JP2006055005A - New urethanase gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for purifying and obtaining a new urethanase and to provide a method for decomposing a urethane compound utilizing a host cell expressing the new urethanase. <P>SOLUTION: A polynucleotide encodes the new urethanase. The method for purifying and obtaining the enzyme comprises expression of the enzyme having the ability to decompose the urethane compound in the host cell comprising the polynucleotide integrated therein. The method for decomposing the urethane compound comprises utilizing the host cell expressing the new urethanase. The gene of the urethanase is obtained and an expression system in Escherichia coli is established. Thereby, decomposability can be imparted by protein engineering modification and the gene can be applied to enzymatic monomer recycling of polyurethanes. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a polypeptide having a novel urethane compound degrading activity (urethane bond degrading activity), that is, a novel uretanase-encoding polynucleotide, and an enzyme capable of decomposing a urethane compound in a host cell incorporating the polynucleotide. Is expressed, and the enzyme is purified and obtained. Furthermore, the present invention relates to a method for decomposing a urethane compound using a host cell expressing a novel uretanase.

Conventional technology polyurethane is used in various fields because of its excellent properties. However, on the other hand, the amount of waste is increasing year by year, causing serious environmental problems such as incinerator damage due to high combustion heat and landfill saturation. As measures against these wastes, attention has been paid to microbial degradation and enzymatic degradation, which are low-cost and energy-saving processes. If polyurethane can be decomposed into monomers by enzymes, it can be recovered and re-synthesized to produce polyurethane that is exactly the same as the primary product, opening the way to recycling.

  The biodegradability of polyurethane has been mainly researched in terms of degradation by microorganisms and in vivo enzymes, and the main theme has been how to prevent biodegradation. Therefore, research on polyurethane-degrading microorganisms themselves and their degrading enzymes has not progressed.

The decomposition of polyurethane is roughly divided into the decomposition of a urethane bond and the decomposition of a polyol part. Of these, the urethane bond is a bond that is common to all polyurethanes. However, there is little knowledge about the decomposition of urethane bonds in polyurethane. Although there are some reports that urethane bonds undergo hydrolysis due to microbial degradation (Non-Patent Documents 1 and 2), the causal relationship between cleavage of urethane bonds and microorganisms or their enzymes is not clear.
B. Jansen et al., Zentralbl Bakteriol., 276, 36 (1991) RT Darby and AM Kaplan, Appl.Microbiol., 16, 900 (1968)

Polyester-type polyurethane-degrading bacteria include Penibacillus amylolyticus TB-13 strain (Japanese Patent Application No. 2002-334162) and Comamonas acidovorans TB-35 strain (FEMS Microbiology Letters, Vol. 129, 39-42, 1995, Non-Patent Document 3) are known, but these decomposing bacteria decompose ester bonds in urethane, but hardly decompose urethane bonds.
T. Nakajima-Kambe, F. Onuma, N. Kimpara and T. Nakahara, Isolation and characterization of a bacterium which uses polyester polyurethane as a sole carbon and nitrogen source. FEMS Microbiology Letters, Vol. 129, 39-42, 1995

  On the other hand, it has already been reported that low-molecular-weight urethane compounds are degraded by microorganisms, but it is known that the degradation is not caused by uretanase but by esterase. Most of them relate to the improvement of liquor varieties and the decomposition and purification of carbamate pesticides (Japanese Patent Laid-Open Nos. 01-300892, 01-240179, 02-128689, 03-175985, 04-104784). , JP 04-325079), which is not a technology that can be used for the decomposition of polyurethane. As a degrading bacterium of a material that can be a polyurethane raw material, fungi have been reported (Japanese Patent Laid-Open No. 09-192633), but there is no bacterium that can be easily cultured in large quantities, and its degrading enzyme is not specified.

In the case of decomposing a non-natural polymer such as polyurethane with high efficiency, it is preferable to use a genetic engineering modification or a large-scale expression form such as Escherichia coli rather than using a natural enzyme as it is. . For this reason, acquisition of the uretanase gene was also required.
Japanese Patent Laid-Open No. 01-300892 Japanese Patent Laid-Open No. 01-240179 Japanese Patent Laid-Open No. 02-126889 Japanese Patent Laid-Open No. 03-175985 Japanese Patent Laid-Open No. 04-104784 Japanese Patent Laid-Open No. 04-325079 JP 09-192633 A

Problem to be Solved by the Invention The present invention relates to a polynucleotide encoding a novel uretanase capable of breaking a urethane bond in a urethane compound, to express uretanase in a host cell incorporating the polynucleotide, and to purify the enzyme It aims to provide a way to do. A further object of the present invention is to provide a method for decomposing a urethane compound using a host cell expressing a novel uretanase.

Means for Solving the Problems In order to solve this problem, the present inventors have used various low molecular weight urethane bond-containing compounds synthesized from aromatic / aliphatic isocyanates and monohydric alcohols used as raw materials for polyurethane synthesis. A gram-positive bacterium Rhodococcus equi TB-60 strain, which can screen microorganisms using soil as a separation source and can decompose the compound with high efficiency, has been found and a patent application has already been filed (Japanese Patent Application No. 2003) -055421). Furthermore, the present inventors purified uretanase produced by Rhodococcus equii strain TB-60 and examined its properties in detail. The uretanase was converted into an aliphatic compound as well as an aromatic compound used as a raw material for polyurethane synthesis. On the other hand, it has been found that it has urethane bond cleavage activity and has already filed a patent application (Japanese Patent Application No. 2004-58475).

The present invention is a polynucleotide comprising a polynucleotide encoding the uretanase or a complementary sequence thereof.
The present invention is a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2, or a polynucleotide comprising the complementary sequence of the nucleotide sequence of SEQ ID NO: 2.

Furthermore, the present invention is a method for purifying and obtaining an enzyme having a urethane compound resolution in a host cell into which the polynucleotide is incorporated.
Furthermore, the present invention is a method for decomposing a urethane compound using a host cell expressing a novel uretanase.

  In the present specification, “polypeptide having urethane compound decomposing activity” and “uretanase” are used synonymously and are enzymes capable of degrading urethane bonds in urethane compounds. In addition, the “urethane compound” in the present specification refers to a compound having a urethane bond, and includes any molecular weight compound.

BEST MODE FOR CARRYING OUT THE INVENTION
Polynucleotide encoding Uretanase The polynucleotide of the present invention is a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, or a complementary sequence thereof. Here, the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is Rhodococcus equii TB-60 strain (Rodococcus equii TB-60 strain deposited with DSMZ, a German patent organism depositary institution on January 24, 2004 ( It is a uretanase produced by DSMZ 16175)).

  The polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2, which is one embodiment of the polynucleotide of the present invention, comprises amino acids such as the genomic DNA of Rhodococcus equi TB-60 and the N-terminal amino acid sequence of uretanase possessed by Rhodococcus equi TB-60 Using a probe prepared based on the sequence, it was obtained through hybridization and cloning steps. A person skilled in the art can obtain a polynucleotide of the present invention from the genomic DNA of Rhodococcus equi strain TB-60 by preparing a suitable probe based on the disclosure of the nucleotide sequence of SEQ ID NO: 2, in a manner well known to those skilled in the art. it can.

  The polynucleotide of the present invention can include its degeneracy. Degeneracy refers to a phenomenon in which one amino acid can be encoded by different nucleotide codons. Thus, the nucleotide sequence of a nucleic acid molecule encoding uretanase comprising the amino acid sequence shown in SEQ ID NO: 1 can be altered by degeneracy.

A polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2, under conditions highly stringent to the complementary sequence of the polynucleotide set forth in SEQ ID NO: 2 [eg, 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), Hybridize to filter-bound DNA in 1 mM EDTA at 65 ° C. and wash at 68 ° C. in 0.1 × SSC / 0.1% SDS (Ausubel, FM et al., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, and John Wily & Sons, New York, p.2.10.3)] Depending on the nucleotide sequence that hybridizes, the encoded protein may also exhibit the same enzymatic activity. Therefore, those polynucleotides are also included in the scope of the present invention as long as they have similar uretanase activity. Furthermore, under conditions moderately stringent to the complementary sequence of the polynucleotide set forth in SEQ ID NO: 2 [eg, washed at 42 ° C. in 0.2 × SSC / 0.1% SDS (Ausubel, et al., 1989 , Supra)], depending on the hybridizing polynucleotide, the encoded protein may also exhibit the same enzymatic activity. Therefore, those polynucleotides are also included in the scope of the present invention as long as they have similar uretanase activity.

According to genetic recombination technology, it is possible to artificially mutate a specific site of a basic polynucleotide without changing the basic characteristics of the polynucleotide or improving the characteristics. . Similarly, a polynucleotide having a natural base sequence provided by the present invention or a polynucleotide having a base sequence different from that of the natural one is artificially inserted, deleted, substituted or added, It is possible to have the same or improved properties as the polynucleotide of the present invention, and the present invention includes such a mutant polynucleotide. That is, the polynucleotide in which a part of the polynucleotide shown in SEQ ID NO: 2 in the sequence listing is inserted, deleted, substituted or added is 20 or less, preferably 10 or less in the sequence shown in SEQ ID NO: 2, Preferably, the polynucleotide is substituted with 5 or less bases. Further, such a polynucleotide and the base sequence shown in SEQ ID NO: 2 have a homology of 70% or more, preferably 80% or more, more preferably 90% or more (homology calculation is performed by, for example, BLAST (Basic (Local Alignment Search Tool) search can be used.) Such a polynucleotide is also included in the scope of the present invention as long as it encodes a polypeptide having the characteristic of having uretanase activity.
Production of Uretanase by Genetically Modified Cells The method for producing uretanase by genetically modified cells is as follows. First, a polynucleotide molecule is inserted into a host cell and inserted into any vector capable of forming a recombinant microorganism. Vectors can be either RNA or DNA, either prokaryotic or eukaryotic, but are typically viruses or plasmids. The vector can be expressed as an extrachromosomal element (eg, a plasmid) or it can be integrated into the chromosome. The integrated polynucleotide molecule can be under the control of a chromosomal promoter, under the control of a natural or plasmid promoter, or under a combination of several promoters. Single or multiple copies of the polynucleotide molecule can be integrated into the chromosome. The vector is then transfected into a host cell to form a recombinant cell. Suitable host cells for transfection include any bacterium, fungus (eg, yeast) that can be transfected. Preferred host cells for use in the present invention include, but are not limited to, any microbial cell suitable for expression of uretanase, including, for example, E. coli, Bacillus subtilis, yeast and the like. Furthermore, a genetically modified cell containing uretanase can be obtained by culturing the host under culture conditions suitable for the host. Suitable culture conditions for the host are well-known to those of skill in the art.

Separation and purification of uretanase from a host cell incorporating the polynucleotide sequence of the present invention can be performed by using a method usually used for separation and purification of a protein from the cell. Specifically, it can be carried out by using commonly used separation and purification means after disrupting the cells. Non-limiting examples of cell destruction include sonication, high-pressure homogenizer treatment, and osmotic shock method. Separation and purification means may be used by appropriately combining methods such as salting out, gel filtration, and ion exchange chromatography. Furthermore, in the production of uretanase by genetic recombination, the recombinant enzyme is produced so that it has a His-tag at the C-terminus, the cultured cells are centrifuged and the periplasmic fraction is extracted by the osmotic shock method. Since the recombinant enzyme has a His-tag at the C-terminus, it can be easily purified by a column chelated with nickel.
Properties and characteristics of uretanase One embodiment of uretanase obtained from a host cell incorporating a polynucleotide sequence of the present invention is a polypeptide having the amino acid sequence of SEQ ID NO: 1.

  In addition to urethane compounds, the uretanase has hydrolytic activity on amides and esters. The uretanase of the present invention also has urethane bond cleavage activity against aromatic and aliphatic compounds used in the synthesis of polyurethane.

  In this specification, polyurethane is a general term for polymer compounds having a urethane bond (—NHCOO—) in the molecule, and is obtained by a reaction between a polyfunctional isocyanate and a hydroxyl group-containing compound. A polymer having a group such as carbamate, and various types of branched or crosslinked polymers can be prepared by changing the functional number of hydroxyl group or isocyanate group, and all of them are included.

The uretanase of the present invention is stable in the pH range of 8-10.
The uretanase is composed of 472 amino acids and has an estimated molecular weight of 50,699 Da.
Method for Decomposing Urethane Compound Further, the present invention provides a method for decomposing a urethane compound by the action of uretanase produced by genetically modified cells. The uretanase used in the method may be a purified enzyme, a crudely purified enzyme, or a liquid obtained by disrupting cells. Cell disruption can be performed by methods known to those skilled in the art.

  The urethane compound that can be decomposed by the uretanase of the present invention is only required to have a urethane bond in the molecular structure. Non-limiting examples include toluene-2,4-carbamic acid dibutyl ester, toluene-2,6-dicarbamic acid dibutyl ester, methylenebisphenyl dicarbamic acid dibutyl ester, hexamethylene-dicarbamic acid dibutyl ester, norbornene dicarbamic acid Examples include dibutyl esters and polyurethanes using them as synthetic raw materials.

The number average molecular weight of the polyurethane resin that can be applied in the decomposition method of the present invention is not particularly limited.
The urethane compound to be decomposed may be added to the solution as an emulsion or in the form of a powder, or may be added as a lump such as a film or a pellet. The amount of the urethane compound added to the solution is preferably 0.01 to 10% by weight. The amount of uretanase to be added may be extremely small, but is preferably 0.01% by weight or more (wet weight) with respect to the urethane compound in consideration of decomposition efficiency. Moreover, the urethane compound to be subjected to decomposition may be one kind or plural kinds. The solution may be a solution obtained by adding a urethane compound to a buffer solution, but may also contain a nitrogen source, an inorganic salt, a vitamin, or the like. Examples of the buffer solution include a phosphate buffer solution.

  The time required for the decomposition of the urethane compound can vary depending on the type, composition, shape and amount of the urethane compound to be decomposed, the relative amount of the urea compound to the urethane compound, pH, temperature and other various decomposition conditions.

  Confirmation of the decomposition of the urethane compound during the decomposition reaction includes, for example, measurement of weight loss of the urethane compound subjected to decomposition, measurement of the amount of residual urethane compound by high performance liquid chromatography (HPLC), or diamine compound which is a urethane-bonded hydrolyzate It can be confirmed by measuring the production of Confirmation of the production | generation of a diamine compound can be performed by, for example, using a diamine compound expected to be produced by thin layer chromatography as a standard substance, or by gas chromatography.

Method for completely decomposing polyurethane As an embodiment of a method for decomposing solid polyurethane, Penibacillus amylolyticus TB-13 strain (accession number FERM P-19104; 334162) and / or the Commamonas acid bolans TB-35 strain, or an enzyme derived from these strains, and the ureatinase of the present invention having urethane binding ability can be used for complete degradation of polyurethane.

EXAMPLES The present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited to these examples.

Cloning procedures for urethanase gene from cloned Rhodococcus equi TB60 strain urethanase gene from Example 1 Rhodococcus equi TB60 strain is shown in Figure 1.

Step A. Preparation of total DNA of Rhodococcus equi strain TB60 Total DNA was extracted from the cells of Rhodococcus equi strain TB60 cultured overnight at 30 ° C. in a gravy liquid medium. The extraction method is as shown in FIG.

Step B. Amplification of uretanase gene by PCR B-1. Determination of N-terminal and internal amino acid sequences of uretanase Using purified uretanase, the N-terminal and internal amino acid sequences were determined. For the determination of the internal sequence, a polypeptide fragment of about 2 kDa obtained by digestion with V8 protease was used. Table 1 shows the determined N-terminal and internal amino acid sequences.

B-2. PCR using TB60 total DNA as template
Primers for amplifying the uretanase gene were designed based on the N-terminal amino acid sequence determined in B-1 (Table 2).

  PCR was performed under the conditions shown in FIG. 3 using N1-2 and N3R as primers and TB60 total DNA as a template.

The DNA fragment of about 1 kbp obtained as a result of PCR was inserted into pGEM-TEasy (Promega), and E. coli DH10B was transformed. As a result, plasmid pN-TA having a 1 kbp insert was obtained.

Step C. Southern hybridization cloning (1)
C-1. Preparation and hybridization of DNA probe Plasmid pN-TA obtained in B-2 was treated with a restriction enzyme to obtain insert DNA. A probe was prepared using this DNA fragment (Probe 1). Gene Image Random-Prime Labeling Module (Amersham Bioscience) was used for probe preparation.

Southern hybridization was performed on the total DNA of the TB60 strain digested with PstI using the prepared probe. CDP-Star Detection Module (Amersham Bioscience) was used for detection. As a result, a signal was detected in a DNA fragment of about 4 kbp.
C-2. Cloning
Total DNA of the TB60 strain was digested with PstI, and a DNA fragment of about 4 kbp was extracted from an agarose gel. The extracted DNA was inserted into pUC19 digested with PstI, and E. coli DH10B was transformed. Using the resulting transformant as a template, colony direct PCR was performed to detect a plasmid having the target region. N1-2 / N3R was used as a primer at that time.

  As a result, a plasmid having a 4.2 kbp fragment, pURE9, was obtained. As a result of nucleotide sequence analysis, it was revealed that this fragment encodes the first half of the uretanase gene (FIG. 4). Therefore, Southern hybridization was performed again in order to clone the latter half of this gene.

Step D. Southern hybridization cloning (2)
D-1. DNA probe preparation and hybridization
A PstI-SalI region (about 0.2 kbp) of pURE9 was amplified by PCR to prepare a probe (Probe 2). The primers SalF / PstR shown in Table 2 were used for PCR. PCR conditions were according to B-2.

Southern hybridization was performed on the total DNA of the TB60 strain digested with BamHI using the prepared probe. The detection method conformed to C-1. As a result, a signal was detected in a DNA fragment of about 2.5 kbp.
D-2. Cloning
The total DNA of TB60 strain was digested with BamHI, and a DNA fragment of about 2.5 kbp was extracted from an agarose gel. The extracted DNA was inserted into pUC19 digested with BamHI, and E. coli DH10B was transformed. Using the resulting transformant as a template, colony direct PCR was performed to detect a plasmid having the target region. SalF / PstR shown in Table 2 was used as a primer at that time.

As a result, a plasmid pURE41 having a fragment of 2.5 kbp was obtained. As a result of nucleotide sequence analysis, it was confirmed that pURE41 has a BamHI (3) -PstI (2) region of pURE9 (FIG. 4).

Step E. Nucleotide sequence analysis
As a result of analyzing the nucleotide sequences of pURE9 and pURE41, one ORF was found in the SacI (2) -SalI (2) region. FIG. 5 shows the entire base sequence and deduced amino acid sequence of this ORF. This ORF consisted of 1419 bp nucleotides and encoded 472 amino acids (presumed molecular weight 50,699 Da). The amino acid sequence of 20 residues from the N-terminus (Met1-Ser20) completely matched the N-terminal amino acid sequence of purified uretanase. The amino acid sequence of Ara300-Met311 was identical to the internal amino acid sequence of the purified enzyme. From the above results, this ORF was shown to encode uretanase of A1 strain. Hereinafter, this ORF is called ureA.

Proteins having homology with this enzyme were searched from a database (DDBJ, GenBank). As a result, it showed homology with several bacterial amidases, but at most 34% of enantioselective amidase (Accession No. A19131) from Rhodococcus sp. No related protein was found.

Step F. Expression F-1. Construction of expression vector
ureA was amplified by PCR using pUR9 and pURE41 as templates. At that time, a primer (Ure-PET-Nde / Ure-PET-Nhe, Table 2) that can add an NdeI site at the 5 ′ end and an NheI site at the 3 ′ end was used. PCR conditions were as shown in FIG.

The amplified 1.4 kb fragment was extracted from an agarose gel and inserted into a pET25b (+) vector (Novagen) cut with NdeI / NheI. Thereby, an expression vector in which a His-Tag was added to the C-terminus of UreA was constructed. The nucleotide sequence of the DNA inserted into the vector was analyzed and confirmed to be identical to ureA. This was designated as pURE-pet.
F-2. Expression using E. coli as a host Expression was performed by the method shown in FIG. E. coli BL21 (DE3) having pURE-pet was inoculated into 50 ml of LB medium (containing 100 μg / ml Ap), and subjected to rotary shaking culture at 30 ° C. overnight. The whole amount was inoculated into 500 ml of LB medium (containing 100 μg / ml of Ap), and subjected to rotary shaking culture at 20 ° C. After 5 hours, 0.5 ml of 1 mM IPTG was added and induction was performed at 20 ° C. for 24 hours. The microbial cells after induction were sonicated and the supernatant obtained by centrifugation was used as a crude enzyme solution (Cell free extract).

F-3. Purification of expression enzyme The crude enzyme solution obtained with F-2 was fractionated by precipitation with 40-60% saturated ammonium sulfate, and then affinity chromatography using HiTrap Chelating HP column (capacity 1 ml, Amersham Bioscience). It was used for. Ni ²⁺ was chelated in advance, and then equilibrated with 20 mM K-phosphate buffer (pH 7.0) containing 0.5 M NaCl. The enzyme was eluted with an imidazole linear gradient of 60-200 mM / 60 min. After collecting the fraction having uretanase activity, desalting and concentration were performed by ultrafiltration. Glycerol was added to 20% and stored at 4 ° C. SDS-PAGE of the purified enzyme is shown in FIG. This enzyme showed a molecular weight equivalent to that of uretanase derived from the TB60 strain.

F-4. Confirmation of Urethane Decomposition Activity Using purified recombinant uretanase, the urethane bond decomposition activity in the urethane compound was confirmed. Toluene dicarbamic acid dibutyl ester (TDCB) and methylenebisphenyldicarbamic acid dibutyl ester (MDCB) were used as substrates. The reaction solution shown in Table 3 was placed in a small test tube and reacted at 30 ° C. for 24 hours. After the reaction, the remaining substrate was removed by centrifugation, and then the degradation product was extracted with the same amount of ethyl acetate as the reaction solution and subjected to GC-MS. The GC-MS conditions are as shown in FIG.

As a result of the decomposition reaction, when TDCB was used as a substrate, a peak of the decomposition product was detected at 3.8 minutes. As a result of mass spectral analysis, this compound was identified as toluenediamine. When MDCB was used as a substrate, a degradation product peak was detected at 8.0 minutes. As a result of mass spectral analysis, this compound was identified as diaminodiphenylmethane. From the above results, it was confirmed that the recombinant enzyme cleaved the urethane bond of TDCB and MDCB. The degradation product was the same as that of TB60 strain-derived uretanase.

Effects of the Invention As an enzyme capable of degrading solid polyurethane, several polyester-type polyurethane degrading enzymes have been reported. However, these enzymes are esterases, and ester bonds in polyurethane are decomposed, but urethane bonds are hardly decomposed, so that low molecular weight urethane compounds remain as final products. By allowing the uretanase of the present invention to coexist with these polyurethane degrading enzymes, complete degradation of the polyurethane becomes possible.

Moreover, since the gene for uretanase was obtained by the present invention and the expression system in Escherichia coli was established, it can be applied to enzymatic monomer recycling of polyurethane by imparting degradability by protein engineering modification.

FIG. 1 shows the cloning procedure of the uretanase gene derived from Rhodococcus equi strain TB60. FIG. 2 shows a method for extracting total DNA from cells of Rhodococcus equii TB60 strain. FIG. 3 shows the PCR conditions. FIG. 4 shows the results of nucleotide sequence analysis, and shows that the 4.2 kbp fragment in the obtained plasmid pURE9 encodes the first half of the target uretanase gene. FIG. 5 shows the entire nucleotide sequence of the uretanase gene ORF and the deduced amino acid sequence. FIG. 6 shows PCR conditions. FIG. 7 shows an expression procedure using E. coli as a host. FIG. 8 shows SDS-PAGE of the purified enzyme. The obtained enzyme showed a molecular weight equivalent to that of uretanase derived from the TB60 strain. FIG. 9 shows the GC-MS analysis conditions and results of analysis of degradation products in the urethane decomposing activity confirmation test.

Claims (14)

  A polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, or a complementary sequence thereof.   The polynucleotide of Claim 1 comprising the nucleotide sequence of SEQ ID NO: 2.   The polynucleotide of Claim 1 comprising a complementary sequence to the nucleotide sequence of SEQ ID NO: 2.   A polynucleotide comprising a polynucleotide that hybridizes to a complementary strand of the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a polypeptide having urethane compound-degrading activity, or a complementary sequence thereof.   A polynucleotide which is a sequence in which a part of the nucleotide sequence of SEQ ID NO: 2 has been deleted, substituted, inserted or added, and which comprises a polynucleotide encoding a polypeptide having urethane compound-degrading activity, or a complementary sequence thereof.   The polynucleotide according to claim 4 or 5, wherein the urethane compound is a low molecular weight compound.   The polynucleotide according to claim 4 or 5, wherein the urethane compound is polyurethane.   A recombinant vector comprising any of the polynucleotides of claims 1-7.   A transformed host cell comprising the recombinant vector of claim 8.   A method for preparing a substantially purified or isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1, wherein the host cell of claim 9 is capable of directing expression of the polypeptide or peptide fragment. Culturing under and recovering the polypeptide or peptide fragment from the cell culture in a substantially purified or isolated form.   A method for decomposing a urethane compound, comprising a step of contacting the host cell of claim 9 expressing a polypeptide having a urethane compound degrading activity with a urethane compound.   The degradation method according to claim 11, wherein the cell is Escherichia coli.   The decomposition method according to claim 11 or 12, wherein the urethane compound is a low molecular weight compound.   The decomposition method according to claim 11 or 12, wherein the urethane compound is polyurethane.

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