JP2007319092A - Method for degrading biodegradable resin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inexpensive polylactic acid-degrading enzyme which can be produced in a large amount and exhibits a polylactic acid-degrading activity even at temperature below 30°C. <P>SOLUTION: This method for degrading a biodegradable resin is characterized by culturing Streptomyces coelicolor A3(2) and then bringing the obtained culture product into contact with the biodegradable resin. A biodegradable resin-degrading protein precursor comprises the following amino acid sequences. (1) An amino acid sequence constituting a chaperone region, (2) an amino acid sequence necessary for cleaving a chaperone region, and (3) an amino acid sequence which is originated from Streptomyces coelicolor A3(2) and gives a protein comprising the amino acid sequence and having a biodegradable resin-degrading activity. A gene encoding the precursor, a vector containing the gene, and a host cell containing the vector are provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for decomposing a biodegradable resin.

Currently, disposal of packaging containers is a problem. Incineration treatment similar to general-purpose resins is not a good method because it emits carbon dioxide directly to the environment. The method of decomposing into microorganisms in the environment such as landfill can be expected to reduce the addition to the environment, but it takes time to decompose and it is difficult to secure a site. Further, even when a biodegradable resin-degrading microorganism isolated from the environment is used, it cannot be rapidly decomposed due to problems such as a small amount of enzyme.
On the other hand, polylactic acid is a polymer that hydrolyzes in an aqueous solution and is applied as a medical and pharmaceutical material. Since polylactic acid can be synthesized from a renewable resource such as starch through lactic acid fermentation, it has attracted attention as a material for biodegradable resins that can replace general-purpose resins that are difficult to environmentally decompose. Examples of polylactic acid include poly L-lactic acid, poly D-lactic acid, poly DL-lactic acid, and copolymers with other monomers, depending on the type of constituent monomers. Polylactic acid is known to be hydrolyzed by enzymes. As such an enzyme, a degrading enzyme of the actinomycete Amycolatopsis sp. K104-1 is known (Non-patent Document 1). However, the enzyme does not exhibit polylactic acid-degrading activity below 30 ° C. In addition, a method of purifying and extracting a large amount of biodegradable resin-degrading enzyme and using it for decomposition is expensive and difficult to realize. For this reason, environmentally friendly system development that applies a method for acquiring a large amount of biodegradable enzyme and a method for acquiring a large amount of biodegradable resin-degrading enzyme is required.

J Bacteriol. 2005 Nov; 187 (21): 7333-40., Appl Environ Microbiol. 2001 Jan; 67 (1): 345-53.

  Accordingly, an object of the present invention is to provide an inexpensive polylactic acid degrading enzyme that can be mass-produced. Another object of the present invention is to provide a degrading enzyme exhibiting polylactic acid-degrading activity even at a temperature below 30 ° C.

The present invention provides a method for degrading a biodegradable resin comprising culturing Streptomyces coelicolor A3 (2) and contacting the resulting culture with a biodegradable resin.
The present invention also provides a biodegradable resin-degrading protein precursor comprising the following amino acid sequence:
(1) an amino acid sequence constituting the chaperone region,
(2) an amino acid sequence necessary for cleaving the chaperone region, and (3) (i) an amino acid sequence represented by SEQ ID NO: 2 or (ii) one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2 An amino acid sequence in which an amino acid is deleted, substituted or added, and a protein comprising this amino acid sequence has biodegradable resin degrading activity.
The present invention also provides a gene encoding the precursor, a vector containing the gene, and a host containing the vector.
The present invention also provides a biodegradable resin solubilizer comprising the precursor or an activated biodegradable resin-degraded protein that can be derived from the precursor.
The present invention also provides a method for solubilizing a biodegradable resin, which comprises bringing a biodegradable resin into contact with the solubilizer.

The method for degrading a biodegradable resin of the present invention includes culturing Streptomyces coelicolor A3 (2) and contacting the resulting culture with the biodegradable resin.
The biodegradable resin may be a resin having biodegradability, and may be a chemically synthesized resin, a microbial resin, a natural product-based resin, or the like. For example, aliphatic polyester, polyvinyl alcohol (PVA), cellulose And the like. Examples of the aliphatic polyester include polylactic acid (PLA) resin and derivatives thereof, polybutylene succinate (PBS) resin and derivatives thereof, polycaprolactone (PCL), polyhydroxybutyrate (PHB) and derivatives thereof, polyethylene adipate (PEA) ), Polyglycolic acid (PGA), polytetramethylene adipate, condensates of diol and dicarboxylic acid, and the like. Examples of celluloses include methyl cellulose, ethyl cellulose, and acetyl cellulose. Moreover, the modified body of the said biodegradable resin, the said biodegradable resin and the mixture with general purpose chemical resin may be sufficient. Polylactic acid resin is preferable.
The polylactic acid resin is a resin having lactic acid as a main constituent unit of the polymer. For example, lactic acid homopolymers such as poly L-lactic acid and poly D-lactic acid, at least one of L-lactic acid and D-lactic acid, and alanine, glycolide, glycine, glycolic acid, glycol, polyethylene glycol, polypropylene glycol, ε-caprolactone, Examples thereof include lactic acid copolymers with at least one of polyvinyl alcohol, saccharides and polyhydric alcohol, and poly D, L-lactic acid. Preferably, it is a lactic acid homopolymer, a lactic acid copolymer of at least one of L-lactic acid and D-lactic acid and alanine or glycolide, and poly D, L-lactic acid, more preferably at least one of L-lactic acid and D-lactic acid. Polylactic acid copolymer of glyceride and glycolide, poly-L-lactic acid. The number average molecular weight of polylactic acid is not particularly limited, but is preferably 5000 to 1 × 10 ⁶ , more preferably 50000 to 4 × 10 ⁵ . The weight ratio of lactic acid units in polylactic acid is not particularly limited, but is preferably 10% or more, more preferably 30% or more.

As a medium for culturing Streptomyces coelicolor A3 (2), a conventionally used medium or the like can be used. For example, PYM medium and CFMM medium.
The culture temperature is not particularly limited as long as polylactic acid-degrading enzyme is produced, but is preferably 10 to 40 ° C, more preferably 20 to 30 ° C.
The pH of the medium is preferably 5.0 to 9.0, more preferably 6.0 to 8.0.
The culture time is not particularly limited and is appropriately selected according to the amount of bacteria used, the medium, and the like. For example, in CFMM medium, it is 50 to 100 hours, preferably 60 to 70 hours.
Moreover, at the time of culture, it is not always necessary, but stirring is preferable. Other culture conditions are appropriately selected in view of the object of the present invention.

  The biodegradable resin is then contacted, preferably immersed, in the resulting culture. The temperature at this time is preferably 10 to 60 ° C, more preferably 20 to 50 ° C. Alternatively, an enzyme may be collected from the culture obtained as described above, and a treatment agent containing this enzyme may be brought into contact with the biodegradable resin. Examples of the method for collecting the enzyme include, for example, a method in which the culture is removed by filtration or the like by a conventional method, and if necessary, purified by dialysis, salting out, ion exchange with an ion exchange resin, gel filtration, affinity chromatography, etc. Is mentioned.

The precursor of the biodegradable resin-degrading protein of the present invention consists of the following amino acid sequences.
(1) an amino acid sequence constituting the chaperone region,
(2) amino acids necessary for cleaving the chaperone region, and (3) (i) the amino acid sequence represented by SEQ ID NO: 2 or (ii) one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2 Is an amino acid sequence in which is deleted, substituted or added, and a protein comprising this amino acid sequence has biodegradable resin degrading activity.
The chaperone region is a sequence necessary for making the structure of the expressed protein a normal structure. Examples of the amino acid sequence constituting the chaperone region include D--PQP (-is an arbitrary amino acid), and DGGPQP (SEQ ID NO: 4) is preferable.
Examples of amino acids necessary for cleaving the chaperone region include Ser, Gly, Thr, Ala, Val and the like, and preferably Ser.
The protein consisting of the amino acid sequence represented by SEQ ID NO: 2 is already known as a protein having protein hydrolytic ability (National Center for Biotechnology Information, registration number CAC36053). However, even when a gene encoding this is introduced into Escherichia coli or the like to express the protein, the protein produced is not solubilized and does not exhibit the degradation activity of the biodegradable resin. In addition, even when a gene encoding a polypeptide consisting of a sequence obtained by adding a chaperone region to the amino acid sequence represented by SEQ ID NO: 2 is introduced into Escherichia coli or the like to express the protein, the protein produced is solubilized. It does not show the biodegradable resin degradation activity. On the other hand, the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 encodes a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 added with a chaperone region and an amino acid necessary for cleaving the chaperone region. It has been found for the first time by research by the present inventors that when a gene is introduced into Escherichia coli or the like to express the protein, the protein produced is solubilized and exhibits the degradation activity of the biodegradable resin. In the solubilization mechanism, 6 residues of DGGPQP (SEQ ID NO: 4) function as a chaperone and promote solubilization. In the activation mechanism, when the N-terminal side of the first D of the precursor is cut off, the C-terminal side of S of DGGPQPS is cleaved by self-reaction.
In the present invention, the biodegradable resin-degrading protein lacks one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2 by a method known to those skilled in the art as long as it has biodegradable resin-degrading activity. It may be lost, replaced or added.
Examples of the gene encoding the precursor include SEQ ID NO: 5.

In the present invention, the protein may be any protein as long as it has polylactic acid-degrading activity, and may be a natural protein, a recombinant protein, or a chemically synthesized protein, and its origin is not particularly limited. As a method for obtaining a natural protein, a microorganism that expresses a target protein is used as a starting material, cultured, and then, if necessary, the target protein is eluted from a microorganism obtained by culturing the target protein. A method for isolating and purifying a target protein using polylactic acid degradation activity as an index by subjecting the product to known methods for protein isolation and purification such as salting out, affinity chromatography, ion exchange chromatography or gel filtration Can be illustrated. Here, for example, when affinity chromatography is used, the target protein can be purified by using a carrier to which an antibody against the protein of the present invention is bound.
As a method for obtaining a recombinant protein, a recombinant expression vector obtained by cloning the gene of the present invention encoding the target protein as described above into a suitable expression vector is transformed into a host (E. coli, yeast, etc.). The target protein can be produced by culturing the transformant under suitable conditions. In order to isolate the target protein, it is generally preferable to secrete the target protein into the culture supernatant, and this can be performed by appropriately selecting the combination of the recombinant vector / host, the culture conditions, and the like. In addition, a person skilled in the art can appropriately perform the chemical synthesis of a protein having a desired amino acid sequence according to the amino acid sequence of the protein having polylactic acid-degrading activity disclosed by the present invention.

According to the present invention, a vector containing a gene encoding a protein having polylactic acid-degrading activity is provided. Here, the type of the vector into which the gene is incorporated is not particularly limited, and an appropriate vector can be selected according to the purpose of the subsequent operation. In general, plasmid vectors, phage vectors and the like can be used, and these are commercially available from many vendors. Moreover, when producing a recombinant protein (protein having polylactic acid degradation activity) encoded by the gene of the present invention, an expression vector is preferably used.
Such an expression vector can be produced, for example, by (1) cutting out a DNA encoding a protein having polylactic acid-degrading activity of the present invention and (2) ligating the DNA downstream of a promoter in an appropriate expression vector. Can do. Examples of vectors include plasmids derived from E. coli (pBR322, etc.), plasmids derived from Bacillus subtilis (pUB110, etc.), plasmids derived from Streptomyces lividans (pIJ702, etc.), plasmids derived from yeast (pSH19, etc.), bacteriophages such as λ phage, Animal viruses such as retrovirus, vaccinia virus and baculovirus are used. The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression.

Moreover, according to this invention, the transformant produced by transforming the said recombinant vector to a host is provided. Any suitable organism can be selected as the host, and microorganisms, eukaryotic microorganisms (animal cells, plant cells, yeast, etc.), prokaryotic microorganisms (E. coli etc.) and the like can be used. For example, E. coli (Escherichia coli, etc.), Bacillus subtilis (Brevibacillus brevis, etc.), actinomycetes (Streptomyces lividans, etc.), yeast (Saccaromyces cerevisiae, etc.), insect or insect cells, animal cells (mouse cells, hamster cells, Monkey cells, human cells, etc.) are used. Brevibacillus brevis is preferable. As a method for transformation, methods known to those skilled in the art can be used, and specifically, calcium phosphate method, electroporation, microinjection, lipofection method and the like can be appropriately used.
By culturing the transformant thus obtained under suitable conditions, the protein having polylactic acid-degrading activity of the present invention can be produced. For the culture, a conventional method can be used depending on the transformant (host). As for the type and composition of the medium used for the culture, a conventional method can be used depending on the transformant (host). For the purpose of isolating the target protein, it is preferable to secrete the polylactic acid-degrading active protein into the culture supernatant, and the recombinant vector / host combination, culture conditions, and the like can be appropriately selected in view of this.
Next, a protein having polylactic acid-degrading activity is recovered from the obtained culture, isolated, and subjected to a step of purification as necessary. The protein is obtained from the culture solution (supernatant after centrifugation) when the target protein is secreted into the culture, or after pulverizing or dissolving the culture (cells) when accumulating in the cells. It can collect | recover from a culture solution (supernatant after a centrifugation process). The obtained culture broth is subjected to a known method for protein isolation and purification such as salting out, affinity chromatography, ion exchange chromatography or gel filtration. Can be isolated and purified. Here, for example, when affinity chromatography is used, the target protein can be purified by using a carrier to which an antibody against the protein of the present invention is bound.

  The biodegradable resin solubilization method of the present invention comprises a biodegradable resin as a solubilizer for a biodegradable resin containing the precursor or an activated biodegradable resin-degradable protein that can be derived from the precursor. Including contacting. The solubilizer may be one in which the precursor or an activated biodegradable resin-degraded protein that can be derived from the precursor is dissolved or suspended in a solvent. A suitable solvent is not particularly limited as long as it contains water necessary for solubilization of the biodegradable resin. Specifically, a medium such as PYM medium or CFMM medium, or an enzyme such as BugBuster Protein Extraction Reagent. Examples thereof include solutions that maintain activity. An aqueous solution is preferred. In addition, the precursor or the activated biodegradable resin-degraded protein that can be derived from the precursor may be supported on fine particles. The fine particles are not particularly limited as long as the contact area with the biodegradable resin to be brought into contact is increased to some extent, but in the shape of granules, powders, etc., it is possible to use compost used for soil or compost. it can. For example, a method in which an adsorption sequence (His-tag) or binding sequence to another substance is fused to the C-terminal side of the gene of the present invention and expressed, and immobilized by a substance that binds to each other, a substance that catalyzes binding, or the like Is mentioned. Specifically, it is obtained by adding or fusion-expressing either the amino acid Gln or Lys to the enzyme or immobilization material of the present invention, and using transglutaminase that catalyzes the binding of Gln and Lys (Biotechnol. Bioeng 2004 May 20; 86 (4): 399-404). Alternatively, a method of expressing on a cell membrane of a microorganism (Appl Microbiol Biotechnol. 2004 Mar; 64 (1): 28-40. Epub 2004 Jan 10.) or a method of adsorbing to an inorganic substance using a microorganism or the like (Biotechnol Bioeng. 2000 Dec 20; 70 (6): 704-9.) And the like.

The solubilizer may comprise the host of the present invention, in which case the precursor or an activated biodegradable resin-degrading protein that can be derived from the precursor is continuously produced from the host. The Further, the precursor produced from the host of the present invention or an activated biodegradable resin-degraded protein that can be derived from the precursor may be separately added to the solubilizer. In this case, sterilization treatment before contacting the biodegradable resin, sterilization treatment performed for the purpose of preventing the diffusion of the recombinant after solubilization, and the like can be omitted. These methods may be appropriately selected in consideration of the properties of the host and the biodegradable resin.
Examples of the method of bringing the biodegradable resin into contact include a method of immersing the biodegradable resin in the solubilizing agent of the present invention, a method of mixing the resin with a treatment that avoids contact with water, the above carrier And a method of mixing them.
The biodegradable resin may be brought into contact with the solubilizer of the present invention as it is, but may be subdivided in advance for the purpose of shortening the decomposition time. For example, the fluid may be subdivided after being mechanically pulverized, cut or heated.
In addition, the biodegradable resin solubilization method of the present invention can solubilize the biodegradable resin in a state including contents that do not inhibit solubilization of the biodegradable resin, such as food. In addition, when the culture temperature of the microorganism that expresses the biodegradable resin-degrading enzyme is different from the solubilization temperature of the biodegradable resin, a separate process may be used (FIG. 6).

（ＰＹＭ培地）

（２×ＹＴ培地）

（ＴＥバッファー）
4mmol/l Tris-HCl（トリス−ヒドロキシメチル−アミノメタン）塩酸
1mmol/l エチレンジアミン四酢酸２ナトリウム２水和物（EDTA）
pH8.0に調整
（ＣＴＡＢ溶液）
10％/l 臭化セチルトリメチルアンモニウム（CTAB）
1mol/l 塩化ナトリウム
６０℃で溶解
（ＴＡＥ電気泳動用ゲル）
1.5％アガロース
4mmol/l Tris-HCl
1mmol/l EDTA
1mmol/l 酢酸
（２×ＹＴ＋Ｇ培地）

（Ｍ９培地）

Hereinafter, the present invention will be specifically described by way of examples. In addition, what does not write the company name after the chemical name is manufactured by Wako Pure Chemical Industries. Unless otherwise specified, the solvent is water, and the medium is subjected to pressure and heat treatment at 121 ° C. for 20 minutes before culturing.
(CFMM medium)

(PYM medium)

(2 x YT medium)

(TE buffer)
4mmol / l Tris-HCl (Tris-hydroxymethyl-aminomethane) hydrochloric acid
1mmol / l Disodium ethylenediaminetetraacetic acid dihydrate (EDTA)
Adjust to pH 8.0 (CTAB solution)
10% / l cetyltrimethylammonium bromide (CTAB)
1mol / l Sodium chloride dissolved at 60 ° C (TAE electrophoresis gel)
1.5% agarose
4mmol / l Tris-HCl
1mmol / l EDTA
1mmol / l acetic acid (2xYT + G medium)

(M9 medium)

Example 1
(1. Culture method of ATCC BAA-471, Streptomyces coelicolor A3 (2))
PYM medium was used and cultured at 28 ° C. for 24 hours.
(2. Genomic extraction method of Streptomyces coelicolor ATCC BAA-471)
The bacterial cells are centrifuged at 6000 × g for 5 minutes, the obtained bacterial cells are suspended in TE buffer, 1/5 volume of 5 mol / l sodium chloride aqueous solution is added and stirred, and then 1/10 volume of 1/10 volume. CTAB solution was added, stirred, and incubated at 60 ° C. for 30 minutes. Chloroform was added in an equal amount of the above mixture and stirred, and centrifuged at 15,000 × g for 5 minutes to remove the aqueous solution layer. This operation was repeated twice. Thereafter, an equal amount of 2-propanol was added, and the precipitate was collected, washed once with 100% and 80% ethanol, dried at room temperature, and then dissolved in TE buffer to obtain a genome solution.

(3. Gene preparation method)
The enzyme products and Kit products shown below were in accordance with the instruction manual unless otherwise specified. Antibiotics necessary for selection are also added to vectors and hosts.
For PCR (Polymerase Chain Reaction), DNA Polymerase Z-taq (Takara Bio) was used, and primers (described below) were used from Invitrogen. As a template, the genome solution of Streptomyces coelicolor ATCC BAA-471 prepared above was used.
PCR reaction was performed at the temperature shown below. The annealing temperatures used for the reaction were 62 ° C. (Forward: Reverse), 65 ° C. (HF: Reverse), and 68 ° C. (SH-F: Reverse) for plasmids (I), (II), and (III), respectively. .
98 ° C, 2 minutes-(i)
98 ° C, 5 seconds-(ii)
Each annealing temperature, 5 seconds − (iii) (ii) to (iv) 30 cycles at 72 ° C., 10 seconds − (iv)
72 ° C, 2 minutes-(v)
4 ℃, ∞-(vi)

After completion of the reaction, TAE electrophoresis gel was used, and electrophoresis (100 V, 50 minutes) was performed using Mupid-ex (advanced company) which is an electrophoresis basket.
Thereafter, the sample was stained with a nucleic acid staining reagent SYBR GreenI (Takara Bio), and the target DNA fragment was extracted from the gel using a DNA extraction kit NucleoSpin Extract Kit (MACHEREY-NAGEL).
The above DNA fragment was ligated with the cloning vector pT7 Blue2 T-vector (Novagen) using the DNA-binding enzyme Ligation Convenience Kit (Nippon Gene) and transformed into the host Escherichia coli DH5α (Takara Bio) (FIG. 1). The culture was performed at 37 ° C. using 2 × YT medium. The plasmid was collected using NucleoSpin plasmid Kit (MACHEREY-NAGEL).
After confirming that the DNA sequence is 100% identical (consigned to Takara Bio Inc.), the expression vector pET26b (+) (Novagen) and the plasmid whose DNA sequence was confirmed were restricted with the restriction enzymes NcoI / XhoI (both restriction) Both enzymes were treated with Takara Bio Inc. (37 ° C., 2 hours), and after electrophoresis, DNA fragments were collected using Nucleospin plasmid Kit. Both were treated with Ligation Convenience Kit, transformed into Escherichia coli DH5α (Takara Bio), and cultured at 37 ° C. using 2 × YT medium.
The resulting plasmid was transformed into the expression host Rosetta2 (Novagen).

The presence or absence of expression was confirmed by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Each plasmid ((I), (II), (III), pET26b (+)) was transformed into Rosetta2 and grown on 2 × YT + G medium plate containing antibiotics at 37 ° C. for 16-24 hours. Thereafter, the cells were grown in a liquid 2 × YT + G medium at 37 ° C. for 12 to 16 hours. Thereafter, 0.1% of the cell culture medium was inoculated into a liquid M9 medium and grown at 37 ° C. until the absorbance at 600 nm (OD ₍₆₀₀₎ ) = 0.6 to 0.8, and isopropyl-β-D (-)-thiogalactoside ( IPTG) (final concentration 1 mM) was added and shaken at 20 ° C. for 24 hours. After that, each E. coli was recovered by centrifugation (15,000 × g, 1 minute), the supernatant on the medium was removed, lysed with the cell disruption solution BugBuster Protein Extraction Reagent (Novagen), and centrifuged (15,000 × g, 15 minutes) ) Got a fine. 10 μl of this supernatant and 20 μl of Laemmli Sample Buffer (BIO-RAD) -mercaptoethanol (95: 5) mixture were mixed and incubated at 98 ° C. for 4 minutes. Then, it was left to reach room temperature. Regarding the plasmid (I), it was confirmed that the protein expressed in the insoluble fraction was contained, but the protein was not contained in the soluble fraction. It was confirmed that the plasmids (II) and (III) contained the protein expressed in the soluble fraction.
Criterion Ready Gel J (BIO-RAD) for electrophoresis gel, Criterion cell (BIO-RAD) for electrophoresis layer, and 10 × Tris / Glycine / SDS Buffer (BIO-RAD) for electrophoresis reagent ), Power Supply Basic (BIO-RAD) was used as the power source, and each sample was subjected to electrophoresis (40 mA, 120 minutes). Thereafter, staining was performed with a staining solution SimplyBlue SafeStain (Invitrogen).
In the activity evaluation method, the following multilayer medium was prepared and evaluated as proteolytic activity.
Upper layer: 2% YT medium suspended in 2% skim milk, 1.5% agarose, and 0.1 mmol / l IPTG
Lower layer: 15 ml of 2 × YT medium containing 1.5% agarose and 0.1 mmol / l IPTG
The following recombinant Escherichia coli was grown at 20 ° C. in this overlay medium. The genes of pET26b (+), (II) and (III) were transformed into Rosetta 2 to obtain recombinant E. coli. The results are shown in FIG.

(4. Preparation method of low molecular weight polylactic acid (PLA) solution)
A 300-ml separable flask equipped with a stirrer, a nitrogen introduction tube, and a Dean-Stark type water separator was reacted with 200 g of DL-lactic acid at 180 ° C., and reacted for about 4 hours in a nitrogen atmosphere while removing the produced water. It was obtained by raising the temperature to 0 ° C. and reacting for 3 hours.
The obtained polylactic acid was dissolved in 0.5 mol / l bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, and the pH was adjusted to 7.2 after dissolution.

(5. Detection of polylactic acid degradation activity)
Each plasmid ((I), (II), (III) (FIG. 2)) was transformed into Rosetta2 and grown on 2 × YT + G medium plate at 37 ° C. for 16-24 hours.
Thereafter, the cells were grown in a liquid 2 × YT + G medium at 37 ° C. for 12 to 16 hours.
Thereafter, 0.1% of the cell culture medium was inoculated into a liquid M9 medium. D. ₍₆₀₀₎ = 0.6 to 0.8, grown at 37 ° C., IPTG (final concentration 1 mM) and polylactic acid solution (final concentration 0.1%) were added, and shaken at 20 ° C. for 24 hours.
After recovering the supernatant, phosphoric acid (final concentration 1%) was added to prepare a sample solution. Detection was performed by high performance liquid chromatography (HPLC: JASCO Gulliver).

2 ml of the sample solution was filtered through a 0.45 μm membrane filter to obtain a sample solution. WATERS Atlantis 4.6 × 250 mm was used for the column, 50 μl of the sample solution was injected, and detection was performed at UV 210 nm. The eluent was separated with a concentration gradient shown in Table 3 at a column temperature of 40 ° C. A standard curve was prepared using lactic acid. The results are shown in FIGS. The horizontal axis represents the polymerization number of lactic acid, and the vertical axis represents the concentration of polylactic acid. The left bar graph is Rosetta2 not induced by IPTG, and the reaction time with polylactic acid is 0 hour. The center bar graph is Rosetta 2 not induced with IPTG, and reacted with polylactic acid for 24 hours. The right bar graph is Rosetta2 in which the expression of the gene of the present invention was induced with IPTG, which was reacted with polylactic acid for 24 hours. As a result, the trimer and lower were increased and the tetramer and higher were decreased. This indicates that the higher molecular weight polylactic acid has changed to a lower molecular weight, and it can be said that the degradation activity of polylactic acid was detected. 4 and 5 are essentially the same diagram, FIG. 4 shows the concentration of 0 to 800 mg / l on the vertical axis, the lactic acid monomer to 12 monomer on the horizontal axis, and FIG. The concentration is 0 to 50 mg / l, and the horizontal axis is lactic acid 9-mer to 12-mer. Also, lactic acid hexamer and octamer could not be detected by the Rosetta2 product.

The schematic diagram of the plasmid used in Example 1 is shown. Details of the target enzyme and the name of the plasmid are shown. Proteinase activity detection in the plasmid of FIG. Degradation behavior of polylactic acid during E. coli expression (1-12mer) Degradation behavior of polylactic acid during E. coli expression (9-12mer) Overview of decomposition and solubilization system

Claims (7)

  A method for degrading a biodegradable resin comprising culturing Streptomyces coelicolor A3 (2) and contacting the resulting culture with a biodegradable resin. A biodegradable resin-degrading protein precursor consisting of the following amino acid sequences:
(1) an amino acid sequence constituting the chaperone region,
(2) an amino acid sequence necessary for cleaving the chaperone region, and (3) (i) an amino acid sequence represented by SEQ ID NO: 2 or (ii) one or several amino acids in the amino acid sequence represented by SEQ ID NO: 2 An amino acid sequence in which an amino acid is deleted, substituted or added, and a protein comprising this amino acid sequence has biodegradable resin degrading activity.   A gene encoding the precursor according to claim 2.   A vector comprising the gene according to claim 3.   A host comprising the vector according to claim 4.   A biodegradable resin solubilizer comprising the precursor according to claim 2 or an activated biodegradable resin degradable protein that can be derived from the precursor.   A method for solubilizing a biodegradable resin, comprising bringing the biodegradable resin into contact with the solubilizer according to claim 6.

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