Novel carbamate hydrolases for enzymatic degradation of polyurethanes

Abstract

The present invention relates to novel carbamate hydrolases for the enzymatic decomposition of polyurethanes and to an enzymatic process for the complete decomposition of polyurethanes into defined monomers.

Description

[0001] The present invention relates to a novel carbamate hydrolase (urethanase) for enzymatic decomposition of polyurethane, and an enzymatic method for completely decomposing polyurethane into specific monomers.

[0002] Polyurethanes are already well-established in many areas of everyday life. For example, they can be found in soft foams (mattresses, sponges, upholstered furniture), rigid foams (insulation materials, building materials), thermoplastics (sports shoes), or coatings (lacquers, inks, adhesives). Due to the ever-increasing demand for these products, they are being produced in ever-increasing quantities. At the same time, there is a growing demand for methods that allow the recycling of unused polyurethane products to be as sustainable as possible and to reuse the structural units of the polymers. To this end, the bonds in the polyurethanes must be specifically cleaved to obtain specific decomposition products and thus make them reusable.

[0003] In addition to the physiological function that enzyme plays in living organisms, enzyme can also be used for catalysis chemical reaction outside this context in a diversified manner. Here, the reaction can be carried out under conditions milder than conventional chemical processes, such as lower temperature, neutral pH value and without using aggressive chemicals. Thus, energy saving, minimization of by-product formation and environmental protection can be achieved, which helps to reduce running costs. In some cases, only by using enzyme, unstable reactant could react (Jaeger, K.-E. & Reetz, MT (1998) Microbial lipases form versatile tools for biotechnology. Trends in biotechnology, 16, 396-403). In addition, enzyme is typically regioselective, stereoselective and enantioselective, which makes the purification of product significantly simplified and therefore can effectively synthesize difficult-to-obtain product (Hasan, F., Shah, AA & Hameed, A. (2006) Industrial applications of microbial lipases. Enzyme and Microbial Technology, 39, 235-251).

[0004] Polyurethane recycling is primarily carried out through thermal recycling. This process is typically carried out in a batch process at very high temperatures and with very long reaction times, using catalysts. Thermal decomposition of the polymer chains during cracking reactions can lead to undesirable and nonspecific decomposition products, or the formation of epoxy rings. This can lead to high odors and undesirable crosslinking of the chains in the recycled raw material, making it impossible to reuse products that come into contact with humans, particularly in the production of foam materials for furniture and mattresses. Alternatively, complete combustion and the resulting energy utilization are also carried out, which, while energy is obtained, does not allow for efficient reuse of the polymer building blocks.

[0005] Polyurethanes are known to be degraded to a certain extent by bacteria and fungi. Polyester polyurethanes are significantly more susceptible to microbial / enzymatic degradation 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] Polyester polyurethanes can be readily decomposed by hydrolysis of ester bonds. The relatively easy decomposition of polyesters is not surprising, as ester bonds in hydrophobic substrates are naturally cleaved during lipid degradation, and polyesters without carbamate bonds can also be relatively easily decomposed by esterases and lipases (Marten, E., Müller, R.-J. & Deckwer, W.-D. (2003) Studies on the enzymatic hydrolysis of polyesters I. Lowmolecular mass model esters and aliphatic polyesters. Polymer degradation and stability, 80, 485-501; Marten, E., Müller, R.-J. & Deckwer, W.-D. (2005) Studies on the enzymatic hydrolysis of polyesters. II. Aliphatic-aromatic copolyesters. Polymer degradation and stability, 88, 371-381). In various literature sources, enzymes used to decompose polyurethanes have been characterized as esterases (Allen, AB, Hilliard, NP & Howard, GT (1999) Purification and characterization of a soluble polyurethanedegrading 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, JR, Campbell, JR, Thompson, L., Walz, SL & Schultz, WW(1994) Biodegradation of a colloidal ester-based polyurethane by soil fungi. International biodeterioration & biodegradation, 33, 103-113; Darby, RT &Kaplan, AM (1968) Fungal susceptibility of polyurethanes. Appliedmicrobiology, 16, 900-905; Howard, GT, Norton, WN & Burks, T. (2012) Growth of Acinetobacter gerneri P7 on polyurethane and the purification and characterization of a polyurethanase enzyme. Biodegradation, 23, 561-573; Kaplan, AM, Darby, RT, Greenberger, M. & Rodgers, M. (1968) Microbialdeterioration of polyurethane systems. Dev Ind Microbiol, 82, 362-371;Kay,M., Morton, L. & Prince, E. (1991) Bacterial degradation of polyesterpolyurethane. International biodeterioration, 27, 205-222; Vega, RE, Main,T. & Howard, GT (1999) Cloning and expression in Escherichia coli of apolyurethane-degrading enzyme from Pseudomonas fluorescens. Internationalbiodeterioration & biodegradation, 43, 49-55). There is no clear evidence for cleavage of carbamate bonds, as in either case the enzyme characterization was not based on the cleavage of molecules with carbamate groups.

[0007] The decomposition of poly(ester)urethanes by fungi or bacteria is described in numerous publications and patents. However, this decomposition is often limited to the relatively easily cleavable ester bonds and is often demonstrated only through macroscopic observation of polymer decomposition. This lacks the controlled decomposition of ester and urethane bonds found in the present invention, and often results in long decomposition times. These publications indicate that carbamate hydrolases are widely used enzymes, but they do not demonstrate their specific capabilities, usage possibilities and group classification as used in the present invention (JP09192633, Tang, YW, Labow, RS, Santerre, JP (2003) Enzyme induced biodegradation of polycarbonate-polyurethanes: dose dependence effect of cholesterol esterase. Biomaterials 24 (12), 2003-2011, Vega, RE, Main, T. & Howard, GT (1999) Cloning and expression in Escherichia coli of a polyurethane-degrading enzyme from Pseudomonas fluorescens. International biodeterioration & biodegradation, 43, 49-55).

[0008] A decomposition process for the enzymatic decomposition of poly(ester)carbamates is known in which, in a first step, poly(ester)carbamate is decomposed from Comamonas acidovorans ( Comamonas acidovorans Esterases are obtained from cultures of bacterial species of the bacterium β-lactamase (β-lactamase). The esterases are isolated in complex workup steps and used batchwise to decompose poly(ester)urethanes. This multi-stage process results in long decomposition times, and specific cleavage of the urethane bonds has not been demonstrated (JP 09201192, JP 10271994).

[0009] Numerous patents and publications describe the decomposition of poly(ester)urethanes using cutinases, esterases, and / or lipases. However, the decomposition described here is limited to the relatively simple cleavage of the ester bond, rather than specifically targeting the carbamate bond. In addition, no specific combinations of enzymes for the targeted controlled decomposition of the ester and carbamate bonds are described. It is believed that the described methods result in little or no cleavage of the carbamate bond. Consequently, the diamine used cannot be effectively recovered (EP 0968300, US 6,180,381).

[0010] WO 2013 / 134801 describes the decomposition of aromatic polyurethanes based on polyether polyols using enzymes of EC class 3. The specific enzyme sequence is not described, so the method described in the patent cited does not demonstrate specificity for the decomposition of specific carbamate bonds, as demonstrated in the present invention, nor does it demonstrate controlled cleavage of ester bonds or isolated cleavage of carbamate bonds. Furthermore, there is no description of adjusting the pH of the mixture during the polymer decomposition process to maintain carbamate hydrolase activity. Furthermore, there is no description of regioselective decomposition, nor of the decomposition of aliphatic poly(ester)carbamates.

[0011] WO 2006 / 019095 describes a carbamate hydrolase and variants thereof obtained by protein engineering. The enzyme can cleave carbamate oligomers based on TDA or MDA. However, the bond is not regioselectively cleaved, nor is there any application in combination with esterases for polymer decomposition. Furthermore, no other carbamate hydrolases from the GatA or Aes family or any other species are described.

[0012] It was therefore an object of the present invention to provide further enzymes which can be used for the enzymatic cleavage of urethane bonds and preferably for the complete enzymatic decomposition of polyurethanes. Furthermore, an enzymatic process which enables the decomposition of polyurethanes into specific monomers should be provided.

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

[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 thereof or a polypeptide having an amino acid sequence according to SEQ ID No. 7 or a variant thereof, characterized in that the polypeptide has carbamate hydrolase activity.

[0015] Reference polypeptides

[0016] SEQ ID No. Internal naming Research naming 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 AesGö56 11 Ref01 SB12 12 Ref02 SB23

[0017] peptides

[0018] 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 each other by peptide bonds. Polypeptides may contain both naturally occurring and synthetic amino acids. They preferably contain known proteinogenic amino acids.

[0019] For SEQ ID Nos. 1 to 5, 9 and 10, variants are obtained by adding, deleting or substituting up to 10%, preferably up to 5%, of the amino acids contained in the respective polypeptide. Preferred variants of SEQ ID No. 7 are obtained by adding, deleting or substituting up to 5% of the amino acids defined in SEQ ID No. 7. Particularly preferred variants of the aforementioned polypeptides are obtained by adding, deleting or substituting 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 substituting up to 3, more preferably up to 2 amino acids. The above modifications can, in principle, be made continuously or discontinuously at all desired positions in the polypeptide. However, they are preferably made only at the N-terminus and / or C-terminus of the polypeptide. However, each variant obtained according to the invention by adding, substituting or deleting amino acids is characterized by the carbamate hydrolase activity as defined in the following application.

[0020] 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 class which is phylogenetically distinct from the only enzyme Ure known hitherto with carbamate hydrolase activity (see Figure 1 ). In this class, no enzymes with corresponding activity are known to date. Polypeptides of this class are also referred to below as "Aes-like".

[0021] Carbamate hydrolase activity

[0022] The term "carbamate hydrolase activity" refers to the ability of a polypeptide to enzymatically catalyze the cleavage of a carbamate group. In this process, one mole of amine, one mole of alcohol, and one mole of CO2 are produced per mole of carbamate groups.

[0023] The carbamate group can be an aromatic or aliphatic bonded carbamate group. In the case of an aromatic bonded carbamate group, the nitrogen atom is directly bonded to the aromatic ring. In the case of an aliphatic bonded carbamate group, the nitrogen atom is bonded to an alkyl group. It is preferably an unbranched alkyl group having 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 carbamate hydrolase activity is capable of enzymatically cleaving aromatic bonded carbamate groups.

[0024] Whether a polypeptide has carbamate hydrolase activity can be examined by cleaving a suitable model substrate.

[0025] For the ability to cleave aromatically bonded carbamate groups, 4-nitrophenylethylcarbamate (ENPC) is preferably used as a model substrate. Cleavage is demonstrated by measuring the increase in the concentration of 4-nitroaniline. This is preferably performed photometrically at a wavelength of 405 nm. The enzyme activity is preferably determined in the presence of 0.2 mg / L ENPC as substrate in a reaction buffer containing 6.25% by volume ethanol and 100 mM KHPO / KHPO, pH 7. The incubation of the enzyme with ENPC in the reaction buffer is preferably carried out at room temperature and preferably for 24 hours.

[0026]

[0027] For the ability to cleave aliphatic bonded carbamate groups, phenylethyl ethyl carbamate (EPEC) is preferably used as a model substrate. Cleavage is demonstrated by measuring the increase in the concentration of phenylethylamine. This is preferably performed by HPLC. The reaction buffer and reaction conditions used preferably correspond to the parameters described above for EPEC.

[0028]

[0029] Enzymatic cleavage

[0030] The term "enzymatic cleavage of a carbamate group" means that the cleavage of the carbamate group in the presence of a polypeptide having carbamate hydrolase activity is more rapid than when incubated under the same reaction conditions using a reaction buffer without the enzyme, or when incubated under the same conditions using a reaction buffer in the presence of an inactive polypeptide. A preferred model for an inactive polypeptide is bovine serum albumin. If, in the presence of the polypeptide to be assayed, the carbamate group is cleaved more rapidly than in an otherwise identical control using BSA, then the polypeptide has carbamate hydrolase activity as understood herein.

[0031] use

[0032] In another 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 thereof, or a GatA-like polypeptide having an amino acid sequence according to SEQ ID No. 7 or a variant thereof, characterized in that the polypeptide has carbamate hydrolase activity for enzymatic cleavage of carbamate bonds.

[0033] Unless expressly defined otherwise, all definitions given above also apply to the embodiments described.

[0034] Carbamate decomposition into low molecular weight decomposition products

[0035] In another embodiment, the present invention is directed to a method for decomposing a polyester polyurethane into low molecular weight decomposition products comprising the steps of:

[0036] a) cleaving ester groups contained in the polyester polyurethane; and

[0037] b) cleaving the carbamate groups contained in the polyester polyurethane with a polypeptide having carbamate hydrolase activity;

[0038] It is a proviso that process steps a) and b) can be carried out in any order or simultaneously.

[0039] Particularly suitable peptides having carbamate hydrolase activity are the peptides described in the present application having an amino acid sequence as defined in the group consisting of SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 and an amino acid sequence having at least 90% sequence identity therewith. Very particular preference is given to peptides having an amino acid sequence as defined in SEQ ID Nos. 3 or 7 and an amino acid sequence having at least 90% sequence identity therewith.

[0040] Therefore, in a particularly preferred embodiment, the present invention relates to a process for decomposing polyester polyurethanes into low molecular weight decomposition products, comprising the following steps:

[0041] a) cleaving ester groups contained in the polyester polyurethane; and

[0042] b) treating the polyurethane with a polypeptide having carbamate hydrolase activity and having an amino acid sequence selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 10 and an amino acid sequence having at least 90% sequence identity thereto;

[0043] It is a proviso that process steps a) and b) can be carried out in any order or simultaneously.

[0044] Process step a) is preferably carried out before process step b).

[0045] Method step a) is preferably carried out using a lipase. The lipase is preferably water-soluble and not present in an immobilized form. In this context, "immobilization" refers to the binding of peptides, in particular antibodies or enzymes, to the surface of blood vessels or to water-insoluble particles, as is generally known in biotechnology.

[0046] Particularly preferred is the use of a lipase capable of cleaving tributyrin. Even more particularly preferred is the use of a polypeptide having an amino acid sequence as defined in SEQ ID No. 11 or SEQ ID No. 12, or an amino acid sequence having at least 90%, preferably at least 95%, sequence identity to either of these sequences, and capable of cleaving tributyrin. Method step a) is preferably carried out under reaction conditions in which the lipase used exhibits activity. Such conditions can be determined by routine experimentation using common biochemical methods.

[0047] Since the polypeptide having carbamate hydrolase activity according to the present invention has its maximum activity in the neutral range, method step b) is preferably carried out at a pH value of 6.0 to 10.0, preferably 6.0 to 8.0. The pH value can be adjusted using all bases known and suitable to those skilled in the art.

[0048] 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, the polyurethane is preferably comminuted before carrying out steps a) and b). This is particularly preferred when the polyurethane is to be used in non-foamed form. Comminution can be carried out in all manner familiar to those skilled in the art, preferably by grinding, flaking, tearing, or cutting.

[0049] The polyurethane comprises at least one aromatic, aliphatic or alicyclic isocyanate as an isocyanate component. The polyurethane preferably comprises only aromatic isocyanates. Preferred aromatic isocyanates are methylene diphenyl isocyanate (MDI), MDI variants with three or more nuclei, naphthalene diisocyanate and toluene diisocyanate. Particularly preferred aromatic isocyanates are methylene diphenyl isocyanate (MDI), MDI variants with three or more nuclei and toluene diisocyanate. MDI variants with three or more nuclei are by-products of the synthesis and may also be contained in the polyurethane. The polyurethane to be decomposed particularly preferably comprises toluene 2,4-diisocyanate and toluene 2,6-diisocyanate.

[0050] The term "polyester polyol" is known to those skilled in the art and refers to 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 contained in the polyurethane to be decomposed particularly preferably have a functionality of 1.5 to 6.0. They contain aromatic and / or aliphatic polyols and aromatic and / or aliphatic polycarboxylic acids in any combination as structural components.

[0051] The low molecular weight decomposition products of polyester-based polyurethane foams preferably have a molecular weight of at most 1000 g / mol.

[0052] (i) an amine derived from the isocyanate used to prepare the polyurethane in question, such as toluene-2,4-diamine in the case of toluene 2,4-diisocyanate; and

[0053] (ii) Alcohols and carboxylic acids used to form polyester polyols used in the synthesis of the polyurethanes in question.

[0054] In the present application, the term "polyol" is understood to mean any compound having at least two hydroxyl groups. The polyol preferably has a molecular weight of up to 300 g / mol. Preferred polyols as low-molecular-weight decomposition 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.

[0055] In the present application, the term "polycarboxylic acid" is understood to mean any compound containing at least two carboxyl groups. The polycarboxylic acid preferably has a molecular weight of up to 300 g / mol. Preferred polycarboxylic acids as low-molecular-weight decomposition products of polyester-based polyurethane foams are selected from the group consisting of succinic acid, glutaric acid, adipic acid, phthalic acid, terephthalic acid, benzene trimectricarboxylic acid, oleic acid, and ricinoleic acid. Particularly preferred polycarboxylic acids as low-molecular-weight decomposition products of polyester-based polyurethane foams are selected from the group consisting of succinic acid, glutaric acid, adipic acid, phthalic acid, terephthalic acid, and benzene trimectricarboxylic acid.

[0056] In the present application, the term "polyamine" is understood to mean any compound containing at least two amino groups. The polyamines preferably have a molecular weight of up to 300 g / mol. Preferred polyamines as low-molecular-weight decomposition products of polyester-based polyurethane foams are selected from the group consisting of methylene-4,4'-diamine, methylene-2,4'-diamine, methylene-2,2'-diamine, toluene-2,4-diamine, toluene-2,6-diamine, hexamethylenediamine, isophoronediamine, xylylenediamine, pentamethylenediamine, p-phenylenediamine, butanediamine, and H12-methylenediamine. More preferably, polyamines are selected from the group consisting of methylene-4,4'-diamine, methylene-2,4'-diamine, methylene-2,2'-diamine, naphthalene-1,4-diamine, naphthalene-1,5-diamine, naphthalene-1,6-diamine, toluene-2,4-diamine, and toluene-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, toluene-2,4-diamine and toluene-2,6-diamine.

[0057] The process according to the invention enables efficient recycling of polyurethanes in two respects: (i) the process itself does not require the use of high energy due to the mild reaction conditions; (ii) the polyurethanes can be utilized in terms of materials due to the formation of specific decomposition products which themselves serve as valuable chemical raw materials.

[0058] In contrast, thermoglycolysis, which is currently the most common chemical decomposition method for recycling polyurethanes and is already in industrial practice, is carried out at very high temperatures. Here, the focus is on obtaining polyols, while amines are separated as interfering factors and are not recycled. In non-enzymatic hydrolysis, both polyols and amines are obtained as products. However, this method is carried out at high temperatures and ambient pressures.

[0059] Overview of the accompanying drawings:

[0060] Figure 1 : Results of phylogenetic analysis of the amino acid sequences disclosed in this application

[0061] The following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the claims in any way. Example

[0062] Assay of enzyme activity using ENPC

[0063] 0.2 mg / ml of ENPC was incubated in 100 mM KH2PO4 / K2HPO4 at pH 7.0 containing 6.25% by volume of ethanol on an "MTS 2 / 4" plate shaker (IKA, Staufen) at room temperature and 900 rpm for 24 hours.

[0064] After filtering the samples, 100 μL of each was transferred to a transparent 96-well plate "UV-Star" with a flat bottom (Greiner Bio-One, Frickenhausen), and the absorbance at 405 and 480 nm was measured. The value at 480 nm was measured because 4-nitroaniline no longer absorbs significantly here, and therefore, if high values ​​appear at these two wavelengths, it is very likely that it is not 4-nitroaniline but another substance that absorbs at 405 nm.

[0065] Upon hydrolysis by carbamate hydrolases, the nearly colorless ENPC is cleaved into 4-nitroaniline, CO2, and ethanol, with 4-nitroaniline being detected at 405 nm in an "Infinite M1000PRO" microtiter plate photometer (Tecan, Mannedorf, Switzerland). The photometer was controlled using "i-control" software, version 3.4.2.0 (Tecan, Mannedorf, Switzerland). 4-Nitroaniline was also detected by HPLC using the "Dabsylamine" method.

[0066] High-pressure liquid chromatography (HPLC)

[0067] High-pressure liquid chromatography (HPLC) was performed on an Agilent Technologies (Santa Clara, USA) 1100 series instrument equipped with an autosampler and a diode array detector (DAD) for the UV and visible regions. A "Zorbax XDB-C18" column (Agilent Technologies, Santa Clara, USA) with a 3.5 μm particle size and dimensions of 4.6 x 75 mm was used for all measurements. For all methods, 5 μL of sample was injected and the column was thermostated to 40°C. The flow rate was typically 1.5 ml / min. Due to the use of reversed-phase columns, elution was performed with increasing concentrations of organic solvent in all methods.

[0068] The "dimethylaminophenylazobenzenesulfonamide" method was used to detect and quantify dimethylaminophenylazobenzenesulfonated aliphatic amines and carbamates. Due to the high intrinsic absorption, aromatic amines and carbamates can be quantified by this method without derivatization. In addition to AcN, 10 mM sodium phosphate buffer, pH 7.0, was used as the eluent, to which 0.005% (w / v) sodium azide was added to prevent microbial growth. To prevent pressure problems caused by contaminated pump valves, 5% (v / v) dd HO was later added to the AcN and the method was adapted ("dimethylaminophenylazobenzenesulfonamide 95"). The amount of MDEC from the enzymatic reaction of 4,4'-MDA with EC was determined using the "dimethylaminophenylazobenzenesulfonamide-12-MeOH" method, in which the aqueous component is acidified and the protonated aromatic amines are eluted very early. The reactions of 4,4′-MDA with DMC, 2,4-TDA with DMC, and 2,4-TDA with EC were studied using the "dimethylaminophenylazobenzenesulfonamide 95-H2O" method, which differs from the "dimethylaminophenylazobenzenesulfonamide 95" method only in the use of pure ddH2O instead of buffer. Data were analyzed using "OpenLAB CDS ChemStationLC" software, version A.02.09

[017] (Agilent Technologies, Santa Clara, USA).

[0069] Dimethylaminophenylazobenzenesulfonamide: Eluent: AcN and 10 mM Na2HPO4 / NaH2PO4, pH 7.0

[0070] t [min] AcN 0 5 6.5 85 8.0 5 10.0 5

[0075] Dimethylaminophenylazobenzenesulfonamide 95 : Eluent: AcN containing 5% (v / v) dd H2O and 10 mM Na2HPO4 / NaH2PO4, pH 7.0

[0076] t [min] AcN(+ 5% (v / v) dd H2O) % 0 5 6.5 90 8.0 5 10.0 5

[0081] Dimethylaminophenylazobenzenesulfonamide-12-MeOH-lang : Eluent: methanol and dd H2O containing 0.1% (v / v) formic acid

[0082] t [min] Methanol % 0 5 2.5 35 8.0 70 8.5 85 10.0 5 12.0 5

[0089] SEQ ID No. Research naming Hydrolysis of ENPC 1 GatA61 + 2 Aes70 + 3 Aes72 + 4 Aes170 + 5 Aes174 + 6 Aes175 + 7 GatA197 + 8 Aes214 + 9 GatA250 + 10 AesGö56 + 11 SB12 + 12 SB23 +

[0090] Testing enzyme activity with EPEC

[0091] The assay was performed as described for ENPC. The formed phenylethylamine was detected by HPLC as described above.

[0092] SEQ ID No. Research naming Hydrolysis of EPEC 1 GatA61 + 2 Aes70 - 3 Aes72 + 4 Aes170 - 5 Aes174 + 6 Aes175 - 7 GatA197 + 8 Aes214 + 9 GatA250 + 10 AesGö56 - 11 SB12 + 12 SB23 +

[0093] Phylogenetic analysis of enzymes

[0094] A phylogenetic tree for carbamate hydrolase relatedness was created using the "MegAlign" program version 10.1.0 (DNASTAR, Madison, USA). The phylogenetic tree was created using "ClustalW" with standard settings.

[0095] The alignment of the different proteins was created using the “Clustal Omega” program (Sievers et al., 2011).

[0096] Database searches of protein sequences were performed using BLASTP (Altschul et al., 1990).

[0097] Open reading frames (ORFs) in sequenced metagenomic sequences were found using the "ORF Finder" online application from NCBI (Wheeler et al., 2007).

[0098] The same hydrolase gene was reduced to one representative and all sequences were checked using ORF to obtain the complete sequence of the gene. Alternative start codons were also allowed in the search. It is apparent that for the gene from pLip214, there were aes Similar N-terminal region, but no start codon could be found. This gene fragment is not located at the edge of the insert fragment of the metagenomic vector, which may explain the truncated gene. For further analysis, aes The carbamate hydrolase gene of the inference that is identified is carried out computer translation, and uses BLASTP to compare with the NCBI database.According to its numbering in the lipase library and with the similarity of GatA or Aes, name the carbamate hydrolase of said inference.

[0099] In order to compare the individual members of the two identified carbamate hydrolase classes (GatA and Aes) with one another, an alignment was formed in each case using the "Clustal Omega" program and, in addition, a pedigree tree was created using the "MegAlign" program, for which a common alignment of the two classes was created. Sequences of enzymes from the literature (Ure, Ana and NfpolyA), all of which are similar to GatA, were also included in the sequence comparison.

[0100] The pedigree tree is as Figure 1 This indicates that the two species are located in different branches, with similarity relationships within the two species being less clear in some cases, as evidenced by smaller bootstrapping values ​​at the nodes. There appears to be greater divergence within the GatA species than within the Aes species, as evidenced by the longer branch lengths within this species. In particular, Aes70 and Aes72, as well as Aes175 and Aes214, show very high similarity, as indicated by both the relatively short branches in the tree and the finding of identical proteins with the greatest similarity in BLASTP searches.

[0101] Preparation of polyurethane foam for decomposition testing

[0102] The raw materials listed below were reacted with each other in a one-step process in a conventional manner for producing polyurethane foams. The apparent density was 38 kg / m 3 (DIN EN ISO 845, October 2009 edition), the compression hardness at 40% compression rate is 3.5 kPa (DIN EN ISO 3386-1, October 2015 edition).

[0103] formula:

[0104] 100 parts Desmophen 2200B

[0105] 3 parts water

[0106] 19 parts Desmodur T80

[0107] 19 parts Desmodur T65

[0108] 0.7 parts N,N'-dimethylpiperazine

[0109] 1 part Tegostab 8325.

[0110] raw material:

[0111] Desmophen® 2200B, Covestro Deutschland AG; branched polyester polyol based on adipic acid, diethylene glycol, and 1,1,1-trimethylolpropane with a hydroxyl number of approximately 60 mg KOH / g.

[0112] Desmodur® T80, Covestro Deutschland AG; an isomer mixture of toluene 2,4- and 2,6-diisocyanate in a mixing ratio of approximately 80:20.

[0113] Desmodur® T65, Covestro Deutschland AG; an isomer mixture of toluene 2,4- and 2,6-diisocyanate in a mixing ratio of approximately 67:33.

[0114] N,N'-dimethylpiperazine, a catalyst from abcr GmbH

[0115] Tegostab® B 8325, foam stabilizer, Evonik

[0116] Water; deionized water.

[0117] The formulation may be made with an index of 90 to 115. The index refers to the molar ratio of isocyanate groups to isocyanate-reactive groups multiplied by 100.

[0118] Decomposition of polyurethane foam

[0119] The substrate used is a polyester polyurethane prepared from toluene diisocyanate. Decomposition occurs in two reaction steps. First, the foam is incubated with lipase. The oligomers produced are then neutralized and cleaved into monomers using carbamate hydrolases.

[0120] In a first step, 1 g of foam was added to a 50 ml centrifuge tube with 20 ml of potassium phosphate buffer at pH 7.0 and approximately 30 mg of CalB lyophilisate "Chirazyme L2" (Roch, Basel, Switzerland) (herein referred to as SEQ ID No. 12) and incubated at 37° C. and 200 rpm for 5 days. The residual foam mass was photographed using an "MH2" microscope (Olympus, Hamburg) by comparison with a negative control without enzyme. The turbid solution was then centrifuged at 25° C. and 4000 rpm in a large-capacity centrifuge for 10 minutes. The clear supernatant was adjusted to pH 7.0 with 1 M NaOH. After approximately 6 hours at room temperature, the slightly lowered pH was re-titrated to 7.0 and the solution was sterile filtered. The soluble oligomer was stored at 4° C. until use.

[0121] In order to further use, this soluble oligomer is added in the 1.5mL reaction vessel, add 20 μ L DMF and 150 μ L buffer (100mM sodium phosphate buffer, be adjusted to pH 6.0 to pH 8.0 for the best buffer for carbamate hydrolase separately) to it.Then add 30 μ L undiluted and purified carbamate hydrolase separately, and shake the mixture on heating block at 30 ℃ and 1000rpm.The mixture containing enzyme storage buffer is used as negative control.After three days, the mixture is filtered by the filter plate (Corning, Kaiserslautern) with PVDF membrane and aperture of 0.2 μ m, and use " dimethylaminophenylazobenzenesulfonamide 95 " method to check the formed toluene 2,4- and 2,6-diisocyanate in the filtrate by HPLC.

[0122] Compared to the negative control without enzyme, it was already macroscopically apparent after the reaction in the mixture containing CalB lyophilizate that the foam had lost its structure and was present as a turbid suspension containing small foam particles. The buffer, which was almost completely absorbed by the foam at the beginning of the experiment, later contained the entire foam mass in the form of disintegrated particles. HPLC analysis showed distinct peaks attributable to the oligomers formed, but no peak indicating the formation of toluenediamine (TDA) (data not shown).

[0123] All expressed carbamate hydrolases and SEQ ID No. 12 were added to the oligomer solution and then examined for TDA formation by HPLC. This was demonstrated for mixtures containing SEQ ID No. 7 and SEQ ID No. 3, where approximately the same amount of 2,6-TDA was measured in both mixtures. However, 2,4-TDA formation was significantly more pronounced in the mixture containing Enz03. Sequence ID No. 3 formed 0.057 g / L of 2,4-TDA and 0.025 g / L of 2,6-TDA, while Sequence 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. Furthermore, both enzymes showed a shift in the oligomer peaks and a general decrease compared to the other mixtures. In the case of SEQ ID No. 7, TDA could be cleaved from the polyester PU foam without prior pretreatment, whereas in the case of SEQ ID No. 3, this was only possible by providing neutralized oligomers after prior ester cleavage. The fact that the peaks identified as oligomers, which were derived from the hydrolysis product of SEQ ID No. 12, were significantly reduced after further treatment with a carbamate hydrolase (where TDA was clearly formed), confirmed that these peaks were oligomers.

[0124] It has also been shown that insoluble TDI-based polyester polyurethane foams can be broken down into their monomers via a combination of two reaction steps. In the first step, the PU foam is predigested by hydrolysis of ester bonds using the lipase CalB. After neutralization, the released oligomers serve as substrates for an overexpressed carbamate hydrolase. Here, the carbamate bonds are hydrolyzed, and monomeric TDA can be detected.

[0125] Finally, it could be shown that the combination of hydrolytic cleavage of the ester bonds by means of lipases, neutralization of the oligomer solution and subsequent hydrolytic cleavage of the urethane bonds allows complete decomposition of the polyurethanes into the specific monomers.

Claims

1. A method for decomposing polyester polyurethane into low molecular weight decomposition products, comprising the following steps: a) cleaving ester groups contained in the polyester polyurethane; and b) cleaving the carbamate groups contained in the polyester polyurethane with a polypeptide having carbamate hydrolase activity; Provided that process steps a) and b) can be performed in any order or simultaneously, The polypeptide having carbamate hydrolase activity consists of the amino acid sequence SEQ ID No.

3.

2. The method according to claim 1, wherein method step a) is carried out before method step b).

3. The process as claimed in claim 1, wherein at least one polyamine selected from the group consisting of toluene-2,4-diamine and toluene-2,6-diamine is formed as a process product.

4. The process according to any one of claims 1 to 3, wherein process step a) is carried out with a lipase.

5. The method of claim 4, wherein the lipase consists of the amino acid sequence in SEQ ID No. 11 or SEQ ID No.

12.

6. Use of a polypeptide consisting of the amino acid sequence SEQ ID No. 3, characterized in that: The polypeptide is used for enzymatic cleavage of carbamate bonds.

7. The use according to claim 6, wherein the urethane bond is aromatic.

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