JP2015156819A - Method for producing styrene monomer from polystyrene polymer, and gene and enzyme used for this method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gene and an enzyme used for producing styrene monomer from polystyrene.SOLUTION: Genes producing styrene monomer from polystyrene according to the present invention bind to the terminal of polystyrene polymer at normal temperature and normal pressure and produce styrene monomer by the reaction of releasing styrene monomer one by one from the terminal. Preferably, the genes are a gene encoding a chitin binding protein and a gene encoding an exo-chitinase enzyme, which are used by simultaneously contacting with polystyrene.

Description

  The present invention relates to a method for producing a styrene monomer by enzymatic decomposition of a styrene polymer such as polystyrene foam and a styrene resin. Further, the present invention can be used to regenerate styrene monomer as a raw material from, for example, discarded polystyrene foam or polystyrene beads reduced in volume by dissolving polystyrene foam.

Styrofoam is produced by allowing polystyrene beads to absorb a gas such as butane and then foaming by applying high-temperature steam at 100 ° C. or higher and pressure. It is classified into expanded polystyrene (EPS), polystyrene paper (PSP), and extruded board (XPS) depending on the production method and application. EPS is mainly used for agricultural and fishery containers and buffer packaging materials, PSP is mainly used for food trays, and XPS is mainly used for heat insulating building materials. Styrofoam is excellent in heat insulation, shock absorption, etc., and it is easy to process, so it will continue to be consumed in the future, but after its mission, it will be discharged as waste in industry and households. Yes. Under these circumstances, as with other fossil fuel-derived plastics, the problem of depletion of raw materials, the reduction of CO ₂ emissions, and the problem of waste disposal of polystyrene foam are becoming increasingly important from the viewpoint of energy saving. There is a need for efforts to recycle polystyrene foam.

  Materials recycling methods include material recycling and thermal recycling. The material recycling of the expanded polystyrene is a recycling method in which the expanded polystyrene resin is reused after reducing the volume of the expanded polystyrene by a volume reducing agent or heat treatment, separating the volume reducing agent.

  Thermal recycling is a recycling method in which combustion heat generated when incinerated styrene foam is used as a heat resource. In 2012, the polystyrene recycling rate in Japan was 56.8% for material recycling and 30.6% for thermal recycling, and a total of 87.4% was recycled. Most of the material recycling is not a recycling method to return to the original expanded polystyrene, but recycling that is reborn to other products. In addition, material recycling from polystyrene (including polystyrene beads) to styrene monomer, which is a raw material of polystyrene, can be performed only by a costly heat treatment method, and is hardly performed.

  Non-Patent Document 1 is a document in which a microorganism that degrades polystyrene and styrene monomer is isolated from the environment by the present inventor and identified as a Bacillus thuringiensis STR-Y-O strain.

  Patent Document 1 provides a method for efficiently recovering high-purity styrene monomer at low cost from waste polystyrene. In this proposal, a thermal decomposition device for recovering oil components from waste polystyrene and an oil component recovery device combined with a condenser are installed at multiple locations separated from each other, and recovered by each oil component recovery device. The present invention relates to the development of a method for recovering styrene monomer by transporting the obtained oil component to a common distillation apparatus and distilling the oil component with the distillation apparatus.

  Patent Document 2 provides a method for recovering styrene monomer from polystyrene resin and an apparatus used therefor. A polystyrene solution made by dissolving a polystyrene resin in a solvent whose main component is a styrene monomer is mixed with heated steam, and then this mixed gas is supplied to a decomposition tank containing a catalyst inside. This is the development of a method for decomposing polystyrene resins to obtain styrene monomers.

  Patent Document 3 provides a method for recovering styrene monomer from impact-resistant polystyrene. This is a method for obtaining a styrene monomer by thermally decomposing impact-resistant polystyrene using a sulfate and / or a manganese dioxide catalyst.

Eisaku Oikawa, Kinchidarin, Takeshi Endo, Masaaki Oikawa, Yoshinobu Ishibashi, Isolation and degradation characteristics of polystyrene-degrading microorganisms for the construction of polystyrene foam zero emission treatment, 2003, Proc. 373-379. Vaaje-Kolstad, G., Horn, JH, van Aalten, DMF, Synstad, B., and AH, EIjsink, BGH: The Non-catalytic Chitin-binding Protein CBP21 from Serratia marcescens is Essential for Chitin Degradation, The J. Biol Chrm, Vol. 280, No. 31, Issue of Auguest 5, pp. 28492-28497, 2005. Sambrook, J., Fritsch. E. F. and Maniatis, T .: Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory Press. 1989. JP 2003-335710 A JP 2001-294708 JP 2001-288124 A

  The present invention provides a method for producing a styrene monomer by introducing a polystyrene degrading gene into Escherichia coli, etc., producing this degrading enzyme, and decomposing the produced enzyme by contacting with polystyrene under normal temperature and normal pressure. An object of the present invention is to provide a gene, an enzyme, a chemical, a styrene monomer production method, and a styrene monomer production apparatus that produce styrene monomer from polystyrene.

  The present inventors have identified a microorganism Bacillus thuringiensis STR-Y-O strain having an ability to decompose a polystyrene foam (polystyrene solution) dissolved in a solvent from the environment (Non-patent Document 1). Then, the molecular weight distribution of the polystyrene (PS) degradation product by this STR-Y-O strain was analyzed using a gel permeation chromatograph analyzer (GPC). As a result, not the decomposition in which the molecular weight distribution shifts to a low molecule, but the decomposition in which only the amount of polystyrene at the peak molecular weight decreases. If the molecular weight distribution is a decomposition that shifts from a polymer to a low molecule, the decomposition occurs randomly inside the polystyrene chain, which is considered to indicate a distribution in which the polystyrene of the polymer moves to the low molecule side. On the other hand, when decomposition occurs at the end of the polystyrene chain, it is considered that the styrene monomer is released one by one, so that the distribution from the polymer to the low molecule is unlikely to occur. Furthermore, if the decomposition is to release styrene monomer one by one from the end, styrene monomer should be produced in the decomposition product. Therefore, the decomposition product was analyzed using a gas chromatograph mass spectrometer. As a result, styrene monomer was not detected in the medium-only control without addition of bacteria, but styrene monomer was detected in the culture solution in which the STR-Y-O strain was planted. From this result, it was speculated that the degrading enzyme was not an endo-type enzyme that decomposes randomly from the inside of the polystyrene chain, but an exo-type degrading enzyme that decomposes from the end of the polystyrene chain.

  Furthermore, partial production of the enzyme was performed from the culture solution of the STR-Y-O strain using the polystyrene degrading enzyme activity as an index. The enzyme activity was measured by synthesizing polystyrene labeled with a fluorescent substance, and the change in the degree of fluorescence polarization caused by enzymatic degradation of the labeled body was measured with a fluorescence polarization degree measuring device. Enzyme purification was performed using ammonium sulfate fractionation, anion exchange membrane, and cation exchange membrane. When the degradation activity was analyzed using this purified enzyme, the degradation activity was lost.

  Furthermore, when this purified enzyme was subjected to polyacrylamide gel electrophoresis and the molecular weight was estimated, a protein was detected at a position of 20 Kda. Therefore, this protein was fragmented using a peptidase enzyme, and the amino acid sequence of the obtained peptide fragment was determined and collated with a database. As a result, this molecule was identified as the Serratia marcescens chitin-binding protein CBP21. Chitin is a sugar polymer containing nitrogen, and is a substance constituting the exoskeleton of arthropods such as crabs and shrimps and crustaceans.

  CBP21 protein binds to chitin according to the study of Vaaje-Kolstad et al. (Non-patent Document 2), but has no chitinolytic activity and works together with chitinase, another chitinolytic enzyme, thereby degrading chitinase degrading enzyme. It was shown to be a protein that works to increase

  The purified CBP21 protein alone did not show polystyrene degradation activity, and CBP21 does not have chitin degradation activity and has a function to enhance chitinase degradation activity. Speculated that in addition to CBP21, chitinase was necessary. Therefore, we searched for CBP21 and chitinase of three strains of Bacillus thuringiensis, including the Bacillus thuringiensis AI Hakam strain, which is the same species as the Bacillus thuringiensis STR-Y-O strain and whose genome is publicly available on the web. As a result, all three strains were shown to have genes encoding CBP21, endo-type chitinase, and exo-type chitinase, respectively.

  From the above studies, it has been speculated that the degradation of polystyrene by the STR-Y-O strain may be due to the synergistic effect of exo-type chitinase and chitin-binding protein, which catalyses the reaction of releasing styrene monomer from the end of the polystyrene chain.

  Therefore, PCR was used to amplify the cbp21 gene encoding CBP21 on the chromosome of the STR-YO strain and the chi36 gene encoding exochitinase Chi36 on the chromosome of the STR-YO strain using oligomer DNA primers prepared with reference to the DNA sequence information of AI Hakam strain. did.

  Next, each amplified gene was introduced into E. coli and expressed to produce an enzyme. Furthermore, after refine | purifying the produced enzyme, in addition to the solution containing polystyrene, the presence or absence of the production | generation of the polystyrene monomer was analyzed with the gas chromatograph mass spectrometer. As a result, when only the CBP21 protein and only the Chi36 enzyme were added, the production of styrene monomer was hardly detected, but when both the CBP21 protein and the Chi36 enzyme were added, the dominant styrene monomer production was detected. From the above experimental results, it was concluded that both chitin-binding protein CBP21 and exochinase Chi36 are enzymes that decompose polystyrene and produce styrene monomers.

  The microorganism Bacillus thuringiensis STR-Y-O strain having the polystyrene-degrading enzyme gene identified in the present invention is stored as the accession number FERM P-19582 at the National Institute of Technology and Evaluation, and is available.

  According to the present invention, a styrene monomer can be purified from microorganisms and enzymes at normal temperature and normal pressure.

It is a graph which shows the experimental result of the polystyrene residual rate by this invention, and has shown the comparison result of the experiment using new EPS, 5-year used EPS, and a culture supernatant. It is a table | surface which shows the primer used for PCR cloning. It is a graph which shows the experimental result of the amount of styrene monomers by this invention. It is a figure which shows the comparison result of the styrene monomer production amount. It is a graph which shows the PS-> SM conversion rate by the combination of CBP21 and Chi36. It is a table | surface which shows the mol weight of the refined styrene monomer per mol of enzyme.

experimental method:
Strains used:
The strains used in this experiment are as follows.
The strain Bacillus thuringiensis STR-YO, which possesses a polystyrene-degrading enzyme gene, is a strain isolated from field soil that has been disposed of for a long time with waste plastics and waste vinyl products from Yamagata City, Yamagata Prefecture. This stock has been deposited with the National Institute of Technology and Evaluation as Accession Number FERM P-19582 and is available.

Medium used:
LB medium was used as a culture medium for microorganisms. The composition of LB medium is tryptone 10 g, yeast extract 5 g, NaCl 5 g, glucose 1 g, agar 15 g (in the case of agar medium), distilled water 1 L (up to 1 L), and this is autoclaved (20 min, 121 ° C., 2 atm) ) And used. When culturing the recombinant Escherichia coli, ampicillin was added to each medium so that the final concentration was 50 μg / ml.
In the polystyrene degradation experiment using the STR-YO strain, LB medium and M9 medium (Non-patent Document 3) were used.

Reagents and materials used:
As a new polystyrene foam, a polystyrene foam produced as a raft float for oyster raft cultivation was used. Styrofoam used for 5 years was used for 5 years on the Seto Inland Sea coast of Kure City after being attached to a raft for raising oyster rafts. These polystyrene foams were broken by hand and dissolved using d-limonene (Wako Pure Chemical Industries) to a final concentration of 0.3 mg / ml.

Experimental device:
For gel permeation chromatographic analysis, JASCO PU-2080Plus was used. The column was Shodex GF-510HQ and the mobile phase was tetrahydrofuran.
JEOL JmsQ1000GC K9 was used for gas chromatograph mass spectrometry. The column used was Spellcoequity 5, inner diameter 0.25 μm, and length 30 m.

Experimental procedure:
(1) PCR cloning of the cbp21 gene First, the STR-YO strain was cultured, and after chromosome adjustment, PCR amplification was performed using two kinds of primers. PCR for use in determining the base sequence of the cbp21 gene was performed using cbp-F0 primer 5′-CAGCATTTGCTGGTGGAGCTGCGA-3 ′) and cbp-R1 primer (5′-GTAGCCAATCTGACTACTTGTC-3 ′). In the case of protein production in E. coli, PCR is performed by cbp-N2 primer (5'-CACGGATCCCATGGATACGTAGAATCACC-3 ') and cbp-R1 lacking the DNA sequence encoding the 5' end signal sequence and introducing the BamHI site. This was performed using a primer (5′-GTAGCCAATCTGACTACTTGTC-3 ′). PCR was performed using Ex-Taq DNA polymerase (Takara Bio) or PRIME-STAR DNA polymerase (Takara Bio). The obtained PCR product for base sequence determination was subjected to agarose gel electrophoresis, DNA of a desired size was recovered, and the recovered DNA fragment was incorporated into a pGEM-T vector (Promega). Thereafter, this recombinant plasmid was introduced into E. coli. A plasmid was prepared from this recombinant E. coli, and the nucleotide sequence of the insert site of this plasmid was determined. The PCR product for protein production by E. coli was cleaved with restriction enzymes BamHI and ScaI, and the resulting 750 bp DNA fragment was recovered by polyacrylamide gel electrophoresis. Finally, this recovered DNA fragment was ligated to the BamHI and SmaI sites of the expression vector pQE81L (Qiagen) and introduced into E. coli DH5a to obtain a transformant DH5a + pQE81L + cbp21. This recombinant Escherichia coli was used for mass production of CBP21.

  The homology of the cloned cbp21 gene with the cbp21 gene of AI Hakam strain is 87.1% (total: 361bp) at the nucleic acid level, and at the amino acid level, 94.6% Total: 187 residues).

(2) chi36 gene PCR cloning First, after culturing STR-YO strain and chromosome adjustment, chi-F1 primer (5'-CCAGCACCYGCTGTGGCTTC-3 ') and chi-R1 primer (5'-GAGAGTYATGTTGAAGGTATG-3') PCR amplification was performed using The obtained 2.5 Kb PCR product was recovered by polyacrylamide gel electrophoresis, incorporated into a cloning vector pGEM-T (Promega), and introduced into E. coli DH5a to obtain a transformant DH5α + pGEM-T + chi36. A plasmid was prepared from this recombinant Escherichia coli, and the nucleotide sequence was determined. Next, PCR amplification was performed using pGEM-T + chi36 as a template and chi-N2 primer (5′-ACGGATCCGCAAACAATTTAGGTTCAAAATTACTCG-3 ′) and vector-side RV primer (5′-CAGGAAACAGCTATGAC-3 ′). This PCR product was purified by High Pure PCR Product Purification Kit (Roche). The purified PCR product was cleaved with restriction enzymes BamHI and SspI. The obtained 1133 bp DNA fragment was recovered by polyacrylamide gel electrophoresis. Finally, this recovered DNA fragment was ligated to the BamHI and SmaI sites of the expression vector pQE81L (Qiagen), and transformed into E. coli DH5a to obtain a transformant DH5a + pQE81L + chi36.

  The homology of the cloned chi36 gene with the chi36 gene of AI Hakam strain was 96.3% at the nucleic acid level and 90% at the amino acid level.

  Based on these results, the recombinant DNA technology will be applied in the future to introduce the gene encoding this chitin-binding protein CBP21 and the gene encoding exo-type chitinase chi36 into Escherichia coli, etc. to enhance the expression of the recombinant enzyme. It is considered that more efficient styrene monomer production can be performed by mass-producing the enzyme and using this enzyme as a styrene monomer production material.

Protein purification
(1) Mass culture and sonication of E. coli transformants
(a) Each of E. coli transformant (DH5a + pQE81 + cbp21) and E. coli transformant (DH5a + pQE81 + chi36) stored in a glycerol solution at -85 ° C, each containing 100 μg / mL ampicillin (Amp) Were streaked on 1 × LB agar medium and incubated at 37 ° C. overnight.
(b) The emerged single colony was inoculated into 1 × LB liquid medium containing 20 mL of Amp, and cultured with shaking at 130 rpm overnight at 37 ° C.
(c) On the next day, 10 mL of the culture solution was transferred to 1 L of 1 × LB liquid medium containing Amp, and cultured with shaking at 110 ° C. and 30 ° C. until the growth degree OD600 nm became 0.5.
(d) IPTG (Takara Bio) was added to a final concentration of 0.3 mM, and further cultured for 2 hours.
(e) Place the culture in a high-speed centrifuge and collect the cells by centrifugation at 4 ° C, 5,000 rpm for 5 minutes. Thereafter, the medium was discarded, the cells were transferred to a tube, and stored at -85 ° C until the next operation.
(f) The operations of (a) to (e) were repeated to collect 10 L of each 1 × LB liquid medium containing Amp.
(h) The bacterial cells were suspended by adding 1 mL of lysis buffer (50 mM NaH ₂ PO ₄ , 100 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM Imidazol) per 1 L of the culture medium.
(i) In (h), cells were disrupted with an ultrasonic crusher. (Conditions of ultrasonic crusher: 10 seconds operation, 60 seconds stop repeated for 15 minutes)
(j) The cell disruption solution was set in a micro high speed centrifuge and centrifuged at 4 ° C., 15,000 rpm for 20 minutes.
(k) The supernatant was transferred to a new tube and quantified using protein assay kit I (BIO-RAD Laboratories).

(2) Purification of protein by Ni-NTA column chromatography The procedure was as follows. (a) 5 mL of Ni-NTA Superflow resin (Qiagen) was poured into a 1 x 10 cm column. (b) The column was equilibrated with 40 mL of lysis buffer. (c) The protein exchanged into the lysis buffer was loaded onto the column at a flow rate of 0.5 mL / min. (d) The column was washed with 40 mL lysis buffer. The column was washed with 40 mL of wash buffer (50 mM NaH ₂ PO ₄ , 100 mM TrisHCl pH 8.0, 300 mM NaCl, 20 mM Imidazol). (e) 40 mL of elution buffer (50 mM NaH ₂ PO ₄ , 100 mM TrisHCl pH 8.0, 300 mM NaCl, 250 mM Imidazol) was added to the column to elute CBP21 or Chi36. Fractions were collected 1 mL each. (f) The fraction in which protein absorption was obtained by absorbance was exchanged and concentrated to Tris-DTT buffer (20 mM Tris-HCL, 0.1 mM DTT) using Centriplus 10 (Amicon, Inc). (g) Protein was quantified using Protein Assay Kit I (BIO-RAD Laboratories). (h) The purified enzyme was stored at 4 ° C. until the polystyrene degradation experiment.

(3) Confirmation by SDS-PAGE of protein electrophoresis
The purity of the purified protein was confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Production experiment of styrene monomer from polystyrene using purified protein
(1) Preparation of reaction solution
(a) As shown in the table, add purified enzyme or buffer solution to a 50 mL glass centrifuge tube, add foamed polystyrene (= 0.3 mg / ml polystyrene solution) dissolved in limonene, and adjust to 10 mL with water. did.
(b) The centrifuge tube was wrapped with aluminum foil to shield it from light, and reacted at 37 ° C. for 4 days with gentle shaking.

(2) Extraction of degradation products
(a) Add 2 mL of dichloromethane to the culture and mix for 2 minutes with a vortex mixer.
(b) Centrifuge at 3,000 rpm for 5 minutes at room temperature.
(c) Using a Pasteur, transfer the dichloromethane layer (lower layer) to a 2 mL microtube containing 0.1 g of anhydrous sodium sulfate.
(d) Mix well by vortexing and leave at room temperature for 10 minutes.
(e) Centrifuge the flash and transfer the supernatant to a vial with a lid. Store at 4 ° C until analysis
(f) The polystyrene degradation product was analyzed using a gel permeation chromatograph (GPC) analyzer to determine the amount of polystyrene degradation product and to analyze the molecular weight distribution. The quantitative analysis of styrene monomer was performed using a gas chromatograph mass (GS-MS) analyzer.

Polystyrene resolution analysis with polystyrene-degrading bacteria STR-YO strain
(a) The STR-YO strain was cultured with shaking at 30 ° C. in a 50 mL Erlenmeyer flask containing 10 mL of 1 × LB medium. 200 μl of this culture solution was transferred into a 100 mL conical stoppered flask containing 20 mL of M9 medium, and further cultured with shaking for 3 days.
(b) In the case of a decomposition experiment using a culture solution containing bacterial cells, 200 μl of EPS dissolved in 0.3 mg / ml limonene was added to the culture solution and shaken at 30 ° C. for 8 days. On the other hand, in the decomposition experiment using the supernatant of the culture solution, the culture solution in the Erlenmeyer flask was transferred to a 50 ml centrifuge tube and centrifuged at 4 ° C. and 15,000 rpm for 15 minutes. The supernatant was transferred to a new Erlenmeyer flask with a stopper, 200 μl of EPS dissolved in 0.3 mg / ml limonene was added, and the mixture was shaken at 30 ° C. for 8 days.
(c) The degradation product in the culture solution was extracted with dichloromethane, and the extracted degradation product was analyzed using a gel permeation chromatograph (GPC) analyzer and a gas chromatograph mass spectrometer.

Discussion of experimental results
(1) Mass purification of CBP21 and Chi36 As a result of polyacrylamide gel electrophoresis of purified CBP21 protein, a band was detected at the predicted 21.9 kDa position by adding the expression inducer IPTG. confirmed. It was also confirmed that CBP21 with artificially added 6 × His-tag could be purified with high purity by passing it through a nickel column. The concentration of purified CBP21 in the solution was 0.832 (μg / μL) before concentration and 0.360 (μg / μL) after concentration, and the total amount finally purified was from 10 L to 600 μg, with a very small amount. It was.
The purified Chi36 protein was subjected to polyacrylamide gel electrophoresis. As a result, a band was detected at the predicted position of 37.7 kDa when IPTG was added, confirming that expression was remarkably induced. Moreover, it was confirmed that Chi36, to which 6 × His-tag was artificially added, was purified with high purity by passing through a nickel column. The concentration of the purified Chi36 in the solution was 0.840 (μg / μl) before concentration and 1.008 (μg / μl) after concentration, and the total amount finally purified was 2400 μg (2.4 mg ) And very small amount. The reason why CBP21 and Chi36 protein production is less than normal may be due to the difference in codon usage between Bacillus and E. coli. In the future, it was considered necessary to perform protein production after returning the DNA sequence to codons frequently used in E. coli.

(2) Production of styrene monomer from polystyrene using a large amount of purified CBP21 and Chi36 Polystyrene (PS) decomposition experiments were conducted using purified CBP21 and Chi36. The amount of enzyme used in the experiment is 100 μg for CBP21 and 400 μg for Chi36.
The amount of styrene monomer (SM) calculated from the results of gas chromatograph mass spectrometry is only 0.1 mg / L (CBP21) to 0.18 when only CBP21 is added and when only Chi36 is added compared to materials without enzyme mg / L (chi36) was detected. On the other hand, when both CBP21 and Chi36 are added, it is about 10 times (up to 14 times) for CBP21 and about 6 times (up to maximum) for CBP21, compared to when CBP21 or Chi36 is decomposed alone. 9 times) of about 1 mg / L of SM was produced.
When both CBP21 and Chi36 were added, the PS → SM conversion rate was 6.5% (maximum 9.1%), and a high conversion rate was obtained. From these results, it was concluded that both CBP21 and Chi36 proteins are enzymes that generate SM from polystyrene PS.
Furthermore, the mol weight of SM produced for each 1 mol of CBP21 and Chi36 was calculated. As a result, CBP21 was calculated to produce 4.70 mol of SM per mol. Chi36 was calculated to produce 1.88 mol of SM per mol. Even though CBP21 alone produces less SM than Chi36 alone, when both CBP21 and Chi36 are added, the molecular weight of SM produced per mol is shown to be higher for CBP21 than for Chi36. It was. Therefore, from the mol ratio, it can be said that CBP21 has a higher contribution to PS production from PS than Chi36.
From these results, as expected, both CBP21 and Chi36 proteins are involved in the reaction that degrades PS to produce SM, and the initial degradation of PS in the STR-YO strain is both CBP21 and Chi36 proteins. It was clarified that this reaction was an SM formation reaction.

(3) Styrene monomer production from polystyrene using culture supernatant of STR-YO strain
Analysis of decomposition of new foamed polystyrene dissolved in limonene by STR-YO and foamed polystyrene used for 5 years confirmed that 20% resolution of polystyrene with an initial concentration equivalent to that of new foamed polystyrene is confirmed even in degraded and damaged foamed polystyrene. It was shown that the resolution can be exhibited regardless of deterioration or damage. Furthermore, when the culture solution of the STR-YO strain was collected by centrifugation and the supernatant was removed, the same experiment was performed. An initial concentration of 20% resolution of polystyrene was confirmed. Furthermore, styrene monomer was detected in this decomposition product. This result can also be said from the results of sequencing of cbp21 and chi36, but both genes were shown to encode a signal sequence at the 5 'end, and the proteins encoded by both genes were secreted outside the cell. Therefore, it was speculated that PS was converted to SM by both secreted proteins.
From this, it is considered that the degrading enzyme is an extracellular secreted enzyme, and PS degradation by STR-YO strain can be handled as a substance (enzyme) without the need for bacterial cells in terms of handling, wild type If the efficiency of enzyme production can be improved by optimizing the culture efficiency of the STR-YO strain, the styrene monomer can be produced at low cost, regardless of the production by recombinant Escherichia coli with the cbp21 and chi36 genes. It is considered that the handling will be simplified, and it may become more practical. A solution of used polystyrene foam in an organic solvent such as limonene is added directly to the culture supernatant of the STR-YO strain to produce SM at room temperature and pressure, and the solution containing this produced SM is distilled. In addition to equipment, a method for purifying SM can be provided.

  The preferred embodiment of the present invention has been described in detail above, but the present invention is not limited to the specific embodiment, and various modifications can be made within the scope of the present invention described in the claims. Deformation / change is possible.

Claims (16)

A gene having a DNA sequence encoding a chitinase enzyme having polystyrene degrading enzyme activity of the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing. A chitinase enzyme that is a gene product of the gene, wherein the enzyme has the amino acid sequence of SEQ ID NO: 1. A gene having a DNA sequence encoding a protein enzyme having a polystyrene-degrading enzyme activity of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing and binding to chitin. A protein enzyme having the chitin-binding property and having the amino acid sequence of SEQ ID NO: 2. The gene of the DNA sequence which codes the fusion protein which the histidine tag was added to the protein of Claim 2 shown by sequence number 3 of a sequence table. The gene of the DNA sequence which codes the fusion protein which the histidine tag was added to the protein of Claim 4 shown by sequence number 4 of a sequence table. A recombinant microorganism used for producing a styrene monomer produced by introducing the gene of any one of claims 1 and 3 into a microorganism. A microorganism having the gene of any one of claims 1 and 3. A method for producing a styrene monomer by simultaneously contacting the enzyme of any one of claims 2 and 4 with polystyrene. A drug such as a carrier, such as a carrier on which the enzyme according to claim 2 or 4 is immobilized, or a medicine for producing a styrene monomer. The styrene monomer manufacturing apparatus which manufactures a styrene monomer using the gene in any one of Claim 1 and Claim 3. The styrene monomer manufacturing apparatus which manufactures a styrene monomer using the enzyme in any one of Claim 2 and Claim 4. A microorganism having polystyrene-degrading activity, chitin-binding protein, and styrene monomer-forming activity. A microorganism having polystyrene degrading activity, exo-type chitinase enzyme activity, and further having styrene monomer-forming activity. A microorganism such as a powder or a tablet, wherein the microorganism according to claim 10 or claim 11, or a material such as a carrier on which the microorganism according to claim 10 and claim 11 is simultaneously immobilized, or a microorganism. An additive in which one of the microorganisms according to claim 10 or claim 11, or a material such as a carrier on which the microorganisms according to claim 10 and claim 11 are simultaneously immobilized, or a microorganism as a powder or a tablet.

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