NOVEL URETHANASES FOR THE ENZYMATIC DEGRADATION OF POLYURETHANES

Abstract

The present invention relates to new urethanases for the enzymatic breakdown of polyurethanes and to an enzymatic process for the complete breakdown of polyurethanes into defined monomers.

Description

[0001] The present invention relates to new urethanases for the enzymatic breakdown of polyurethanes and to an enzymatic process for the complete breakdown of polyurethanes into defined monomers.

[0002] Polyurethanes are established in many areas of normal life. They can be found, for example, in soft foams (mattresses, sponges, upholstered furniture), hard foams (insulation materials, building materials), thermoplastics (sports shoes) or coatings (varnishes, paints, adhesives). The constantly increasing demand for products means that ever greater volumes are being produced. At the same time, there is a growing need for methods that maximize the sustainable recycling of polyurethane products that are no longer needed and so allow the building blocks of the polymers to be reused. For this, the bonds in the polyurethanes must be selectively cleaved in order to be able to obtain defined breakdown products, thereby making them recyclable.

[0003] In addition to the physiological functions that enzymes perform in living organisms, enzymes can be used in a diversity of ways for the catalysis of chemical reactions outside this context. Such reactions can be carried out under milder conditions than conventional chemical processes, for example lower temperature, neutral pH, and without the use of aggressive chemicals. Through this it is possible to save on energy, minimize the formation of by-products, and protect the environment, which helps to reduce operating costs. In some cases, it is only through the use of enzymes that it is possible for labile starting materials to be used as reaction feedstocks (Jaeger, K.-E. & Reetz, M. T. (1998) Microbial lipases form versatile tools for biotechnology. Trends in biotechnology, 16, 396-403). Moreover, enzymes are often regio-, stereo- and enantioselective, which makes the purification of the products substantially easier, which can permit the efficient synthesis of products that are otherwise difficult to obtain (Hasan, F., Shah, A. A. & Hameed, A. (2006) Industrial applications of microbial lipases. Enzyme and Microbial Technology, 39, 235-251).

[0004] The recycling of polyurethanes is primarily carried out through thermal recycling. This process generally takes place at very high temperatures and with very long reaction times in a batch process, as well as involving the use of catalysts. What can happen in such processes is that thermal breakdown of the polymer chains in cracking reactions leads to undesired and undefined breakdown products or else the formation of epoxy rings occurs, which results in a high odor nuisance and disadvantageous crosslinking of the chains in the recycled raw material, which can make it impossible to reuse said materials in products in particular with close human contact, particularly in the production of foams for use in furniture and mattresses. An alternative option is for complete combustion and thus energy recovery to be carried out, which generates energy, but does not allow efficient reuse of the polymer building blocks.

[0005] It is known that polyurethanes can be broken down to a certain degree by bacteria and fungi. Polyester polyurethanes are considerably more susceptible to such microbial/enzymatic breakdown than polyether polyurethanes (Nakajima-Kambe, T., Shigeno-Akutsu, Y., Nomura, N., Onuma, F. & Nakahara, T. (1999) Microbial degradation of polyurethane, polyester polyurethanes and polyether polyurethanes. Applied microbiology and biotechnology, 51, 134-140).

[0006] The breakdown of polyester polyurethanes can be readily accomplished by hydrolysis of the ester linkages. The relatively simple breakdown of polyesters is not surprising, given that ester linkages in hydrophobic substrates in nature must also be cleaved when lipids are broken down and polyesters without urethane linkages can likewise be broken down relatively easily by esterases and lipases (Marten, E., Muller, R.-J. & Deckwer, W.-D. (2003) Studies on the enzymatic hydrolysis of polyesters I. Low molecular mass model esters and aliphatic polyesters. Polymer degradation and stability, 80, 485-501; Marten, E., Muller, R.-J. & Deckwer, W.-D. (2005) Studies on the enzymatic hydrolysis of polyesters. II. Aliphatic-aromatic copolyesters. Polymer degradation and stability, 88, 371-381). Enzymes used to break down polyurethane have been characterized as esterases in various literature sources (Allen, A. B., Hilliard, N. P. & Howard, G. T. (1999) Purification and characterization of a soluble polyurethane degrading enzyme from Comamonas acidovorans. International biodeterioration & biodegradation, 43, 37-41; Blake, R., Norton, W. & Howard, G. (1998) Adherence and growth of a Bacillus species on an insoluble polyester polyurethane. International biodeterioration & biodegradation, 42, 63-73; Crabbe, J. R., Campbell, J. R., Thompson, L., Walz, S. L. & Schultz, W. W. (1994) Biodegradation of a colloidal ester-based polyurethane by soil fungi. International biodeterioration & biodegradation, 33, 103-113; Darby, R. T. & Kaplan, A. M. (1968) Fungal susceptibility of polyurethanes. Applied microbiology, 16, 900-905; Howard, G. T., Norton, W. N. & Burks, T. (2012) Growth of Acinetobacter gerneri P7 on polyurethane and the purification and characterization of a polyurethanase enzyme. Biodegradation, 23, 561-573; Kaplan, A. M., Darby, R. T., Greenberger, M. & Rodgers, M. (1968) Microbial deterioration of polyurethane systems. Dev Ind Microbiol, 82, 362-371; Kay, M., Morton, L. & Prince, E. (1991) Bacterial degradation of polyester polyurethane. International biodeterioration, 27, 205-222; Vega, R. E., Main, T. & Howard, G. T. (1999) Cloning and expression in Escherichia coli of a polyurethane-degrading enzyme from Pseudomonas fluorescens. International biodeterioration & biodegradation, 43, 49-55). There is no clear demonstration therein of cleavage of the urethane linkage, since there were no instances of enzyme characterization being carried out on the basis of cleavage of a molecule having a urethane group.

[0007] The breakdown of poly(ester urethane)s with fungi or bacteria is described in many publications and patents. However, the breakdown mostly targets only the relatively easily cleaved ester linkages and is mostly demonstrated only by macroscopic observation of polymer breakdown. There is no controlled breakdown here of ester and urethane linkages as in the present invention, and long breakdown times often result. These publications show that urethanases are commonly found enzymes, but provide no demonstration of the specific capabilities, potential uses, and grouping thereof, as employed in the present invention. (JP09192633, Tang, Y. W., Labow, R. S., Santerre, J. P. (2003) Enzyme induced biodegradation of polycarbonate-polyurethanes: dose dependence effect of cholesterol esterase. Biomaterials 24 (12), 2003-2011, Vega, R. E., Main, T. & Howard, G. T. (1999) Cloning and expression in Escherichia coli of a polyurethane-degrading enzyme from Pseudomonas fluorescens. International biodeterioration & biodegradation, 43, 49-55)

[0008] A breakdown process for the enzymatic breakdown of poly(ester urethane)s is known, the first step of which is to obtain an esterase from a culture of Comamonas acidovorans strains by using only poly(ester urethane) as the carbon source. In a complicated purification step, the esterase is separated and used for the breakdown of poly(ester urethane)s in a batch process. This gives rise to long breakdown times in a multistage process and no demonstration of specific cleavage of the urethane linkages (JP 09201192, JP 10271994).

[0009] The breakdown of poly(ester urethane)s with cutinases, esterases, and/or lipases is described in various patents and publications. However, the breakdown here targets only the relatively simple cleavage of the ester linkages, but not specifically the urethane linkages. In addition, no specific combination of enzymes that cleave ester and urethane linkages is described for the selective control of the breakdown. It can be assumed that the described processes result in little or no cleavage of the urethane linkage. This means that diamines used cannot be recovered efficiently (EP 0968300, U.S. Pat. No. 6,180,381).

[0010] WO 2013/134801 describes the breakdown of aromatic polyurethanes based on polyether polyols using an enzyme of class EC 3. No specific enzyme sequences are stated, consequently neither the specificity of the process in the breakdown of particular urethane linkages, nor the controlled cleavage of ester linkages and separate cleavage of urethane linkages, as shown in the present invention, are demonstrated in the cited patent. Moreover, there is no description of the regulation of the pH of the mixture during polymer breakdown in order to maintain urethanase activity. Moreover, no regioselective breakdown is described, nor breakdown of aliphatic poly(ester urethane)s.

[0011] WO 2006/019095 describes a urethanase and variants of this enzyme obtained by protein engineering. The enzyme can cleave urethane oligomers based on TDA or MDA. However, bonds are not cleaved regioselectively here, neither is there any application in combination with esterases for the breakdown of polymers. Furthermore, no other urethanases from the GatA or Aes families or any other group are described.

[0012] It was thus an object of the present invention to provide further enzymes that can be used for the enzymatic cleavage of urethane linkages and preferably for the complete enzymatic breakdown of polyurethanes. Furthermore, an enzymatic process should be provided that allows the breakdown of polyurethanes into defined monomers.

[0013] This object is achieved by the embodiments disclosed in the claims and in the description below.

[0014] In a first embodiment, the present invention relates to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 and variants of said polypeptides or to a polypeptide having an amino acid sequence in accordance with SEQ ID No. 7 or a variant thereof, characterized in that the polypeptide has urethanase activity.

Reference for the Polypeptides Mentioned

TABLE-US-00001

[0015] SEQ ID No. Internal designation Designation in study 1 Enz01 GatA61 2 Enz02 Aes70 3 Enz03 Aes72 4 Enz04 Aes170 5 Enz05 Aes174 6 Enz06 Aes175 7 Enz07 GatA197 8 Enz08 Aes214 9 Enz09 GatA250 10 Enz10 AesGo56 11 Ref01 SB12 12 Ref02 SB23

Polypeptide

[0016] The term "polypeptide" is well known to those skilled in the art. It refers to a chain of at least 50, preferably at least 70, amino acids linked to one another by peptide linkages. A polypeptide may comprise both naturally occurring and synthetic amino acids. It preferably comprises the known proteinogenic amino acids.

[0017] For SEQ ID Nos. 1 to 5, 9, and 10, a variant is obtained by adding, deleting or exchanging up to 10%, preferably up to 5%, of the amino acids present in the respective polypeptide. A preferred variant of SEQ ID No. 7 is obtained by adding, deleting or exchanging up to 5% of the amino acids defined in SEQ ID No. 7. Particularly preferred variants of the abovementioned polypeptides are obtained by adding, deleting or exchanging up to 20, preferably up to 10, and more preferably up to 5, amino acids of the disclosed sequences. Preferred variants of SEQ ID No. 6 and SEQ ID No. 8 are obtained by adding, deleting or exchanging up to 3, more preferably up to 2, amino acids. The abovementioned modifications may in principle be executed continuously or discontinuously at any desired point in the polypeptide. However, they are preferably executed only at the N-terminus and/or at the C-terminus of the polypeptide. Each variant obtained by adding, exchanging or deleting amino acids according to the invention is, however, characterized by urethanase activity as defined in this application hereinbelow.

[0018] The polypeptides as defined by SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, and SEQ ID No. 10. form a group that is phylogenetically different from the sole enzyme having urethanase activity that is known to date, Ure (see FIG. 1). No enzymes having corresponding activity were previously known from this group. This group of polypeptides is also referred to herein below as "Aes-like".

Urethanase Activity

[0019] The term "urethanase activity" refers to the ability of a polypeptide to enzymatically catalyze the cleavage of a urethane group. In this process, each mole of urethane group gives rise to one mole of amine, one mole of alcohol, and one mole of CO.sub.2.

[0020] The urethane group may be an aromatically or aliphatically attached urethane group. In the case of an aromatically attached urethane group, the nitrogen atom is attached directly to an aromatic ring. In the case of an aliphatically attached urethane group, the nitrogen atom is attached to an alkyl radical. The alkyl radical is preferably unbranched and composed of at least one, more preferably at least two, and most preferably at least three, carbon atoms. In a preferred embodiment of the present invention, the polypeptide having urethanase activity is capable of enzymatically cleaving an aromatically attached urethane group.

[0021] Whether a polypeptide has urethanase activity can be checked through the cleavage of suitable model substrates.

[0022] The model substrate for the ability to cleave aromatically attached urethane groups is preferably ethyl 4-nitrophenyl carbamate (ENPC). Cleavage is demonstrated by determining the increase in the concentration of 4-nitroaniline. This is done preferably photometrically at a wavelength of 405 nm. The enzyme activity is determined preferably in a reaction buffer containing 100 mM of K.sub.2HPO.sub.4/KH.sub.2PO.sub.4, pH 7 with 6.25% by volume of ethanol in the presence of 0.2 mg/L of ENPC as substrate. Incubation of the enzyme with ENPC in the reaction buffer is carried out preferably at room temperature and preferably for 24 hours.

##STR00001##

[0023] The model substrate for the ability to cleave aliphatically attached urethane groups is preferably ethyl phenethyl carbamate (EPEC). Cleavage is demonstrated by determining the increase in the concentration of phenethylamine. This is done preferably by HPLC. The reaction buffer used and the reaction conditions preferably correspond to the parameters described above for ENPC.

##STR00002##

Enzymatic Cleavage

[0024] The term "enzymatic cleavage of a urethane group" indicates that the cleavage of a urethane group described above proceeds more rapidly in the presence of a polypeptide having urethanase activity than it does when incubated with the reaction buffer without enzyme under the same reaction conditions or when incubated with the reaction buffer under the same conditions in the presence of an inactive polypeptide. The preferred model for an inactive polypeptide is bovine serum albumin. If, in the presence of a polypeptide being tested, the cleavage of the urethane group proceeds more rapidly than in an otherwise identical control with BSA, said polypeptide possesses urethanase activity as understood in this application.

Use

[0025] In a further embodiment, the present invention relates to the use of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 and variants of said polypeptides or to a GatA-similar polypeptide having an amino acid sequence in accordance with SEQ ID No. 7 or a variant thereof, characterized in that the polypeptide has urethanase activity in the enzymatic cleavage of urethane linkages.

[0026] Unless explicitly defined otherwise, all definitions given above apply to this embodiment too.

Breakdown of Urethanes into Low-Molecular-Weight Breakdown Products

[0027] In a further embodiment, the present invention relates to a process for breaking down polyester polyurethanes into low-molecular-weight breakdown products, comprising the steps of

[0028] a) cleaving the ester groups present in the polyester polyurethane; and

[0029] b) cleaving the urethane groups present in the polyester polyurethane with a polypeptide that has urethanase activity;

[0030] with the proviso that process steps a) and b) may be carried out in either order or else in parallel.

[0031] Particularly suitable as peptides having urethanase activity are the peptides described in this application having amino acid sequences as defined in the group consisting of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and amino acid sequences having at least 90% sequence identity with the abovementioned sequences. Very particular preference is given to peptides having amino acid sequences as defined in SEQ ID No. 3 or 7 and amino acid sequences having at least 90% sequence identity with the abovementioned sequences.

[0032] Consequently, in a particularly preferred embodiment, the present invention relates to a process for breaking down polyester polyurethanes into low-molecular-weight breakdown products, comprising the steps of

[0033] a) cleaving the ester groups present in the polyester polyurethane; and

[0034] b) treating the polyurethane with a polypeptide that has urethanase activity and has an amino acid sequence selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 10 and amino acid sequences having at least 90% sequence identity with the abovementioned sequences; with the proviso that process steps a) and b) may be carried out in either order or else in parallel.

[0035] Preference is given to carrying out process step a) before process step b).

[0036] Process step a) is preferably carried out with a lipase. This lipase is preferably water-soluble and not present in an immobilized form. "Immobilized" here refers to the attachment of peptides that is generally known in biotechnology, particularly the attachment of antibodies or enzymes, to the surface of vessels or to water-insoluble particles.

[0037] Particular preference is given to using a lipase capable of cleaving tributyrin. Even more particular preference is given to using a polypeptide that has an amino acid sequence as defined in SEQ ID No. 11 or SEQ ID No. 12 or that has an amino acid sequence having at least 90%, preferably at least 95%, sequence identity with one of the two abovementioned sequences and which is capable of cleaving tributyrin. Process step a) is preferably carried out under reaction conditions in which the employed lipase shows activity. Such conditions can be determined by routine experiments using common biochemical methods.

[0038] Since the polypeptides having urethanase activity according to the invention have their maximum activity in the neutral range, process step b) is preferably carried out at a pH between 6.0 and 10.0, preferably between 6.0 and 8.0. The pH may be adjusted using all suitable bases known to those skilled in the art.

[0039] The term "polyester polyurethane" refers to a polyurethane formed from one or more polyester polyols and one or more isocyanates. The polyurethane may be foamed or non-foamed. It is preferably foamed. To increase the specific surface area, it is preferable to comminute the polyurethane before carrying out process steps a) and b). This is particularly preferable when polyurethane is to be used in non-foamed form. Comminution may be done in any way familiar to those skilled in the art, preferably by milling, slicing, tearing or cutting.

[0040] The polyurethane comprises as the isocyanate component at least one aromatic, aliphatic or cycloaliphatic isocyanate. The polyurethane preferably comprises only aromatic isocyanates. Preferred aromatic isocyanates are methylene diphenyl isocyanate (MDI), MDI variants having three or more aromatic rings, naphthylene diisocyanate, and tolylene diisocyanate. Particularly preferred aromatic isocyanates are methylene diphenyl isocyanate (MDI), MDI variants having three or more aromatic rings, and tolylene diisocyanate. MDI variants having three or more aromatic rings are synthesis by-products and may also be present in polyurethanes. The polyurethane to be broken down particularly preferably comprises tolylene 2,4-diisocyanate and tolylene 2,6-diisocyanate.

[0041] The term "polyester polyol" is known to those skilled in the art and describes polyesters containing an average of at least 1.5, preferably at least 1.8, and more preferably at least 2.0, hydroxyl groups per molecule. The polyester polyols present in the polyurethane to be broken down particularly preferably have functionality of between 1.5 and 6.0. They contain as structural elements aromatic and/or aliphatic polyols and also aromatic and/or aliphatic polycarboxylic acids in any combination.

[0042] The low-molecular-weight breakdown products of the polyester-based polyurethane foams preferably have a molecular weight of not more than 1000 g/mol. These are preferably

[0043] (i) amines derived from the isocyanates used in the production of the polyurethane concerned, for example tolylene-2,4-diamine in the case of tolylene 2,4-diisocyanate; and

[0044] (ii) alcohols and carboxylic acids used to form the polyester polyols employed in the synthesis of the polyurethane concerned.

[0045] A "polyol" is in this application understood as meaning any compound having at least two hydroxyl groups. Said polyol preferably has a molecular weight of not more than 300 g/mol. Preferred polyols that are low-molecular-weight breakdown products of polyester-based polyurethane foams are selected from the group consisting of ethylene glycol, diethylene glycol, 1,4-butanediol, triethylene glycol, propylene glycol, 1,2-dipropylene glycol, neopentyl glycol, glycerol, 1,1,1-trimethylolpropane, sucrose, sorbitol, and pentaerythritol.

[0046] A "polycarboxylic acid" is in this application understood as meaning any compound containing at least two carboxyl groups. Said polycarboxylic acid preferably has a molecular weight of not more than 300 g/mol. Preferred polycarboxylic acids that are low-molecular-weight breakdown products of polyester-based polyurethane foams are selected from the group consisting of succinic acid, glutaric acid, adipic acid, phthalic acid, terephthalic acid, benzenetricarboxylic acid, oleic acid, and ricinoleic acid. Particularly preferred polycarboxylic acids that are low-molecular-weight breakdown products of polyester-based polyurethane foams are selected from the group consisting of succinic acid, glutaric acid, adipic acid, phthalic acid, terephthalic acid, and benzenetricarboxylic acid.

[0047] A "polyamine" is in this application understood as meaning any compound containing at least two amino groups. Said polyamine preferably has a molecular weight of not more than 300 g/mol. Preferred polyamines that are low-molecular-weight breakdown products of the polyester-based polyurethane foams are selected from the group consisting of methylene-4,4'-diamine, methylene-2,4'-diamine, methylene-2,2'-diamine, tolylene-2,4-diamine, tolylene-2,6-diamine, hexamethylenediamine, isophorone diamine, xylylenediamine, pentamethylenediamine, para-phenylenediamine, butyldiamine, and H12-methylenediamine. Further preference is given to polyamines selected from the group consisting of methylene-4,4'-diamine, methylene-2,4'-diamine, methylene-2,2'-diamine, naphthylene-1,4-diamine, naphthylene-1,5-diamine, naphthylene-1,6-diamine, tolylene-2,4-diamine, and tolylene-2,6-diamine. Particular preference is given to polyamines selected from the group consisting of methylene-4,4'-diamine, methylene-2,4'-diamine, methylene-2,2'-diamine, tolylene-2,4-diamine, and tolylene-2,6-diamine.

[0048] The process according to the invention allows effective recycling of polyurethanes in two ways: (i) The process itself operates under mild reaction conditions and so does not require a high input of energy and (ii) it allows the polyurethane to be recycled, because defined breakdown products are formed that are themselves valuable chemical raw materials.

[0049] By comparison, thermal glycolysis, which is currently the most common chemolysis for recycling polyurethane and has already been put into practice industrially, is carried out at very high temperatures. The focus here is on extraction of the polyols, whereas the amines are separated as an interfering species and are not recovered. In non-enzymatic hydrolysis, both polyols and amines are obtained as products. However, this process is carried out at high temperatures and high ambient pressures.

OVERVIEW OF THE FIGURES

[0050] FIG. 1: Result of the phylogenetic analysis of the amino acid sequences disclosed in the present application

[0051] The working examples that follow serve merely to elucidate the invention. They are not intended to limit the scope of the claims in any way.

EXAMPLES

[0052] Test of Enzyme Activity with ENPC

[0053] 0.2 mg/ml of ENPC was incubated for 24 hours in 100 mM KH.sub.2PO.sub.4/K.sub.2HPO.sub.4 at pH 7.0 containing 6.25% by volume of ethanol at room temperature and 900 rpm on the "MTS 2/4" plate shaker (IKA, Staufen).

[0054] After filtering the samples, 100 .mu.L of each was transferred to transparent flat-bottom 96-well "UV-Star" plates (Greiner Bio-One, Frickenhausen) and the absorbance at 405 and 480 nm determined. The value at 480 nm was measured, because 4-nitroaniline does not show any significant absorption and, if high values are observed at both wavelengths, it is highly likely that is not 4-nitroaniline but another substance that was responsible for the absorbance at 405 nm.

[0055] Hydrolysis by urethanases causes cleavage of the almost colorless ENPC into 4-nitroaniline, CO.sub.2, and ethanol, resulting in the detection of 4-nitroaniline at 405 nm in the "Infinite M1000PRO" microtiter plate photometer (Tecan, Mannedorf, Switzerland). The photometer was controlled using the "i-control" software (Tecan, Mannedorf, Switzerland), version 3.4.2.0. 4-Nitroaniline was additionally detected by HPLC using the "dabsylamine" method.

High Pressure Liquid Chromatography (HPLC)

[0056] High-pressure liquid chromatography was carried out on an Agilent Technologies (Santa Clara, USA) 1100 series instrument equipped with an autosampler and DAD (diode array detector) for UV and the visible light region. All measurements were carried out using a "Zorbax XDB-C18" column having a particle size of 3.5 .mu.m and dimensions of 4.6.times.75 mm (Agilent Technologies, Santa Clara, USA). In all methods, a 5 .mu.L sample was injected into a column heated to 40.degree. C. The flow was generally 1.5 ml/min. The use of a reverse-phase column means that elution in all methods is with increasing concentrations of organic solvent.

[0057] Detection and quantification of dabsylated aliphatic amines and urethanes was done using the "dabsylamine" method. This method allows the quantification of aromatic amines and urethanes without derivatization on account of their high intrinsic absorption. Also used as eluent in addition to AcN was 10 mM sodium phosphate buffer pH 7.0, to which 0.005% (w/v) sodium azide was added to protect against microbial growth. In order to prevent pressure problems caused by contaminated pump valves, 5% (v/v) of dd H.sub.2O was later added to the AcN and the method adjusted ("Dabsylamin95"). The MDEC formed from the enzyme-catalyzed reactions of 4,4'-MDA with EC was quantified using the "Dabsylamin-12-MeOH" method, in which the aqueous component is acidified and the protonated aromatic amines thereby generated elute very early. The reactions of 4,4'-MDA with DMC, 2,4-TDA with DMC, and 2,4-TDA with EC were investigated using the "Dabsylamin95-H2O" method, which differs from "Dabsylamin95" only in that pure dd H.sub.2O is used instead of buffer. The data were analyzed using the "OpenLAB CDS ChemStationLC" software, version A.02.09 [017] (Agilent Technologies, Santa Clara, USA).

Dabsylamine: Eluent: AcN and 10 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4, pH 7.0

TABLE-US-00002 t [min] AcN 0 5 6.5 85 8.0 5 10.0 5

Dabsylamin95: Eluent: AcN Containing 5% (v/v) dd H.sub.2O and 10 mM Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4, pH 7.0

TABLE-US-00003 t [min] % AcN (+5% (v/v) dd H.sub.2O) 0 5 6.5 90 8.0 5 10.0 5

Dabsylannin-12-MeOH-Lang: Eluent: Methanol and dd H.sub.2O Containing 0.1% (v/v) Methanoic Acid

TABLE-US-00004 t [min] % methanol 0 5 2.5 35 8.0 70 8.5 85 10.0 5 12.0 5

TABLE-US-00005 SEQ ID No. Designation in study Hydrolysis of ENPC 1 GatA61 + 2 Aes70 + 3 Aes72 + 4 Aes170 + 5 Aes174 + 6 Aes175 + 7 GatA197 + 8 Aes214 + 9 GatA250 + 10 AesGo56 + 11 SB12 + 12 SB23 +

Test of Enzyme Activity with EPEC

[0058] The test was carried out as described for ENPC. The phenethylamine formed was detected by HPLC as described above.

TABLE-US-00006 SEQ ID No. Designation in study Hydrolysis of EPEC 1 GatA61 + 2 Aes70 - 3 Aes72 + 4 Aes170 - 5 Aes174 + 6 Aes175 - 7 GatA197 + 8 Aes214 + 9 GatA250 + 10 AesGo56 - 11 SB12 + 12 SB23 +

Phylogenetic Analysis of the Enzymes

[0059] Phylogenetic trees showing the relatedness of the urethanases were created using the "MegAlign" software (DNASTAR, Madison, USA), version 10.1.0. The phylogenetic trees were created with the default settings using "ClustalW".

[0060] Alignments of the different proteins were created using the "Clustal Omega" software (Sievers et al., 2011).

[0061] Database searches for protein sequences were carried out using BLASTP (Altschul et al., 1990).

[0062] Open reading frames (ORFs) in sequenced metagenome sequences were located using the online application "ORF Finder" from the NCBI (Wheeler et al., 2007).

[0063] Identical hydrolase genes were reduced to a single representative and all sequences examined with ORFs in order to obtain the complete sequences of the genes. Alternative start codons were also allowed in the search. It was evident here that the gene from pLip214 included an N-terminal region with similarity to aes, but without a start codon having been identified. This gene segment was not located on the edge of the insert of the metagenome vector, which could explain a truncated gene. For further analyses, the region with similarity to aes but without a start codon was used as the sequence for this gene. The identified putative urethanase genes were translated in silico and compared with the NCBI database using BLASTP. The putative urethanases were named on the basis of their number in the lipase bank and the similarity to GatA or Aes.

[0064] In order to compare the individual members of the two identified urethanase groups (GatA and Aes), an alignment was in each case created with the "Clustal Omega" software and a phylogenetic tree additionally created with the "MegAlign" software, with a common alignment of the two groups created for the phylogenetic tree. The sequence comparison also included the sequences for the enzymes from the literature (Ure, Ana, and NfpolyA), which all showed similarity with GatA.

[0065] The phylogenetic tree is shown in FIG. 1. This shows that the two groups are located in different branches, the similarities within the two groups being not so clear in some instances, as can be seen from the lower bootstrapping values at the nodes. Within the GatA group there seem to be greater differences than within the Aes group, as can be seen from the longer branch lengths in this group. In particular Aes70 and Aes72 and also Aes175 and Aes214 show very high similarity, as manifested both by the relatively short branches in the phylogenetic tree and by the same protein with greatest similarity having been found in the BLASTP search.

Production of the Polyurethane Foam for the Breakdown Tests

[0066] The starting materials listed below were reacted in the manner of processing customary for the production of polyurethane foams in the one-step process.

[0067] The bulk density was 38 kg/m.sup.3 (DIN EN ISO 845 in the version of October 2009), the compressive strength at 40% compression was 3.5 kPa (DIN EN ISO 3386-1 in the version of October 2015)

Formulation:

TABLE-US-00007

[0068] 100 parts Desmophen 2200B 3 parts water 19 parts Desmodur T80 19 parts Desmodur T65 0.7 parts N,N'-dimethylpiperazine 1 part Tegostab 8325

Raw Materials:

[0069] Desmophen.RTM. 2200B, Covestro Deutschland AG; branched polyester polyol based on adipic acid, diethylene glycol and 1,1,1-trimethylolpropane having a hydroxyl value of approx. 60 mg KOH/g.

[0070] Desmodur.RTM. T80, Covestro Deutschland AG; isomer mixture comprising tolylene 2,4- and 2-6-diisocyanate in a mixture ratio of approx. 80:20.

[0071] Desmodur.RTM. T65, Covestro Deutschland AG; isomer mixture comprising tolylene 2,4- and 2-6-diisocyanate in a mixture ratio of approx. 67:33.

[0072] N,N'-Dimethylpiperazine, catalyst from abcr GmbH

[0073] Tegostab.RTM.B 8325, foam stabilizer, from Evonik

[0074] Water; deionized water

[0075] The formulation may be executed with indices of 90 to 115. The index is defined as the molar ratio of isocyanate groups to isocyanate-reactive groups multiplied by 100.

Breakdown of Polyurethane Foam

[0076] The substrate used was a polyester polyurethane produced with tolylene diisocyanate. Breakdown took place in two reaction steps. First, the foam was incubated with a lipase. The resulting oligomers were neutralized and then cleaved into monomers with a urethanase.

[0077] In the first step, 1 g of the foam was transferred to a 50 ml centrifuge tube with 20 ml of potassium phosphate buffer pH 7.0 and approx. 30 mg of CalB lyophilizate ("Chirazyme L2" from Roche, Basel, Switzerland) (here referred to as SEQ ID No. 12) and incubated at 37.degree. C. and 200 rpm for 5 days. Fragments of the foam residues were photographed with a "MH2" microscope (Olympus, Hamburg) by comparison with a negative control without enzyme. The turbid solution was then centrifuged for 10 minutes at 25.degree. C. and 4000 rpm in a large-capacity centrifuge. The clear supernatant was adjusted to pH 7.0 with 1 M NaOH. After about 6 hours at room temperature, the slight fall in pH was retitrated to 7.0 and the solution underwent a sterilizing filtration. The soluble oligomers were stored at 4.degree. C. until use.

[0078] For further use, the soluble oligomers were transferred to 1.5 mL reaction vessels and mixed with 20 .mu.L of DMF and 150 .mu.L of the optimal buffer for the respective urethanase (100 mM sodium phosphate buffer, adjusted to the respective optimal pH for the urethanase in the pH 6.0 to pH 8.0 range). To each was then added 30 .mu.L of the undiluted, purified urethanase and the mixtures were shaken on the heating block at 30.degree. C. and 1000 rpm. A mixture containing enzyme storage buffer was used as the negative control. After three days, the batches were filtered through filter plates with a PVDF membrane and a pore size of 0.2 .mu.m (Corning, Kaiserslautern) and the filtrate was analyzed by HPLC using the "Dabsylamine95" method in respect of the tolylene 2,4- and 2-6-diisocyanate formed.

[0079] After the reaction in the mixture containing the CalB lyophilizate, it was already macroscopically evident by comparison with a negative control without enzyme that the foam had lost all structure and was present as a turbid suspension containing small particles of foam. The buffer, which had been almost completely absorbed by the foam at the start of the experiment, subsequently contained the entire foam mass in the form of broken-down particles. HPLC analysis showed clear peaks that were assigned to the oligomers formed, but no peaks pointing to the formation of tolylenediamine (TDA) (data not shown).

[0080] The oligomer solution was treated with all of the expressed urethanases and with SEQ ID No. 12 and then examined by HPLC for the formation of TDA. This was demonstrated for the mixtures containing SEQ ID No. 7 and SEQ ID No. 3, with the measured amount of 2,6-TDA being approximately the same in the two mixtures and the formation of 2,4-TDA in the mixture containing Enz03 found to be markedly more pronounced. SEQ ID No. 3 afforded 0.057 g/L of 2,4-TDA and 0.025 g/L of 2,6-TDA, whereas SEQ ID No. 7 resulted in the formation of 0.0075 g/L of 2,4-TDA and 0.024 g/L of 2,6-TDA. In addition, in contrast to the other mixtures, the oligomer peaks for these two enzymes showed changes and a general reduction in size. In the case of SEQ ID No. 7, TDA was cleaved from the polyester PU foam even without prior pretreatment, whereas in the case of SEQ ID No. 3 this was possible only by providing neutralized oligomers after prior ester cleavage. The fact that the product peaks identified as oligomer peaks from the hydrolysis with SEQ ID No. 12 were dramatically smaller after further treatment with urethanases, this being accompanied by significant TDA formation, confirmed that these were oligomer peaks.

[0081] It was also demonstrated that insoluble TDI-based polyester-polyurethane foam can be cleaved into its monomers by a combination of two reaction steps. In a first step, the PU foam was predigested using the lipase CalB through hydrolysis of the ester linkages. After neutralization, the liberated oligomers served as a substrate for the overexpressed urethanases. This was accompanied by hydrolysis of the urethane linkages and the detection of TDA in monomeric form.

[0082] In conclusion, it can be seen that a combination of hydrolytic cleavage of the ester linkages by means of lipases, neutralization of the oligomer solution, and subsequent hydrolytic cleavage of the urethane linkages permits the complete breakdown of polyurethanes into defined monomers.

Sequence CWU 1

1

121469PRTUnknownDerived from metagenome 1Met Met Gly Gly Val Gly Val Arg Glu Glu Leu Ala Thr Trp Thr Ala1 5 10 15Val Arg Leu Ala Glu His Ile Arg Lys Lys Glu Leu Ser Pro Val Glu 20 25 30Val Thr Asp Tyr Phe Leu Arg Arg Ile Glu Ala Leu Asn Pro Ala Val 35 40 45Asn Ala Phe Cys Thr Val Asp Ala Asp Gly Ala Met Arg Ala Ala Lys 50 55 60Ala Ala Glu Gln Arg Leu Met Ala Gly Glu Thr Pro Pro Leu Leu Gly65 70 75 80Val Pro Val Ala Ile Lys Asp Leu Thr Pro Thr Lys Gly Ile Arg Thr 85 90 95Thr Tyr Gly Ser Arg Leu Phe Ala Asp Asn Val Pro Glu Ala Asp Ala 100 105 110Val Leu Val Thr Arg Leu Lys Gln Ala Gly Ala Ile Ile Val Gly Lys 115 120 125Thr Asn Thr Pro Glu Phe Gly His Ala Gly Val Thr Asp Asn Arg Leu 130 135 140Phe Gly Arg Thr Asn Asn Pro Trp Asp Leu Ser Arg Ile Ala Gly Gly145 150 155 160Ser Ser Gly Gly Ser Asp Gly Gly Gly Ser Ile Arg Ile Pro Ala Ser 165 170 175Cys Cys Gly Ile Phe Gly Phe Lys Pro Thr Phe Gly Arg Val Pro His 180 185 190Asp Thr Gly Ala Thr Ala Phe Ser Ile Thr Ala Pro Phe Leu His His 195 200 205Gly Pro Met Ser Arg Thr Val Glu Asp Ser Val Leu Met Leu Ala Ala 210 215 220Met Gln Gly Pro Asp Gly Cys Asp Pro Phe Ser Leu Pro Leu Pro Gly225 230 235 240Ile Asp Trp Pro Leu Ser Ala Glu Ile Lys Pro Phe Ser Gln Trp Arg 245 250 255Ile Ala Tyr Ser Pro Asn Leu Asp Phe Tyr Ala Ile Asp Pro Ala Val 260 265 270Arg Gln Val Met Glu Gln Ala Val Ser Ala Leu Gln Gly Leu Gly Cys 275 280 285Arg Val Glu Glu Val Arg Leu Gly Leu Glu Glu Gly Lys Thr Leu Val 290 295 300Leu Glu Thr Phe Ala Arg Leu Trp Ala Val His Tyr Ala Ala Phe Tyr305 310 315 320Glu Glu Leu Leu Glu Arg Glu Ala Glu Leu Ser Lys Gly Phe Val Ala 325 330 335Thr Ile Arg Tyr Gly Gln Gln Phe Ser Ala Val Glu Tyr Lys Arg Leu 340 345 350Glu Arg Pro Arg Ala Val Val Tyr Glu Arg Val Glu Asn Val Phe Ala 355 360 365Lys Tyr Asp Leu Leu Ile Thr Pro Thr Leu Ala Val Pro Pro Phe Ala 370 375 380His Asp Cys Pro Pro Arg Glu Ile Asp Gly Lys Ala Val Asn Pro Tyr385 390 395 400Asn Glu Trp Met Leu Thr Ser Ile Phe Asn Leu Thr Gly His Pro Val 405 410 415Ala Ser Ile Pro Ala Gly Phe Ser Pro Glu Gly Leu Pro Ile Gly Met 420 425 430Gln Ile Val Gly Pro Arg Leu Ala Asp Ala Ala Val Leu Glu Phe Ala 435 440 445Tyr Leu Phe Glu Gln Thr Val Ala Pro Arg Arg Pro Tyr Pro Cys Asp 450 455 460Asp Val Arg Leu Asn4652264PRTUnknownDerived from metagenomemisc_feature(252)..(252)Xaa can be any naturally occurring amino acid 2Leu Asp Tyr Leu Gly Gly Phe Ser Pro Leu Glu Ser Asp Val Thr Val1 5 10 15Glu Lys Thr Arg Ile Ala Gly Val Pro Gly Glu Trp Ile Ser Thr Pro 20 25 30Asp Ala Arg Lys Asp Arg Val Leu Phe Tyr Leu His Gly Gly Ala Tyr 35 40 45Cys Phe Gly Ser Cys Asp Ser His Arg Gly Leu Val Ser Arg Leu Ala 50 55 60Arg Ala Cys Gly Ser Arg Ala Leu Leu Ile Glu Tyr Arg Leu Ala Pro65 70 75 80Glu His Pro Phe Pro Ala Ala Leu Glu Asp Ser Thr Ala Ala Tyr Arg 85 90 95Glu Leu Ile Arg Ser Gly Val Arg Pro Glu Asn Leu Val Ile Ala Gly 100 105 110Asp Ser Ala Gly Gly Gly Leu Thr Met Ala Thr Leu Leu Thr Leu Arg 115 120 125Asp Glu Gly Asp Pro Leu Pro Ser Ala Ala Val Leu Leu Ser Pro Trp 130 135 140Thr Asp Leu Glu Gly Thr Gly Glu Ser Met Lys Thr Lys Ala Asp Val145 150 155 160Glu Pro Trp Leu Asp Pro Glu Lys Ser His Leu Leu Ala Lys Leu Tyr 165 170 175Leu Gly Asp Leu Asp Pro Arg His Pro Leu Val Ser Pro Ile His Ala 180 185 190Asp Leu Asn Asn Leu Pro Pro Leu Leu Val His Val Gly Ser Asp Glu 195 200 205Cys Leu Leu Asp Asp Ser Val Arg Leu Val Glu Arg Ala Lys Ser Ala 210 215 220Gly Val Glu Thr Glu Phe Lys Ile Cys Asp Glu Met Trp His Val Phe225 230 235 240His Gly Phe Pro Ile Pro Glu Ala Gln Gln Ala Xaa Glu Glu Ile Gly 245 250 255Ala Phe Val Arg Ala Arg Leu Pro 2603297PRTUnknownDerived from metagenome 3Met Ala Ser Pro Gln Ser Glu Ala Ile Arg Gln Met Leu Arg Glu Gln1 5 10 15Lys Glu Ala Ala Lys Lys Gly Ala Pro Ser Ile Glu Glu Gln Arg Arg 20 25 30Gln Leu Asp Tyr Leu Gly Gly Phe Ser Pro Leu Glu Ser Asp Val Thr 35 40 45Val Glu Lys Thr Arg Ile Ala Gly Val Pro Gly Glu Trp Ile Ser Thr 50 55 60Pro Asp Ala Arg Lys Asp Arg Val Leu Phe Tyr Leu His Gly Gly Ala65 70 75 80Tyr Cys Phe Gly Ser Cys Asp Ser His Arg Gly Leu Val Ser Arg Leu 85 90 95Ala Arg Ala Cys Gly Ser Arg Ala Leu Leu Ile Glu Tyr Arg Leu Ala 100 105 110Pro Glu His Pro Phe Pro Ala Ala Leu Glu Asp Ser Thr Ala Ala Tyr 115 120 125Arg Glu Leu Ile Arg Ser Gly Val Arg Pro Glu Asn Leu Val Ile Ala 130 135 140Gly Asp Ser Ala Gly Gly Gly Leu Thr Met Ala Thr Leu Leu Thr Leu145 150 155 160Arg Asp Glu Gly Asp Pro Leu Pro Ser Ala Ala Val Leu Leu Ser Pro 165 170 175Trp Thr Asp Leu Glu Gly Thr Gly Glu Ser Met Lys Thr Lys Ala Asp 180 185 190Val Glu Pro Trp Leu Asp Pro Glu Lys Ser His Leu Leu Ala Lys Leu 195 200 205Tyr Leu Gly Asp Leu Asp Pro Arg His Pro Leu Val Ser Pro Ile His 210 215 220Ala Asp Leu Asn Asn Leu Pro Pro Leu Leu Val His Val Gly Ser Asp225 230 235 240Glu Cys Leu Leu Asp Asp Ser Val Arg Leu Val Glu Arg Ala Lys Ser 245 250 255Ala Gly Val Glu Thr Glu Phe Lys Ile Trp Asp Glu Met Trp His Val 260 265 270Phe His Gly Phe Pro Ile Pro Glu Ala Gln Gln Ala Ile Glu Glu Ile 275 280 285Gly Ala Phe Val Arg Ala Arg Leu Pro 290 2954311PRTUnknownDerived from metagenome 4Met Ala Asp Pro Gln Leu Glu Ala Val Leu Val Gly Leu Ala Gln Ala1 5 10 15Ser Ala Gly Ala Gln Gly Pro Ala Thr Val Glu Gly Phe Arg Val Ala 20 25 30Leu Arg Glu Leu Thr Arg Met Leu Asp Phe Arg Asp Ile Pro Val Gly 35 40 45Arg Val Glu Asn Arg Met Ile Pro Gly Pro Asp Gly Glu Ile Gly Ile 50 55 60Arg Ile Tyr Thr Pro Ile Ala Ala Gly Ala Arg Met Leu Glu Thr Leu65 70 75 80Ile Tyr Phe His Gly Gly Gly Phe Val Ala Gly Asp Leu Glu Thr His 85 90 95Asp Thr Leu Cys Arg Gly Leu Thr Ala Arg Ser Gly Cys Arg Val Ile 100 105 110Ser Val Asp Tyr Arg Leu Ala Pro Glu His Pro Phe Pro Ala Ala Ile 115 120 125Asp Asp Ser Tyr Ala Ala Leu Arg Trp Ile Glu Ala Asn Ala Thr Thr 130 135 140Leu Gly Val Asp Ser Asn Arg Ile Ala Val Gly Gly Asp Ser Ala Gly145 150 155 160Gly Asn Ile Ala Ala Val Val Ala Gln Leu Ala Arg Gly Ala Gly Asn 165 170 175Pro Val Val Arg Phe Gln Leu Leu Ile Tyr Pro Val Val Gln Trp Asp 180 185 190Val Ala Thr Pro Ser Arg Gln Gln Phe Ala Glu Asp Pro Ile Ile Pro 195 200 205Arg Asp Val Ile Asp Met Cys Ala Arg Asn Tyr Phe Gly Pro Met Val 210 215 220Pro Ala Thr Asp Phe Arg Ala Ala Pro Leu Ala Ala Ser Asp Leu Ala225 230 235 240Gly Leu Pro Pro Ala Tyr Val Ile Thr Ala Gly Leu Asp Pro Leu Arg 245 250 255Asp Glu Gly Ala Gln Tyr Ala Glu Lys Leu Arg Glu Ala Gly Val Ala 260 265 270Val Glu His Val Gly Tyr Asp Asp Met Ile His Gly Phe Met Ser Met 275 280 285Ser Asn Ala Leu Asp Thr Ala Lys Leu Ala Ile Glu Arg Ala Gly Asp 290 295 300Ala Leu Arg Asn Ala Leu Arg305 3105316PRTUnknownDerived from metagenome 5Met Ser Leu Asp Pro Lys Ala Arg Glu Leu Leu Ala Met Val Tyr Arg1 5 10 15Val Asn Ala Pro Arg Phe His Glu Leu Ser Val Ser Gln Ala Arg His 20 25 30Ala Thr Gln Lys Leu Met Phe Ala Phe Arg Pro Glu Ala Pro Ala Val 35 40 45Ala Ser Thr Thr Glu Val Pro Ile Pro Arg Pro Asp Gly Ser Val Leu 50 55 60Phe Ala Arg Leu Tyr Arg Pro Leu Gly Cys His Ala Ser Glu Asp Leu65 70 75 80Gly Leu Leu Ile Tyr Phe His Gly Gly Gly Trp Cys Thr Gly Asp Leu 85 90 95Pro Gly Tyr Asp Val Leu Cys Arg Glu Leu Ala Asn Gln Ser Gly Ala 100 105 110Ala Val Leu Ser Val Asp Tyr Arg Leu Ala Pro Glu His Arg Phe Pro 115 120 125Ala Ala Val His Asp Ala Ser Leu Ala Phe Glu Trp Ser Thr Glu Asn 130 135 140Ala Ser Leu Leu Gly Val Asp Ala Glu Arg Ile Ala Leu Gly Gly Asp145 150 155 160Ser Ala Gly Gly Asn Leu Ala Ile Val Ala Ala Leu Glu Ala Arg Asp 165 170 175Arg Ala Ala Arg Met Pro Arg Ala Leu Ala Leu Ile Tyr Pro Ser Thr 180 185 190Gln Ile His Ser Glu Arg Ser Ser Arg Glu Thr Phe Ala Asp Gly Tyr 195 200 205Phe Leu Asp Arg Glu Ser Leu Arg Trp Phe Tyr Glu His Tyr Phe Ala 210 215 220Asp Pro Ala Gln Ala Gln Ser Trp Gln Ala Ser Pro Met Leu Ala Ala225 230 235 240Ser Leu Ala Gly Leu Pro Pro Ala Ile Leu Ile Thr Ala Gly Cys Asp 245 250 255Pro Leu Thr Asp Asp Cys Val Ala Phe Ala Glu Arg Met Val Ala Asp 260 265 270Gly Gly Leu Val Val Arg His His Phe Glu Gly Met Val His Gly Phe 275 280 285Leu Pro Leu Gly Lys Phe Phe Ala Gln Ala Asn Glu Ala Val Arg Cys 290 295 300Val Ser Ser Tyr Leu Arg Glu Ala Leu Gln Ala Ser305 310 3156297PRTUnknownDerived from metagenomemisc_feature(216)..(216)Xaa can be any naturally occurring amino acid 6Met Ser Leu Glu Glu Leu Ala Val Val Arg Gln Leu Leu Ala Gly Leu1 5 10 15Val Thr Gly Glu Ala Arg Ser Leu Glu Asp Phe Arg Thr Ser Tyr Asp 20 25 30Glu Ala Gly Lys Ala Phe Gly Leu Pro Glu Gly Val Thr Val Thr Pro 35 40 45Val Ser Ala Gly Gly Val Pro Gly Glu Trp Leu Ala Pro Ala Ala Gly 50 55 60Ala Gly Lys Arg Val Leu Leu Tyr Leu His Gly Gly Gly Tyr Ala Leu65 70 75 80Gly Ser Leu Asp Ser His Arg His Leu Ala Ala His Thr Ala Leu Ala 85 90 95Leu Asn Gly Arg Val Leu Leu Ile Asp Tyr Arg Arg Ser Pro Glu His 100 105 110Pro Phe Pro Ala Ala Val Asp Asp Ala Leu Ala Ala Tyr Arg Trp Leu 115 120 125Thr Glu Thr Gly Val Asp Pro Ala Lys Leu Ala Val Ala Gly Asp Ser 130 135 140Ala Gly Gly Gly Leu Thr Val Ala Val Leu Leu Ala Ala Arg Asp Ala145 150 155 160Gly Leu Arg Leu Pro Ala Ala Ala Val Cys Ile Ser Pro Trp Ala Asn 165 170 175Leu Glu Asn Lys Gly Ala Ser Tyr Gly Ala Lys Ala Asn Val Asp Pro 180 185 190Met Val Arg His Ala Asp Leu Glu Leu Trp Thr Ala Ala Tyr Leu Gly 195 200 205Thr Ser Thr Pro Arg Arg Ala Xaa Leu Ala Ser Pro Val Tyr Ala Asp 210 215 220Leu Asn Gly Leu Pro Pro Phe Leu Ile Gln Val Gly Ser Ser Glu Val225 230 235 240Leu Leu Ser Asp Ser His Leu Leu Ala Asp Arg Leu Lys Glu Ala Gly 245 250 255Val Ser Val Asp Leu His Val Trp Pro Glu Met Ile His Val Trp His 260 265 270Trp Phe Ala Pro Val Leu Ser Glu Gly Arg Ala Ala Ile Asp Glu Met 275 280 285Ala Ser Phe Leu Asp Thr Lys Leu Gly 290 2957490PRTUnknownDerived from metagenome 7Met Thr Gly Leu His Phe Arg Ser Ala Ser Glu Leu Gly Arg Met Ile1 5 10 15Arg Arg Gly Glu Ile Ser Ser Ala Glu Leu Thr Asp His Phe Ile Gln 20 25 30Arg Ile Glu Thr Leu Asp Gly Lys Thr Asn Ala Val Val Ala Arg Asp 35 40 45Phe Asp Arg Ala Arg Ala Leu Ala Lys Glu Ala Asp Ala Ala Gln Ala 50 55 60Arg Gly Ala Ser Leu Gly Ala Leu His Gly Leu Pro Phe Thr Ile Lys65 70 75 80Asp Ala Tyr Glu Val Glu Gly Ile Val Ser Thr Gly Gly Asn Pro Thr 85 90 95Trp Lys Asp His Val Pro Thr Ser Ser Ala Thr Ala Val Glu Arg Leu 100 105 110Gln Arg Ser Gly Ala Ile Val Met Gly Lys Thr Asn Val Pro Tyr Leu 115 120 125Ser Gly Asp Leu Gln Thr Tyr Asn Asp Ile Tyr Gly Thr Thr Asn Asn 130 135 140Pro Trp Ala Leu Asp Cys Gly Pro Gly Gly Ser Ser Gly Gly Ser Ala145 150 155 160Ala Ser Leu Ala Ala Gly Phe Ala Ala Ala Glu Phe Gly Ser Asp Ile 165 170 175Gly Gly Ser Ile Arg Thr Pro Ala His Leu Cys Gly Val Phe Gly His 180 185 190Lys Pro Ser Phe Gly Ile Val Pro Lys Arg Gly His Leu Ser Pro Pro 195 200 205Pro Gly Cys Leu Ser Glu Gly Asp Leu Ser Val Ala Gly Pro Leu Ala 210 215 220Arg Ser Ala Glu Asp Leu Lys Leu Leu Leu Ser Leu Thr Ala Gly Pro225 230 235 240Asp Trp Ala Asp Ala Ala Gly Trp Lys Leu Asp Leu Pro Pro Ala Arg 245 250 255Ala Arg Thr Pro Arg Glu Leu Arg Ala Ala Val Trp Ile Asp Asp Glu 260 265 270Phe Cys Asp Ile Asp Arg Glu Ser Ala Asp Leu Leu Arg Asn Ala Ala 275 280 285Lys Ala Leu Gln Asp Ala Gly Ala Asn Val Asp Trp Asn Ala Arg Pro 290 295 300Asp Phe Thr Leu Ala Glu Ile Thr Glu Cys Tyr Leu Ile Leu Leu His305 310 315 320Ser Gln Ile Gly Ala Gly Met Pro Gln Ser Ile Arg Asp His Trp Ala 325 330 335Glu Met Lys Lys Gly Phe Ala Pro Asp Asp Lys Ser His Ala Ala Leu 340 345 350Gln Ala Ile Gly Gly Thr Leu Ser Leu Ala Glu Arg Ala Val Trp Lys 355 360 365Glu Val Gln Ala Gln Leu Arg Trp Lys Trp His Thr Phe Phe Lys Ser 370 375 380Tyr Asp Val Val Leu Ser Pro Val Leu Met Arg Pro Ala Phe Glu His385 390 395 400Asn His Gln Ser Asn Trp His Lys Arg Glu Leu Asp Val Asn Gly Val 405 410 415Lys Arg Pro Tyr Met Asp Val Leu Ile Trp Ala Gly Pro Ala Val Val 420 425 430Ser Tyr Leu Pro Ala Thr Ala Ala Pro Val Gly Val Thr Ser Glu Gly 435 440 445Lys Pro Val Gly Ile Gln Ile Ile Gly Pro His Leu Glu Asp Tyr Thr 450 455 460Thr Ile Ala Val Ala Gly Met Phe Glu Glu Ile Leu Gly Gly Phe Lys465 470

475 480Pro Pro Lys Gly Trp Ala Ala Ala Leu Glu 485 4908177PRTUnknownDerived from metagenome 8Cys Ala Cys Cys Leu Ser Leu Val Asp Arg Asp Gly Arg Arg Pro Gly1 5 10 15Glu Leu Ala Val Ala Gly Asp Ser Ala Gly Gly Gly Leu Thr Val Ala 20 25 30Val Leu Leu Ala Ala Arg Asp Ala Gly Leu Arg Leu Pro Ala Ala Ala 35 40 45Val Cys Ile Ser Pro Trp Ala Asn Leu Glu Asn Lys Gly Ala Ser Tyr 50 55 60Gly Ala Lys Ala Asn Val Asp Pro Met Val Arg His Ala Asp Leu Glu65 70 75 80Leu Trp Thr Ala Ala Tyr Leu Gly Thr Ser Thr Pro Arg Arg Ala Pro 85 90 95Leu Ala Ser Pro Val Tyr Ala Asp Leu Asn Gly Leu Pro Pro Phe Leu 100 105 110Ile Gln Val Gly Ser Ser Glu Val Leu Leu Ser Asp Ser His Leu Leu 115 120 125Ala Asp Arg Leu Lys Glu Ala Gly Val Ser Val Asp Leu His Val Trp 130 135 140Pro Glu Met Ile His Val Trp His Trp Phe Ala Pro Val Leu Ser Glu145 150 155 160Gly Arg Ala Ala Ile Asp Glu Met Ala Ser Phe Leu Asp Thr Lys Leu 165 170 175Gly9491PRTUnknownDerived from metagenome 9Leu Glu Arg Ser Asp Leu Asp Tyr Ala Ser Ala Thr Glu Ile Ala Arg1 5 10 15Leu Val Arg Thr Arg Gln Ile Ser Ala Ala Asp Val Thr Glu His Ala 20 25 30Ile Ser Arg Ile Glu Ala Arg Asn Gly Ser Leu Asn Ala Phe Val Tyr 35 40 45Thr Asp Phe Glu Gln Ala Arg Ser Arg Ala Lys Asp Leu Asp Thr Arg 50 55 60Ile Ser Ala Gly Glu Asp Val Gly Pro Leu Ala Gly Val Pro Thr Ala65 70 75 80Ile Lys Asp Leu Phe Asn Phe Tyr Pro Gly Trp Pro Ser Thr Leu Gly 85 90 95Gly Ile Arg Cys Leu Arg Asp Phe Lys Leu Asp Val Lys Ser Arg Tyr 100 105 110Ala Thr Lys Met Glu Glu Ala Gly Ala Val Val Leu Gly Ile Thr Asn 115 120 125Ser Pro Val Leu Gly Phe Arg Gly Thr Thr Asp Asn Asp Leu Tyr Gly 130 135 140Pro Thr Arg Asn Pro Phe Asp Leu Ser Arg Asn Ser Gly Gly Ser Ser145 150 155 160Gly Gly Thr Ser Ala Ala Val Ala Asp Gly Leu Leu Pro Ile Gly Asp 165 170 175Gly Thr Asp Gly Gly Gly Ser Ile Arg Ile Pro Ala Ala Trp Cys His 180 185 190Val Phe Gly Phe Gln Ala Ser Pro Gly Arg Ile Pro Leu Ala Ile Arg 195 200 205Pro Asn Ala Phe Gly Ala Ala Ala Pro Phe Ile Tyr Glu Gly Pro Ile 210 215 220Thr Arg Thr Val Glu Asp Ala Ala Leu Ala Met Ser Val Leu Ala Gly225 230 235 240Ser Asp Pro Ala Asp Pro Phe Ser Leu Asn Asp Arg Leu Asp Trp Leu 245 250 255Gly Ala Val Asp Gln Pro Ile Thr Ser Leu Arg Ile Gly Phe Thr Pro 260 265 270Asp Phe Gly Gly Phe Pro Val Glu Pro Ala Val Ala Ala Thr Ile Ala 275 280 285His Ala Val Arg Ala Phe Glu Gln Ala Gly Ala Lys Ile Val Pro Leu 290 295 300Lys Leu Asp Phe Gly Tyr Thr His Asp Glu Leu Ser Gln Leu Trp Cys305 310 315 320Arg Met Ile Ser Gln Gly Thr Ile Ala Val Val Asp Ser Phe Ala Glu 325 330 335Asn Gly Leu His Leu Glu Pro Asp Phe Pro Ala Pro Val Met Glu Trp 340 345 350Ala Gln Lys Ala Lys Asn Ala Thr Pro Leu Asp Leu His Arg Asp Gln 355 360 365Val Met Arg Thr Lys Val Tyr Asp Val Leu Asn Ala Ala Phe Ser Gln 370 375 380Val Asp Leu Ile Ala Gly Pro Thr Thr Thr Cys Leu Pro Thr Pro Asn385 390 395 400Gly Glu Arg Gly Met Thr Val Gly Pro Ser Glu Ile Ala Gly Thr Pro 405 410 415Ile Asn Arg Leu Ile Gly Phe Cys Pro Thr Phe Leu Thr Asn Phe Thr 420 425 430Gly Asn Pro Ala Ala Ser Leu Pro Ala Gly Leu Ala Asp Gly Leu Pro 435 440 445Val Gly Leu Met Leu Ile Gly Pro Arg Arg Asp Asp Leu Thr Val Leu 450 455 460Ser Ala Ser Ala Ala Phe Glu Arg Val Gln Pro Trp Ala Asp Ser Tyr465 470 475 480Arg Ile Pro Ala Ala Arg Pro Leu Gly Ser Gln 485 49010330PRTUnknownDerived from metagenome 10Met Arg Pro Arg Ser Arg Pro His Ala Arg Ala Arg Gly Ala Pro Thr1 5 10 15Ile Leu Arg Asp Pro Ala Thr Met Ala Leu His Arg Thr Pro Arg Arg 20 25 30Asn Asp Met Ala Asp Arg Gly Ile Glu Val Val His Ala His Leu Ala 35 40 45Lys Leu Pro Pro Ala Asp Ser Leu Thr Val Ala Glu Arg Arg Ala Gln 50 55 60Tyr Glu Arg Ala Glu Lys Val Phe Pro Leu Ser Pro Asp Val Lys Val65 70 75 80Glu Arg Val Thr Ala Gly Ala Ala Pro Ala Glu Trp Leu Arg Pro Pro 85 90 95Ser Ala Arg Ala Gly His Val Val Leu Tyr Leu His Gly Gly Gly Tyr 100 105 110Val Ile Gly Ser Pro Arg Ser His Arg His Leu Ala Ala Ala Ile Ala 115 120 125Gly Ala Ala Gly Thr Asn Ala Leu Leu Leu Asp Tyr Arg Leu Ala Pro 130 135 140Glu His Pro Phe Pro Ala Ala Leu Asp Asp Ala Val Ala Ala Tyr Arg145 150 155 160Trp Leu Leu Asp Gln Gly Ile Ala Ala Glu His Ile Ala Val Ala Gly 165 170 175Asp Ser Ala Gly Gly Gly Leu Thr Val Ala Thr Leu Leu Ala Leu Arg 180 185 190Asp Ala His Leu Pro Arg Pro Ala Ala Gly Val Cys Ile Ser Pro Trp 195 200 205Val Asp Leu Thr Cys Ser Gly Gly Ser Tyr Gln Ser Lys Ala Gly Val 210 215 220Asp Pro Ile Val Arg Gln Ala Gly Val Ala Glu Met Ala Arg Ala Tyr225 230 235 240Leu Gly Ala Thr Asp Pro Arg Ser Pro Leu Ala Ser Pro Leu Phe Ala 245 250 255Asp Leu Arg Gly Leu Pro Pro Leu Leu Ile His Val Gly Ser Asp Glu 260 265 270Val Leu Leu Asp Asp Ala Ile Gly Leu Ala Glu Arg Ala Lys Ala Ala 275 280 285Gly Val Asp Ala Thr Leu Glu Gln Trp Asp Arg Met Ile His Val Trp 290 295 300His Trp Phe Leu Pro Met Leu Asp Glu Ala Gln Thr Ala Val Glu Ser305 310 315 320Ile Gly Arg Phe Val Arg Ala Arg Thr Ala 325 33011544PRTPig 11Gly Gln Pro Ala Ser Pro Pro Val Val Asp Thr Ala Gln Gly Arg Val1 5 10 15Leu Gly Lys Tyr Val Ser Leu Glu Gly Leu Ala Gln Pro Val Ala Val 20 25 30Phe Leu Gly Val Pro Phe Ala Lys Pro Pro Leu Gly Ser Leu Arg Phe 35 40 45Ala Pro Pro Gln Pro Ala Glu Pro Trp Ser Phe Val Lys Asn Thr Thr 50 55 60Ser Tyr Pro Pro Met Cys Cys Gln Asp Pro Val Ala Gly Gln Met Thr65 70 75 80Ser Asp Leu Phe Thr Asn Arg Lys Glu Arg Leu Ile Pro Glu Phe Ser 85 90 95Glu Asp Cys Leu Tyr Leu Asn Ile Tyr Thr Pro Ala Asp Leu Thr Lys 100 105 110Arg Gly Arg Leu Pro Val Met Val Trp Ile His Gly Gly Gly Leu Val 115 120 125Val Gly Gly Ala Ser Thr Tyr Asp Gly Leu Ala Leu Ala Ala His Glu 130 135 140Asn Val Val Val Val Ala Ile Gln Tyr Arg Leu Gly Ile Trp Gly Phe145 150 155 160Phe Ser Thr Gly Asp Glu His Ser Arg Gly Asn Trp Gly His Leu Asp 165 170 175Gln Val Ala Ala Leu His Trp Val Gln Glu Asn Ile Ala Asn Phe Gly 180 185 190Gly Asp Pro Gly Ser Val Thr Ile Phe Gly Glu Ser Ala Gly Gly Glu 195 200 205Ser Val Ser Val Leu Val Leu Ser Pro Leu Ala Lys Asn Leu Phe His 210 215 220Arg Ala Ile Ser Glu Ser Gly Val Ala Phe Thr Ala Gly Leu Val Arg225 230 235 240Lys Asp Met Lys Ala Ala Ala Lys Gln Ile Ala Val Leu Ala Gly Cys 245 250 255Lys Thr Thr Thr Ser Ala Val Phe Val His Cys Leu Arg Gln Lys Ser 260 265 270Glu Asp Glu Leu Leu Asp Leu Thr Leu Lys Met Lys Phe Phe Ala Leu 275 280 285Asp Leu His Gly Asp Pro Arg Glu Ser His Pro Phe Leu Thr Thr Val 290 295 300Val Asp Gly Val Leu Leu Pro Lys Met Pro Glu Glu Ile Leu Ala Glu305 310 315 320Lys Asp Phe Asn Thr Val Pro Tyr Ile Val Gly Ile Asn Lys Gln Glu 325 330 335Phe Gly Trp Leu Leu Pro Thr Met Met Gly Phe Pro Leu Ser Glu Gly 340 345 350Lys Leu Asp Gln Lys Thr Ala Thr Ser Leu Leu Trp Lys Ser Tyr Pro 355 360 365Ile Ala Asn Ile Pro Glu Glu Leu Thr Pro Val Ala Thr Asp Lys Tyr 370 375 380Leu Gly Gly Thr Asp Asp Pro Val Lys Lys Lys Asp Leu Phe Leu Asp385 390 395 400Leu Met Gly Asp Val Val Phe Gly Val Pro Ser Val Thr Val Ala Arg 405 410 415Gln His Arg Asp Ala Gly Ala Pro Thr Tyr Met Tyr Glu Phe Gln Tyr 420 425 430Arg Pro Ser Phe Ser Ser Asp Lys Lys Pro Lys Thr Val Ile Gly Asp 435 440 445His Gly Asp Glu Ile Phe Ser Val Phe Gly Ala Pro Phe Leu Arg Gly 450 455 460Asp Ala Pro Glu Glu Glu Val Ser Leu Ser Lys Thr Val Met Lys Phe465 470 475 480Trp Ala Asn Phe Ala Arg Ser Gly Asn Pro Asn Gly Glu Gly Leu Pro 485 490 495His Trp Pro Met Tyr Asp Gln Glu Glu Gly Tyr Leu Gln Ile Gly Val 500 505 510Asn Thr Gln Ala Ala Lys Arg Leu Lys Gly Glu Glu Val Ala Phe Trp 515 520 525Asn Asp Leu Leu Ser Lys Glu Ala Ala Lys Lys Pro Pro Lys Ile Lys 530 535 54012342PRTCandida sp. 12Met Lys Leu Leu Ser Leu Thr Gly Val Ala Gly Val Leu Ala Thr Cys1 5 10 15Val Ala Ala Thr Pro Leu Val Lys Arg Leu Pro Ser Gly Ser Asp Pro 20 25 30Ala Phe Ser Gln Pro Lys Ser Val Leu Asp Ala Gly Leu Thr Cys Gln 35 40 45Gly Ala Ser Pro Ser Ser Val Ser Lys Pro Ile Leu Leu Val Pro Gly 50 55 60Thr Gly Thr Thr Gly Pro Gln Ser Phe Asp Ser Asn Trp Ile Pro Leu65 70 75 80Ser Thr Gln Leu Gly Tyr Thr Pro Cys Trp Ile Ser Pro Pro Pro Phe 85 90 95Met Leu Asn Asp Thr Gln Val Asn Thr Glu Tyr Met Val Asn Ala Ile 100 105 110Thr Ala Leu Tyr Ala Gly Ser Gly Asn Asn Lys Leu Pro Val Leu Thr 115 120 125Trp Ser Gln Gly Gly Leu Val Ala Gln Trp Gly Leu Thr Phe Phe Pro 130 135 140Ser Ile Arg Ser Lys Val Asp Arg Leu Met Ala Phe Ala Pro Asp Tyr145 150 155 160Lys Gly Thr Val Leu Ala Gly Pro Leu Asp Ala Leu Ala Val Ser Ala 165 170 175Pro Ser Val Trp Gln Gln Thr Thr Gly Ser Ala Leu Thr Thr Ala Leu 180 185 190Arg Asn Ala Gly Gly Leu Thr Gln Ile Val Pro Thr Thr Asn Leu Tyr 195 200 205Ser Ala Thr Asp Glu Ile Val Gln Pro Gln Val Ser Asn Ser Pro Leu 210 215 220Asp Ser Ser Tyr Leu Phe Asn Gly Lys Asn Val Gln Ala Gln Ala Val225 230 235 240Cys Gly Pro Leu Phe Val Ile Asp His Ala Gly Ser Leu Thr Ser Gln 245 250 255Phe Ser Tyr Val Val Gly Arg Ser Ala Leu Arg Ser Thr Thr Gly Gln 260 265 270Ala Arg Ser Ala Asp Tyr Gly Ile Thr Asp Cys Asn Pro Leu Pro Ala 275 280 285Asn Asp Leu Thr Pro Glu Gln Lys Val Ala Ala Ala Ala Leu Leu Ala 290 295 300Pro Ala Ala Ala Ala Ile Val Ala Gly Pro Lys Gln Asn Cys Glu Pro305 310 315 320Asp Leu Met Pro Tyr Ala Arg Pro Phe Ala Val Gly Lys Arg Thr Cys 325 330 335Ser Gly Ile Val Thr Pro 340

Claims

1.-11. (canceled)

12. A process for breaking down polyester polyurethanes into low-molecular-weight breakdown products, comprising the steps of a) cleaving the ester groups present in the polyester polyurethane; and b) cleaving the urethane groups present in the polyester polyurethane with a polypeptide that has urethanase activity; with the proviso that process steps a) and b) may be carried out in either order or else in parallel.

13. The process as claimed in claim 12, wherein the polypeptide that has urethanase activity has an amino acid sequence selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 10, and amino acid sequences having at least 90% sequence identity with the sequences SEQ ID No. 1 to SEQ ID No. 10.

14. The process as claimed in claim 12, wherein process step a) is carried out before process step b).

15. The process as claimed in claim 12, wherein polyols, polycarboxylic acids, and polyamines are formed as process products.

16. The process as claimed in claim 15, wherein at least one polyamine selected from the group consisting of methylene-4,4'-diamine, methylene-2,4'-diamine, methylene-2,2'-diamine, naphthylene-1,4-diamine, naphthylene-1,5-diamine, naphthylene-1,6-diamine, tolylene-2,4-diamine, and tolylene-2,6-diamine is formed.

17. The process as claimed in claim 15, wherein at least one polyol selected from the group consisting of ethylene glycol, diethylene glycol, 1,4-butanediol, triethylene glycol, propylene glycol, 1,2-dipropylene glycol, neopentyl glycol, glycerol, 1,1,1-trimethylolpropane, sucrose, sorbitol, and pentaerythritol is formed.

18. The process as claimed in claim 15, wherein at least one polycarboxylic acid selected from the group consisting of succinic acid, glutaric acid, adipic acid, phthalic acid, terephthalic acid, benzenetricarboxylic acid, oleic acid, and ricinoleic acid is formed.

19. The process as claimed in claim 12, wherein process step a) is carried out with a lipase.

20. The process as claimed in claim 19, wherein the lipase has an amino acid sequence as in SEQ ID No. 11 or SEQ ID No. 12 or a variant of the abovementioned sequences having at least 90% sequence identity with SEQ ID No. 11 or SEQ ID No. 12.

21. A method comprising utilizing a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID No. 3, SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 and variants of said polypeptides or of a GatA-similar polypeptide having an amino acid sequence in accordance with SEQ ID No. 7 or a variant thereof, wherein the polypeptide has urethanase activity in the enzymatic cleavage of urethane linkages.

22. The method according to claim 21, wherein the urethane group is aromatically attached.