA for screening.
Many biodegradable plastics such as PBS and PBSA are commercially available, and those skilled in the art can arbitrarily select an appropriate type for screening, and the degree of polymerization, molecular weight, etc. are not limited. For example, when using an emulsion, Bionole Emulsion EM-301 (PBSA) (Showa Polymer Co., Ltd.) can be used.

The screening method used in the method of the present invention may be any method known in the art. For example, an agar plate medium in which a biodegradable plastic emulsion (for example, PBSA emulsion: molecular weight of about 10,000, 1%) is suspended. Above, examine the bacteria that dissolve the emulsion to form a clear zone, or actually a biodegradable plastic film on a flat plate medium (eg PBSA and PBS multifilm: molecular weight about 140-150,000, thickness It is also possible to examine the degradation by bringing yeast or the like into contact with 2 μm). When a plastic film is actually used, the decomposition can be examined by image analysis, for example, by measuring the brightness of the film.
Alternatively, the liquid medium containing the biodegradable plastic emulsion may be cultivated in a liquid medium to confirm that the liquid medium becomes transparent. Furthermore, these known screening methods can be modified and used by those skilled in the art.
In addition, more effective screening can be performed by combining these screening methods. For example, a method is known in which, on an agar plate medium in which a biodegradable plastic emulsion is suspended, the emulsion is dissolved to isolate bacteria that form a transparent zone, and the solid plastic degradation activity is examined by liquid culture (Uchida H et al., J. Biosci, Bioeng. 93 p. 245 247 (2000)).

  In the present invention, PBSA emulsion (for example, molecular weight of about 10,000, 1%) degrading bacteria is selected on an agar plate medium, and then a multi-film of PBSA and PBS (for example, molecular weight of about 140 to 150,000 is selected from these bacteria. It is preferable to use a two-stage screening method that selects bacteria that degrade 2 μm in thickness. By adopting this screening method, efficient screening can be performed. The amount of multi-film decomposition can be evaluated by, for example, imaging a film and calculating a numerical value by calculating a luminance change rate per certain area. In one embodiment of the present invention, a biodegradable plastic degrading enzyme is added to a culture medium by adding a natural fat (eg, soybean oil) or a non-carbohydrate carbon source such as glycerol, ethanol, lactic acid or amino acid as a carbon source. It is possible to guide efficiently. In the present invention, microorganisms such as yeast are preferably cultured by adding soybean oil or glycerol, particularly preferably glycerol, to the medium.

  By the above first and second steps of the present invention, it is possible to efficiently isolate bacteria having high activity and secretion amount of biodegradable plastic degrading enzyme. For example, in bacteria using soil as a separation source, it was said that about 1% of emulsion-degrading bacteria decompose solid biodegradable plastics. In one embodiment of the method of the present invention (two-stage screening is performed), 30% (PBS) to 46% (PBSA) of the emulsion-degrading filamentous fungi degraded the biodegradable plastic multifilm. As described above, in the method of the present invention, microorganisms such as yeast that degrade biodegradable plastics can be efficiently selected, so that it is possible to provide a wide variety of bacteria and enzymes that degrade biodegradable plastics.

  In the third step of the method for producing a biodegradable plastic degrading enzyme of the present invention, the screened yeast, filamentous fungus or bacterium is cultured in a medium containing the biodegradable plastic to produce the biodegradable plastic degrading enzyme as a culture solution. To secrete. In the second step of the present invention, the bacteria exhibiting the biodegradable plastic degrading activity such as PBS and / or PBSA are cultured under the conditions for producing the biodegradable plastic degrading active enzyme as in the screening, The enzyme is secreted into the extracellular medium. Conditions for cultivating yeast and the like can be arbitrarily set by those skilled in the art. A liquid medium based on the medium used for screening is prepared, and the inoculum is cultured and shaken to proliferate the bacteria. Thus, the biodegradable plastic degrading enzyme can be secreted into the medium. For example, Kamini NR et al. Production, purification and characterization of an extracellular lipase from the yeast, Cryptococcus Process Biochemistry 36 p. 317-324 (2000) and Kolattukudy PE et al. Cutinases from fungi and pollen Methods in Enzymology 71 652-664 (1981) or a modified method thereof can be used. Moreover, the method of extracting from the solid culture using the plant body as a support | carrier generally used for the enzyme production by a filamentous fungus, or foaming support | carriers, such as a urethane foam, can also be used.

  Similarly to the screening, non-sugar carbon sources such as natural fats and oils (for example, soybean oil) and glycerol can be used as the carbon source. By using these carbon sources, biodegradable plastic degrading enzymes can be efficiently induced. In particular, when glycerol is used, there is an advantage that the enzyme is hardly inactivated through the subsequent purification step, and the enzyme activity can be kept high.

  In the fourth step of the method for producing the biodegradable plastic degrading enzyme of the present invention, the biodegradable plastic degrading enzyme is purified from the culture solution. Purification of the enzyme can be performed by a method known in the art, and the literature cited in the third step can be referred to again here. For example, ammonium sulfate may be added to the supernatant of the culture solution to obtain a precipitate, or the culture solution may be concentrated using a ultrafiltration method using Asahi Kasei Microza or the like. Further, the precipitate may be dissolved in 20 mM Tris-HCl buffer and then dialyzed against 20 mM Tris-HCl buffer using a dialysis tube such as spectrapor MWCO 12000-14000, and then passed through a DEAE sepharose column. The existing protein may be removed, and the enzyme may be further purified through an SP sepharose column if necessary. Moreover, you may combine said operation suitably.

When the molecular weight of the biodegradable plastic degrading enzyme produced by the method of the present invention is measured by SDS electrophoresis, it shows a molecular weight of about 22-28 kDa.
In addition, the enzyme produced by the method of the present invention exhibits PBS and / or PBSA degrading activity. The activity of the purified biodegradable plastic degrading enzyme is, for example, by suspending PBSA emulsion in 20 mM Tris-HCl (pH 6.8) and adding 1.8 ml of an aqueous solution having an absorbance (OD660) of about 0.5 to a test tube with a 13 mm diameter. It can be determined by adding 200 μl of crude enzyme solution or microbial culture supernatant and measuring the absorbance using an apparatus such as Spectronic 20A (Shimadzu).
The biodegradable plastic enzyme of the present invention purified from P. antarctica JCM10317 includes the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. As long as the protein has biodegradable plastic degradation activity, one or several amino acids may be deleted, substituted or added.
Furthermore, when tested on plate and liquid media containing polylactic acid (PLA) emulsion, the enzyme has polylactic acid degrading activity. The degradation activity of polylactic acid is, for example, an emulsion of polylactic acid (Miyoshi oil: Randy PL-1000 (particle size 4.3 μm), Randy PL-2000 (particle size 2.3 μm)) in 20 mM Tris-HCl (pH 6.8). It can be measured by adding a dialysate of an ammonium sulfate precipitate of a microorganism culture solution to a suspended aqueous solution having an absorbance (OD660) of about 0.5, and measuring the absorbance using Spectronic 20A (Shimadzu) or the like. It is also possible to confirm by dissolving the polylactic acid emulsion to form a transparent zone on a plate medium containing the polylactic acid emulsion.

  In addition, a biodegradable plastic degradation preparation containing the microorganisms screened in step 2 of the method of the present invention can be produced. The biodegradable plastic degradation preparation of the present invention containing microorganisms can be prepared by a person skilled in the art by a well-known method, and may be a microorganism alone or may be mixed with well-known solvents, additives and the like.

  In addition, a step of transforming an appropriate host cell with a polynucleotide encoding the protein of the present invention or a vector incorporating the polynucleotide of the present invention, and culturing the host cell under conditions for expressing the polynucleotide A biodegradable plastic degrading enzyme can be obtained by the method for producing a biodegradable plastic degrading enzyme including the step of: Combinations of vectors and host cells suitable for gene expression by vector introduction are known in the art, and those skilled in the art can appropriately select vectors and host cells used in the present invention. Examples of host cells include yeast, filamentous fungi such as koji mold, and Escherichia coli. Among them, yeasts such as Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris are preferable. When the yeast is used as a host cell, a budding yeast expression vector pYES2.1 (Invitrogen) and a plasmid PEPGK41 or a chromosome introduction vector can be used as an expression vector. In addition, the culture conditions can determine the composition of the medium and the culture temperature according to the type of the vector and the host cell. For example, when the yeast is used, it can be cultured at 25 to 40 ° C., preferably 30 ° C., in a Wickerham synthetic medium.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the scope of the present invention is not limited to these.
Isolation of biodegradable plastic-degrading bacteria:
First, PBSA emulsion (molecular weight about 10,000, 1%)-degrading bacteria were selected on an agar plate medium, and then a PBSA and PBS multi-film (molecular weight about 140-150,000, thickness 2) was selected from these bacteria. A two-stage screening method was used to select for microbes).

(Screening for biodegradable plastic-degrading yeast)
Inducible enzyme producing strain selection medium for yeast (FMM: Fungi Minimum Medium)
Underlayer

Upper layer B
About 15 ml of the lower layer plate medium was prepared in a 9 cm petri dish, and after setting, it was stored in a 45 ° C. incubator. Meanwhile, about 5 ml of the upper layer B dissolved and sterilized in an Erlenmeyer flask containing a stirrer was overlaid. At this time, the upper layer B was stirred with a stirrer so that the oil was uniformly mixed. Chloramphenicol as an antibiotic was added at a final concentration of 40 ppm.

First, eight Pseudozyma yeast strains (P. antarctica JCM3941 (source: brown rice), P. aphidis JCM10318, P.fusiformata JCM3931 (source: cauliflower), P. parantarctica JCM11752, P. plolifica JCM 10319 (source: Himekuroabragaya), P. ruglosa JCM10323 (source: corn leaf), P. thailandica JCM11753, P. tsukubaensis JCM10324 (source: flower), and yeast Cryptococcus luteolus NBRC0411 After inoculating a medium containing emulsion (FMM, FMZ (composition will be described later), Malt Agar (Difco)), 6 strains dissolved PBSA on the FMM medium. The emulsion contained in the upper layer of the plate medium was decomposed to confirm that a clear zone was formed around the bacteria.
Pseudozyma genus strains (P. antarctica JCM3941, P. antarctica JCM 10317, P. aphidis JCM10318, P. floculosaJCM10321 (separation source: red clover)), P. fusiformata JCM3931, P. parantarctica JCM11752, P plolifica JCM 10319, P. ruglosa JCM10323, P. thailandica JCM11753, P. tsukubaensis JCM10324) and Cryptococcus luteolus NBRC0411 strain were applied on FMM medium and cultured at 30 ° C the next day, the surface on which the bacteria had grown was made of PBSA and PBS. (2 μm, 2 cm film) was placed and kept at 30 ° C. As a result of peeling the membrane after one week, the P. antarctica JCM10317, P. antarctica JCM3941, P. ruglosaJCM10323 and P. parantarctica JCM11752 strains showed high degradation activity.
About 4 strains (P. antarctica JCM10317, P. antarctica JCM3941, P. ruglosaJCM10323 and P. parantarcticaJCM11752 strains) with high degrading activity, the same experiment was carried out, and each multifilm was peeled off once a day, and the degree of degradation Was observed (FIG. 1).
In addition, when yeast was actually collected from rice collected from two places in the paddy field and screened in the same manner on FMM medium, each yeast strain that decomposed the PBSA emulsion was isolated. Both yeasts degraded PBSA and PBS multifils. These yeasts were identified as P. antarctica from the rDNA base sequence (rice isolates A and B).

  As described above, microorganisms with high biodegradable plastic degrading ability could be separated from plants at high frequency. It was found that most of the yeasts of the genus Pseudozyma were isolated as resident bacteria on the plant surface. It has been clarified that many microorganisms such as yeasts that can inhabit the leaves and fruits of healthy plants and the surface of flowers have the ability to decompose biodegradable plastics.

Enzyme activity measurement and purification Enzyme purification method:
Place 50 ml of FMM liquid medium (carbon source 6% glucose) in a 500 ml Erlenmeyer flask and add P. antarctica JCM10317 strain, P. parantarctica JCM11752, or P. antarctica strain 2 isolated from rice to PDA (potato agarose agar, Difco) slant. 1 platinum ear was inoculated and cultured at 30 ° C. and 200 rpm / min for 17 hours with rotary shaking. 500 μl of this seed culture solution is inoculated into 50 ml of FMM liquid medium (carbon source 1% soybean oil or 6% glycerol: both Wako Pure Chemicals), and is shaken at 30 ° C. and 200 rpm / min in a 500 ml Erlenmeyer flask. Cultured at last. The culture broth was collected, centrifuged, and the supernatant from which the cells were removed was measured for PBSA degrading activity.
The enzyme activity was determined by suspending PBSA emulsion in 20 mM Tris-HCl (pH 6.8), placing 1.8 ml of an aqueous solution with an absorbance (OD660) of about 0.5 in a test tube with a diameter of 13 mm, and adding 200 μl of the microorganism culture supernatant. In addition, the absorbance was measured using Spectronic 20A (Shimadzu). The test tube was shaken at 30 ° C. and 120 rpm, removed at regular intervals, and the amount of increase in absorbance was measured to determine enzyme activity.
Assuming that a sufficient amount of enzyme was secreted in the culture broth whose absorbance (OD660) had fallen to the range of about 0.3 after 1 hour from the initial stage (about 0.5), it proceeded to the next step.

The culture solution was centrifuged at 10000 g for 15 minutes at room temperature to separate the cells and the culture supernatant. Ammonium sulfate was added to the culture supernatant so as to be 50% saturation and dissolved. After gently stirring at 4 ° C. for 17 hours, the mixture was centrifuged at 4 ° C. and 25000 g for 15 minutes to collect the precipitate. The precipitate was dissolved in 20 mM Tris-HCl buffer. The solution was sealed in a dialysis tube (spectrapor MWCO 12000-14000) and dialyzed with 20 mM Tris-HCl buffer to obtain a crude enzyme solution.
Purification of the enzyme was advanced using the crude enzyme solution of P. antarctica JCM10317 strain.

A crude enzyme solution (3 ml) was obtained from 6 L of the culture solution. 3 ml of the crude enzyme solution was concentrated to 500 μl by ultrafiltration (Kurabo, Centricut Ultramini, fraction 10,000). This was subjected to DEAE sepharose column and SP sepharose column (both GE Healthcare Bioscience) equilibrated with 50 mM Tris-HCl (pH 6.8). Active fractions 1.5 ml obtained from them were concentrated by ultrafiltration (Kurabo, Centricut Ultramini, fraction 10,000), and impurities were removed to make the enzyme solution 100 μl. A gel filtration column (Tosoh Corporation TSK-gel G3000SW _XL , flow rate 0.5 mL / min, detection wavelength 220 nm) was equilibrated with 50 mM Tris-HCl (pH 6.8) containing 0.3 M NaCl. 80 μl of 100 μl of the concentrated sample was injected into this, and 30 eluates of 30 mL were collected and the activity of each fraction was measured.
A wavelength peak was detected at a retention time of 23.391 minutes. In addition, a clear activity was observed in the fractions obtained between 23.00 minutes and 26.99 minutes, of which the fractions from 24.00 minutes to 24.99 minutes and 25.00 minutes to 25.99 minutes showed the highest activity. FIG. 2 shows the PBS degradation activity after each chromatography step.

  The biodegradable plastic degrading enzyme purified as described above was confirmed by SDS-PAGE (FIG. 3). Two bands are confirmed in the SP-adsorbed fraction, but the sizes of several cutinases already reported in the oil culture solution are all 20-30 kD, and the band density and enzyme when the culture conditions are changed. From the comparison with the activity, the smaller molecular weight (22-28 kDa) was estimated as the target enzyme. Further purification was performed by gel filtration. Fractions 1 and 2 both became one band on SDS-PAGE. When the enzyme activity of this fraction was measured, the activity was confirmed (FIG. 2), and thus the enzyme was identified as a protein having a molecular weight of 22 to 28 kDa.

  In addition, for P. parantarctica JCM11752 strain and rice isolate A, the induction of the target enzyme was confirmed by changing the carbon source added to the liquid medium. The enzyme showing biodegradable plastic degradation activity includes soybean oil and glycerol. It was confirmed that it was efficiently induced in the medium (FIG. 4).

In addition, the internal amino acid sequence of the biodegradable plastic degrading enzyme purified from the supernatant of the P. antarctica JCM10317 strain glycerol 6% -FMM culture solution was determined.
Among the enzymes decomposed with trypsin and separated on an HPLC column under the following conditions, the No. 1 and No. 2 peak N-terminal sequences were identified (see FIG. 5).

HPLC conditions
No. 1: IVAQVK (SEQ ID NO: 1)
No. 2: XAXGXANIVAQV (SEQ ID NO: 2)
3: No. XTSEPQGPSVGF (SEQ ID NO: 3)
(X is not identified. However, the amino acid at position 2-5 can be T.)

Example of enzyme degrading PBSA emulsion and PBSA membrane
When 50 ml of P. antarctica JCM10317, P. parantarctica JCM11752 and 50 ml of P. antarctica strain isolated from rice were precipitated with ammonium sulfate and the precipitated 20 mM Tris-HCl buffer pH6.8 dialysate was used as the crude enzyme, PBSA The activity of dissolving the emulsion was confirmed (FIG. 6). Further, as a result of SDS-PAGE gel electrophoresis, a protein band having the same molecular weight as P. antarctica JCM10317 was confirmed from any of the bacteria. PBSA (Showa Polymer Bionore PBS3020) (100 mg) was dissolved in 5 ml of dichloromethane, and developed in a glass petri dish (9 cm in diameter) in a draft, and the dichloromethane was evaporated to form a PBSA film. This membrane was cut into 1 cm ² . This membrane was added in the presence of 2 ml of 20 mM Tris-HCl buffer pH6.8 and 50 μl of a crude enzyme solution, and shaken at 30 ° C. and 50 rpm for 5 days. As a result, it was confirmed that the PBSA membrane was decomposed according to the obtained enzyme activity amount.

Further analysis was performed to identify the entire amino acid sequence and gene sequence of the above-mentioned plastic degrading enzyme derived from P. antarctica JCM10317.
When the enzyme was purified as described above and its N-terminal amino acid sequence was examined, the sequence of AG * SSYVIINT (SEQ ID NO: 4) was obtained. Further, when the internal amino acid sequence was examined, the following sequences were further obtained.
IVAQVK
* A * G * ANIVAQV
* TSE (P / T) QGPSVGF
AG * SSYVIINTR *** (SEQ ID NO: 5)
GVILIGNPEHKPNLA * NVDG (SEQ ID NO: 6)
SAVSGGSEYDTVYPAGIDQN (SEQ ID NO: 7)
GISAAFTQGVPSNWVSK *** (SEQ ID NO: 8)

  Among the above, based on the sequences of AG * SSYVIINTR *** and GISAAFTQGVPSNWVSK *** (* indicates an unidentifiable amino acid), primer PaE3F (5'-tacgtSatcatcaacacVcg-3 ') (SEQ ID NO: 9) and PaE6R (5′-gacgccctgBgtgaaNgc-3 ′) (SEQ ID NO: 10) was synthesized. On the other hand, RNA was extracted using the hot phenol method under the condition that P. antarctica JCM10317 strain was producing raw plastic degradation enzyme, and mRNA was purified using Oligotex dT30 (Takara Bio Inc.). As a result of RT-PCR reaction using Prime Script RT-PCR kit (Takara Bio Inc.) based on the designed primer sequences (PaE3, PaE6R) using the extracted mRNA as a template, amplification of about 370 nt A fragment (PaE3F-PaE6R amplified fragment) was obtained. The base sequence of the PaE3F-PaE6R amplified fragment was decoded, and based on the results, primers PaE134R (5'-atgcgggtgttcatggtgcg-3 ') (SEQ ID NO: 11) and PaE308F (5'-atgcccttcctcagcttactg-3') (SEQ ID NO: 12) ) Was synthesized. Next, genomic Southern hybridization of P. antarctica JCM10317 strain was performed using the PaE3F-PaE6R amplified fragment as a probe. As a result, one band hybridized to the chromosomal DNA digested with the restriction enzyme BamHI was detected, and it was estimated that the corresponding gene was one in the chromosome.

  The target gene was isolated using inverse PCR. PCR was carried out using the primers PaE308F and PaE134R using chromosomal DNA digested with BamHI circularized by self-ligation reaction as a template. One PCR amplified fragment was cloned into a TA vector, and the nucleotide sequence was decoded. As a result, the cloned DNA fragment (SEQ ID NO: 13) contained the entire gene sequence of the raw plastic degradation enzyme. After substitution with the amino acid sequence, the sequence of the ORF and secreted enzyme (SEQ ID NO: 14) was determined based on the result of the N-terminal amino acid sequence.

  Furthermore, using the BLAST search method at the National Institute of Genetics DNA data bank of Japan (DDBJ), a registered sequence having high homology with the DNA sequence and amino acid sequence of the raw plastic degradation enzyme obtained from P. antarctica JCM10317 above. I did a search. The search conditions were top 10 search conditions, expected value 10, gap 1 and filter 1. Comparison was made by three methods: “DNA-DNA comparison”, “amino acid sequence-amino acid sequence comparison”, “DNA sequence → DNA sequence translated from amino acid sequence → DNA sequence translated from amino acid sequence”. As a result, in the “DNA-DNA comparison”, the one that showed the highest homology in each search was CS406466 | CS406466.1 Sequence 36 from Patent WO2006042156 score: 46 E-value: 0.087, “Amino acid sequence-amino acid sequence comparison” "Tr | Q4PBK7 | Q4PBK7_USTMA Putative uncharacterized protein score: 286 E-value: 6e-76," DNA sequence → DNA sequence translated from amino acid sequence → DNA sequence translated from amino acid sequence "is EA136855 | EA136855.1 Sequence 1170 from patent US 7214786. Score 294 E-value 2e-77. From the above, it was speculated that the DNA sequence and amino acid sequence specified this time are novel sequences different from known gene sequences and amino acid sequences.

  Subsequently, gene expression in a heterologous yeast was attempted in order to confirm that the gene separated as described above was a plastic degrading enzyme. Based on the sequence obtained by the inverse PCR method, primers PaE-20F (CCCTTCCGATCTATCTCAAGA) (SEQ ID NO: 15) and PaE + 16R (ATGGACGAGGCTACGATCTG) (SEQ ID NO: 16) were designed and chromosomal DNA of P. antarctica JCM10317 strain Was used as a template to amplify the gene by PCR. The amplified fragment was cloned into the budding yeast expression vector pYES2.1 (Invitrogen) and transformed into the budding yeast BY4741 strain. In this plasmid, the target gene is inserted downstream of the GAL1 promoter induced by galactose. The budding yeast transformed with this plasmid dissolved the PBSA emulsion in a synthetic medium supplemented with galactose, but did not dissolve in glucose (FIG. 11). In addition, when cloned into the plasmid PEPGK41 constitutively expressed in Kluyveromyces lactis and transformed into the K. lactis MW98-8C strain, the PBSA emulsion was constitutively dissolved (same as above). From the above results, it was confirmed that the cloned gene was a gene of plastic degrading enzyme.

Polylactic acid degradation test Based on the enzyme activity measurement method described above, the polylactic acid (PLA) degradation activity of the biodegradable plastic degrading enzyme of P. antarctica JCM10317 was examined. Specifically, an emulsion of polylactic acid (Miyoshi oil and fat: Randy PL-1000 (particle size 4.3 μm), Randy PL-2000 (particle size 2.3 μm)) was suspended in 20 mM Tris-HCl (pH 6.8). 1.98 ml of an aqueous solution having an absorbance (OD660) of about 0.5 was placed in a test tube having a diameter of 13 mm, 20 μl of dialysate of ammonium sulfate precipitate from P. antarctica JCM10317 was added, and the absorbance was measured using Spectronic 20A (Shimadzu). It was. The test tube was shaken at 30 ° C. and 120 rpm, removed at regular intervals, and the amount of increase in absorbance was measured to determine enzyme activity. As a result, it was found that the enzyme also decomposes polylactic acid (FIGS. 7 and 8).

Only 1% emulsion of polylactic acid (Miyoshi oil: Randy PL-2000 (particle size 2.3 μm)) in FMM medium, or 1.5% containing 1% polylactic acid and 1% soybean oil, or 1% polylactic acid and 6% glycerol Three types of plate culture medium overlaid with an agar medium were prepared, and 9 strains of the genus Pseudozyma were inoculated thereon and incubated at 30 ° C. The strains in which the polylactic acid emulsion was dissolved to form a clear zone were P. antarctica (JCM10317, JCM3941) and P. tsukubaensis only in a medium containing P. antarctica and glycerol isolated from rice. JCM10324 (FIG. 9).
Although polylactic acid is a plastic derived from biological materials, it is generally very low in biodegradability, and few microorganisms have polylactic acid degrading activity especially at room temperature. However, it has been found that yeasts screened by the method of the present invention include those that produce enzymes that degrade polylactic acid at room temperature at a high frequency.

  Furthermore, degradation of the polylactic acid film by the enzyme purified from the strains of P. antarctica JCM10317 and P. tsukubaensis JCM10324 was confirmed. P. antarctica JCM10317, JCM3941, P. antarctica2 strain isolated from rice and P. tsukubaensis JCM10324 strain, 1.5% agar medium containing 1% polylactic acid emulsion (Randy PL-2000) and glycerol (6%) in FMM medium Was inoculated into a plate medium overlaid and cultured at 30 ° C. overnight. Polylactic acid film 1cm painting on this [terramac draining garbage bag (Unitika commerce: 97% polylactic acid fiber, 3% biodegradable polyurethane) 0.1g dissolved in 3ml dichloromethane, spread on a glass petri dish (9cm caliber) and draft The solvent was volatilized inside and a polylactic acid film was made by itself. ] And kept warm at 30 ° C. When the membrane was peeled off on days 3, 6, 10, and 14 and the degree of membrane degradation was observed, P. antarctica JCM10317 strain and P. tsukubaensis JCM10324 strain showed high degradation activity. The membrane used also contains polyurethane, but two weeks after the start of the culture, the membrane was clearly degraded by more than half, and it was confirmed that this bacterium decomposes polylactic acid. (FIG. 12).

(Separation of biodegradable plastic-degrading filamentous fungi)
Selection of filamentous fungi that degrade biodegradable plastics was performed in the same manner as in yeast using the following medium.
Inducible enzyme producing strain selection medium for filamentous fungi (FMZ: Czapek-Dox Minimum Medium)
Underlayer

Upper layer B
A plate medium was prepared in the same manner as the selection medium for yeast. Chloramphenicol was added as an antibiotic at a final concentration of 40 ppm.

The filamentous fungi that had been separated and identified by the inventor from the leaves of rice and wheat and stored were subjected to selection with PBSA emulsion. Using a medium in which an emulsion was layered on FMZ medium, 71 strains capable of degrading emulsion were isolated from 1227 strains. Of these, 26 were selected from 539 strains isolated from wheat, 27 were selected from 380 from barley, and 17 were selected from 308 from rice. When the resolution of PBSA and PBS multifilm was examined by secondary selection of plastic emulsion-degrading bacteria isolated from various samples, only 50 PBSA multifilms were degraded, but both PBS and PBSA multifilms were degraded. Twenty-one strains of bacteria that degrade the film were isolated.
On the other hand, biodegradable plastic-degrading microorganisms were separated from biodegradable plastics embedded in various leaves and fruit surfaces, soil, and soil according to the screening manual, and 64 strains of bacteria capable of degrading emulsion were obtained. . Of these, in the second selection, 37 strains decomposed only PBSA multifilm, but 20 strains that decompose both PBS and PBSA multifilm were isolated. Among these plastic emulsion-degrading bacteria, 64 strains were analyzed for morphology and analysis of the ITS region of the rDNA base sequence, and filamentous fungi such as Cladosporium, Penicillium and Alternaria as shown in the table below. Was identified.
There was a difference in the amount of degradation between the two films. The total number of strains that showed even a slight resolution in PBSA was 87, and 41 strains that showed a slight resolution in PBS.
Table 1: Genus names of filamentous fungi that showed biodegradable plastic degrading activity and the number of bacteria isolated


Among the above, filamentous fungi such as Alternaria alternata, Cladosporium cladosporioides, Cladosporium oxysporum, and Penicillium pinophilum were specifically identified.

About Leptosphaeria sp. (Strain name 44-2), PBS and PBSA decomposition | disassembly in the culture medium of a clear zone formation strain and the degradation degree were analyzed (FIG. 10). The film solubility was 48.3% for the PBSA film and 34.9% for the PBS film.
Analysis was performed according to the following method:
“Transparent original positive film mode scans the film at 300 dpi. Use Aquacosmos ver2.0 for analysis and save the image as a TIFF file (grayscale). Open the file on Aquacosmos and select the toolbar in the measurement window. , Drag to enclose a picture of plastic film Select “Create same measurement window as last time” and select all the films on the captured image as independent measurement windows of the same size. (At this time, the unprocessed film and the white spot without film are also enclosed in the same way as a control.) The luminance of each area is calculated from “Area Analysis” in the analysis menu. Based on the control, calculate the amount of membrane degradation. "
(Measured film brightness-brightness of undegraded film) / (white screen brightness-brightness of undegraded film) X100 = decomposition (%)

(Separation of biodegradable plastic-degrading bacteria)
Selection of bacteria for degrading biodegradable plastics was performed in the same manner as in yeast using the following medium.

Constitutive enzyme production strain selection medium (NA)
Underlayer

Upper layer A
A plate medium was prepared in the same manner as the selection medium for yeast. Cycloheximide as an antibiotic was added at a final concentration of 50 ppm.

Preserved bacteria were isolated from the leaves of rice, etc. in Tsukuba Agricultural Research Field. A test bacterium was transplanted into NA-PBSA medium, and a strain that formed a clear zone with growth was selected as a degrading bacterium. As a secondary selection, sterilized PBSA and polybutylene succinate (PBS) multifilms were placed on NA medium and the plastic membrane resolution was investigated. As a result, eight strains of bacteria that constitutively degrade the PBSA emulsion and 20 strains that inductively degrade were obtained from the 488 strains isolated from rice.
When plastic degradation bacteria separated from various samples were secondarily selected, PBS multifilm was placed in a 2% yeast extract (Difco) solution and cultured with shaking, and the resolution was examined. Indicated. Among them, one strain having high activity was identified as Bacillus pumilus from the rDNA base sequence.

(Example of separating biodegradable plastic-degrading microorganisms from insects)
Collect 2 individuals of Drosophila that were on the surface of citrus citrus fruit, put them in a suitable container with 10 mM sodium phosphate buffer (pH 7.0) and beads, and shake at 1500 rpm with cooling. Microorganisms were extracted. This extract was applied to an FMM or FMZ medium overlaid with PBSA emulsion and 1% soybean oil according to the method described above. Bacteria that formed a clear zone were isolated, and PBS and PBSA film degrading activity were examined according to the method described above. Galactomyces geotrichum separated with FMM medium was only PBSA membrane, Acremonium genus separated with FMM medium was PBSA and PBS. The film was decomposed efficiently. The fungus was identified from the base sequence and morphological observation of rDNA.
At five sites in the Kanto region, we collected a large number of larvae of two species of the Coleoptera stag beetle (Madaraku stag beetle and Nebuta stag beetle) from brown decayed wood with a high lignin content. These two kinds of larvae were obtained specifically for brown rot wood. Microorganisms were cultivated aerobically from the digestive tract (midgut and hindgut) and body surface of stag beetles larvae. According to the above-mentioned method, PBSA resolution of each microorganism was examined using a medium containing PBSA emulsion as a nutrient source. From the middle intestine of Nepalese stag beetle and Nebuto stag beetle, bacteria having PBSA resolution of 2 and 11 respectively. The strain was isolated. In addition, fungi with PBSA-degrading ability were isolated from the midgut, nebutococcus body surface, and midgut of the red stag beetle, respectively. When these bacteria and fungi were examined for PBS resolution using PBS Multi according to the method described above, one fungal strain derived from the midgut of Nebutococcus showed high resolution. This fungus was identified as a fungus belonging to the genus Phaeosphaeriopsis from the base sequence of 18S rDNA.

Biodegradable plastic film degradation by yeast is shown (upper: PBSA, lower: PBS, numbers are days). The PBS decomposition | disassembly activity after each chromatography process of the biodegradable plastic degradation enzyme of P. antarctica JCM10317 strain is shown. The SDS-PAGE electrophoretic diagram of the gel filtration fraction of the biodegradable plastic degradation enzyme of P. antarctica JCM10317 strain is shown. P. parantarctica JCM11752 strain and rice isolate A (P. antarctica) biodegradable plastic-degrading enzymes are induced in a medium containing glycerol or soybean oil (SDS-PAGE 14.1% Gel, silver stain) . The chromatogram when the biodegradable plastic-degrading enzyme purified from P. antarctica JCM10317 strain was digested with trypsin and separated by HPLC. The degradation of PBSA emulsion by enzymes of the genus Pseudozyma is shown. A photograph of the degradation of polylactic acid by a biodegradable plastic degrading enzyme purified from P. antarctica JCM10317 strain is shown. It shows a decrease in turbidity due to polylactic acid degradation by a biodegradable plastic degrading enzyme purified from P. antarctica JCM10317. Figure 2 shows degradation of polylactic acid (PLA) by various microorganisms screened according to the present invention. For the filamentous fungus Leptosphaeria sp. (Strain name 44-2), the degradation of PBS and PBSA film in the culture medium of the clear zone forming strain is shown. The dissolution of PBSA emulsion by heterologous yeast expressing a gene isolated from P. antarctica JCM10317 strain is shown. +/- indicates the presence or absence of gene transfer. The degradation of polylactic acid films by enzymes purified from P. antarctica JCM10317 and P. tsukubaensis JCM10324 strains is shown.

Claims (16)

A method for producing a biodegradable plastic degrading enzyme, comprising the following steps.
1) Pseudozyma genus or Cryptococcus genus yeast, Acremonium genus, Alternaria genus, Aspergillus genus, Aureobasidium genus, Cladosporium genus, Epicocccum genus, Fusarium genus, Leptosphaeria genus, Penicillium genus or Phoma genus fungus that can live on the leaves of plants Or preparing a screening sample collected from bacteria of Bacillus pumilus ,
2) screening the biodegradable plastic from the yeast, filamentous fungus or bacteria,
3) A step of culturing the screened yeast, filamentous fungus or bacterium in a medium containing biodegradable plastic to secrete biodegradable plastic degrading enzyme into the culture solution,
4) A step of purifying the biodegradable plastic degrading enzyme from the culture solution. Step 1) is a yeast that can inhabit the leaf surface of a plant, and is collected from a yeast selected from the group consisting of P.antarctica JCM10317, P.antarctica JCM3941, P.ruglosaJCM10323, P.tsukubaensis JCM10324 and P.parantarctica JCM11752 The method according to claim 1, which is a step of preparing a screened screening sample. Step 1) is a step of preparing a screening sample collected from a filamentous fungus selected from the group consisting of Alternaria alternata, Cladosporium cladosporioides, Cladosporium oxysporum and Penicillium pinophilum The method of claim 1, wherein: Biodegradable plastic is PBS or PBSA, any one method according to claim 1-3. The method according to any one of claims 1 to 4 , wherein the leaves of the plant are wheat or rice leaves. The method according to any one of claims 1 to 5 , wherein the medium contains glycerol. A biodegradable plastic-degrading enzyme obtained by the method of claim 1, SEQ ID NO: 1 comprises the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: 3, about 22~28kDa molecular weight by SDS electrophoresis A biodegradable plastic degrading enzyme having PBS and PBSA degrading activity . Furthermore, the biodegradable plastic degrading enzyme of Claim 7 which has PLA degradation activity. Comprising contacting a biodegradable plastic-degrading enzyme according to claim 7 or 8, wherein the biodegradable plastics, the method for decomposing a biodegradable plastic. A biodegradable plastic-degrading preparation comprising the yeast or filamentous fungus screened in step 2 of the method according to claim 2 or 3 . A biodegradable plastic-degrading enzyme obtained by the method of claim 1, SEQ ID NO: 13 biodegradable plastic-degrading enzyme encoded by 501 to 1172 of the nucleotide sequence of. A biodegradable plastic-degrading enzyme obtained by the method according to claim 1, comprising the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 95% homology with said sequence, Biodegradable plastic-degrading enzyme with activity. A biodegradable plastic degrading enzyme obtained by the method according to claim 1, comprising the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 95% homology with the sequence, and PBS and PBSA degradation Biodegradable plastic-degrading enzyme with activity.   A polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 14. The polynucleotide according to claim 14 , which is represented by the nucleotide sequence at positions 501 to 1172 of SEQ ID NO: 13. Transforming a polynucleotide encoding the biodegradable plastic degrading enzyme according to claim 12 or 13 or a vector incorporating the polynucleotide according to claim 14 or 15 into an appropriate host cell; and A method for producing a biodegradable plastic degrading enzyme, comprising a step of culturing the host cell under conditions for expressing a polynucleotide.